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Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

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Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

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Content CHARACTERIZATION OF A FRAGMENT IN SECRETED HSP90α WITH
POTENTIAL THERAPEUTIC BENEFITS IN WOUND HEALING AND CANCER

by

Divya Sahu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)

August 2013

Copyright 2013 Divya Sahu

ii
EPIGRAPH

“Great scientists don’t do different things; they do things differently.”
-- Modified Anonymous saying

iii
DEDICATION

I dedicate this work to my parents who have been pillars to me in all my endeavours and
who always inspired me to put in my best. Without the sacrifices made by them, I would
not have been able to pursue a career in science.

iv
ACKNOWLEDGEMENTS

I would like to thank the following people who made the work in this thesis
possible:
Dr. Wei Li, my PI, for his invaluable guidance and support throughout the course
of this work. He taught me how to work and think scientifically and emphasized attention
to the finer details, which make all the difference between success and failure. Working
with him trained me not only in how to conduct research, but also to be mentally strong
and never give up.
Dr. Mei Chen, who is indispensable for the smooth running of both Li and Chen
labs. Her scientific input and practical approach has been invaluable during my research.
Drs. Zhengwei Zhao, Cheih-Fang Cheng and Jianhua Fan, for their mentoring
during my initial days in the lab as well as support and help during this work.
My fellow graduate students Dr. Arum Han, Priyamvada Jayaprakash, Katheryn
O’Brien, Ayesha Bhatia and Fred Tsen as well as Xinyi Wang, Yingping Hou and Dr.
Brian Hwang from Chen Lab who were all great colleagues.
My past and present committee members, Drs. Michael Stallcup, Agnieszka
Kobielak, Gerhard Coetzee and Young Kong Hong, for their support and feedback.
My scientific collaborators, Dr.Susan Ou, Dr. Jennifer Isaacs, Michelle Macveigh,
Alan Situ and Daniel Gerke, for all their assistance.
My friends and family, especially my parents, brother and Shikhar, for their
unyielding support and encouragement throughout this process.

v

TABLE OF CONTENTS

Epigraph ii
Dedication iii

Acknowledgements iv

List of Figures vii

Abstract x

Chapter 1: Secreted Heat Shock Protein 90α in Wound Healing and Cancer 1
Introduction 1
eHsp90, secreted by living cells or leaked by dead cells? 3
Induced secretion for normal cells and constitutive secretion for tumor 6
cells
Secretion of Hsp90α via non-classical exosomal protein secretory
Pathway 8
eHsp90 does not need ATPase for function 9
Downstream targets of eHsp90 12
Is eHsp90 a design of Mother Nature? 15
How was eHsp90 connected to wound healing? 20
How was eHsp90 connected to cancer and to what types of cancer? 22
Is eHsp90 a more effective and less toxic target than intracellular Hsp90
for treatment of the tumors? 25
Wound healing and tumor progression: similar strategy used by
peacemaker and terrorist 26
Conclusion and Perspective 29

Chapter 2: A Fragment of Secreted Hsp90α Carries Properties that Enable it
to Effectively Accelerate both Acute and Diabetic Wound Healing
in Mice 30
Introduction 30
Materials and Methods 35
Results 41
Identification of a minimum promotility epitope in secreted
Hsp90α, within its linker region and middle domain 41
F-5 fragment of Hsp90α accelerates acute wound closure more
Strongly than becaplermin/PDGF-BB in mice 44
F-5 is superior to becaplermin gel/PDGF-BB in promoting diabetic

vi
wound healing 48
Three unique properties of F-5 — a common motility factor, a
TGF-β– resistant factor, and a hyperglycemia-resistant factor —
and all absent from conventional growth factors 52
Discussion 57

Chapter 3: A Potenially Common Peptide Target in Secreted Heat Shock
Protein 90α for Hypoxia Inducible Factor-1α positive tumors 63
Introduction 63
Materials and Methods 68
Results 75
Constitutively expressed HIF-1α is essential for invasiveness of
breast cancer cells 75
Deregulated HIF-1α causes constitutive Hsp90α secretion 80
Identification of the key element in secreted Hsp90α that mediates
deregulated HIF-1α–driven invasion 87
Interruption of Hsp90α–LRP-1 signaling in MDA-MB-231 cells
blocks their ability for lung colonization and tumor formation 90
Regulationtion of Hsp90α secretion in MDA-MB-231 cells by
Hif-1α 97
Discussion 100

Chapter 4: Target Validation of Role of Secreted Hsp90α in Wound Healing
and Cancer 106
Introduction 106
Materials and Methods 111
Results 114
Confirmation of cell impermeability of DMAG-N-oxide 114
NPGA inhibits secreted Hsp90α mediated skin cell migration
in vitro 114
NPGA delays wound healing in mice 119
F-5 maintains its native structure in Hsp90α 123
Preparation of F-5 antigen for immunization 123
Screening the tail bleeds from F-5 immunized mice 125
Discussion 132

Chapter 5: Summary and Conclusions 136

References 146

vii
LIST OF FIGURES

Figure 1.1 A schematic distinction of the functional elements for intracellular
vs. extracellular Hsp90α 11

Figure 1.2 eHsp90α-driven, not growth factor-driven, cell migration
overrides TGFβ inhibition 18

Figure 2.1 Expression of different recombinant Hsp90α proteins/peptides
(wild type and mutants) 42

Figure 2.2 F-5 peptide retains the full promotility activity of full-length
Hsp90α 43

Figure 2.3 F-5 is superior to FDA-approved becaplermin/PDGF-BB in
acute wound healing 46

Figure 2.4 Measurements of F-5 versus becaplermin in acute wound healing 47

Figure 2.5 F-5 promotes reepithelialization of the wound 49

Figure 2.6 F-5 is superior to becaplermin/PDGF-BB in recruiting dermal cells
in diabetic wound healing 51

Figure 2.7 F-5 is a common promotility factor and overrides inhibition by
TGF-β 53

Figure 2.8 F-5 rescues cell migration under hyperglycemia 56

Figure 3.1 Breast cancer cells have deregulated HIF-1α 76

Figure 3.2 HIF-1α and HIF-1β expression is required for breast cancer cell
migration 78

Figure 3.3 Down-regulation of HIF-1α or HIF-1β inhibits MDA-MB-231
cell invasion 79

Figure 3.4 Deregulated HIF-1α uses secreted Hsp90α for migration and
invasion 81

Figure 3.5 Expression of exogenous HIF-1α in HIF-1α-depleted
MDA-MB-231 cells rescues their cell migration and invasion 83

viii
Figure 3.6 Secreted Hsp90α is essential for migration of MDA-MB-231
cells under serum-free conditions 85

Figure 3.7 Secreted Hsp90α is essential for invasion of MDA-MB-231 cells
under serum-free conditions 86

Figure 3.8 Expression of various recombinant Hsp90α peptides that retain
either a full or partial promotility activity of full-length Hsp90α 88

Figure 3.9 F-5 epitope in secreted Hsp90α rescues invasion defect of
HIF-1α–down-regulated MDA-MB-231 cells 89

Figure 3.10 F-5 epitope in secreted Hsp90α rescues invasion defect of
HIF-1β–down-regulated MDA-MB-231 cells 91

Figure 3.11 LRP-1 mediates is essential for secreted Hsp90α-induced
invasion of MDA-MB-231 cells 92

Figure 3.12 α2-macroglobulin does not affect Hsp90α rescue of invasion
defect of HIF-1α–down-regulated MDA-MB-231 cells 93

Figure 3.13 Hsp90α signaling is essential for MDA-MB-231 cell lung
colonization and tumor formation in vivo. 95

Figure 3.14 Construction of HIF-1α mutants for studying effect on Hsp90α 99
secretion

Figure 3.15 A model of secreted Hsp90α as a potential target for
HIF-1α–positive cancers 102

Figure 4.1 NPGA is a non-permeable inhibitor of Hsp90α 115

Figure 4.2 NPGA inhibits Hsp90α, but not growth factor induced migration 117

Figure 4.3 NPGA inhibits hypoxia induced migration 118

Figure 4.4 NPGA delays wound healing 121

Figure 4.5 NPGA delays wound healing by retarding re-epithelialization 122

Figure 4.6 F-5 retains its native structure 124

Figure 4.7 Preparation of F-5 antigen for immunization 126

ix

Figure 4.8 F-5 immunized mice serum has primarily IgG antibodies 128

Figure 4.9 F-5 immunized mice serum has Hsp90β cross-reactivity 129

Figure 4.10 F-5 immunized mice serum recognizes F- 5 by Western Blot 130

Figure 4.11 F-5 immunized mice serum inhibits hypoxia induced migration 131

x
ABSTRACT

Heat shock protein 90alpha (Hsp90α) is an abundant molecular chaperone. We
have recently shown that normal cells under stresses such as hypoxia secrete Hsp90α
which promotes migration, signaling through LDL receptor-related protein-1 (LRP-1). In
this study, we investigated the mechanism behind role of secreted Hsp90a in wound
healing and cancer.
Using systematic mutagenesis, we identified the minimum functional element in
Hsp90α, F-5, required for cell migration. Topical application of F-5 enhances closure of
acute and diabetic wounds in mice more effectively than PDGF-BB, the only FDA-
approved therapy. The superior efficacy of F-5 is due to its ability to 1) recruit both
epidermal and dermal cells through universally expressed LRP-1 receptor, 2) override
inhibition of dermal cell migration by TGFβ and 3) override anti-migration effects of
hyperglycemia. Therefore, this study challenges the long-standing paradigm for
developing growth factor-based therapies and identifies a novel wound-healing agent.
Like wounds, hypoxia is common in cancer and ~40% of solid tumors
overexpress hypoxia-inducible factor-1alpha (Hif-1α). We show that lentiviral knock-
down of Hif-1α blocks Hsp90α secretion and invasion/migration of breast cancer cells
while reintroducing active Hif-1α rescues these effects. Neutralization of secreted
Hsp90α by inhibitor or antibody or knock-down of LRP-1 reduces tumor cell invasion in
vitro and tumor formation in nude mice. Introduction of Hsp90α F-5 bypasses the Hif-1α
depletion blockade and rescues cancer cell invasion. Since normal cells do not secrete

xi
Hsp90α under physiological conditions, our data suggest that drugs targeting secreted
Hsp90α F-5 region should be more effective and less toxic for treatment of Hif-1α
positive tumors.
In light of the clinical relevance of extracellular Hsp90α, we conducted further
target validation studies. We used a non permeable inhibitor of Hsp90α, DMAG-N-oxide,
to show that secreted Hsp90α is important for the wound healing process. We are also
developing a monoclonal antibody against the F-5 region of Hsp90α. It would be useful
in validating the role of pro-motility effect of Hsp90α on wound healing and cancer. It
can potentially be further developed into an anti cancer therapeutic agent.

1
CHAPTER 1

SECRETED HEAT SHOCK PROTEIN-90 (HSP90) IN
WOUND HEALING AND CANCER

INTRODUCTION
Since the discovery of the first “growth factor” in the 70s, it has become widely
believed that local growth factors are the driving force for wound healing, i.e. the lateral
migration and proliferation of epidermal keratinocytes over the wound bed to close the
wound and the inward migration and growth of dermal fibroblasts and microvascular
endothelial cells into the wound bed to remodel the damaged tissue and to build a new
vascularized neodermis (Martin, 1997; Singer and Clark, 1999; Werner and Grose, 2003).
However, after two decades of extensive studies and clinical trials on a handful of growth
factors alone or in combination (Grose and Werner, 2004; Werner and Grose, 2003), only
recombinant human platelet-derived growth factor-BB (PDGF-BB) has received US FDA
approval for topical treatment of diabetic ulcers (Regranex/becaplermin gel, 0.01%,
Ortho-McNeil Pharmaceutical, Raritan, NJ) (LeGrand, 1998; Steed, 1995; Wieman et al.,
1998). Since then, however, its modest efficacy, high cost and risk of causing cancer in
patients (who receive three tubes or more of the treatment) have limited the use of PDGF-
BB in clinical practice (Mandracchia et al., 2001; Nagai and Embil, 2002). While these
rather unexpected outcomes have clearly implicated that conventional growth factors are
not the critical force of wound closure as they were hoped for, this disappointing reality

2
has been continuously overlooked or simply ignored. In 2007, Li and colleagues
proposed that the factor that is primarily responsible for promoting the initial wound
closure comes from secretion of the stressed skin cells at thewound edge. Fromthe
secreted proteins of primary human keratinocytes and human dermal fibroblasts, these
authors found that secreted Hsp90α promotes wound closure in mice farmore strongly
than the becaplermin gel (Cheng et al., 2008; Cheng et al., 2011; Li et al., 2007). Why is
secreted Hsp90α chosen? Is secreted Hsp90α, instead of growth factors, responsible for
the initial wound closure? No one would have imagined and believed this.
Going back a decade, Csermely and colleagues raised a question “Why do we
need constitutively so much of Hsp90 (in a cell)?” They argued that the major cellular
function of Hsp90 is not entirely as an intracellular chaperone. Instead, it is due to some
other unrecognized functions that would require such a large amount of Hsp90 protein
stored in the cell (Csermely et al., 1998). In the late 1970s, a number of laboratories have
repeatedly reported expression of Hsp90 on the cell surface, either as a tumor antigen or a
protein that assists antigen presentation to antigen-presenting cell (Cheng et al., 2010;
Csermely et al., 1998; Tsutsumi and Neckers, 2007). Then, secreted form of Hsp90 was
reported to cause activation of ERK1/2 in rat vascular smooth muscle cells (Liao et al.,
2000) and stimulation of growth in lymphoid cells (Kuroita et al., 1992). However,
results of these earlier studies on extracellular Hsp90 were largely viewed as
experimental artifacts for good reasons (see later sections). Since 2004, new evidence has
started to accumulate and begun to shed light on the Csermely's prediction. These new
studies argue that the purpose for the steady-state storage of such a large amount of

3
Hsp90 in cells by Mother Nature is for normal cells to launch a rapid protective response
to environmental insults, including heat, hypoxia, UV, gamma-irradiation, reactive
oxygen species (ROS), injury-released growth factors (Cheng et al., 2010). Tumor cells
also recognize the usefulness of secreted Hsp90 in tissue invasion and metastasis
(Schmitt et al., 2007; Tsutsumi and Neckers, 2007). In this review, we provide an
updated analysis of extracellular Hsp90, focusing on its role in skinwound healing and
cancer progression.We are not going to discuss the antigen-presenting role of cell-
released Hsp90, which has been well covered in recent reviews (Basu and Srivastava,
2000; Binder et al., 2004).

eHsp90, secreted by living cells or leaked by dead cells?
Although the first observation was made three decades ago, the notion of
“surface-bound” and “secreted” Hsp90 by living cells has only recently gained traction in
the field with specific attributions of a number of review articles by experts then and now
(Cheng et al., 2010; Csermely et al., 1998; Schmitt et al., 2007; Tsutsumi and Neckers,
2007). Even though, skepticisms remain among many others as whether these
extracellular Hsp90 proteins are results of pathophysiological processes or of a leak from
a small number of dead cells in culture. Multhoff and Hightower tried to address this
critical issue 15 years ago with a number of Hsp and non-Hsp examples in the studies that
used various pharmacological approaches and argued that eHsp90 was no artifact
(Multhoff and Hightower, 1996). The primary reason for this skepticism is that Hsp90
does not fit into any of the conventional categories of the actively secreted proteins, such

4
as growth factors, extracellular matrices (ECMs) and matrix metaloproteinases (MMPs).
First, Hsp90 has neither any signal peptide (SP) for secretion via the endoplasmic
reticulum (ER)/Golgi protein secretory pathway nor a recognizable transmembrane
sequence for membrane anchoring. Second, there had already been reports that Hsp90
could be released to extracellular environment following cell necrosis (Cheng et al.,
2010). In the latter case, the released Hsp90 binds and helps antigen recognition and
triggers innate immune responses. Therefore, it was arguable that eHsp90 found in
conditioned media of cultured cells may come from a small portion of dead cells
(Csermely et al., 1998). Recent studies by independent groups have provided stronger
arguments that the cells secrete Hsp90 for purpose. First, quiescent normal cells do not
secrete Hsp90 (Cheng et al., 2008; Li et al., 2007; Liao et al., 2000). However, many
pathological and stress cues trigger normal cells to secrete Hsp90, including reactive
oxygen species (ROS) (Tsutsumi and Neckers, 2007), heat (Clayton et al., 2005;
Hightower and Guidon, 1989), hypoxia (Li et al., 2007), gammairradiation (Yu et al.,
2006) and tissue injury-released cytokines, such as TGFα (Cheng et al., 2008). For
instance, TGFα is low or undetectable in intact skin and only appears in the wound
following skin injury. TGFα is known to increase cell survival and cell number rather
than causing cell death in human keratinocytes. Interestingly, Cheng et al. showed that
TGFα stimulation causes rapid membrane translocation and secretion of both endogenous
and an exogenously expressed GFP-tagged Hsp90α with defect in ATPase (Cheng et al.,
2008). More specifically, Tsutsumi and colleagues reported that a conserved hydrophobic
motif in a beta-strand at the boundary between the N-terminal domain and charged linker

5
of Hsp90 is required for Hsp90 secretion (Tsutsumi et al., 2009). Wang et al. showed that
a C-terminal EEVDmotif and the Thr-90 phosphorylation both play a regulatory role
inHsp90 secretion (Wang et al., 2009). On one hand, these findings strongly support the
notion that Hsp90 secretion is a regulated process. On the other hand, it is hard to
imaginewhy Hsp90 needs multiple distinct sequence motifs across the entire protein for
regulation of its secretion, not to mention how these motifs relate to the most reported
exosome trafficking pathway for secretion of Hsp90 and other Hsp proteins (see later
section). More on regulation of Hsp90 secretion, Li and colleagues identified a key
upstream regulator of Hsp90α secretion, the hypoxia-inducible factor-1alpha (HIF-1α) in
human dermal fibroblasts and keratinocytes (Li et al., 2007; Woodley et al., 2009).
Dominant negative mutant of HIF-1α (DN-HIF-1α) blocks the secretion, whereas a
constitutively active mutant of HIF-1α (CA-HIF-1α) makes the cells to secrete Hsp90α
even under normoxia (Woodley et al., 2009). The same mechanism appears to take place
in tumor cells. Depletion of the constitutively expressed HIF-1α or HIF-1β from breast
cancer cells, MDA-MB-23, completely blocked the secretion of Hsp90α, which could be
rescued by exogenously re-introducing the CA-HIF-1α, but not DN-HIF-1α (Sahu et al.).
Despite the above remarkable findings, direct support for releasing Hsp90 on purpose by
living cells would require additional studies on the detailed routes of its membrane
translocation and secretion in response to environmental cues.

6
Induced secretion for normal cells and constitutive secretion for tumor cells
Normal cells do not secrete Hsp90 unless being triggered by environmental
insults. Hightower and Guidon reported first that heat-shocked rat embryonic cells secrete
Hsp90 and Hsp70. This secretion could not be blocked by monensin or colchicine, two
inhibitors of the conventional ER/Golgi protein secretory pathway (Hightower and
Guidon, 1989). Clayton and colleagues used proteomic approach to analyze the peptide
contents of B cell-secreted proteins under either physiological temperature (37 °C) or
heat shock (44 °C for 3h). They found that heat shock induced Hsp90α to go into the
nano-vesicles called exosomes and then be secreted outside the cells (Clayton et al.,
2005). Liao et al. reported that treatment of rat vascular smooth muscle cells with
LY83583, an oxidative stress generator, caused secretion of Hsp90α. The eHsp90α in
turn induced a late phase activation of the ERK1/2 pathway (Liao et al., 2000). Yu and
colleagues found that γ irradiation induced secretion ofHsp90β, butnotHsp90α, in a p53-
dependent fashion via exosomes, proposing a “DNA damageNp53NHsp90β secretion”
pathway (Yu et al., 2006). Cheng et al. showed that TGFα-induced Hsp90α membrane
translocation and secretion to culture medium in primary human keratinocytes were
sensitive to inhibitors of the exosome protein trafficking, but not the conventional
ER/Golgi protein trafficking, pathway (Cheng et al., 2008). Finally, Li and colleagues
showed that hypoxia (1% O2) induced Hsp90α secretion via HIF-1α. Blockade of
eHsp90α function by neutralizing antibodies completely inhibited hypoxia-induced cell
motility (Li et al., 2007). In particular, the identification of HIF-1α as a key
upstreamregulator of Hsp90α secretion has an important implication in cancer. Hypoxia

7
is a known micro-environmental stress that is connected to the growth, invasion, and
metastasis of many solid tumors (Simon and Keith, 2008). Under constant hypoxia,
cancer cells are forced to adapt, via HIF-1α, alternative and selfsupporting mechanisms
for continued survival and expansion. Overexpression of HIF-1α has been estimated to
occur in approximately 40% of the tumors in humans (Semenza, 2007a). Therefore,
surface expression and/or secretion of Hsp90α should become constitutive in those HIF-
1α-overexpressing tumors.While this prediction remains to be formally tested, many
tumor cell lines have been reported to secrete Hsp90. Kuroita and colleagues reported
purification of Hsp90α from conditioned media of human hybridoma SH-76 cells
(Kuroita et al., 1992). Eustace et al. reported Hsp90α, but not Hsp90β, in conditioned
media of HT-1080 tumor cells (Eustace et al., 2004). Wang et al. reported secretion of
Hsp90α by MCF-7 human breast cells (Wang et al., 2009). Suzuki and Kulkarni found
Hsp90β secreted by MG63 osteosarcoma cells (Suzuki and Kulkarni) [45]. Chen and
colleagues reported secretion of Hsp90αby colorectal cancer cell line, HCT-8 (Chen et
al., 2010). Work by Tsutsumi and colleagues implied secretion of Hsp90α by a variety of
tumor cell lines (Tsutsumi et al., 2008). Finally, recent study from our laboratory
demonstrated that breast cancer cells, MDA-MB-231 and MDA-MB-468, overexpress
HIF-1α that causes constitutive secretion of Hsp90α in a HIF-1-dependent fashion (Sahu
et al.). Fig. 1 summarizes what triggers Hsp90α secretion in normal cells versus tumor
cells, in which HIF-1α is a central regulator. However, the signaling steps between HIF-
1α and Hsp90α secretion machinery (such as exosomes) remain entirely unknown.

8
Secretion of Hsp90α via non-classical exosomal protein secretory pathway
How does Hsp90 travel through the cell membrane? There are several cellular
protein trafficking machineries. First, the classical ER/Golgi protein secretory pathway
requires the to-be-secreted protein to have a 15–30 amino acid signal peptide (SP) at its
amino terminus and to use it as the “permit” for going out of the cell. The second protein
secretory pathway ismediated by secreted nano-vesicles, called exosomes, which are used
for secreting proteins that do not have any SP sequences. Exosomes, also called
‘intraluminal vesicles’ (ILVs), are non-plasma-membrane-derived vesicles that are 30–90
nm in diameter and initially contained within the multivesicular bodies (MVB). A well
known function of MVB is to serve as an intermediate station during degradation of the
proteins internalized from the cell surface or sorted from the trans Golgi organelle
(Denzer et al., 2000; Thery et al., 2002). However, theMVB-derived exosomes can also
fuse with the plasma membrane to release their cargo proteins into the extracellular
space. This release process was reported to include 1) sorting into smaller vesicles; 2)
fusing with the cell membrane; and 3) release of the vesicle to the extracellular space. All
the proteins that have been identified in exosomes are located in the cell cytosol or
endosomal compartments, but never in the ER, Golgi apparatus, mitochondria or nucleus.
Making use of two chemical inhibitors, brefeldin A (BFA) that selectively blocks the
classical ER/Golgi protein secretory pathway and dimethyl amiloride (DMA) that blocks
the exosome protein secretory pathway, several groups showed that DMA selectively
inhibits the membrane translocation and secretion of Hsp90α or Hsp90β by various types
of cells (Cheng et al., 2008; Hegmans et al., 2004; Lancaster and Febbraio, 2005; Mignot

9
et al., 2006; Savina et al., 2003). In the same experiments, BFA had little inhibition of
Hsp90 secretion (Cheng et al., 2008). There have been no reports that BFA or DMA
causes more or less cell death. An unanswered question, however, is how stress signals,
such as HIF-1α, are connected to this novel protein secretory pathway and what are the
relationships between the reported secretion motifs in Hsp90 and exosomes. However,
once Hsp90α proteins are secreted outside the cells, they appear to be “naked”, instead of
continuing being wrapped in the exosomes, since neutralizing anti-Hsp90α antibodies
were able to completely block their actions (Cheng et al., 2008; Li et al., 2007; Sahu et
al.; Woodley et al., 2009).

eHsp90 does not need ATPase for function
Eustace and colleagues studied the role of eHsp90α found in conditioned media of
HT-1080 fibrosarcoma cells by using two approaches. First, they used the fluorophore-
assisted light inactivation (FALI) technique, in which fluorophore fluorescein induces
antibody-coupled FITC to generate short-lived hydroxyl radicals that cause damage of
proteins within 40 Å. Therefore, the damage is often specific on the antibodybound
protein, i.e. Hsp90α, but not surrounding proteins. This technique damages the whole
molecule. Second, they used DMAG-N-oxide, a geldanamycin/ 17-AAG-derived but
cell-impermeable Hsp90 inhibitor that targets the Hsp90's ATPase activity. They reported
an importance of eHsp90α in the tumor cell invasion (Eustace et al., 2004). Tsutsumi and
colleagues also tested the effect of DMAG-N-oxide on invasion of several cancer cell
lines in vitro and lung colonization by melanoma cells in mice and reported inhibition of

10
cell invasion in vitro and/or tumor formation in vivo by DMAG-N-oxide (Tsutsumi et al.,
2008). The results of these studies suggested that the N′-terminal ATP-binding and
ATPase of Hsp90α are still required for function of eHsp90α outside the cells. Focusing
on the pro-motility activity of eHsp90α on primary human skin cells, Cheng and
colleagues undertook mutagenesis approach to address the same issue. First, they
compared recombinant proteins of the wild type and E47A, E47D, and D93N mutants of
Hsp90α for pro-motility activity on human keratinocytes. As previously reported,
Hsp90α-wt has a full ATPase activity, Hsp90α-E47D mutant loses half of the ATPase
activity, whereas Hsp90α-E47A and Hsp90α-D93Nmutants lose the entire ATPase
activity (Young et al., 2001). Cheng et al. found that all the ATPase mutant proteins
retained a similar pro-motility activity as theHsp90α-wt. Second, they used sequential
deletion mutagenesis to narrow down the pro-motility domain to a region between the
linker region (LR) and the middle (M) domain of human Hsp90α (Cheng et al., 2008).
Finally, their latest study has identified a 115-amino acid fragment, called F-5 (aa-236 to
aa-350), that promotes skin cellmigration in vitro andwound healing in vivo as effectively
as the full-length Hsp90α-wt (Cheng et al., 2011). Collectively, these findings
demonstrate that the N-terminal ATPase domain and the C-terminal dimer-forming and
co-factor-binding domain are dispensable for eHsp90α to promote cell migration. A
schematic representation of the structure and function requirements for intracellular
Hsp90α and eHsp90α is shown in Fig. 1.1A. It should be pointed out that stimulation of
cell migration might not be the only function reported for eHsp90. The 115 amino acid

11

Figure 1.1 A schematic distinction of the functional elements for intracellular vs.
extracellular Hsp90β. (A) The intracellular chaperone function of Hsp90 requires almost
the entire molecule, especially the amino terminal (green) and the carboxyl terminal
(blue) domains. The extracellular pro-motility function of Hsp90α depends on less than a
115 amino acid fragment (F-5) located at the boundary between the LR and the M
domains. This epitope appears at the surface of Hsp90 protein [ref. [13]]. (B) F-5 is
highly conserved during evolution of Hsp90 genes. Green, identify; Yellow: similarity;
Red, distinction.

12
sequence of F-5 is highly conserved during evolution, as shown in Fig. 1.1B. However,
no more than 20% identity of F-5 was found in other Hsp family genes. On the other
hand, the observations made by Eustace, Tsutsumi and their colleagues were
independently confirmed by studies of others. Cheng and colleagues in collaboration with
Isaacs's group verified that the DMAG-N-oxide inhibitor could indeed block the full-
length Hsp90α-stimulated human skin cell migration. However, as expected, DMAG-N-
oxide showed little inhibition of the F-5 peptide-induced cell migration [Cheng, C-F, J.
Isaacs andW. Li, unpublished] or migration induced by the middle domain of eHsp90α
(Cheng et al., 2008; Gopal et al., 2011).While the reason for the apparent discrepancy
remains unknown,we suggest that binding of DMAG-N-oxide to the N′-terminal ATPase
domain of the full-length eHsp90α may cause a conformational change in eHsp90α, so
that the real functional epitope within eHsp90α, i.e. the F-5 region, becomes
cryptic.While this hypothesis remains to be tested, it can be concluded that eHsp90 is no
chaperone.

Downstream targets of eHsp90
How eHsp90α promotes cell migration has just begun to be appreciated. Eustace
and colleagues reported that Hsp90α, but not Hsp90β, promotes cancer cell migration and
invasion by binding and activating thematrixmetalloproteinase 2 (MMP2) (Eustace et al.,
2004). Two independent groups have recently provided additional support for this
observation (Sidera et al., 2004; Song et al., 2010; Stellas et al., 2010) and, furthermore,
showed that the M domain (aa-272 to aa-617) of Hsp90α is responsible for the activation

13
(Song et al., 2010). Following their identification of eHsp90α in conditioned media of
human skin cells in 2007 (Li et al., 2007), Li and colleagues have also been attempting to
identify a target that is essential for hypoxia- and eHsp90α-stimulated human skin
cellmigration. First, they wanted to verify the involvement of MMP2 or any other MMPs
by utilizing two broad MMP inhibitors, GM6001 (N-[(2R)-2-
(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide, or
Galardin) and MMP Inhibitor III (hydroxamido-carbonylmethyl)-4-methylpentanoyl-L-
tryptophan. However, they found that presence of either GM6001 orMMP inhibitor III
showed little inhibitory effect on human recombinant Hsp90α-stimulated human
keratinocyte migration (Cheng et al., 2008). Whether this discrepancy is due to
differences in cellular contexts between the normal and the tumor cells remains unclear.
Besides MMP2, Sidera and colleagues showed that a pool of cell membrane-bound
Hsp90α interacts with the HER-2 tyrosine kinase receptor in breast cancer cells, leading
to increased cell motility and invasion (Sidera et al., 2008; Sidera et al., 2004; Stellas et
al., 2010). Suzuki and Kulkarni reported that eHsp90β blocks the conversion from latent
TGFβ to its active form, leading to decreased tumor suppressing effect of TGFβ (Suzuki
and Kulkarni). McCready et al. reported that eHsp90αexogenously delivered by added
exosomes participated in plasminogen activation and tumor cell migration (McCready et
al.). Moreover, Chung et al. showed that eHsp90 secreted by stressed vascular SMCs
regulates transcription of IL-8 gene (Chung et al., 2009). While we are still distant from
understanding the mechanisms of action, these results at the least suggest that eHsp90 has
multiple downstream targets in distinct extracellular environment. Among all the studies

14
on eHsp90 targets since 2004, the investigation of LRP-1 as a cell surface receptor for
eHsp90α has gone into relatively more details and, therefore, is selected out here for
mentioning with a few extra sentences. Cheng et al. estimated that eHsp90α could readily
reach the optimal working concentrations of 0.05–0.1 μM that maximally stimulates cell
migration in vitro (Cheng et al., 2008). In their cell migration assays, human recombinant
Hsp90α exhibited a saturating and subsequently declined effect on human skin cells,
when increasing amounts of eHsp90α were added. This was an important observation that
suggests that eHsp90α acts by binding to a receptor-like molecule on the cell surface with
certain Km (50% of equilibrium) and Kd (dissociation constant) values. Then, Cheng et
al. used four independent approaches (neutralizing antibodies, RAP (LRP-1-associated
protein) inhibitor, RNAi, and somatic LRP-1 mutant cell line), to prove that the widely
expressed cell surface receptor, LRP-1, mediates the eHsp90α signaling to promote cell
migration. In vitro, GST-eHsp90 directly pulled down LRP-1 via its pro-motility
fragment between the LR and the M domain of Hsp90α (Cheng et al., 2008). More
convincingly, when these authors re-introduced an RNAi-resistant mini-LRP-1 cDNA
into the LRP1-downregulated cells, they were able to rescue the migration response of
the cells to either eHsp90α or hypoxia. These findings led the investigators to propose the
following working model: “HypoxiaNHIF-1αNHsp90α secretionNLRP-1Ncell motility”
autocrine pathway for tissue repair and tumor invasion and metastasis (Tsutsumi et al.,
2009), as previously shown in Fig. 1. However, it is equally important to point out the
complexity of LRP-1 receptor signaling. First, LRP-1 (also called CD91) is a large
heterotrimeric protein consisting of a 515-kDa extracellular domain and n 85-kDa trans-

15
membrane subunit (Herz and Strickland, 2001). Its expression was initially found in
monocytes, hepatocytes, fibroblasts, and keratinocytes (Kristensen et al., 1990; Strickland
et al., 1990; Van Leuven et al., 1993). Second, LRP-1 is a bona fide cell surface
receptor/co-receptor (with other receptors) for signal transduction across the membrane
(Herz and Strickland, 2001). However, the complexity of LRP-1 signal transduction
mainly comes from its large ligand binding repertoire. Besides gp96 and eHsp90, other
extracellular heat shock proteins that also bind LRP-1 include calreticulin, eHsp60 and
eHsp70 (Basu et al., 2001; Habich et al., 2002; Ogden et al., 2001; Vandivier et al.,
2002). In addition, opposite roles for LRP-1 signaling have been reported. For instance,
LRP-1 has been shown to play a critical role in PDGF-BB-stimulated ERK1/2 activation
and cell proliferation and also in TGFβ-stimulated anti-proliferation (Lillis et al., 2005;
Lillis et al., 2008). Cheng et al. reported that, while eHsp90α dramatically increased
cellmigration, human recombinant Hsp70, gp96 and calreticulin exhibited either a modest
or no stimulation of cell migration (Cheng et al., 2008). There is a clear need to identify
the specific binding site in LRP-1 for eHsp90α, in order to understand its mechanism of
action.

Is eHsp90 a design of Mother Nature?
Whenever the extracellular conditions become less than normal, such as heat shock, the
cells down-regulate overall protein synthesis and yet selectively up-regulate Hsp
expressions. This selective increase in chaperones has long been interpreted as helping
deal with protein folding and stability. In fact, there has been little evidence to support

16
this long-standing claim. Csermely and colleagues argued that evolution would not have
tolerated such an abundant storage of Hsp90 in the cells, if the function of Hsp90 had had
only been an intracellular chaperone (Csermely et al., 1998). Therefore, many suspected
that those “extra” Hsp90 serve the similar role as cytoskeletal proteins. Based on recent
studies on eHsp90, the $64,000 question is whether Mother Nature purposely designed
the increase in an already high level of intracellular Hsp90 to supply the “eHsp90 pool”
for the cells to deal with environmental stress. Currently, there has been limited
knowledge for or against this previously unthinkable possibility, either. The answer to the
above question for sure will not come in the near future. If we jump one step forward to
assume the “eHsp90 hypothesis” is correct, why had Mother Nature chosen Hsp90
(instead of the numerous known cell migration-promoting factors) for this job? What
unique qualifications was eHsp90 given by Mother Nature and those qualifications are
absent from those conventional pro-motility factors, such as growth factors. A possible
answer to these questions merged from a surprising finding of our group a few years ago.
It is known for long that TGFβ family cytokines block conventional growth factor-
stimulated proliferation in many cell types and migration in certain cell types. For
instance, Bandyopadhay et al. found that TGFβ3 blocks growth factor-stimulated human
dermal fibroblast and endothelial cell, but not human keratinocyte, migration
(Bandyopadhyay et al., 2006). This inhibition by TGFβ3 was believed to be a major
reason for why conventional growth factor therapy in wound healing has largely failed
(Cheng et al., 2011). However, our laboratory found that TGFβ3 was unable to inhibit
eHsp90α-induced migration of any type of human skin cells (Cheng et al., 2008). This

17
unique property of eHsp90α is schematically represented in Fig. 1.2. Such a superior
effect of eHsp90α over growth factors was also reflected in in vivo wound healing
studies. Topical application of recombinant Hsp90α proteins to diabetic mouse wounds
dramatically shortened the time of wound closure from35 days to ~18 days, significantly
stronger than becaplermin gel (PDGF-BB) treatment (Cheng et al., 2011). These authors
proposed that it is eHsp90α, but not the conventionally claimed growth factors (such as
PDGF-BB), that drives inward migration of the dermal cells into the wound against the
TGFβ inhibition. As previously emphasized, moving into the wound by these dermal
cells is essential for the wound remodeling and new blood vessel formation.
Similarly, cancer has also a love-and-hate relationship with TGFβ, which is
regarded as both tumor suppressor and tumor promoter (Bachman and Park, 2005).
Hanahan and Weinberg in their most cited review of 2000 organized the traits of cancer
into six distinct yet overlapping events; 1) self-sufficiency in growth signals; 2)
insensitivity to anti-growth signals; 3) tissue invasion and metastasis; 4) unlimited
proliferative potential; 5) sustained angiogenesis; and 6) preventing apoptosis (Hanahan
and Weinberg, 2000). They cited that one of the most recognized anti-growth signals is
TGFβ. TGFβ prevents an inactivating phosphorylation of the tumor suppressor gene
product Rb in cells, thereby blocking the cell's advance through the G1 phase of the cell
cycle. In this case, TGFβ induces increased gene expression of p15INK4B and p21,
which in turn inhibit cyclin:CDK complex's kinase activity responsible for the
inactivating phosphorylation of Rb. In certain cell types, TGFβ also suppresses
expression of the c-myc proto-oncogene, a positive regulator of the cell cycle

18

Figure 1.2 eHsp90α-driven, not growth factor-driven, cell migration overrides
TGFβ inhibition. (Upper part) Conventional growth factor-induced cell migration is
sensitive to the anti-motility signal of TGFβ,which is abundantly present inwounded
tissues or at tumor site. Unlessmutations take place along the TGFβ signaling pathway,
growth factors will not be able to be effective on the cells in vivo. (Lower part) eHsp90 is
the first ligand-like peptide that is able to continue promoting cellmigration in the
presence of TGF-beta. The mechanism of action remains to be studied. This finding
explains 1) why eHsp90 is more effective than growth factors in skin wound healing and
2) cancers without mutation in TGFβ signaling pathway progress.

19
progression. These inhibitory capabilities of TGFβ are obviously bad news for cancer
cells (Bachman and Park, 2005). To sabotage the tumor suppressing effect of TGFβ, a
number of tumors choose to mutate either the type II (TβRII) or type I (TβRI) TGFβ
receptor. Other tumors choose to eliminate downstreamsignalingmolecule, Smad4, which
forms complex with activated Smad2/3 to regulate gene expression in the nucleus. These
TGFβ pathway-mutated tumors include gastrointestinal and colorectal cancer
(Papadopoulos et al., 1995), gastric ovarian cancer (Chen et al., 2001; Pinto et al., 2003),
breast cancer (Papadopoulos et al., 1995) and pancreative cancer (Hahm et al., 2000;
Howe et al., 1998). Those alterations in TGFβ signaling pathway have presumably made
the cancer cells no longer sensitive to the anti-proliferation and anti-migration signals of
TGFβ. However, in many other human cancers, no mutations in TGFβ signaling
components are found. How could these cancers bypass the TGFβ's inhibitory signals?
There have been few studies that address this question. If we put the importance of HIF-1
in cancer and the recent finding of the “HIF-1NHsp90α secretionNLRP-1Ntumor cell
invasion” axis into perspective, one may extrapolate a possible answer to the puzzle from
the following facts: 1) Approximately 40% of all human tumors have constitutively
elevated expression ofHIF-1α, the critical subunit of the master transcription factor for
tissue oxygen homeostasis (Semenza, 2007a); 2) HIF-1α is a central controller of Hsp90α
secretion (Li et al., 2007; Woodley et al., 2009); and 3) eHsp90α is required for cancer
cell invasion in vitro (Wang et al.) and tumor formation in vivo (Sahu et al.; Stellas et al.,
2010; Tsutsumi et al., 2008). Therefore, it is conceivable that secretion ofHsp90αis an

20
alternative strategy for cancer cells to bypass the anti-motility of TGFβ without mutating
TGFβ signaling components.

How was eHsp90 connected to wound healing?
What is the natural driving force of skin wound healing? For decades, the
conventional wisdom has been that cell type-specific growth factors represent Mother
Nature's design for repairing wounds (Singer and Clark, 1999; Werner and Grose, 2003).
These growth factors often appear only when skin is wounded or their concentrations rise
significantly from a basal level in response to injury, such as TGFα and KGF (FGF7) for
keratinocytes, PDGF-BB for dermal fibroblasts and VEGF-A for dermal microvascular
endothelial cells. Based on this belief, more than 30 growth factors have been subjected
to extensive pre-clinical studies and/or clinical trials alone or in combinations (Grose and
Werner, 2004). Despite these enormous efforts, the in vivo functions for many of these
growth factors remained unconfirmed and their efficacy in human trials mostly fell short
of providing significant clinical benefits. Among them all, only recombinant PDGF-BB
received the FDA approval (becaplermin gel) for treatment of diabetic ulcers, as
previously mentioned. Subsequent studies found becaplermin gel haslow efficacy and
higher risks of causing cancer in patients, resulting in its declined use in clinical practice
(http://www.medicalnewstoday.com/releases/110442.php). This significant side-effect
may not be surprising to cancer researchers, since it was already known years before the
FDA approval of becaplermin gel that overexpression of PDGF-BB (c-sis) or autocrine
presence of its viral form, v-sis, will cause cell transformation and that yet the

21
recommended dosage of PDGF-BB in becaplermin gel is more than 1000 fold higher
than the range of the physiological PDGF-BB levels in human circulation. The reason for
this stern reality has never been investigated, even though it challenges whether
continued emphasis on growth factors is the right thing to do for wound healing. In 2006,
Badyopadhay and colleagues made a surprising observation that had ultimately led to the
discovery of an unconventional wound-healing factor, the eHsp90α. Having noticed that
fetal bovine serum (FBS) has been used to culture human cells for years, the researchers
argued that human cells are never in contact with FBS in reality and, instead, it is the
human serum that represents the main soluble environment in wounded skin. They
compared the effect of FBS versus human serum on migration of three major human skin
cell types, epidermal keratinocytes and dermal fibroblasts and microvascular endothelial
cells. They found that FBS non-discriminatively stimulated migration of all three types of
human skin cells, as expected. However, they were surprised to find that human serum
only promoted keratinocyte migration, whereas it halted migration of the two dermal cell
types. They further revealed that the blocking signal in human serum comes from TGFβ3
(not TGFβ1or TGFβ2) and the selective sensitivity of the human dermal cells to TGFβ3
is due to their 7–15 fold higher levels of TβIIR than that in epidermal keratinocytes
(Bandyopadhyay et al., 2006). Animportant message of this finding was that conventional
growth factors may not be able to induce proliferation and migration of dermal
fibroblasts and microvascular endothelial cells in vivo, as they do in vitro, due to
abundant presence of TGFβ3 in human serum. These authors speculated that this defect
in growth factors represents a reason for why the majority of the growth factor trials in

22
the past failed to show any promising efficacy. If it were not the action of growth factors,
what would be the factor that drives dermal cell migration against TGFβ3 inhibition in
the wound and where would it come from? The researchers reasoned that this critical
factor does not come from human serum, because the latter inhibits dermal cell
migration. Instead, they suggested that it comes from secretion of the migrating
keratinocytes in response to injury-generated stress signals, such as hypoxia, and factors
that promote keratinocyte migration, such as TGFα (Li et al., 2006; O'Toole et al., 1997).
More importantly, they proposed that this factor must satisfy the following two criteria:
1) being a common pro-motility factor for all three types of human skin cells and 2) being
able to override TGFβ3 inhibition. First, they detected a strong pro-motility activity in
serum-free conditioned medium of TGFα-stimulated keratinocytes and hypoxia-treated
dermal fibroblasts, which drives migration of all three types of human skin cells. In
subsequent 18 months, from 10 L of serum-free conditioned medium of primary human
keratinocytes, protein purification allowed them to discover eHsp90αresponsible for the
entire pro-motility activity in the conditioned medium (Cheng et al., 2008; Li et al.,
2007). That was a total surprise to these researchers.

How was eHsp90 connected to cancer and to what types of cancer?
Although eHsp90 was reported as a cell surface-bound tumor antigen back in
1970s, the first direct evidence for eHsp90 in tumor cell invasion in vitro and tumor
formation in nude mice came from two relatively recent studies. Both of these studies
used membrane impermeable 17-AAG inhibitors to block the action of eHsp90 and

23
reported for the first time requirement of eHsp90 for tumor progression (Eustace et al.,
2004; Tsutsumi et al., 2008). Stellas et al. used a mAb, 4C5, against Hsp90α to confirm
the importance of eHsp90 in breast cancer cell “deposits” in nude mice (Stellas et al.,
2010). Sahu and colleagues found that permanent down-regulation of the LRP-1 receptor
inMDA-MB-231 cells dramatically reduced lung colonization of the cells in nude mice
(Eustace et al., 2004). However, all these studies suffered from technical limitations that
should have made the investigators refrain from making any definitive conclusions, like
those they had had made. First, DMAG-N-oxide treated melanoma cells were reported to
have decreased lung colonization in vivo (Tsutsumi et al., 2008). However, it is hard to
understand how a single pre-treatment of the cells with the drug in vitro could have had
the reported long lasting effect after the cells were injected into mice. Second, Patsavoudi
and colleagues reported that mixing breast cancer cells with 4C5 in vitro prior to injection
into nude mice resulted in reduced lung deposits of the cells within hours following
injection (Stellas et al., 2010). Under these conditions, there would have been no way to
know whether the co-injected 4C5 could have worked by continuously binding and
neutralizing constantly secreted Hsp90α and Hsp90β by the tumor cells for the entire
period of the experiments. It would make more sense to inject and maintain a steady-state
amount of DMAG-Noxide or 4C5 in circulation, prior to injection with the tumor cells.
For DMAG-N-oxide, stability in the animals was cited as the reason for excluding
injection into blood. Third, while the data was convincing that breast cancer cells lacking
the LRP-1 receptor were unable to effectively form tumors in nude mice, the effect of
down-regulation of LRP-1 may not necessarily be due to specific blockade of eHsp90α

24
signaling. LRP-1 is known to bind a number of other ligands (Basu et al., 2001). Thus, to
prove the essential role of eHsp90, there is a need to develop more specific and stable
inhibitors against the pro-motility function of eHsp90. Nonetheless, it has come to the
clinically important question if tumors do or do not secreteHsp90. So far, at least one
critical regulator of Hsp90 secretion has been established. Li et al. showed that HIF-1α
mediates hypoxia-induced Hsp90α secretion in human dermal fibroblasts and
keratinocytes. Forced expression of a constitutively activated HIF-1α, CA-HIF-1α, was
sufficient to replace hypoxia to cause Hsp90α secretion (Li et al., 2007; Woodley et al.,
2009). This finding is relevant to cancer, since HIF-1α overexpression is associated with
increased patient mortality in approximately 40% of human solid tumors, independent of
other specific mechanisms (Semenza, 2007a). Taking breast cancer as an example, Dales
et al. carried out anti-HIF-1αimmunohistochemical assays on frozen sections of 745
breast cancer samples and found that the levels of HIF-1α expression correlated to poor
prognosis, lower overall survival and high metastasis risk among both node-negative and
node-positive patients (Dales et al., 2005). By using HIF-1α expression as a marker, it
was estimated that approximately 25–40% of all invasive breast cancer samples are
hypoxic, suggesting that HIF- 1α may be used as a broader marker for breast cancers
(Basu et al., 2001). Sahu et al. have recently shown that down-regulation of the
endogenous and constitutively expressed HIF-1α in breast cancer cell lines, MDA-MB-
231 and MDA-MB-468, completely blocked Hsp90α secretion, and the secretion could
be rescued by re-introducing RNAi-insensitive WT-HIF-1α and CA-HIF-1α, but not DN-
HIF-1α, genes (Sahu et al.). These data establish that HIF-1α is a crucial and direct

25
upstream regulator of Hsp90α secretion. If we extrapolate these findings and numbers on
HIF-1α and cancer progression, it suggests that eHsp90α plays an important role at least
in those HIF-1α-overexpressing (≥40%) tumors in humans.

Is eHsp90 a more effective and less toxic target than intracellular Hsp90 for
treatment of the tumors?
In many, but not all, tumor cells, Hsp90 has been found either quantitatively
overexpressed or qualitatively overactivated (with similar expressing levels of normal
cells) (Kamal et al., 2003). In either case, these seemingly “cancer-related” Hsp90
proteins are believed to bind and protect the stability and, therefore, oncogenecity of the
oncogene products (Lundgren et al., 2007). Such higher degrees of protection of the
oncoproteins by these Hsp90 proteins in tumor cells than that of cellular proteins in
normal cells are viewed as an opportunity for anti-tumor drugs. Geldanamycin (GM, or
benzoquinone ansamycin) and its derivatives inhibit the ATP binding and ATP
hydrolysis functions of Hsp90 and have been the focus of anti-tumor drug development
for two decades (Neckers and Neckers, 2002; Whitesell et al., 1994). Many of the earlier
trials did not advance. Several newer generations of chemically modified and less toxic
GM-related drugs are being developed and tested in a dozen new clinical trials (Solit and
Chiosis, 2008; Trepel et al., 2010). An obvious key hurdle of these trials is how to
minimize their potential interference with the physiological chaperone function of Hsp90
in surrounding normal tissue and cells and selectively harm the “cancer-related” Hsp90
proteins in tumor cells embedded next to the normal cells. It has proven difficult for

26
Hsp90 inhibitors of this nature (Trepel et al., 2010). In contrast, no physiological function
has been reported for the action of eHsp90, which requires the F-5 epitope within the
highly charged linker region and part of the middle domain in Hsp90α. Using gene rescue
experiments, Picard and colleagues showed that a highly charged linker region in yeast
Hsp90 (Hsp82), which overlaps with the F-5 epitope of human Hsp90α, was dispensable
for viability in yeast (Louvion et al., 1996). This genetic datum, together with all the
studies for the past 7 years, suggests that secretion of Hsp90 by normal cells is an
emergency response of the cells to environmental insults, such as normal cells in a
wounded tissue or cancer cells in a hypoxic environment. Furthermore, eHsp90 does not
require the ATPase region, which is the target for the GM-related inhibitors and the
reason for the drug-caused cytotoxicity. Therefore, the F-5 epitope in eHsp90α may
represent an excellent target for design of safer, effective and more specific inhibitors for
treatment of HIF-1α-positive tumors. Therefore, we propose that new anti-cancer drugs
should i) selectively inhibit eHsp90 (not its intracellular counterpart) and ii) specifically
target the pro-motility activity located at the F-5 region. Drugs that bear both properties
should achieve a higher efficacy and pose minimum toxicity to cancer patients. A
schematic representation of this simplified thought is depicted in Fig. 5.

Wound healing and tumor progression: similar strategy used by peacemaker and
terror
What is the relationship between wound healing and cancer? Wound healing is a
physiological repair process by epithelial, fibroblastic and endothelial cells that is only

27
activated in response to injury or a surgeon's knife. Cancer is in manyways a similar
process by a similar group of cells (called tumor stroma cells as a distinction) in response
to the “injury” that is caused by the invading tumor cells.Wound healing usually has a
beginning and an ending, whereas cancer has a beginning but often an open ending
(patient death) if left untreated. In his seminarconverted analytical article published in
The New England Journal of Medicine, Dvotak listed lines of similarities (and
distinctions) between wound healing and tumor stroma generation and suggested that
tumor stroma formation is a subversion of the normal wound healing process. Therefore,
he called tumors “wounds that do not heal” (Dvorak, 1986). Such a reverse relationship
of “healing” and “no healing” between wound healing and cancer should be taken into
account, whence eHsp90 becomes a clinical target. For instance, topical application of F-
5 peptide to wounds has to consider if the peptide goes into the blood circulation and
travels to the site where an early-stage tumor is in progress. Under this circumstance, F-5
might aid the tumor cell invasion and speed up its growth. On the other hand,
administration of inhibitors (a mAb, for instance) of F-5 for blockade of the tumor
growth might interfere with the normal wound healing process in the same patient. This
is a legitimate and realistic concern. For example, numerous studies showed that the
people with type II diabetes are more likely to die from cancer than non-diabetic people.
Therefore, for a diabetic patient who has cancer and a foot ulcer, if the patient is treated
with inhibitors of F-5 by an oncologist to slow the cancer progression, the administered
inhibitor could interfere with the healing process of the chronic wound managed by a
wound specialist. The reverse is true. If the diabetic ulcer is treated with F-5 peptide, the

28
peptide may travel through the blood circulation to the tumor site and to aid invasion and
metastasis of the tumor. It is important to knowwhat patients should receive the
treatments and what patients should not.

29
CONCLUSION AND PERSPECTIVE
It is becoming clear that Hsp90 has two Mother Nature assigned roles to play.
One is as an intracellular chaperone and the other is as an extracellular tissue-repairing
factor, both of which seem to be designed for the cells to cope with environmental
changes, such as tissue injury. Both functions have been taken advantage of by tumor
cells during invasion and metastasis. A central controller of Hsp90 secretion inside the
cells is HIF-1α, which is overexpressed in more than 40% solid tumors in humans. LRP-1
is essential for eHsp90 signaling to promote cellmigration and tumor invasion.More
importantly, the recognition of eHsp90's existence and its role in tissue repair and cancer
invasion has revealed a new line of therapeutic intervention. Evidence-based advantages
of targeting eHsp90 over conventional growth factors for wound healing have been
shown in pre-clinical studies. Predicted advantages of targeting eHsp90 over intracellular
Hsp90 in prevention of tumor progression remain to be tested, upon availability of
specific inhibitors. A crucial tool for the future studies is to develop inhibitors that
specifically target the F-5 region of eHsp90 for both in vitro and in vivo experiments.
Meanwhile, for the next 5 years, important questions such as mechanisms of Hsp90
secretion and eHsp90 actionwill continue keeping researchers busy and challenged.

30

CHAPTER 2

A FRAGMENT OF SECRETED HSP90α CARRIES PROPERTIES THAT
ENABLE IT TO ACCELERATE EFFECTIVELY BOTH ACUTE AND
DIABETIC WOUND HEALING IN MICE

INTRODUCTION
According to the Wound Healing Society, about 15% of older adults in the US
suffer from chronic wounds, including predominantly venous stasis ulcers, pressure
ulcers (bedsores), and diabetic (neuropathic) foot ulcers (Crandall, 2003; Sen et al.,
2009). Every year 2 to 3 million more Americans are diagnosed with various types of
chronic wounds. For instance, an estimated 18% of diabetic patients over the age of 65 in
the US have nonhealing foot ulcers (Falanga, 2005). In this particular patient population,
the number of wound infection-caused leg amputations is approaching 100,000 per year.
Worldwide, it is estimated that a lower limb is lost every 30 seconds as result of diabetic
wound infection (Sen et al., 2009). The surgical procedure, hospitalization, and aftermath
of wound care can cost US taxpayers $100,000 per patient (in a 24-month period) and,
not to mention, compromise quality of the patients’ lives. The collective health care cost
of the various chronic wounds exceeds $25 billion annually, a rapid increase due to
increasing health care cost, an aging population, and a rise in the incidence of diabetes

31
and obesity in the US. On top of the above, the continued lack of effective treatments of
chronic wounds has further contributed to the scope of this devastating problem.
Since the discovery of the first growth factor in the late 1970s, it has become
conventional wisdom that locally released growth factors in an injured tissue constitute
the main driving force to heal the wound (Grose and Werner, 2004; Werner and Grose,
2003). Specifically, under this assumption, growth factors are responsible for promoting
the lateral migration of epidermal keratinocytes to close the wound, the inward migration
of dermal fibroblasts to remodel the damaged tissue, and migration of microvascular
endothelial cells to rebuild vascularized neodermis in the wounded space (Martin, 1997;
Singer and Clark, 1999). Since the first report of the EGF clinical trial on wound healing
in 1989 (Brown et al., 1989), more than a dozen growth factor trials have been
conducted. The list includes (a) EGF on partialthickness wounds of skin grafts (Brown et
al., 1989), on traumatic corneal epithelial defects (Pastor and Calonge, 1992), on
tympanic membrane with chronic perforation (Ramsay et al., 1995), and on advanced
diabetic foot ulcers (Fernandez-Montequin et al., 2007; Mohan, 2007); (b) bFGF on
partialthickness burn wounds of children (Greenhalgh and Rieman, 1994), on second-
degree burns (Fu et al., 1998), and on diabetic ulcers (Uchi et al., 2009); (c) acidic FGF
on partial-thickness burns and skin graft donor sites (Ma et al., 2007); (d) GM-CSF plus
bFGF on pressure ulcers (Robson et al., 2000); and (e) PDGF-BB on chronic pressure
and diabetic ulcers (LeGrand, 1998; Pierce et al., 1992; Smiell et al., 1999; Steed, 1995;
Wieman et al., 1998). Despite the fact that most of these doubleblinded trials reported
promising clinical efficacies in humans, only the human recombinant PDGF-BB has

32
received US FDA approval for treatment of limb diabetic ulcers (Regranex, becaplermin
gel 0.01%, Ortho-McNeil Pharmaceutical) (LeGrand, 1998). After its approval in 1997,
multicenter, randomized, parallel trials showed that becaplermin, at 100 μg/g of PDGF-
BB, improved, at best, 15% of complete wound closures (50% treated versus 36%
placebo) (LeGrand, 1998; Smiell et al., 1999; Steed, 1995; Wieman et al., 1998). These
results are not considered to be a cost-effective benefit for clinical practice (Mandracchia
et al., 2001; Nagai and Embil, 2002). In 2008, the US FDA added a black box warning
regarding increased risks for cancer mortality in patients who need extensive treatments
(≥3 tubes) of becaplermin gel. This significant side effect may not be surprising to cancer
researchers, since it was already known, years before the FDA approval of becaplermin
gel, that overexpression of PDGF-BB (c-sis) or autocrine presence of its viral form, v-sis,
will cause cell transformation (Bejcek et al., 1992), and yet the recommended dosage of
PDGF-BB in becaplermin gel is more than 1,000-fold higher than the range of the
physiological PDGF-BB levels in human circulation (van den Dolder et al., 2006).
So, what was against the conventional wisdom? Initially, in an entirely isolated study of
ours, we noticed that FBS or its equivalent has been widely used in studies of human skin
cells and wound healing. However, these human cells are never in contact with FBS in
reality, instead they are bathed in human serum in the wound. We challenged the
assumption that FBS and human serum share completely interchangeable factors for
human skin cell migration. Results of our experiments showed that, while FBS equally
stimulated migration of human epidermal and dermal cells, human serum only promoted
human keratinocyte (HK) migration and halted human dermal fibroblast (HDF) and

33
human microvascular endothelial cell (HDMEC) migration (Bandyopadhyay et al., 2006;
Henry et al., 2003). We further identified TGF-β3 (not TGF-β1 or TGF-β2) in human
serum that was responsible for the inhibitory effect of human serum on migration of the
human dermal cells, which express 7- to 18-fold higher levels of the type II TGF-β
receptor (TβRII) than human epidermal keratinocytes (Bandyopadhyay et al., 2006). In
this case, however, TGF-β3 is no troublemaker; instead it controls the “traffic” of the
epidermal and dermal cell migration to ensure a speedy and proper closure of the wound
(Bandyopadhyay et al., 2006). An important implication of these findings is that the
conventional growth factors, such as PDGF-BB for dermal fibroblasts and VEGF-A for
endothelial cells, may not be able to do the job, as they were hoped, in human wounds,
because of the copresence of TGF-β3. We speculated that the source of the molecule
driving the wound closure comes from secreted proteins by human skin cells at the
wound edge in response to the injury. Protein purification allowed us to discover a novel
wound healing-promoting factor, the secreted form of Hsp90α, from both HDFs and HKs
(Cheng et al., 2008; Li et al., 2007). It is now clear that normal cells do not secrete
Hsp90α in the absence of tissue stress (Cheng et al., 2010). However, when the cells
encounter pathophysiological conditions, such as cancer (Tsutsumi and Neckers, 2007),
or stress cues from the environment, including hypoxia (Li et al., 2007), heat shock
(Clayton et al., 2005; Hightower and Guidon, 1989), reactive oxygen species (Liao et al.,
2000), gamma-irradiation (Yu et al., 2006), or tissue injury-released cytokines (Cheng et
al., 2008), they respond by sending out, via the unconventional exosome pathway (Cheng
et al., 2010), their abundantly stored Hsp90α for tissue repair or tissue invasion (tumors).

34
In this study, we take the study of the effect of secreted Hsp90α on wound healing to
what we believe to be a new preclinical level by systematically analyzing secreted
Hsp90α treatment versus FDA-approved conventional growth factor treatment of acute
and diabetic wounds in mice. More importantly, we provide 3 mechanisms to explain
why a 115-aa fragment from secreted Hsp90α (F-5) may represent the bona fide driving
force for the initial wound closure and a new generation of treatment for diabetic wounds.

35
MATERIALS AND METHODS
Cell culture and Reagents
Primary human neonatal HKs, HDFs, and HDMECs were purchased from
Clonetics. HKs were cultured in EpiLife medium with added HK growth supplements.
HDFs were cultured in DMEM supplemented with 10% FBS. HDMECs were cultured in
growth factor-supplemented Medium 131 (Cascade Biologics). The third or fourth
passages were used in cell migration assays. rhPDGF-BB, rhTGF-α, and rhTGF-β3 were
purchased from R&D Systems. Regranex (becaplermin 0.01% gel, Ortho-McNeil
Pharmaceutical) was prescribed and purchased from USC Medical Plaza Pharmacy solely
for this study. Antibodies against PDGFRα and PDGFRβ were from Santa Cruz
Biotechnology Inc. (SC-338) and Genzyme (1263-00), respectively. Anti-LRP1/CD91
antibody was purchased from Progen Biotechnik. Mouse monoclonal antibody (pan)
against Keratin (ab8068) was from Abcam Inc. PECAM-1 (M-20) goat polyclonal
antibody (sc-1506) was from Santa Cruz Biotechnology Inc. Monoclonal mouse anti-
mouse SMA antibody was from Dako Denmark A/S. Biotinylated rabbit antigoat IgG and
biotinylated horse anti-mouse IgG were from Vector Laboratories. Chromagon system,
Dako Liquid DAB+ substrate, was from Dako Denmark A/S. Mouse antibody on mouse
tissues (mouseon- mouse) detection system, VECTOR M.O.M. Immunodetection Kit,
and the Immunoperoxidase System, VECTORSTAIN Elite ABC Kit, were from Vector
Laboratories. Carboxymethylcellulose (CMC) sodium salt (C5678) (pH measured at
7.14) was from Sigma-Aldrich. Rat type I collagen was purchased from BD Biosciences.
Anti –β-actin antibody and anti-GAPDH antibody were from Cell Signaling Technology.

36
The RAP construct in bacteria was a gift of Guojun Bu (University of Washington, St.
Louis, Missouri, USA). XL-10 Gold Ultra competent cells were from Stratagene. Stat
Strips (adhesive bandages) were from Notro Max Products. 3M Coban (self-adhesive
wrap) was from 3M.

Subcloning, production, and purification of Hsp90α fragments
The cDNAs encoding the full-length and various fragments (F-1 to F-6) of
Hsp90α were generated by PCR using human Hsp90α cDNA as the template. PCR
fragments were subcloned into the His-tag pET15b vector (EMD Biosciences Inc.) at
BamH1 or Bam H1 and NdeI sites. DNA sequences of the fragments were verified by
DNA sequencing. The pET15b-Hsp90α constructs were transformed into BL21-
codonPlus (DE3)-RP competent cells (Stratagene) following a manufacturer-provided
protocol. Protein production and purification were described previously (Cheng et al.,
2008). Proteins were confirmed by Western blots concentrated in Centricons YM (10 to
50) to a final concentration of 1 mg/ml, and stored in 10% glycerol in DPBS at –80°C.

Cell migration assays
The colloidal gold migration assay, the in vitro wound healing (scratch) assay,
and the transwell assay were as modified and described previously by us (Li et al., 2004;
Lindquist and Craig, 1988). Data from independent experiments (n ≥ 3) were averaged
and calculated as percentage, OD reading, or fold increase in response to corresponding
stimulus over the baseline control (mean +/- SD; P < 0.05).

37

The FG-12 lentiviral system, RNAi against LRP-1, lentiviral production, and
infection
Details regarding these methods have been published by our laboratory and by
others (Li et al., 2007; Qin et al., 2003; Woodley et al., 2009).

Preparation of CMC gel with Hsp90α peptides
CMC powder (viscosity 50 –200 cps, purity 99.5%, sodium salt) was dissolved in
double-distilled H2O in a tissue culture hood at 5% concentration (w/v), heated for 4
hours at 37°C, placed in a shaker for 24 hours at 4°C, and brought back to room
temperature. This CMC solution was mixed in a 1:1 ratio (vol/vol) with a desired
concentration of FPLC-purified and -filtered (0.22 μm) Hsp90α peptide (in DPBS). The
CMC gel diluted with DPBS or mixed with DPBS containing Hsp90α peptides was
applied topically to the wounds.

Wound healing in mice
Thymic hairless (Foxn1) mice and BKS.Cg-m+/+Leprdb/J mice were obtained
from The Jackson Laboratory and housed 4 per cage prior to or 1 per cage during
experiments. Athymic hairless mice of 8 to 10 weeks of age were anesthetized with
isoflurane prior to the wound-creating surgery. Full-thickness excision wounds (1 cm × 1
cm) were created by marking the area of the wound in the mid-back with a fine marker
and a ruler, lifting the skin with a pair of a forceps and excising the full-thickness skin

38
along the lines with a pair of surgical scissors. The db/db mice of 6 weeks of age were
subjected to the similar procedure (but with back shaved) to create 1.2 cm × 1.2 cm
wounds. Immediately after the surgery on day 0, the wounds were topically treated with
100 μl of either 5% CMC gel (placebo) or the same gel containing an Hsp90α peptide or
becaplermin gel (100 μg/g PDGF-BB) at its clinically recommended dosage. Each wound
was covered with a bandage and a self-adherent wrap (Coban) to prevent desiccation and
infection while the wound was exposed. Bandages and Cobans were changed every 3
days after the initial 4 days. Standardized digital photographs were taken of the wounds,
with the same distance between camera and preanesthetized animal for each animal. The
photographs were examined using planimetry for objective evaluation for degree of
wound healing (Bandyopadhyay et al., 2006; Woodley et al., 2007). The open wound
areas were determined with an image analyzer (AlphaEase FC version 4.1.0, Alpha
Innotech Corporation). The total pixels that cover the unhealed areas were drawn onto the
digital photographs using a pattern overlay in ImageJ (http://rsbweb.nih.gov/ij/). The
number of pixels covering an open wound area on a given day was divided by the number
of pixels spreading over the initial wound on day 0 to obtain the percentage of closure.
Percentage, rather than actual distance (e.g., mm), of wound closure was calculated from
the measured wound areas (pixel density). This method allows more accurate
measurements of the wounds, considering the fact that certain margin of errors during
surgical procedures may exist among different experimental groups or even among the 3
mice within the same group. The percentage of wound healing was based on changes in
the same wound on the same mouse for indicated time points, instead of changes among

39
different mice. Thus, the calculated mean for the wound healing (percentage) among the
3 mice in a group was independently obtained, and the statistics of 3 groups of 3
independent experiments were calculated. We defined and presented the healing in 2
ways: (a) the open area of the healing wound on a given day/the open area of the original
wound × 100 for the indicated days and (b) comparison between the healed area of the
wounds with Hsp90α treatment with the healed areas of the wounds with placebo
treatment on each specific day.

H&E and IHC staining
The F-5–untreated or –treated mice were euthanized either 7 (nude mice) or 14
days (db/db mice) after the surgery (wounding). The wounds, together with unwounded
skin margins, were excised and put into 10% formaldehyde. The H&E staining was
carried out as previously described (van den Dolder et al., 2006). In order to show the
wound of placebotreated mice compared with that of F-5–treated mice, multiple
overlapping pictures were taken under a microscope (Nikon, Eclipse TE2000-U, ×4) and
used to reconstitute the entire wound. Standard IHC staining procedure was carried out.
All antibodies were used in 1:100 dilutions. The M.O.M. kit was used for mouse
antibodies (pan-keratin and SMA) on mouse tissues to control background staining.

40
Statistics
Data are presented as mean +/- SD. Statistical significance for comparisons was
determined by the Student ’s 2-tailed t test. A P value of equal or less than 0.05 was
considered statistically significant.

Study approval
All animal studies were conducted using protocols approved, prior to initiation of
this study, by the University of Southern California Institutional Animal Use Committee.

41
RESULTS
Identification of a minimum promotility epitope in secreted Hsp90α, within
its linker region and middle domain
In order to gain further insights into how secreted Hsp90α heals wounds, we
attempted to identify the minimum size of the therapeutic peptide in secreted Hsp90α that
still retains the full promotility activity of the full-length Hsp90α in vitro and its full
capability of promoting wound healing in vivo. Deletion mutagenesis, as schematically
summarized in Figure 2.1A, was used to obtain the various fragments of human Hsp90α
largely according to its previously defined domains (Lindquist and Craig, 1988).
Recombinant proteins were produced in the pET15b protein expression system, purified
sequentially by Ni+ affinity column chromatography and fast protein liquid
chromatography (FPLC), and confirmed on SDS-PAGE (Figure 2.1B). In cell motility
assays, with full-length Hsp90α and PDGF-BB as positive controls (Figure 2.2A), we
found that F-2 (Figure 2.2D) and F-5 (Figure 2.2C) stimulated HDF migration as
effectively as the full-length Hsp90α protein (Figure 2.2A), although higher
concentrations of F-2 and F-5 were needed. However, as the 115-aa F-5 fragment
continued to shorten, we observed declines in promotility activity in those peptides. For
instance, the 54-aa peptide, F-6, showed a significantly reduced promotility activity
(Figure 2.2D). The F-1, F-3, and F-4 peptides, which represent the N-terminal, middle
(alone), and C-terminal regions of Hsp90α, respectively, showed low or little stimulation
of cell migration (data not shown). Note that we initially reported a moderate promotility
activity from both the middle and C-terminal domain fragments on HKs

42

Figure 2.1 Expression of different recombinant Hsp90α proteins/peptides (wild type
and mutants). (A) A schematic representation of 7 human Hsp90α proteins/peptides
(wild type and mutants). Each cDNA fragment by PCR was subcloned into pET15b and
expressed in BL-21 bacteria, according to the manufacturer’s protocol. Protein was
sequentially purified by Ni+ column and finally FPLC, prior to in vitro motility assays
and in vivo wound healing assays. (B) A Coomassie Blue-stained SDS-PAGE gel to
show the proteins after FPLC (~3 μg/lane). The first lane on far left is molecular weight
markers. Mr, molecular weight.

43

Figure 2.2 F-5 peptide retains the full promotility activity of full-length Hsp90α. (A–
D) Data of colloidal gold migration assay of serum-starved HDFs in response to PDGF-
BB (15 ng/ml) and the full-length and the various fragments of Hsp90α, with the
indicated concentrations, are presented. The negative results of F-1, F-3, and F-4 are not
shown. Only the migration index of the migration experiments (n = 4, *P < 0.05) is
shown. Similar results were observed when in vitro wound healing scratch and the
transwell assays were used. FL, full length.
A B
C D

44
(Cheng et al., 2008), which now appears to be due to differences in protein purity
between that and the current study. Moreover, the current results were confirmed in 3
independent cell migration assays: (a) the colloidal gold migration assay that detects
migration of individual cells; (b) the in vitro wound healing (scratch) assay that measures
migration of a cell population; and (c) the transwell assay that tests 3-dimensional
chemotaxis (Li et al., 2004). Similar results were also obtained from cell migration assays
with HKs and HDMECs (see below). Thus, we conclude that F-5 is the smallest peptide
that retains an equivalent promotility activity as the full-length Hsp90α protein.

F-5 fragment of Hsp90α accelerates acute wound closure more strongly
than becaplermin/PDGF-BB in mice
Next, we tested how these peptides translate their in vitro promotility activity to in
vivo wound healing capability. In these experiments, we compared them with the only
FDA-approved growth factor therapy, becaplermin gel (PDGF-BB), in athymic hairless
mice. The primary reasons to choose athymic hairless mice are (a) to minimize the host
innate immune response to a human peptide and, therefore, immune response-caused
wound contraction and (b) to minimize the effect of inflammatory response after the
injury and to detect the specific effect of a topically applied peptide. We first carried out
large-scale screening tests (4 concentrations of each peptide) in mice to identify the
optimal concentration for each peptide that showed the strongest promotion of full-
thickness excision wound closure after a single application on day 0 (data not shown).
The peptides in their optimized concentrations were subjected to comparisons with the

45
becaplermin gel for promoting wound closure over time. Representative images of the
wounds are presented in Figure 2.3. Treatment with full-length Hsp90α (Figure 2.3A),
392-aa F-2 (Figure 2.3B), and 115-aa F-5 (Figure 2.3C) all strongly accelerated the
wound closure, in comparison with placebo. Among these peptides, interestingly, F-5
showed the strongest effect. In contrast, the shorter 54-aa F-6 showed a dramatic decline
in promoting wound healing (Figure 2.3D). Nonetheless, these in vivo results are
consistent with their in vitro promotility activities. To our surprise, we found that the
becaplermin gel treatment showed limited acceleration of the acute wound closure
(Figure 2.3E). Computer-assisted planimetry of 3 independent experiments confirmed a
superior effect of the Hsp90α peptides to that of becaplermin gel in promoting acute
wound closure (Figure 2.4). When promotion of wound closure was compared on the
same day, it was observed that F-5 showed the strongest effect (Figure 2.4C vs. Figure
2.4, A and B). F-6 (Figure 2.4D) and becaplermin gel (Figure 2.4E) showed a
comparable, but significantly smaller, effect compared with the full-length Hsp90α, F-2,
and F-5. Using the previously described methodology (Greenhalgh et al., 1990), we also
estimated the percentage of the unhealed areas of the F-5–treated wounds versus that of
placebo-treated wounds on each day over that of the wounds on day 0. F-5 treatment
shortened the time of complete wound closure from approximately 17 days (placebo) to
10 days (Figure 2.4F). We examined the possibility that multiple treatments would
further shorten the time of complete wound closure. However, repeated treatments of the
wounds with F-5 did not show significant advantage over a single treatment. The possible
reasons, albeit technical, will be discussed (see Discussion).

46

Figure 2.3 F-5 is superior to FDA-approved becaplermin/PDGF-BB in acute wound
healing. Full-thickness skin wounds (1 cm × 1 cm) in athymic nude mice were treated
with either placebo (10% CMC gel) or the gel containing an optimized concentration of
(A) full-length Hsp90α, (B) F-2, (C) F-5, (D) F-6 (n = 3 mice per peptide, per
experiment), or (E) becaplermin (100 μg/g of PDGF-BB). Plus signs indicate treated
mice, and minus signs indicate placebo mice. The images of 1 representative experiment
are shown.

47

Figure 2.4 Measurements of F-5 versus becaplermin in acute wound healing. (A–E)
Percentage of the accelerated wound closure at days 0, 4, 7, 10, 12, and 14 after
treatments of the various Hsp90α fragments or becaplermin versus placebo (mean ± SD).
(F) A single treatment with F-5 shortened the wound closure time from 17 days to 10
days. *P ≤ 0.05, compared with placebo.

48
It is arguable that mouse skin wounds heal largely via contraction, due to the nature of
loose skin with dense hair follicles. Whereas, human skin wounds heal through lateral
migration of keratinocytes, i.e., reepithelialization, followed by inward migration of the
dermal cells (Coulombe, 2003). To examine F-5 –treated wounds, wedge biopsies of the
day 7 wounds were subjected to H&E staining, microscopic analyses, and measurements.
The boundary between unwounded skin and newly healed skin is marked with a green
arrow (Figure 2.5). The F-5–treated wound (Figure 2.5B) showed a much smaller,
unhealed area overall in comparison with that of the placebo-treated wound (Figure 2.5A,
dotted red lines). Moreover, the F-5–treated wound exhibited substantially more
reepithelialization than the placebo-treated wound (Figure 2.5, dotted yellow lines). The
reepithelialized tongue (ReT) can be clearly visualized in the enlarged images on both
ends. These results suggest that promotion of reepithelialization is a mechanism of action
by F-5.

F-5 is superior to becaplermin gel/PDGF-BB in promoting diabetic wound healing
A critical question is whether F-5 promotes healing ofchronic wounds as well.
Among the 3 major types of chronic ulcers, pressure, venous, and diabetic ulcers, animal
models are only available for studying wound healing in diabetes-equivalent conditions.
Among a handful of murine models for diabetes, the db/db mouse is a commonly used
model for type II diabetes, with a plasma glucose level of 300 –500 mg/dl by 6 to 8 weeks
of age (in humans, >180 mg/dl is diabetic hyperglycemia). These mice were reported to
take more than 50 days to heal a 1.5 cm × 1.5 cm full-thickness wound, in comparison

49

Figure 2.5 F-5 promotes reepithelialization of the wound. On day 7, wedge biopsies of
full-thickness wounded skin (i.e., including a portion of unwounded skin), with either (A)
placebo or (B) the F-5 peptide treatment, were H&E stained and photographed under a
light microscope. Independently photographed images with identical magnifications were
reconstituted to show the unhealed areas of the wounds. Red dotted lines indicate
unhealed wound space. Yellow dotted lines mark the newly reepithelialized epidermis.
Green lines with arrows point out unwounded skin areas. The fronts of newly
reepithelialized epidermis were enlarged, as shown in higher-magnification images. Scale
bars: 0.33 mm; 0.01 mm (left and right).

50
with 20 days of similar wounds in normal littermates (Olerud, 2008). Moreover, healed
wounds in db/db mice show a considerably higher degree of reepithelialization rather
than wound contraction compared with healed wounds of nondiabetic mice (Gibran et al.,
2002).
While PDGF-BB was reported to cause little improvement in acute wound
healing in mice (Lynch et al., 1987), it accelerates wound healing in db/db mice
(Greenhalgh et al., 1990). Therefore, we made a side-by-side comparison of placebo, F-5,
and becaplermin on full-thickness wound healing in db/db mice. While becaplermin gel
is recommended for daily use on diabetic ulcers in humans, we continued to use it with
our single treatment protocol for the reasons previously mentioned (also see Discussion)
and measured the rate of the wound closure for up to 18 days. The F-5–treated wounds
showed complete closure between 14 and 18 days (Figure 2.6A, left panels). The
placebo-treated wounds healed at a much slower rate, with more than 25% unhealed area
on day 18 (Figure 2.6A, middle panels). Becaplermin gel treatment caused faster wound
closure than the placebo (Figure 2.6A, right panels). However, the effect of becaplermin
gel was substantially weaker than that of F-5, with a significant unhealed area on day 18.
Quantitation of the data from 3 inde- pendent experiments is shown in Figure 2.6B. We
conclude that F-5 is a more effective agent for both acute and diabetic wounds than
conventional growth factor therapy in mice. This work was done in collaboration with
Fred Tsen.

51

Figure 2.6 F-5 is superior to becaplermin/PDGF-BB in recruiting dermal cells in
diabetic wound healing. (A) Full-thickness excision wounds (1.2 cm × 1.2 cm) were
created on the backs of db/db mice and treated with placebo (10% CMC gel) or F-5 or
becaplermin. The images of 1 out of 3 representative experiments are shown from day 0
and the day of complete closure of the F-5–treated wounds. (B) Percentage of the wound
closure on day 5 to day 18 in reference to the day 0 wounds (mean ± SD). A single
treatment with F-5 on day 0 shortened the wound closure time from approximately 35
days to 14 days. *P ≤ 0.05, compared with placebo.
A B

52
Three unique properties of F-5 — a common motility factor, a TGF-β– resistant
factor, and a hyperglycemia-resistant factor — all absent from conventional growth
factors
Having demonstrated the superior effect of the F-5 fragment in secreted Hsp90α
over that of FDAapproved conventional growth factor therapy on both acute and diabetic
wound healing, we asked what made F-5 more effective than conventional growth
factors, such as PDGF-BB. First, we reasoned that, ideally, a single factor-based wound
healing agent should be a molecule that is able to recruit both epidermal and dermal cells
into the wound bed. In contrast, if an agent only selectively acts on some, but not all, the
skin cell types, it might be less effective in the wound healing process that requires
multiple cell types of wound healing. Thus, we analyzed the effect of F-5 on migration of
the 3 major human skin cell types, HKs, HDFs, and HDMECs. In the absence of any
stimulus, all 3 types of skin cells exhibited limited levels of motility (Figure 2.7A, first
column). Interestingly, PDGF-BB was only able to promote migration of HDFs but not
HKs or HDMECs (Figure 2.7A, second column). In contrast, F-5 was able to promote
migration of all 3 types of cells (Figure 2.7A, third column). A computer-assisted
quantitation of the cell migration is shown in Figure 2.7B (bars 4–9 vs. bars 1–3). Thus,
the first unique property of F-5 is that it is a common promotility factor for skin cells.
What is the molecular basis for the difference between F-5 and PDGF-BB? We
focused on the presence or absence of the receptors for PDGF-BB (i.e., PDGFRα and
PDGFRβ) and secreted Hsp90α (i.e., LDL receptor-related protein-1 [LRP-1]). We found

53

Figure 2.7 F-5 is a common promotility factor and overrides inhibition by TGF-β.
Primary HKs, HDMECs, and HDFs were serum-starved overnight and subjected to
colloidal gold migration assays and either untreated or treated with indicated growth
factors (15 ng/ml of PDGF-BB) or F-5 (2.3 μM) in the absence or presence of TGF-β3
(1.5 ng/ml). (A) Comparisons of F-5 peptide with PDGF-BB on stimulation of HK,
HDMEC, and HDF migration in either the absence (second and third columns versus the
first column) or presence (forth and fifth columns) of TGF-β3). Images of cell migration
from 1 representative experiment are shown. The dotted circles point out the averaged
migration tracks under indicated conditions. The arrows point to cell migration inhibited
by the presence of TGF-β3. Original magnification, ×40. (B) Migration index of the
tracks is shown (n = 3; *P < 0.05, compared with serum-free control). Arrow indicates
Tβ3-inhibited cell migration. (C) Lysates of HKs, HDMECs, and HDFs were analyzed by
Western blot with anti-PDGFRα, anti-PDGFRβ, or anti–LRP-1 antibodies as indicated.
Equal sample loading control is indicated by anti-GAPDH antibody blot.

54
that the lack of response to PDGF-BB from HKs and HDMECs was due to the total
absence of both PDGFRα and PDGFRβ on these cells (Figure 7C). As expected, only
HDFs expressed the 2 PDGFRs. In contrast to the selective expression of PDGFRsonly in
HDFs, HKs, HDFs, and HDMECs all express comparable levels of LRP-1, the receptor
for secreted Hsp90α signaling to promote cell motility (Brownlee, 2001; Cheng et al.,
2008). If we extrapolate these in vitro findings to equivalent wound healing events in
mice, it suggests that PDGF-BB cannot have a direct role in recruitments of HKs for
wound reepithelialization and HDMECs for wound neovascularization. Second, we have
previously shown that TGF-β3 that is present in human serum (the main soluble
environment of an acute wound) selectively blocks growth factor-induced HDF and
HDMEC migration, due to the higher levels of TβRII expression on these dermal cells
than the epidermal cells (Bandyopadhyay et al., 2006). This finding suggests that
conventional growth factors might not even be able to recruit the dermal cells to the
wound, which contains an abundant amount of TGF-β throughout the healing process.
Therefore, we tested whether or not F-5 could override the inhibition of TGF-β to
promote dermal cell migration. In the presence of TGF-β3, the PDGF-BB – induced
migration of HDF was completely inhibited (Figure 2.7A, forth column, arrows).
Intriguingly, however, even in the presence of TGF-β3 (Figure 2.7A, far right column),
F-5 remained equally effective on stimulation of migration of all 3 cell types.
Quantitation of these results is shown in Figure 2.7B (bars 10 –15). This second unique
property of F-5 provides another explanation for why F-5 heals wounds faster than
becaplermin. Third, we asked what made F-5 more effective in promoting diabetic wound

55
healing. It is known that all forms of diabetes are characterized by chronic hyperglycemia
in circulation, which is blamed for delayed healing of diabetic wounds (Brownlee, 2001).
Reportedly, hyperglycemia was able to destabilize HIF-1α protein (Botusan et al., 2008),
the key regulator of Hsp90α secretion in HKs and HDFs (Li et al., 2007; Woodley et al.,
2009). We specifically tested whether hyperglycemia blocks hypoxia-induced HDF
motility and whether F-5 is able to bypass the blockage of hyperglycemia and rescue
HDF migration. Hypoxia strongly promoted HDF migration under normal glucose (5
mmol/l) medium, and hypoxia plus F-5 showed a slightly higher stimulation of cell
migration (Figure 2.8A, top row). However, hypoxia failed to do the same under
hyperglycemia (25 mmol/l glucose) (Figure 2.8A, middle bottom panel). We also found
that hyperglycemia even blocked PDGF-BB –stimulated HDF migration (data not shown).
However, the addition of F-5 “rescued ” the cell migration under hyperglycemia (Figure
2.8A, bottom right panel). Quantitation of the migration data is shown in Figure 2.8B.
This finding provides the third explanation for why F-5 showed a stronger effect on
accelerating diabetic wounds healing.

56

Figure 2.8 F-5 rescues cell migration under hyperglycemia. (A) Primary HDFs were
cultured in medium containing either 5 mmol/l glucose (normal glycemia) or 25 mmol/l
glucose (hyperglycemia) for 2 weeks, serum-starved overnight, and subjected to colloidal
gold migration assay under either normoxia (21% O2) or hypoxia (1% O2) in the
presence or absence of F-5 (2.3 μM). Images of cell migration from 1 representative
experiment are shown. The circles point out the averaged migration tracks under the
indicated conditions. Original magnification, ×40. (B) Migration index of the cell
migration tracks is shown (n = 3; *P < 0.05, compared with serum-free control).

57
DISCUSSION
For more than 3 decades, the conventional wisdom has been that serum factors —
collectively called growth factors — represent the primary force in Mother Nature ’s
design for wound healing (Heldin, 1996; Martin, 1997; Singer and Clark, 1999). These
often cell-type –specific growth factors either appear only when tissue is wounded or rise
significantly from their basal concentrations in response to injury, such as TGF-α and
KGF (FGF7) for HKs, PDGF-BB for HDFs, and VEGF-A for HDMECs. Since the mid
1970s, more than 30 growth factors have been subjected to extensive in vitro, preclinical,
and clinical studies alone or in combinations (Grose and Werner, 2004). Despite
enormous efforts, in vivo functions for many of these growth factors remained
unconfirmed, and their efficacy in human trials fell short of providing significant clinical
benefits (Martin, 1997; Werner and Grose, 2003). These rather unexpected statistics
argue against the long-standing paradigm that growth factors are the critical driving force
of wound closure. We speculated that there must be fundamental reasons underlining the
ineffectiveness of conventional growth factors in wound healing. We have since
undertaken 2 mutually complementary approaches to (a) examine the physiological
barriers for conventional growth factor actions and (b) identify a new generation of
wound healing factors. In this study, we have provided several lines of evidence for why
conventional growth factor therapies, such becaplermin/PDGF-BB, could not have been
as effective as they were hoped to be. First, not all skin cell types express a common
receptor for a given growth factor. For example, HKs and HDMECs completely lack the
PDGFR and, therefore, do not respond to becaplermin. Second, the abundant presence of

58
the TGF-β family cytokines throughout the early phase of wound healing blocks any
growth factor-stimulated migration of the dermal cells (note, not epidermal cells; see
(Bandyopadhyay et al., 2006)) and, therefore, their recruitment into the wound bed
(Bandyopadhyay et al., 2006; Brown et al., 1995; Eskild-Jensen et al., 1997; Harrington
et al., 2000; O'Kane and Ferguson, 1997; Roberts, 1996). Third, additional
pathophysiological conditions, such as hyperglycemia in diabetes, add layers that block
the effectiveness of growth factors in diabetic wounds (Botusan et al., 2008; Brownlee,
2001). More importantly, we have identified a more effective wound healing agent, F-5, a
fragment from secreted Hsp90α. In contrast to conventional growth factors, F-5 equally
promotes migration of all 3 types of human skin cells that are essential for wound
healing; F-5 overrides the inhibition of human dermal cell migration by TGF-β; and F-5
resists hyperglycemia to promote cell migration. Topical treatment of acute and diabetic
wounds with F-5 greatly accelerates wound closure through increased reepithelialization.
Based on these findings, we propose a new paradigm for what drives epidermal and
dermal cell migration to close the wound. Prior to injury, cell motility remains undetected
in intact skin. Within hours after skin injury, HKs start to migrate laterally across the
wound (possibly induced by hypoxia-driven Hsp90α autocrine signaling or TGF-α; see
(Brownlee, 2001)) and to secrete Hsp90α. At the same time, however, HDFs and
HDMECs at the wound edge are not able to immediately move into the wound bed due to
the presence of TGF-β3. Once the secreted Hsp90α reaches the threshold concentration of
100 nM (Cheng et al., 2008; Li et al., 2007), it triggers the dermal cells to migrate into
the wound bed from the surrounding wound edge, even in the presence of TGF-β3).

59
Finally, the migrating HKs completely close the wound, and the newly moved-in HDFs
start to remodel the wounded tissue and HDMECs to rebuild new blood vessels. We
propose here that injury-induced secretion ofHsp90α, instead of the conventional growth
factors, is the initial driving force of wound closure. After the initial wound closure, the
dermal remodeling neovascularization processes would take many months to complete.
Many other factors, including conventional growth factors, may play roles in the later
events of wound healing, when the TGF-β levels decrease (Hocevar et al., 1999). The
capability of F-5 to strongly accelerate diabetic wound closure is consistent with previous
studies on a recognized cause for diabetic wounds, hyperglycemia. One of the critical
environmental stimuli for wound healing is relative hypoxia (Elson et al., 2000; Hocevar
et al., 1999; Knighton et al., 1981; Tandara and Mustoe, 2004). HIF-1α is a master
transcription factor that regulates tissue adaptive responses to environmental hypoxia
(Semenza, 2007b) and is expressed throughout the multistage processes of acute wound
healing. Impaired response, i.e., lack of HIF-1α accumulation in the cells, to hypoxia in
diabetic ulcers isa known contributor to the delayed wound healing (Botusan et al., 2008).
In vivo, lower levels of HIF-1α protein were reported in foot ulcer biopsies in patients
with diabetes (Catrina et al., 2004). In vitro studies showed that hyperglycemia impairs
HIF-1α protein stability and function via the Von Hippel Lindau pathway (Botusan et al.,
2008; Catrina et al., 2004; Fadini et al., 2006; Gao et al., 2007). Botusan et al. have
demonstrated that forced stabilization of HIF-1α was necessary and sufficient to resume
diabetic wound healing (Botusan et al., 2008). In parallel, we have previously shown that
HIF-1α is a key upstream regulator of Hsp90α secretion. The secreted Hsp90α in turn

60
promotes human epidermal and dermal cell migration via a novel “HIF-1α > Hsp90α
secretion > LRP-1 ” signaling pathway (Li et al., 2007; Woodley et al., 2009). Results of
these two previously unrelated studies together point out the possibility that
hyperglycemia destabilizes HIF-1α, blocks hypoxia-driven Hsp90α secretion and delays
diabetic wound healing. The addition of F-5 bypasses the hyperglycemia-caused damage
at HIF-1α and jump-starts migration of the cells that otherwise cannot respond to the
environmental hypoxia. Our data indicated that F-5 is more effective than the full-length
Hsp90α in vivo, but requires higher concentrations to maintain that effect. Our current
understanding of this phenomenon is largely at the level of speculation. It is conceivable
that without possibly steric interferences by the 235-aa N-terminal domain and the 381-aa
C-terminal domain, F-5 can fully reveal its effect of promoting cell motility. On the other
hand, without the N-terminal and C-terminal domains, the shorter peptide may be
compromised on binding affinity and even stability and, therefore, show the requirement
for higher concentration to maintain an equivalent promotility activity as the full-length
protein. Our experiments show that even a single application of F-5 could lead to a
remarkable acceleration of the wound closure in db/db mice. If both such efficacy and
duration of the F-5 action could translate into humans, it may significantly improve
patient life and help to reduce the overall cost of diabetic wound clinic as well. The high
cost of the currently available care mostly comes from home visits by physicians with
various specialties and daily passive assistance of nurses, due to unavailability of
effective treatments (Harrington et al., 2000; Woodley et al., 2007). On the other hand,
we expect that multiple treatments with F-5 should result in more prominent healing

61
effects. Becaplermin gel, for instance, is recommended for daily applications to achieve
its clinical effect. In the current study, we focused on a single treatment in our animal
experiments for 2 technical reasons. First, for experiments that involve a large number of
mice, it is hard to ensure that the procedures on all wounds are performed universally.
Second, frequent opening and closing a healing wound for new treatments will risk
damaging the on-going healing tissue (the newly generated epidermal layer in particular)
and add extra stress and discomfort to the animals. Nonetheless, there have been reported
options to deal with these technical limitations. Covering the wound with Tegaderm and
multiple applications of the tested agent by injecting it through the Tegaderm with a
gauge needle was reported as a way to minimize these technical concerns (Qin et al.,
2003). The fact that extracellular Hsp90α is a motogen but not a mitogen (i.e., it does not
stimulate cell proliferation) makes physiological sense (Cheng et al., 2008; Li et al.,
2007). First, keratinocyte migration occurs almost immediately after skin injury and plays
a critical role in closing the wound. After the initial epidermal closure, completion of the
subsequent dermal neovascularization and remodeling processes would take many
months. Second, when a cell is migrating toward the wound area, it cannot proliferate at
the same time. In addition, growth factor-stimulated proliferation of both epidermal and
dermal cells would be inhibited by TGF-β that appears in the injured skin
(Bandyopadhyay et al., 2006). Third, cell migration precedes cell proliferation during
wound healing. While the cells at the wound edge are moving toward the wound bed,
they leave behind “empty space” between themselves and the cells behind them. The
cells that are located behind the migrating cells start to proliferate after losing contact

62
inhibition with the front moving cells. The stimuli of the cell proliferation likely come
from plasma growth factors in the surrounding unwounded blood vessels, in which TGF-
β levels are low or undetectable. Thus, cell proliferation appears to refill the space
generated by the front-migrating cells. The role of secreted Hsp90α appears to promote
the initial wound closure as quickly as possible.
Finally, proof of the relevance of animal model research to humans is the ultimate
standard, especially considering the fact that many animal models for human diseases do
not exactly reflect the genetic setting in humans. Many believe that this is the main
reason for the majority of the therapeutic agents in the past, which show great promise in
animal studies, to have ultimately failed in humans. For instance, human diabetes is a
polygenic disease, whereas the db/db mouse is a monogenic (i.e., mutation in a single
gene) diabetic model. Therefore, whether or not F-5 has similar effect on human diabetic
wounds remains to be seen.

63
CHAPTER 3

A POTENTIALLY COMMON PEPTIDE TARGET IN
SECRETED HEAT SHOCK PROTEIN-90α FOR
HYPOXIA-INDUCIBLE FACTOR-1α -POSITIVE TUMORS

INTRODUCTION
In normal cells under normoxia ( ∼8% oxygen level in tissues), the hypoxia-
inducible factor-1α (HIF-1α) protein is constantly synthesized and immediately subjected
to an O2-dependent prolyl hydroxylation. This modification then targets HIF-1α to the
ubiquitination-proteasome machinery for degradation (Semenza, 2003). As a result, the
overall steady-state level of HIF-1α is kept low. Under hypoxia, however, HIF-1α
hydroxylation and subsequent degradation are suppressed, resulting in a rise in the HIF-
1α level in the cells. The increased HIF-1α then forms a functional heterodimer with the
constitutively present HIF-1β (ARNT), the master transcriptional complex, called HIF-1.
HIF-1 translocates into the nucleus and regulates expression of hypoxia response
element–containing genes in a p300/CBP-dependent manner (Arany et al., 1996). In
contrast, HIF-1α proteins are kept at a constitutive level in many tumors. This is caused
by the tissue hypoxia generated by outgrowth of the rapidly proliferating tumor cells over
the surrounding vascular network, creating a distance that is longer than the reach of the
oxygen supply from the nearest blood circulation (Bertout et al., 2008). Under constant
ischemia, the tumor cells then undergo genetic and epigenetic changes to adapt

64
alternative and self-supporting mechanisms for continued survival, expansion, and
progression until neovascularization around them is complete. Thus action of oncogenes,
inhibition of tumor suppressor genes, and deactivation of the enzymes involved in HIF-
1α ubiquitination and degradation could all contribute to the deregulated expression of
HIF-1α in tumor cells (Majmundar et al., 2010). The deregulated HIF-1α plays a crucial
role in tumorigenesis in animal models. Down-regulation of deregulated HIF-1α expres-
sion or inhibition of the HIF-1α action slows tumor growth and renders the tumor more
susceptible to killing by radiotherapy and chemotherapy (Majmundar et al., 2010). In
humans, the constitutively expressed HIF-1α is linked to large tumor size, high grade,
and lymph node–negative metastasis, which make the tumor less accessible to
radiotherapy and chemotherapy (Hutchison et al., 2004). Therefore the constitutively
expressed HIF-1α in tumor cells has become a marker to predict possible outcomes of
patients with tumor metastasis. Whereas sabotaging the deregulated HIF-1α in tumor
cells could in concept prevent tumor progression, directly targeting the intracellularly
located HIF-1α or the enzymes that regulate HIF-1α stability is challenging (Poon et al.,
2009).
The human heat shock protein-90 (Hsp90) chaperone family includes four
confirmed members—the cytosolic Hsp90α and Hsp90β, the endoplasmic reticulum
GRP94, and the mitochondrial TRAP1, which are encoded by distinct genes (Chen et al.,
2005). As with the overexpression (accumulation) of HIF-1α in tumor cells, Hsp90α has
also been found either quantitatively overexpressed or qualitatively overactivated in a
variety of tumors (Kamal et al., 2003). These “extra” or “overactive” Hsp90α proteins are

65
believed to bind and protect the stability of oncogene products inside the cell (Grenert et
al., 1997; Neckers and Neckers, 2002; Welch and Feramisco, 1982). Such a seemingly
higher degree of protection by Hsp90α in tumor cells than their proto-oncoprotein
counterparts in surrounding normal cells has been taken as the basis for a strategy for
developing anticancer drugs (Trepel et al., 2010; Whitesell et al., 1994). Geldanamycin
(GM, or benzoquinone ansamycin) and its derivatives, which bind and block the ATP-
binding and ATP hydrolysis functions of Hsp90, have been the focus of drug
development for more than a decade (Neckers and Neckers, 2002). GM proved to be too
toxic even in animal models (Supko et al., 1995). A modified form of GM, benzoquinone
ansamycin 17-allylaminogeldanamycin (17-AAG), showed promising efficacy at a dose
range with tolerable toxicity in preclinical studies and has entered several phase 1 and
phase 2/3 clinical trials since 1999 (Solit and Chiosis, 2008; Trepel et al., 2010). Several
newer generations of chemically modified and less toxic GM-related drugs are being
developed in ongoing clinical trials. However, the main hurdle for these drugs remains as
how to selectively target the oncogene-protecting activity of Hsp90 in tumors and spare
the physiological function of Hsp90 in normal cells.
The figure of 1–2% of the total cellular proteins has been widely used to describe
the unusual abundance of Hsp90 protein inside most types of mammalian cells. If one
takes ∼7000 proteins per cell, that content of Hsp90 proteins would be 70- to 150-fold
higher than that of any of other cellular proteins. Csermely et al. (1998) argued that, if
intracellular chaperoning were the only assigned function for Hsp90, such an
overproduction of a single protein in cells would not be well tolerated by evolution

66
(Csermely et al., 1998). They speculated that the major cellular function of Hsp90 might
be another, yet-unrecognized one that would require such an abundant storage of the
protein. Recent studies have discovered a surprising need for normal cells to secrete the
“overstocked” Hsp90α for tissue repair (Li et al., 2011) and for tumor cells to
constitutively secrete Hsp90α for invasion and metastasis (Tsutsumi and Neckers, 2007).
Secretion of Hsp90 and/or its role in invasion and/or metastasis have been reported in
more than a dozen human tumors (Li et al., 2011). Kuroita et al. (1992) reported
purification of Hsp90α from conditioned media of human hybridoma SH-76 cells
(Kuroita et al., 1992). Eustace et al. (2004) reported Hsp90α, but not Hsp90β, in
conditioned media of HT-1080 fibrosarcoma cells (Eustace et al., 2004). Wang et al.
(2009) reported secretion of Hsp90α by MCF-7 human breast cells (Wang et al., 2009).
Suzuki and Kulkarni (2010) found Hsp90β secreted by MG63 osteosarcoma cells (Suzuki
and Kulkarni). Chen et al. (2010) reported secretion of Hsp90α by the colorectal cancer
cell line HCT-8 (Chen et al., 2010). Work by Tsutsumi and colleagues implied secretion
of Hsp90α by a variety of tumor cell lines (Tsutsumi et al., 2008).
What is the relationship between HIF-1α and secretion of Hsp90? HIF-1α is a key
upstream regulator of Hsp90α secretion (Li et al., 2007; Woodley et al., 2009). Because
constitutive accumulation of HIF-1α occurs in >40% of the tumors in humans (Dales et
al., 2005; Poon et al., 2009), the secreted Hsp90α could be a new and effective target for
treatment of these HIF-1α–positive tumors. In the present study, we have tested this
possibility by using the estrogen receptor (ER)–negative and aryl hydrocarbon (Ah)–
nonresponsive breast cancer cell line MDA-MB-231. We proved the importance of the

67
“HIF-1α > Hsp90α secretion” axis in control of cancer cell migration and invasion for the
first time. More important, we identified a critical 115–amino acid epitope, F-5, in
secreted Hsp90α that provides potentially a new therapeutic target for HIF-1α–positive
breast cancers and likely beyond.

68
MATERIALS AND METHODS
Cell Culture and Reagents
Cell lines screened for deregulated HIF-1α expression included the following:
HBl-100 human breast epithelial cell line, four human breast cancer cell lines (MDA-
MB-231, MDA-MB-468, MDA-MB-435, and MCF-7), M24 and M21 human melanoma
cell lines, U251 and U87 human glioma cell lines, A172 human glioblastoma cell line,
PC3 human prostate cancer cell line, and A431 human skin carcinoma cell line. Native
rat-tail type I collagen was from BD Biosciences (Bedford, MA). Colloidal gold (gold
chloride, G4022) was purchased from Sigma-Aldrich (St. Louis, MO). The cDNAs that
encode HIF-1α (wt), HIF-1αCA5 (constitutively active), and HIF-1αΔNBΔAB (dominant
negative) were provided by Gregg Semenza (Johns Hopkins University, Baltimore, MD).
We subcloned them into the lentiviral vector pPPLsin.MCS-Deco (Li et al., 2007).
ShRNAs against human HIF-1α and HIF-1β were cloned in the lentiviral FG-12 system,
as previously described (Woodley et al., 2009). Anti–HIF-1α antibody (610958) and anti-
HIF-1β antibody (611078) were from BD Transduction Laboratories (Lexington, KY).
Anti-Hsp90α antibodies for Western analysis (SPA-840) and for neutralizing function
(SPS-771) were from Stressgen (Victoria, Canada). XL-10 Gold Ultra competent cells
(XL-10 Gold) were from Stratagene (La Jolla, CA). The pET system (pERT15b) for
protein production in Escherichia coli was purchased from Novagen (Madison, WI).
Brefeldin A and dimethyl amiloride were purchased from Sigma-Aldrich. Matrigel
invasion chambers (354480) and protocols were purchased from BD Biosciences.

69
Athymic nude mice (4–6 wk of age; Harlan, Livermore, CA) were used in tumor
formation assays.

Hypoxia treatment and preparation of serum-free conditioned media
The OxyCycler C42 from BioSpherix (Redfield, NY) was used as oxygen content
controller throughout this study. This equipment allows creation of any oxygen profile
with full-range oxygen (0.1–99.9%) and CO2 control (0.1–20.0%). More important, all
media used for hypoxia experiments were preincubated in hypoxia chambers with the
designated oxygen content for 16 h prior to their use to replace normoxic culture media
(Li et al., 2007). Preparation of serum-free conditioned media was carried out as
previously described (Cheng et al., 2008).

Lentiviral systems for up- or down-regulation of target genes
The pRRLsinhCMV system was used to overexpress exogenous HIF-1α. The FG-
12 RNAi delivery system was used to deliver shRNAs against HIF-1, as previously
described (Li et al., 2007; Woodley et al., 2009). To measure protein levels of either
endogenous or exogenous gene products, equal amounts (50 μg of total cellular proteins
per sample) of cell lysate proteins (measured by Bio-Rad Protein Assay, Hercules, CA)
were subjected to antibody Western blot analyses. The results were visualized by ECL
reactions. Films with unsaturated exposure were used for scanning densitometry. Means
from three different exposures of the same experiment were calculated (Cheng et al.,
2008).

70
Three cell migration assays and serum-free conditioned medium
Updated protocol for the colloidal gold cell motility assay, including data and
statistical analysis, and a modified protocol for in vitro wound-healing assay, including
precoating with an ECM, plating scratching, and quantifying migration data, were as
described previously (Li et al., 2004). Transwell assay was performed according our
previously published protocol (Li et al., 2007). Preparation of serum-free conditioned
medium was as described in detail previously (Cheng et al., 2008). Zymography gel
analysis was carried out as previously described (Zhou et al., 2009).

Invasion assay
The procedures are described in detail in the manufacturer’s instruction for the
BD BioCoat Matrigel Invasion Chamber (354480; BD Biosciences).

Densitometry with Alpha Innotech FluorChem SP
The Alpha Innotech FluorChem SP (ProteinSimple, Santa Clara, CA) is a 4-
megapixel charge-coupled device (CCD) specializing in fluorescence,
chemiluminescence, or visible imaging. A 12-bit and 4–million pixel cooled camera is
attached to a manual fixed lens in a MultiImage FC Light Cabinet (DE500FC) with
interference filter and ML-26 dual-wavelength UV transilluminator. It uses FluorChem
AlphaEase FC 32-bit software for image acquisition, enhancement, archiving,
documentation, and analysis (ProteinSimple). We used 1-min exposure with an aperture

71
set at 1.2, zoom at 20, and focus at 1.9 with an open filter and normal sensitivity and high
resolution.

Recombinant Hsp90α production and purification
The coding regions of seven of the eight distinct domains (five, designated N′, M-
1, M-2, C′-1, and C′-2, were as previously reported; (Cheng et al., 2008)) were subcloned
into the histidine (His)-tag pET15b vector (EMD Biosciences, San Diego, CA) at BamH1
using a PCR-based cloning technique. The eighth 27–amino acid peptide, F-8, was a
synthetic peptide. The pET15b-Hsp90α constructs were transformed into BL21-
codonPlus (DE3)-RP competent cells (Stratagene) following the manufacturer-provided
protocol. Protein synthesis was induced by the addition of 0.25 mM isopropyl-β-d-
thiogalactoside (I5502-09; Sigma-Aldrich) to the bacterium culture (OD ≈ 0.8) and
incubation for additional 5 h at 25°C. These His-tagged proteins were first purified by
nickel-nitriloacetic acid column with the HisBind purification kit (EMD Biosciences)
according to the manufacturer’s procedure. The purified proteins were concentrated in
Amicon Ultra (10× or 50×; Millipore, Billerica, MA) to ∼4 ml, filtered (0.22 μm) prior to
load onto a Superdex-200 or 75 HiLoad gel filtration column (GE Healthcare,
Piscataway, NJ), and separated by FPLC. The peptides were eluted by Dulbecco’s phos-
phate-buffered saline (DPBS) buffer (1.2 ml/min), concentrated in a Centricon YM-50 or
YM-10 to 1 mg/ml, and stored in 10% glycerol–DPBS at −70°C.

72
Circular dichroism spectroscopy
F-5 was exchanged into 20 mM K2HPO4/KH2PO4, pH 7.4, 25 mM KCl solution
by four ultrafiltration–dilution cycles (1:10 dilution) and adjusted to a concentration of 20
mM employing e280 nm = 15,470 M−1 cm−1 (Gill and Vonhippel, 1989). CD
measurements were carried out at 25°C on a JASCO (Easton, MD) J-810 spec-
tropolarimeter by acquiring spectra from 190 to 260 nm in a quartz cell of 1-cm path
length. Sixteen scans, recorded in 0.1-nm steps at a rate of 50 nm/min with 0.1-nm
bandwidth and 0.5-s integration time, were accumulated. Spectra were corrected for
solvent contributions. The observed ellipticity in millidegrees, Q, was converted into the
mean residue ellipticity, [Q]MRW, using [Q]MRW = (MRW × Q)/(10dc), where d is the
path length in cm, c is the protein concentration in mg/ml, and MRW (mean residue
weight) is equal to MW/(n − 1), with MW denoting the molecular weight of the
polypeptide chain in daltons and n representing the number of amino acids in the chain.
Peptide secondary structure content was estimated using the CONTIN-LL program
(Provencher and Glockner, 1981) via the DichroWeb interface (Lobley et al., 2002;
Whitmore and Wallace, 2004).

Tumor formation and bioluminescence imaging in mice
Athymic nude mice (4–6 wk of age; Harlan, Livermore, CA) were implanted with
three different cell lines (n = 7 per cell line) to determine the role of LRP-1/CD91
signaling in lung colonization of MDAMB-231 with control shRNA (against Lac-Z),
MDAMB-231 LRP-1-RNAi, and LRP-1−/− MDAMB-468. Mice were anesthetized with

73
2% isoflurane inhalant gas anesthesia using a vaporizer and injected with 1 × 106
cells/mouse intravenously via the tail vein using custom-made catheters to create the
metastatic model. All animal experiments were performed in accordance with the
protocol approved by the University of Southern California Institutional Animal Care and
Use Committee (IACUC). Optical imaging was performed using the IVIS 200 Imaging
System (Xenogen, Alameda, CA), which uses a cooled CCD camera for optimized
sensitive, low-light-level in vivo imaging. Mice were anesthetized throughout the study
using 2% isoflurane inhalant gas anesthesia, followed by an intraperitoneal injection of
the luciferase substrate d-luciferin (50 mg/kg; Caliper Life Sciences, Alameda, CA).
Distribution of the substrate occurred for 12 min, followed by bioluminescence imaging
with the following settings: 1 min/scan; bin, 8; field of view, 13.1 cm; and f-stop, 1. Mice
were imaged sequentially in both dorsal and ventral views during the bioluminescence
signal plateau phase and analyzed using Living Image 3.1 Software (Xenogen). Mice
were imaged weekly until IACUC endpoints were met. All images were normalized to
the same image pseudocolor scale, and circular regions of interest were drawn over the
chest area to quantify the bioluminescence signal. Data from the MDAMB-231-vector
group were compared using a Wilcoxon rank-sum test for statistical significance (p <
0.05) and qualitatively compared with the distribution of signal from the MDAMB-231-
LRP-1-RNAi and LRP-1−/− MDAMB-468 groups. The experiment was repeated three
times.

74
Histochemistry
Whole lung with or without primary tumors were dissected, fixed in 10%
Formalin (Sigma-Aldrich), embedded in paraffin, cut into 6-μm sections throughout the
entire lung, and stained with H&E. Slides were analyzed and the tumors counted through
the whole-lung section on slides using a microscope and quantified.

Statistical analyses
Cell migration and invasion data are presented as mean ± SD. All the in vitro cell
experiments were analyzed with a two-tailed Student’s t test with a confidence interval of
>90%. Analysis of the lung colonization experiment data (photons/second) was
performed using the two-tailed nonparametric Mann–Whitney test. p < 0.05 was
considered statistically significant.

75
RESULTS
Constitutively expressed HIF-1α is essential for invasiveness of breast cancer cells
We wanted to identify a tumor cell line with deregulated expression of HIF-1α
and use this cell model for identifying new downstream effectors of the deregulated HIF-
1α essential for cell invasion in vitro and tumor formation in vivo. After screening
various tumor cell lines (listed in Materials and Methods), we focused on the triple
negative breast cancer cell line MDA-MB-231, previously isolated from pleural effusion
obtained from 51-year-old patient with invasive and metastatic cancer (Cailleau et al.,
1974). This choice also reflected the clinical data showing that ∼30% of invasive breast
cancer samples are hypoxic (Dales et al., 2005; Lundgren et al., 2007). The human
primary dermal fibroblasts HDF were included as a control. As shown in Figure 3.1A, in
HDF cells, the HIF-1α level was almost undetectable under normoxia. A time-dependent
accumulation of HIF-1α protein was detected from the cells under hypoxia (Figure 3.1B).
Under identical conditions (equal protein loading, side-by-side operations, and enhanced
chemiluminescence [ECL] processes), however, a constitutive basal level of HIF-1α ex-
pression could be detected in MDA-MB-231 cells even under normoxia (Figure 3.1A).
Whereas hypoxic treatment of the cells caused a transient increase in HIF-1α, the level
reached a plateau between 3–6 h and then declined to the basal level by 12 h. Although
the significance of this short-term induction of HIF-1α in response to hypoxia in MDA-
MB-231 cells remains to be studied, the constitutive presence of HIF-1α is consistent

76

Figure 3.1 Deregulated HIF-1α is critical for breast cancer cell migration and
invasion. Western blot analysis of the HIF-1α levels in human dermal fibroblasts (HDF)
and breast cancer cells (MDA-MB-231) under either normoxia (21% O2, lane 1) or
hypoxia (1% O2, lanes 2–5) (A) with or without starvation overnight (B) over the
indicated time points. Note: Equal loadings of all samples and all procedures side by side.
HIF-1α
GAPDH
N or Hyp Nor Hyp Nor Hyp Nor Hyp
Unstarved Starved Unstarved Starved
HDF MDA-MB 231
HIF-1α
GAPDH
HIF-1α
GAPDH
Hyp(hr) 0 1 3 6 12 24 48 48(N)
HDF
MDA-MB
231
A
B

77
with the increased anti–HIF-1α antibody staining of many human breast tumor tissue
specimens (Dales et al., 2005; Lundgren et al., 2007).
The constitutive presence of HIF-1α alone in MDA-MB-231 cells is sufficient for
maintaining the cells’ high motility and invasiveness even under serum-free conditions (a
mimic of the hypoxic tumor environment in vivo). We used the lentiviral system FG-12
to deliver a U6 promoter–driven short hairpin RNA (shRNA) against human HIF-1α or
HIF-1β into MDA-MB-231 cells. This system enables >80% gene transduction efficiency
in these cells, as indicated by expression of a cytomegalovirus (CMV) promoter–driven
green fluorescent protein (GFP) gene in the same vector (Figure 3.2A, right vs. left).
Under this system, as shown in Figure 3.2B, we achieved nearly complete down-
regulation of HIF-1α or HIF-1β independently, in comparison to a control shRNA against
LacZ (lanes 1). Moreover, neither of the two shRNAs cross-reacted nonspecifically
between HIF-1α and HIF-1β. When these HIF-1α- or HIF-1β- down-regulated cells were
subjected to cell motility (“scratch”) assays (Figure 3.2C). As shown Figure 3.2D, the
control MDA-MB-231 cells exhibited a constitutively high motility even under serum-
free conditions (Figure 3.2D, b vs. a). However, down-regulation of HIF-1α or HIF-1β
paralyzed the cell motility (Figure 3.2D, d and f vs. c and e). Similar results were
obtained for in vitro invasiveness of the same cells. As shown in Figure 3.3, control
MDA-MB-231 cells strongly penetrated a Matrigel barrier (consisting of laminin, type IV
collagen, heparan sulfate proteoglycan, and nidogen) under serum-free conditions (Figure
3.3, a). However, the HIF-1α– or HIF-1β–down-regulated cells exhibited a dramatic

78

Figure 3.2 HIF-1α and HIF-1β expression is required for breast cancer cell
migration and invasion. (A) The efficiency of FG-12 lentiviral infection in MDA-MB-
231 cells, as indicated by expression of an in-cis CMV-driven GFP gene, followed by
FACS analyses. The same field was shown with either phase contrast (left) or
fluorescence lens (right). (B) Specific down-regulation of HIF-1α (a) or HIF-1β (d)
proteins by FG-12-delivered shRNA, as indicated by Western blot analyses. (C) A
schematic showing the principle of scratch migration assay. (D) Twelve-well tissue
culture plates were precoated with type I collagen (20 μg/ml, 2 h). Serum-starved cells
were plated (250,000 cells/well) in serum-free medium, and >90% of the cells attached
within 2 h. The wound closure at 16 h was photographed and quantified as average gap
(AG; Li et al., 2004). n = 3, p < 0.05.
GAPDH
HIF-1α
GAPDH
HIF-1β
Scratch Migration Assay
sh-lacZ sh-HIF-1α sh-HIF-1β
a.
d.
e.
f.
c.
b.
0hr
24hr
A B
C D

79

Figure 3.3 Down-regulation of HIF-1α or HIF-1β inhibits MDA-MB-231 cell
invasion. Invasion potential of HIF-1α or HIF-1β knockdown and control MDA-MB-231
cells was examined through a Matrigel barrier (b and c vs. a), in accordance with the
manufacturer’s protocol. Note: OD reading (Bio-Rad Protein Assay at 590 nm) on the
penetrated cells only. The data are expressed as means ± SD (n = 4, p < 0.05).
Matrigel Invasion Assay
c.
a.
b.
c.
sh-lacZ
sh-HIF-1α
sh-HIF-1β
O.D.(595 nm)
0.12 0.07
0.018 0.009
0.020 0.01
A B

80
reduction in invasion (Figure 3.3, b and c vs. a). These results establish the MDA-MB-
231 cell line as an adequate tumor cell model to study actions of the deregulated HIF-1α.

Deregulated HIF-1α causes constitutive Hsp90α secretion
We used the following three criteria to find a key downstream and “druggable”
target directly regulated by deregulated HIF-1α in MDA-MB-231 cells: 1) the protein is
constitutively secreted by the cells; 2) the secretion is under direct control of the
deregulated HIF-1α; and 3) function of the protein is essential for invasiveness of the
tumor cells. We focused on the secreted heat shock protein-90α (Hsp90α). First, many
tumor cells constitutively secrete Hsp90α (Li et al.). Second, hypoxia causes various
types of cells to secrete Hsp90α (Li et al., 2007; Li et al.; Woodley et al., 2009). Third,
secreted Hsp90α is essential for hypoxia-driven normal cell migration (Woodley et al.,
2009). Therefore we tested the possibility that the deregulated HIF-1α causes constitutive
Hsp90α secretion, which is crucial for the invasiveness of MDA-MB-231 cells. Serum-
free conditioned media (CM) of HDF and MDA-MB-231 cells cultured under either
normoxia or hypoxia were analyzed for the presence of Hsp90α. The specificity of the
anti-Hsp90α was con¬firmed by experiments showing that under the same conditions this
antibody did not cross-react with Hsp90β or Grp94 (Figure 3.4A), two proteins highly
related to Hsp90α. As shown in Figure 3.4B, secreted Hsp90α was detected from the CM
of HDF cells incubated under hypoxia but not normoxia. In contrast, an equal amount of
secreted Hsp90α was detected from CM of MDA-MB-231 cells cultured under either
normoxia or hypoxia. The constitutive secretion of Hsp90α was caused by the

81

Figure 3.4 Deregulated HIF-1α uses secreted Hsp90α for migration and invasion.
(A) Approximately 400 ng each of recombinant Hsp90α, Hsp90β, and GRP94 were
resolved on duplicate SDS gels and subjected to either Coomassie blue staining (top) or
Western blot using the same anit-Hsp90α antibody (bottom). This antibody is specific
against Hsp90α. (B) Serum-free conditioned medium (CM; 25 μl of 10× concentrated) of
HDF (lanes 1 and 2) or MDA-MB-231 (lanes 3 and 4) cells incubated under normoxia
(N) or hypoxia (H) for 14 h was analyzed for the presence of Hsp90α proteins by
Western blotting. (C) CM of HIF-1α– or HIF-1β–down-regulated cells was analyzed for
the presence of Hsp90α (a, lanes 2, 3 vs. lane 1). A 1.0-ml amount of 1× CM was
concentrated 20 times and subjected to zymography gel analysis (Materials and Methods;
b, lanes 1–3). (D) Reintroduction of wt and CA mutant (a, lanes 2, 3), but not dominant-
negative mutant (b, lanes 4), of HIF-1α rescued Hsp90α secretion in HIF-1α–down-
regulated MDA-MB-231 cells (c, lanes 2 and 3 vs. lanes 1 and 4).
Nor H yp Nor Hyp
Secreted
Hsp90α
HDF MDA-MB 231
Conditioned Media (CM) of
WB : anti-Hsp90α
Stain : Coomassie Blue
75
150
50
100
250
Lac-Z HIF-1α HIF-1β
Secreted
Hsp90
CM of MDA-MB 231 w/ shRNA against
Secreted
MMP9
Vec. wt CA DN
Rescuing Hsp90 secretion
In HIF1α-knock-down 231
GAPDH
Wt
CA
HA-DN
Secreted
Hsp90
A
D C
B

82
deregulated HIF-1α in the cells. It is shown in Figure 3.4C that, whereas Hsp90α
secretion remained unaffected in control RNAi–infected MDA-MB-231 cells (Figure
3.4C, a, lane 1), secreted Hsp90α was undetectable from the CM of either HIF-1α– or
HIF-1β–down-regulated MDA-MB-231 cells (Figure 3.4C, a, lanes 2 and 3). This
inhibition appeared to be specific, since under identical conditions, secretion of matrix
metalloproteinase 9 (MMP9) by the cells was rather slightly increased.. Note that, unlike
intracellular proteins (like β-actin or glyceraldehyde-3-phosphate dehydrogenase for
intracellular protein standards), there are few reliable loading control markers for secreted
proteins. The equal loadings of CM were justified by taking equal volumes of CM from
the same number of cells cultured under identical conditions.
To validate the specific control of Hsp90α secretion by HIF-1α, we carried out
HIF-1α gene rescue experiments. As shown in Figure 3.4D, we exogenously reintroduced
wild-type (wt) and constitutively active (CA) HIF-1α into endogenous HIF-1α–depleted
MDA-MB-231 cells, as detected by anti–HIF-1α antibody immunoblotting analysis.
Because DN-HIF-1α has a large deletion that most commercial anti-HIF-1α antibodies
recognize (Li et al., 2007), we instead proved the expression of the 34-kDa DN-HIF-1α
by using anti–hemagglutinin tag antibody blotting. From CM of these cells, we found that
only wt-HIF-1α and CA-HIF-1α were able to rescue Hsp90α secretion, but not DN-HIF-
1α. These results indicated that secreted Hsp90α is a direct downstream target for
deregulated HIF-1α. More important, the wt-HIF-1α and CA-HIF-1α (Figure 3.5, d and f
vs. b), but not DN- HIF-1α (Figure 3.5, h vs. b), were also able to rescue the blocked cell

83

Figure 3.5 Expression of exogenous HIF-1α in HIF-1α-depleted MDA-MB-231 cells
rescues their cell migration and invasion. Twelve-well tissue culture plates were coated
with type I collagen (40 μg/ml, 2 h). Unattached collagens were removed by washing
with Hank’s balanced salt solution buffer. Serum-starved (18 h) MDA-MB-231 cells
were plated (250,000 cells/well) under serum-free medium so that the cell density
reached 90% confluence within 2 h. The “wounds” were made with a p-200 pipette tip.
The wound closure was photographed and quantitation (AG) carried out as described (Li
et al., 2004). The results shown here were reproducible in three independent experiments
(n = 3, p < 0.05).

84
motility of the endogenous HIF-1α–depleted cells. This work was done in collaboration
with Dr. Zhengwei Zhao.
Next we examined whether secreted Hsp90α mediates deregulated HIF-1α–driven
MDA-MB-231 cell migration and invasion. We used a neutralizing antibody against
secreted (not intracellular) Hsp90α. We applied the colloidal gold migration assay
(Materials and Methods), which measures individual (instead of a population) cell
motility and is more relevant to Hsp90α autocrine signaling. As shown in Figure 3.6,
even under serum-free conditions the MDA-MB-231 cells exhibited constitutive motility
(Figure 3.6, a). The addition of control immunoglobulin G (IgG) showed little effect
(Figure 3.6, b vs. a). However, the addition of increasing amounts of the neutralizing
antibody against Hsp90α into the medium inhibited the motility of the cells in a dose-
dependent manner (Figure 3.6, c–e). This inhibition was Hsp90α specific, since the
addition of excess amounts of recombinant Hsp90α protein reversed the inhibition of cell
migration by the anti-Hsp90α neutralizing antibody (Figure 3.6, f). Besides its inhibitory
effect on cell migration, the same neutralizing antibody also blocked the ability of MDA-
MB-231 cells to invade through a Matrigel barrier. As shown in Figure 3.7, the addition
of increasing amounts of the antibody blocked the cell invasion in a dose-dependent
manner (Figure 3.7, c–e), in comparison to medium alone (Figure 3.7, a) or medium with
control IgG (Figure 3.7, b). Similarly, the addition of excess amounts of recombinant
Hsp90α protein reversed the inhibition of invasion by the anti-Hsp90α antibody (Figure
3.7, f). The experiments with neutralizing antibody were carried out by Dr. Zhengwei
Zhao. Taken together, the results lead us to conclude that deregulated HIF-1α in the

85

Figure 3.6 Secreted Hsp90α is essential for migration of MDA-MB-231 cells under
serum-free conditions. Colloidal gold migration assays show that anti-Hsp90α
neutralizing antibodies blocked individual MDA-MB-231 cell motility in a dose-
dependent manner (c–e vs. a, b). The addition of excess amount of recombinant Hsp90α
reversed the inhibition by the antibodies (f). MI, migration index (%) (Li et al., 2004).
Untreated Control IgG (10 μg) anti-Hsp90α (1 μg)
MI (%): 20 2.7 22 3.1 14.3 2.9
d.
b
anti-Hsp90α (3 μg) anti-Hsp90α (10 μg)
MI (%): 6.1 1.7 3.6 2.1 18 4.1
f. e.
anti-Hsp90 (10 μg) +
Hsp90α (30μg)
c. b. a.

86

Figure 3.7 Secreted Hsp90α is essential for invasion of MDA-MB-231 cells under
serum-free conditions. Anti-Hsp90α neutralizing antibodies blocked MDA-MB-231 cell
invasion through a Matrigel in a dose-dependent manner (c–e vs. a, b). The addition of
excess amount of recombinant Hsp90α reversed the inhibition of invasion by the
antibodies (f). The OD reading is the same as in Figure 3.1.
a. b. c.
Serum-Free Control IgG (10 μg) anti-Hsp90α (1 μg)
anti-Hsp90α (3 μg)
d.
anti-Hsp90α (10 μg)
e.
anti-Hsp90α (10 μg)
+ Hsp90α (30 μg)
f.
O.D: 0.14 0.03 0.15 0.05 0.08 0.01
O.D.: 0.04 0.005 0.017 0.007 0.12 0.04

87
tumor cells causes constitutive Hsp90α secretion, which is crucial for migration and
invasion in the absence of any exogenous growth factors.

Identification of the key element in secreted Hsp90α that mediates deregulated HIF-
1α–driven invasion
We used systematic mutagenesis to identify the minimum functional element in
human Hsp90α. Initially, eight recombinant peptides of Hsp90α were generated and
tested for stimulation of MDA-MB-231 cell motility under serum-free conditions. Five of
the eight peptides (FL, F-2, F-5, F-6, and F-8), which retained various degrees of the full
promotility of the full-length Hsp90α, are schematically shown in Figure 3.8A. The
purity of the fast protein liquid chromatography (FPLC)–purified peptides was revealed
by SDS–PAGE and staining (Figure 3.8B). F-8 is a 27–amino acid synthesized peptide
(too small to show on SDS gel). Because down-regulation of the endogenous HIF-1α in
MDA-MB-231 cells blocked Hsp90α secretion and reduced invasiveness of the cells, we
reasoned that exogenous supplementation of functional peptides of Hsp90α should
bypass the blockade of HIF-1 down-regulation and rescue the invasion defect of the cells.
This rescue approach would allow us to identify the minimum functional element in
secreted Hsp90α. As shown in Figure 3.9A, the parental and control LacZ-RNAi–
infected MDA-MB-231 cells showed strong invasion (Figure 3.9A, a and b), whereas
HIF-1α-down-regulated cells were unable to invade (Figure 3.9A, c). Supplementation of
BSA had little effect on the invasion defect (Figure 3.9A, d). However, FL, F-2, and F-5
under their optimized concentrations were able to partially rescue the invasion defect of

88

Figure 3.8 Expression of various recombinant Hsp90α peptides that retain either a
full or partial promotility activity of full-length Hsp90α. (A) A summary of truncated
peptides of Hsp90α that retain either a full or partial promotility activity of full-length
Hsp90α. Cell motility data are summary (%) of colloidal gold motility and “scratch”
assays combined (n = 3, each assay, p < 0.05). (B) FPLC-purified, full-length, F-2, F-5,
and F-6 were visualized in SDS gel stained with Coomassie brilliant blue (lanes 1–4),
with indicated amounts of BSA as controls (lanes 5–7). F-8 is a synthetic peptide.

89

Figure 3.9 F-5 epitope in secreted Hsp90α rescues invasion defect of HIF-1α–down-
regulated MDA-MB-231 cells. (A) The five peptides with their optimized
concentrations were tested for rescuing invasion defect of HIF-1α–down-regulated MDA-
MB-231 cells. (B) Quantitation of the invasion data (n = 3, p < 0.05) in A.

c. b. d.
e. f. h.
a.
g.
sh-HIF1α + F-6 sh-HIF1α + F-5 sh-HIF1α + F-2 sh-HIF1α + FL
sh-LacZ parental sh-HIF1α sh-HIF1α + BSA A
B

90
the cells (Figure 3.9A, e–g). In contrast, F-6 (Figure 3.9A, h) and F-8 (data not shown)
showed significantly weaker rescuing effects, even if they still retained promotility
activity. Quantitation of the data, as shown in Figure 3.9B, revealed that FL, F-2, and F-5
were equally effective but F-6 was virtually unable to rescue. Similar results were
obtained in HIF-1β–down-regulated MDA-MB-231 cells, in which F-5 was the shortest
peptide that rescued the invasion defect of the cells (Figure 3.10). To further confirm that
the rescuing mechanism by these peptides was to bypass the blockade of HIF-1α down-
regulation, we found that supplementation of F-5 was unable to rescue the invasion defect
of MDA-MB-231 cells with down-regulated LRP-1, the receptor for extracellular Hsp90α
signaling (Cheng et al., 2008; Woodley et al., 2009). As shown in Figure 3.11A,
complete down-regulation of LRP-1 by the FG-12 system resulted in dramatic reduction
of the cell invasion (Figure 3.11B, b vs. a). However, the addition of F-5 was unable to
rescue the LRP-1 depletion–caused invasion de fect (Figure 3.11B, c). We also found that
α-2 macroglobulin, another natural ligand for LRP-1, did not affect Hsp90α-driven
MDA-MB-231 cell invasion, suggesting that these two proteins bind to distinct regions at
the extracellular domain of LRP-1 (Figure 3.12).

Interruption of Hsp90α–LRP-1 signaling in MDA-MB-231 cells blocks their ability
for lung colonization and tumor formation
Colonization of various secondary organs by breast cancer depends on productive
interactions between the tumor cells and the stromal microenvironment. The lung
colonization assay in nude mice is an accepted model to test such ability of tumor cells.

91

Figure 3.10 F-5 epitope in secreted Hsp90α rescues invasion defect of HIF-1β–down-
regulated MDA-MB-231 cells. The five peptides with their optimized concentrations
were tested for rescuing the invasion defect of the endogenous HIF-1β-downregulated
MDA-MB-231 cells. Quantitation of the invasion data shown in O.D. (n=4, p < 0.05).

sh-HIF1 β + F-6 sh-HIF1 β + F-5 sh-HIF1 β + F-2 sh-HIF1 β + FL
sh-LacZ parental sh-HIF1 β sh-HIF1 β + BSA
c. b. d.
e. f. h.
a.
g.

92

Figure 3.11 LRP-1 mediates is essential for secreted Hsp90α-induced invasion of
MDA-MB-231 cells. (A) Lysates of MDA-MB-231 cells infected with vector alone (lane
1) or lentivirus carrying shRNA against LRP-1 receptor (lane 2) were analyzed by
Western blotting with anti–LRP-1 antibody. (B) LRP-1–down-regulated cells were
unable to invade as the control cells (b vs. a), and Hsp90α was unable to rescue the
invasion defect of the LRP-1–down-regulated cells (n = 4, p < 0.05).
a.
Vector
sh-LRP1
O.D. (595 nm)
0.14 0.02
0.034 0.01
Infected w/
a.
b.
0.019 0.07
c.
sh-LRP1
+
F-5
LRP1
GAPDH
B A

93

Figure 3.12 α2M (α2-macroglobulin) did not affect Hsp90α rescue of invasion defect
of HIF-1α–down-regulated MDA-MB-231 cells. Hsp90α was tested for rescuing
invasion defect of HIF-1α–down-regulated MDA-MB-231 cells in the absence or
presence of α2-macroglobulin (A2M) Panels: (a) ) Lac-Z-RNAi-infected cells ; (b) HIF-1
α -RNAi-infected cells; Hsp90α full-length protein (FL) added to HIF-1 α -RNAi-
infected cells, without (c) or with (d to f) increasing concentrations of A2M did not affect
Hsp90α rescue of HIF-1 α -RNAi-infected cells.Quantitation of the invasion data shown
in O.D.
sh-HIF1α + FL sh-LacZ sh-HIF1α
sh-HIF1α + FL
+ A2M 25nM
sh-HIF1α + FL
+ A2M 50nM
sh-HIF1α + FL
+ A2M 75nM
c. b.
d. e. f.
a.
O.D: 0.19 0.07 0.03 0.07 0.13 0.06
O.D: 0.15 0.09 0.17 0.04 0.16 0.07

94
There is lack of effective and specific inhibitors against secreted Hsp90α actions in vivo.
Genetic knockdown of the entire Hsp90α would not distinguish between intracellular and
extracellular Hsp90α. Membrane-impermeable GM-based inhibitors were unstable and
toxic to the animal when they were injected into the circulation (Stellas et al., 2010;
Tsutsumi et al., 2008). Having considered these limitations, we took an alternative
approach to target an immediate downstream effector of secreted Hsp90α, the LRP-1
receptor. We used the FG-12 system to permanently knock down LRP-1 in MDA-MB-
231 cells. As shown in Figure 3.13A, complete down-regulation of LRP-1 was verified in
the exact MDA-MB-231 cells 24 h prior to injection into nude mice. In addition, we
found that another breast cancer cell line, MDA-MB-468, lost endogenous LRP-1
expression (Figure 3.13B, lane 4). Of interest, like MDA-MB-231 cells, MDA-MB-468
cells maintain constitutive HIF-1α expression and constitutive Hsp90α secretion (Figure
3.13C. Thus we used MDA-MB-468 cells as natural LRP–/– cells. All the cell lines,
preengineered to stably express a luciferase gene, were injected via (Figure 3.13D, 4–6).
In contrast, LRP-1–knockdown MDA-MB-231 cells showed much reduced ability of
lung colonization (Figure 3.13D, 10–12), which occurred in only two out of seven mice
per group. MDA-MB-468 cells showed little lung colonization in all mice (Figure 3.13D,
16–18). Dissection of the mice on day 70 revealed visible tumors only in lungs of the
mice injected with vector MDA-MB-231, but not with LRP-1–knockdown MDA-MB-
231 cells or MDA-MB-468 cells. Hematoxylin and eosin (H&E) staining of sections
across the entire lung tissue, showed large, invading tumors in the lungs of mice injected
with vector MDA-MB-231, smaller and fewer tumors in lungs of mice

95

Figure 3.13 Hsp90α signaling is essential for MDA-MB-231 cell lung colonization and
tumor formation in vivo. (A) Lentiviral system, FG-12, mediated shRNA-LRP-1 delivery
LRP-1
GAPDH
LRP-1
GAPDH
DF 231 HK 468 435 PC3
GAPDH
HIF-1α
Nor Hyp
Secreted
Hsp90α
MDA-MB-468
B A C
D

96
and down-regulation of endogenous LRP-1 in MDAMB-231 cells (lane 2 vs. lane 1). (B)
Western blot screening of normal and cancer cell lines for expression of LRP-1 receptor.
(C) Constitutive expression of HIF-1α in MDA-MB-468 cells under either normoxia (N)
or hypoxia (H; a). Constitutive secretion of Hsp90α by MDA-MB-468 cells (c). (D)
Approximately 1 × 106 luciferase-engineered MDAMB-231 cells infected with either
vector only (a) or vector carrying shRNA-LRP-1 (b) or LRP-1−/− MDAMB-468 cells (c)
were injected into the tail vein of SCID mice (n = 7 per group). Whole-body
bioluminescence imaging of the mice was performed once a week. Representative images
(two per group) of the mice show lung colonization of the injected cells at days 1, 14, 28,
56, and 70.

97
injected with LRP-1–knockdown MDA-MB-231 cells, and no visible tumors in lungs of
mice injected with MDA-MB-468 cells (data not shown). LRP-1 (also called CD91) is a
large heterotrimeric protein consisting of a 515-kDa extracellular domain and an 85-kDa
transmembrane subunit (Herz and Strickland, 2001). Besides Hsp90α, several other
extracellular heat shock proteins were reported to also bind LRP-1 (Basu et al., 2001).
Therefore the effect of LRP-1 knockdown on tumor formation did not necessarily prove
that the reduced tumor formation was specifically due to blockade of secreted Hsp90α
signaling through LRP-1. To address this issue, we tested whether recombinant Hsp70,
gp96, and CRT, which have also been shown to bind LRP-1, promote cell migration and
whether they could rescue, like Hsp90α, the invasion defect of HIF-1α–down-regulated
MDA-MB-231. In both migration and invasion assays, Hsp70, gp96, and CRT showed
limited effects (data not shown). Taken together, these findings support the hypothesis
that the “HIF-1 > secreted Hsp90α > LRP-1” autocrine loop plays a critical role in
deregulated HIF-1α–driven tumor progression. The LRP-1 knockdown experiments were
done by Dr. Chieh-Fang Cheng and the lung colonization assay was done by him in
collaboration with Conti lab.

Regulation of Hsp90α secretion in MDA-MB-231 by Hif-1α
Since HIF-1α is essential for Hsp90α secretion, we wanted to investigate how it
regulates this process. Hif regulates transcription oof its target genes by binding to the
Hypoxia Response Elements (HREs) in their promoter regions. However Hsp90α does
not contain any HREs in its promoter. To find out which, if any, steps of HIF-1α

98
transcriptional regulation, i.e. dimerization, DNA binding or transactivation, are required
for Hsp90α secretion, we made deletion mutants of HIF-1α as described in Jiang et al.
(Jiang et al., 1996). The 4-27 aa deletion mutant is deficient in DNA binding, even
though it can dimerize. The 1-813 mutant is unable to transactivate transcription even
though it can dimerize and bind to DNA. 1-390 mutant is sufficient to dimerize. WE also
made a mutant for the second half of HIF-1α, 391-826, as well as a shorter fragment,
521-826, which constitutes the second half of the CA fragment (1-390/521-826). These
mutants are schematically shown in Figure 3.14A. The latter three mutants are fused to
gfp protein. These constructs were cloned into pRRLsinhCMV vector using
BamH1/Xba1 sites. Figure 3.14B shows the constructs before and after digestion. These
constructs will be used to overexpress these mutants after which Hsp90α secretion would
be assessed. This would help elucidate how HIF-1α regulates Hsp90α secretion.

99

Figure 3.14 Construction of Hif-1α mutants for studying effect on Hsp90α secretion.
(A) A schematic of Hif-1α mutants. (B) The Hif-1α mutant constructs were cloned into
pRRLsinhCMV vector. They are shown before (left panel) and after (right panel)
digestion with BamH1/Xba1.

100
DISCUSSION
Under constant hypoxia, cancer cells are forced to adapt, via HIF-1α, alternative
and self-supporting mechanisms for continued survival and expansion. Surface
expression and secretion of Hsp90 should be constitutive in these HIF-1α–positive
tumors. In fact, many types of tumor cells have recently been shown to secrete Hsp90 (Li
et al.). Therefore targeting the secreted Hsp90 for treatment could break the boundary of
tumor type and subtype specificities. Take breast cancers as an example; they are
traditionally divided into three major subtypes: ER+/PR+, HER2+ and TN (triple
negative). The death rate from the disease has dropped modestly over the past decade due
to the availability of multiple treatment choices: surgery, radiation, hormonal therapy,
and chemotherapy. However, there remains a growing sense of frustration among breast
cancer experts over side effects and unsustainable results of current treatments, such as
resistance of hormone receptor–positive breast cancers to endocrine therapies. It appears
that every breast cancer is genetically unique. Many of the genetic differences among
individual tumors influence the likelihood that the cancer will recur (Rosman et al.,
2007). Dales et al. (2005) carried out anti–HIF-1α immunohistochemical assays on
frozen sections of 745 breast cancer samples and found that the levels of HIF-1α
expression correlated with poor prognosis, lower overall survival, and high metastasis
risk among both node-negative and node-positive patients (Dales et al., 2005). By using
HIF-1α expression as a marker, it was estimated that 25–40% of all invasive breast
cancer samples are hypoxic, suggesting that HIF-1α can be used as a broader marker for
breast cancers. In the present study, we identified a critical downstream effector of HIF-

101
1α in breast cancer cells—the secreted Hsp90α. Of the 732–amino acid polypeptide, we
narrowed down a 115–amino acid epitope called F-5 in Hsp90α that is necessary and
sufficient for mediating HIF-1α–driven breast cancer cell invasion. We propose a shift of
paradigm for anticancer drugs targeting Hsp90 from inhibiting the intracellular Hsp90α at
its ATPase to targeting the secreted Hsp90α at the F-5 region, as schematically
summarized in Figure 3.15.
Toxicity versus efficacy has been a long-standing challenge for anticancer drugs
targeting the ATPase of intracellular Hsp90 in clinical trials. These inhibitors are
expected to selectively harm the overexpressed or overly active Hsp90 in cancer cells that
embed in normal tissues while minimizing potential damages in the physiological
chaperone functions of Hsp90 in the surrounding normal cells and, in fact, the entire
body. Difficulties in satisfying these obviously contradicting demands have prevented
drug developments of this type from reaching the Food and Drug Administration as fast
as they were hoped for. In contrast, as far as the secreted Hsp90α is concerned, no
physiological roles (regulation of gene expression, metabolism, proliferation, and
development) have been reported; instead, secretion of Hsp90α appears to be an
emergency response of normal cells to tissue injury, hypoxia, or irradiation, just to men-
tion a few. However, regardless the environmental status, tumor cells maintain a
constitutive level of HIF-1α and use it to trigger Hsp90α secretion for invasion and
metastasis in an otherwise nonlivable environment for normal cells (Cheng et al., 2010).
Therefore we propose that new anticancer drugs that selectively target the F-5 region of
Hsp90α should achieve higher efficacy and pose less toxicity to cancer patients.

102

Figure 3.15 A model of secreted Hsp90α as a potential target for HIF-1α–positive
cancers. The severe hypoxia often found at the center of a tumor causes constitutive
accumulation of HIF-1α. The deregulated HIF-1α triggers secretion of Hsp90α via
exosomes. The secreted Hsp90α binds, via F-5 epitope, to cell-surface LRP-1 receptor
and promotes motility and invasion of tumor cells in an autocrine manner. Whereas
current clinical trials focus on intracellular HIF-1α or Hsp90α, we propose that targeting
the F-5 epitope of secreted Hsp90α would be more effective and safer in the treatment of
cancer patients.
Secreted-Hsp90 
More effective and safer anti-tumor target?
EEKEDK…..RRAPFD
(F-5)
236
350
Intra-Hsp90 
HIF-1α
(de-regulated in
many tumors)
p
current trials
(e.g. 17-AAG)
?
LRP-1
ATPase
Invasion and metastasis
Hif1  Inhibitors

103
What are the downstream targets of secreted Hsp90α? Eustace et al. (2004) reported that
Hsp90α, but not Hsp90β, promotes cancer cell migration and invasion by binding to and
activating MMP2 (Eustace et al., 2004). Sidera et al. (2004, 2008) showed that a pool of
cell membrane–bound Hsp90α interacts with HER-2 tyrosine kinase receptor in breast
cancer cells, leading to increased MMP2 activation, cell motility, and invasion (Sidera et
al., 2008; Sidera et al., 2004). Gopal et al. (2011) recently reported that extracellular
Hsp90α stimulates a binding of LRP-1 to EphA2 receptor during glioblastoma cell
invasion (Gopal et al., 2011). Cheng et al. (2008) used four independent approaches
(neutralizing antibodies, RAP inhibitor, RNAi, and somatic LRP-1–negative mutant cell
line) to demonstrate that the widely expressed cell surface receptor LRP-1 mediates the
extracellular Hsp90α signaling (Cheng et al., 2008). In the present study, we show that
Hsp90α failed to stimulate migration and invasion of LRP-1–down-regulated MDA-MB-
231 cells in vitro. Consistently, Song et al. (2009) reported that LRP-1 is required for
glioblastoma cell migration and invasion in vitro (Song et al., 2009). Furthermore, LRP-
1–down-regulated MDA-MB-231 or LRP-1–null MDA-MB-468 cells exhibited
dramatically reduced lung colonization and tumor formation in vivo. Understanding how
each of the target proteins contributes to Hsp90α-stimulated invasion would require
simultaneous studies of these molecules in a common cell system.
Despite the various experimental approaches, direct demonstration of secreted
Hsp90α in tumor progression in vivo still requires the availability of more-stable and
more-specific inhibitors. Tsutsumi and colleagues used DMAG-N-oxide, a
geldanamycin/17-AAG–derived and cell membrane–impermeable Hsp90 inhibitor, to

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pretreat melanoma cells to block extracellularly located Hsp90α, prior to injecting them
into nude mice. They reported that DMAG-N-oxide–treated cells showed decreased
motility and invasion of the cells in vitro and reduced lung colonization in vivo (Tsutsumi
et al., 2008). There are several limitations of this approach. First, 17-AAG binds and
inhibits the ATPase activity of Hsp90. However, Cheng et al. (2008) demonstrated that
the ATPase domain is dispensable for the extracellular action of Hsp90 (Cheng et al.,
2008). Therefore the inhibitory effect of 17-AAG is not a direct inhibition of the
functional epitope in extracellular Hsp90s. Second, it is hard to understand how a single
pretreatment of the cells with the drug in vitro (due to the drug’s structurally instability in
vivo) could have had the reported long-lasting effect after the cells were injected into
mice. Patsavoudi and colleagues reported development of a monoclonal antibody, 4C5,
that neutralizes secreted Hsp90α and Hsp90β in vitro . They reported that melanoma or
breast cancer cells mixed with 4C5 in vitro showed reduced lung colonization, in
comparison to mixing with a control antibody, after the cells were injected into nude
mice (Sidera et al., 2008; Stellas et al., 2010). However, it is hard to imagine that the
coinjected 4C5 could have worked by continuously binding and neutralizing the
constantly secreted Hsp90α and Hsp90β by the tumor cells for the entire period of the
multiweek experiment. It would make more sense to inject and maintain a steady-state
amount of 4C5 in circulation prior to injection with tumor cells. We showed that breast
cancer cells lacking the LRP-1 receptor were unable to effectively form tumors in nude
mice. As pointed out earlier, the effect of down-regulation of LRP-1 may not necessarily
be due to a specific blockade of secreted Hsp90α signaling, since LRP-1 may potentially

105
bind other, unidentified ligands. Therefore identification of the F-5 peptide in secreted
Hsp90α provides an excellent target for the design of new, effective, and more-specific
inhibitors for studying the role of tumor-secreted Hsp90α.

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CHAPTER 4

TARGET VALIDATION OF ROLE OF SECRETED Hsp90α IN WOUND
HEALING AND CANCER

INTRODUCTION
Heat Shock Protein 90alpha (Hsp90α) is an abundant molecular chaperone which
is essential for the proper folding of its substrates or client proteins. Many of these clients
are oncoproteins which are either overexpressed or mutated in cancer cells and regulate
key processes essential for cancer cell survival. Therefore Hsp90α has gained a lot
interest as an anti-cancer target with an aim to simultaneously target all these pathways.
Several Hsp90α inhibitors, starting with Geldanamycin (GA) have been tested. They all
bind to the ATPase domain of Hsp90α targeting its intracellular chaperone function and
have shown great promise at the bench but have miserably failed in clinical trials because
the high doses required for their efficacy pose toxicity issues (Jhaveri BBA 2012).
In recent years, Hsp90α has also been reported to be secreted by human
keratinocytes (HKCs) (Cheng et al., 2008), dermal fibroblasts (HDFs) (Li et al., 2007),
endothelial cells (Song et al, BBRC 2010), neurons (Sidera et al., 2004) and macrophages
(Basu et al., 2001). Except the latter, where Hsp90α is involved in infections, secreted
Hsp90α has primarily been associated with cell migration. Our lab discovered that
Hsp90α is secreted by HKCs (Cheng et al., 2008) and HDFs (Li et al., 2007), under
hypoxia and is responsible for hypoxia induced migration, a process controlled by

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Hypoxia Inducible Factor–1alpha (Hif-1α). We thus hypothesized that secreted Hsp90α is
the key driver of migration of skin cells post wounding, not growth factors, as was
previously believed. This makes Hsp90α a novel candidate as a wound healing agent. We
have shown that recombinant Hsp90α as well as F-5, which is the minimum Hsp90α
fragment required for its pro-motility effect, is able to promote wound healing in nude
mice, diabetic mice and pigs (Cheng et al., 2011; Li et al., 2007).
In contrast to its secretion under stress in normal cells, Hsp90α is secreted out in a
constitutive fashion by tumor cells. Kuroita et al reported Hsp90α secreted from SH-76
hybridoma cells as early as 1992 (Kuroita et al., 1992). Eustace et al found Hsp90α in
conditioned media of HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cell lines,
but not Hsp90β (Eustace et al., 2004). Wang and group detected Hsp90α in conditioned
media of MCF-7 breast cancer cells (Wang et al., 2009). In the past 2 years, work by
several groups has implicated the presence of Hsp90α on the surface of many tumor cells
including bladder, prostate and colorectal cancers, melanoma and glioblastoma (Li et al.,
2012). In cancer, this secreted Hsp90α plays a key role in cancer invasion and metastasis.
The key pro-motility fragment of Hsp90α, F-5, is sufficient to drive Hif-1α mediated
breast cancer cell invasion (Sahu et al., 2011). Wang and colleagues have further shown
an essential role of Hsp90α in tumor invasion in vivo by using an anti-Hsp90α antibody
which resulted in reduced stromal invasion in mice (Wang et al., 2009). Additionally,
they also reported that cancer patients have elevated plasma levels of Hsp90α compared
to normal people or those with benign tumors and that breast and liver cancer patients
with metastasis have higher plasma levels of Hsp90α compared to metastasis-free

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patients. Chen et al observed that colorectal cancer patients had higher Hsp90α levels in
serum compared to normal volunteers (Chen et al., 2010). Since Hsp90α is secreted by
normal cells only under a stress response, extracellular Hsp90α represents a promising
anti tumor target which would be bereft of the toxicity issues faced with targeting
intracellular Hsp90α.
Since secreted Hsp90α plays an essential role in processes involving cell motility,
namely skin wound healing and cancer, there is considerable therapeutic potential for its
use as a wound healing agent and an anti-cancer target. Hence, to bring Hsp90α based
therapies from the bench to the clinic, target validation approaches against extracellular
Hsp90α are high priority. Tsutsumi and colleagues reported a small molecule derivative
of the Hsp90α inhibitor Geldanamycin, DMAG-N-oxide, which is non-permeable and
therefore, unlike its predecessors, would be specific for extracellular Hsp90α. Treatment
of cells with NPGA in vitro has also been reported to inhibit cell migration suggesting its
efficient targeting of secreted Hsp90α.
Given the importance of secreted Hsp90α in cancer cell migration and invasion,
NPGA would potentially be a very good candidate as an anti-cancer target. Tsutsumi and
colleagues showed that NPGA can effectively inhibit melanoma, bladder and prostate
cancer invasion and melanoma cell migration (Tsutsumi et al., 2008). However to test the
effect of NPGA in vivo, instead of administering the drug, they treated the melanoma
cells with NPGA before injecting them in mice. The authors claimed that structural
instability of NPGA in vivo prevented them from administering the drug to the mice.
Even though decrease in lung colonization was reported in this study, it is difficult to

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explain how a single treatment could inhibit the constitutive secretion of Hsp90α by the
cells in vivo. Thus, while NPGA is a good candidate for functional validation of role of
secreted Hsp90α in cell migration and invasion both in wound healing and cancer, its
stability issues limit its use in in vivo studies and as a therapeutic target for the clinic. For
this reason, better antagonists of secreted Hsp90α are needed. Monoclonal antibodies are
a good choice because they are both stable and cell impermeable. The Patsavoudi group
has developed an antibody 4C5 against Hsp90α which reduces cell migration, however it
also recognizes Hsp90β (Sidera et al., 2004). We are thus developing a monoclonal
antibody specific to F-5, which is the minimum fragment carrying the pro-motility effect
of Hsp90α.
In this study, we show that NPGA inhibits exogenous Hsp90α induced cell
migration as well as hypoxia induced secreted Hsp90α mediated cell migration. We also
show that NPGA delays wound healing through decreased re-epithelialization. These data
show that secreted Hsp90α is intrinsically required for the process of wound healing and
provides support for a shift from growth factor based wound healing therapeutics towards
Hsp90α based ones. NPGA has also previously been shown to reduce cancer cell motility
in vitro indicating its promise as an anti-cancer agent. But its instability in vivo has
limited its prospective use in clinic. Thus to develop a better antagonist specific for
extracellular Hsp90α which could be used in clinic as an anti-cancer drug, we are
developing a neutralizing monoclonal antibody against F-5, the key pro-motility epitope
of Hsp90α, which would specifically neutralize extracellular Hsp90α’s pro-motility
function. We propose that F-5 is a better and more specific anti-cancer target and

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therapies targeted against the F-5 region of Hsp90α would be specific against its pro-
motility activity. To achieve this objective, we injected F-5 into Balb/c mice and found
that it elicited a strong immune response. Serum from mice immunized with F-5
exhibited high antibody titers for Hsp90α of primarily IgG subtype. These sera
effectively inhibited hypoxia induced migration in vitro indicating efficient targeting of
secreted Hsp90α. We plan to now use these mice to produce hybridomas which would be
then screened for clones producing monoclonal antibodies specific to Hsp90α. Such an
antibody would be a very useful tool for determining the role of secreted Hsp90α in
cancer invasion and wound healing both in vitro and in vivo

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MATERIALS AND METHODS

Cell culture and Reagents
Primary human neonatal HDFs were purchased from Clonetics and cultured in
DMEM supplemented with 10% FBS. The third or fourth passages were used in cell
migration assays. DMAG-N-oxide (NPGA) and GA were provided by Jennifer Isaacs
(Medical University of South Carolina, Charleston, SC). rhPDGF-BB was purchased
from R&D Systems. Carboxymethylcellulose (CMC) sodium salt (C5678) (pH measured
at 7.14) was from Sigma-Aldrich. Rat type I collagen was purchased from BD
Biosciences. Anti-Hsp90α antibody was from Calbiochem, anti-Akt antibody was from
Upstate and anti-GAPDH antibody was from Cell Signaling Technology. Whole
molecule mouse IgG was from Santa Cruz Biotechnology Inc. p-NPP substrate was from
Biorad. Stat Strips (adhesive bandages) were from Notro Max Products. 3M Coban (self-
adhesive wrap) was from 3M.

Purification and His-tag cleavage of F-5
His-tagged F-5 was produced and purified using a pET system as described
earlier. They were subjected to Thrombin cleavage for His-tag removal using a Thrombin
Cleave kit (Invitrogen) using manufacturer’s instructions. Gel Filtration was carried out
using PD10 columns (GE, 170851-01).

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Cell migration assays
The colloidal gold migration assay, the in vitro wound healing (scratch) assay,
and the transwell assay were as modified and described previously by us (Li et al., 2004;
Lindquist et al., 1988).

Wound healing in mice and H&E staining
For details regarding these procedures, please refer to Chapter 2.

Study approval
All animal studies were conducted using protocols approved, prior to initiation of
this study, by the University of Southern California Institutional Animal Use Committee.

Circular dichroism spectroscopy
F-5 was exchanged into 20 mM K2HPO4/KH2PO4, pH 7.4, 25 mM KCl solution
by four ultrafiltration–dilution cycles (1:10 dilution) and adjusted to a concentration of 20
mM employing e280 nm = 15,470 M−1 cm−1 (Gill and Vonhippel, 1989). CD
measurements were carried out at 25°C on a JASCO (Easton, MD) J-810
spectropolarimeter by acquiring spectra from 190 to 260 nm in a quartz cell of 1-cm path
length. Sixteen scans, recorded in 0.1-nm steps at a rate of 50 nm/min with 0.1-nm
bandwidth and 0.5-s integration time, were accumulated. Spectra were corrected for

113
solvent contributions. The observed ellipticity in millidegrees, Q, was converted into the
mean residue ellipticity, [Q]MRW, using [Q]MRW = (MRW × Q)/(10dc), where d is the
path length in cm, c is the protein concentration in mg/ml, and MRW (mean residue
weight) is equal to MW/(n − 1), with MW denoting the molecular weight of the
polypeptide chain in daltons and n representing the number of amino acids in the chain.
Peptide secondary structure content was estimated using the CONTIN-LL program
(Provencher and Glockner, 1981) via the DichroWeb interface (Lobley et al., 2002;
Whitmore and Wallace, 2004).

Indirect ELISA
Appropriate amounts of antigen in PBS were coated on plates overnight at room
temperature, with gentle agitation. Next day, the wells were washed with washing buffer
- PBS / 0.05% Tween 20, and then blocked for an hour with 0.1% BSA / PBS / 0.05%
Tween 20. The primary antibody diluted in blocking buffer was added and incubated for
30min. After washing, an alkaline phosphatase conjugated secondary antibody diluted in
blocking buffer was added and incubated for 30min. The wells were washed in washing
buffer followed with a final rinse with water. Substrate was added and color was allowed
to develop for 15min. The plates were then read at 450nm wavelength in an Odyssey
MicroWin plate reader.

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RESULTS

Confirmation of cell impermeability of DMAG-N-oxide
DMAG-N-oxide has been reported to be a cell impermeable derivative of
Geldanamycin (GA). However it is seen that there are batch variations in the purity of the
drug. Therefore before using it for further studies, we wanted to establish its cell
impermeability and efficacy. Intracellular Hsp90α chaperone modulates the folding of
many client proteins. If DMAG-N-oxide is cell permeable, it will antagonize intracellular
Hsp90α and destabilize its client proteins. We therefore treated human dermal fibroblasts
(HDFs) with DMAG-N-oxide, side by side with its cell permeable parent drug,
Geldanamycin (GA), as control, and examined the levels of Akt, a well-established client
protein of Hsp90α, in cell lysates. We observed that GA caused a dose dependent
decrease in the levels of Akt while DMAG-N-oxide did not (Fig. 4.1). GA also caused an
increase in the levels of Hsp90α, as expected, due to its dissociation from Heat Shock
Factor, Hsf, whose activity is negatively regulated by Hsp90α. Thus, free of inhibition, it
up-regulates expression of heat shock proteins, including Hsp90α (Tsutsumi et al., 2008;
Whitesell et al., 1994). On the other hand, DMAG-N-oxide did not cause any change in
Hsp90α levels. These data establish DMAG-N-oxide as a non-permeable form of GA and
it will be referred henceforth as NPGA.

NPGA inhibits secreted Hsp90α mediated skin cell migration in vitro
Extracellular Hsp90α has been previously shown by us as a pro-motility factor
which stimulates migration of skin cells (Li Embo 2007, Woodley JCS 2009), Cheng

115

Figure 4.1 NPGA is a non-permeable inhibitor of Hsp90α. Primary HDFs were treated
with indicated concentrations of either NPGA (A) or GA (B) for 24hours and
intracellular levels of Hsp90α and Akt by western blotting. GAPDH was loading control.

Hsp90α
GAPDH
AKT
Hsp90α
GAPDH
AKT
0 0.1 0.5 1 3 uM 0 0.1 0.5 1 3 uM
GA NPGA
A B

116
MCB 2008). Other groups have shown similar role of extracellular Hsp90α in neuronal
cell migration. (Sidera et al, 2004) We wanted to examine if NPGA, which is an
antagonist of extracellular Hsp90α, could abrogate its function and have anti-motility
effects in a colloidal gold migration assay. We treated HDFs with NPGA in the presence
of different stimuli, FBS, PDGF-bb (a known motogen for HDF) and recombinant
Hsp90α. GA was included as a control. As shown in Fig. 4.2A, NPGA inhibited Hsp90α
stimulated migration but did not affect FBS or PDGF-bb stimulated migration. On the
other hand, GA, which also inhibits intracellular Hsp90α, inhibited migration under all
three conditions, concomitant with the fact that client proteins of intracellular Hsp90α
play an important role in mediating migration through different pathways. This
experiment proves three key aspects of NPGA activity - Firstly, it confirms that NPGA
inhibits extracellular secreted Hsp90α function, validating its role as an Hsp90α
antagonist. Secondly, it establishes that NPGA is indeed cell-impermeable since it does
not affect FBS or PDGF-bb stimulated migration. Thirdly, it also shows that NPGA does
not cause any cell toxicity as the cells are able to respond to different stimuli and are able
to turn on respective pathways, leading to migration. Hypoxia is a major stress caused by
wounding. Due to damaged blood vessels, cells on the wound edge are subjected to
hypoxic environment, causing up-regulation of Hypoxia Inducible Factors (Hif). Hif-1α
induces skin cell migration through secretion of Hsp90α (Li et al., 2007). Thus we
wanted to investigate if NPGA can inhibit HDF migration under physiological stimuli,
such as hypoxia. As shown in Fig. 4.3, NPGA inhibited hypoxia stimulated migration,

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Figure 4.2 NPGA inhibits Hsp90α , but not growth factor induced migration. (A)
Primary HDFs were serum-starved overnight and subjected to colloidal gold salt assay
either untreated or treated with indicated growth factors FBS(10%), PDGF-BB (15ng/ml)
or Hsp90α (15μg/ml) with or without 1μM of GA or NPGA. Images of cell migration
from 2 representative experiments are shown. Dotted circles point out the averaged
migration tracks under indicated conditions. (B) Migration index of the tracks is shown.
A
serum free FBS PDGF-BB Hsp90α
GA
-
NPGA
Migration Index
-
1um GA
1um NPGA
B

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Figure 4.3 NPGA inhibits hypoxia induced migration. Primary HDFs were serum-
starved overnight and subjected to colloidal gold salt assay either under normoxia(21%
O2) or hypoxia (1%O2) either untreated or treated with 1μM of NPGA with or without
indicated growth factors PDGF-BB (15ng/ml), F-5 (30μg/ml). Images of cell migration
from one representative experiment is shown. Dotted circles point out the averaged
migration tracks under indicated conditions.

Hyp
Nor - NPGA NPGA+PDGF-bb NPGA +F-5

119
which could not be rescued by PDGF-bb, in accordance with previous studies showing
hypoxia stimulated migration being carried out primarily through secreted Hsp90α.
In contrast to its activity on full length Hsp90α, NPGA could not inhibit activity
of the F-5 peptide, the minimum 115 amino acid fragment within Hsp90α which we have
previously shown is responsible for its pro-motility effect (Cheng et al., 2011). F-5
constitutes part of the linker region and middle domain of Hsp90α. NPGA has been
shown to bind to the N-terminal ATPase domain of Hsp90α which leads to its inhibition.
Thus, lack of activity of NPGA on F-5 might be due to the absence of N-terminal ATPase
domain.

NPGA delays wound healing in mice
Recombinant Hsp90α can stimulate migration of all 3 types of skin cells
associated with wound healing- keratinocytes, fibroblasts and endothelial cells, and can
accelerate wound healing in mice (Cheng et al., 2011; Li et al., 2007). Damage to blood
capillaries caused by wounding leads to hypoxia in the wound bed. Hypoxia has been
shown to induce secretion of Hsp90α leading to migration of both human keratinocyte
(HKC) and human dermal fibroblast (HDF) (Cheng et al., 2008; Li et al., 2007). Secreted
Hsp90α is essential for endothelial cell migration (Wang et al., 2009). We therefore
wanted to investigate if NPGA, cell-impermeable inhibitor of Hsp90α which is specific
for secreted Hsp90α can delay wound healing in vivo. Full–thickness 1cm x 1cm wounds
were created on the back of athymic nude mice and NPGA mixed with CMC gel or gel
alone (placebo) were topically applied on the wound, right after surgery. Representative

120
images of the wounds are shown in Fig 4.4A. Four different concentrations of NPGA
were used and the optimal concentration was chosen (data not shown). Computer-assisted
planimetry was used to assess the open wound area. Percentage of wound area from
original wound on Day 0 was used instead of absolute values to negate the effect of
minor variations in wound size during surgery. A single application of NPGA on the
wound on Day 0 delayed wound healing with placebo treated wounds healing by day 13-
14 while NPGA treated wounds healing by day 17-18 (Fig 4.4B). Further, since
contraction plays a considerable role in mouse skin healing, we investigated the actual
reason for the difference in wound closure at microscopic level. We wedge biopsied the
wounds on day 14 and examined them by H&E analyses. As seen in Fig 4.5, in contrast
to placebo treated wound, which is completely re-epithelialized, NPGA treated wound
show an unhealed area (red line). The boundary between unwounded and wounded skin
is marked by green arrows. Yellow dotted lines show newly healed skin, characterized by
lack of skin appendages compared to unwounded skin. The insets show the re-
epithelialization tongues (Ret) very clearly on both edges of the wound. This shows that
NPGA delays wound healing by retarding re-epithelialization, which is consistent with
our findings that Hsp90α accelerates wound healing through increased re-
epithelialization. These data provide further evidence that endogenous secreted Hsp90α is
required for wound healing and Hsp90α is a good candidate for wound healing therapies.

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Figure 4.4 NPGA delays wound healing. (A) Full–thickness 1cm x 1cm excision
wounds were created on the back of athymic nude mice and treated with 10% CMC gel
(placebo) or optimized concentration of NPGA mixed with the gel Representative images
of wounds from day 0 till closure of placebo treated wounds are shown (n=3 mice per
group, per experiment). (B) Percentage of wound closure on days 0, 4, 7, 10 and 14, with
or without NPGA are plotted.

M
i
g
r
a
t
i
o
n

I
n
d
e
x
Day 0 Day 4 Day 7 Day 11 Day 14 18uM
+ - + - + - + - + -
A
B
M
i
g
r
a
t
i
o
n

I
n
d
e
x
Day 0 Day 4 Day 7 Day 11 Day 14 18uM
+ - + - + - + - + -
A
B

122

Figure 4.5 NPGA delays wound healing by retarding re-epithelialization. (A and B)
H&E sections of day 14 full–thickness wounds treated with (A) placebo or (B) NPGA
were analyzed. Independent photographs of the same tissue section under identical
magnifications were reconstituted to show the unhealed areas of the wounds. Red line
indicates unhealed space. Yellow dotted lines show newly re-epithelialized epidermis.
Green lines and arrows mark the unwounded skin areas. The front of newly re-
epithelialized epidermis was enlarged to show ReT. Scale bars: 0.33 mm (center), 0.01
mm (left and right).

Placebo
NPGA 18uM
Unhealed
ReT
ReT
B
A

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F-5 maintains its native structure in Hsp90α
CD spectroscopy was performed to evaluate the secondary structure content of the F-5
fragment of human Hsp90α, which is highly conserved in mammals. The first half of F-5
is characterized by consecutive Glu and Lys sequence elements (Figure 4.6A), which are
part of the linker between the N-terminal and middle domains of human Hsp90α and
which we expect to form a dynamically disordered structure. For the second half of F-5
(shown in bold), similar structural propensities as revealed in a recent crystal structure of
residues 293–732 of human Hsp90 (Li et al.) may be anticipated (Figure 4.6B). These
expectations were borne out, as shown in Supplemental Figure 4.6C. Approximately half
of the F-5 fragment is disordered, and the detected secondary structure content correlates
with the Hsp90 crystal structure, which commences with its middle domain. Thus the F-5
peptide recapitulates the structural properties of full-length Hsp90. This work was done
in collaboration with Ulmer lab.

Preparation of F-5 antigen for immunization

Secreted Hsp90α plays an important role in cancer cell migration and invasion,
representing an effective anti-cancer target. We have shown previously that NPGA can
effectively inhibit activity of secreted Hsp90α. However, the instability of this drug in
vivo has limited its possible use in clinic. In order to develop an effective in vivo
antagonist of secreted Hsp90α, we decided to develop a monoclonal antibody against F-5,
the minimum fragment within Hsp90α required for its pro-motility effects. F-5 His-
tagged peptide was purified by Ni-NTA resin as described previously. To avoid possible

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Figure 4.6 F-5 retains its native structure. (A) Amino acid sequence of F-5 with those
in bold representing overlap with fragment whose crystal structure has been determined
(B) CD spectroscopy of F-5 (C) Analysis of secondary structure of F-5.

125
unspecific effects of the His-tag, it was cleaved off using thrombin immobilized on
agarose beads. The time of cleavage reaction was optimized to achieve 100% cleavage.
As shown in Fig 4.7A, 24hr incubation time was required for this purpose and was
chosen as the optimum incubation time for subsequent experiments. The cleaved peptide,
however, was unstable and had a tendency to precipitate out of solution in DPBS. This is
not uncommon as the His-tag is known to increase the solubility of proteins. The cleaved
peptide was thus stabilized in a high salt buffer (see Materials and Methods) and all
downstream processing was done in this buffer. It was made sure that the final salt
concentration for in vivo experiments (it is diluted 1:1 in CMC gel) is close to
physiological salt concentration i.e. 150 mM. The reaction mixture after cleavage was
subjected to FPLC to get rid of the His-tag as well as any bacterial endotoxin. The
peptide was finally stored in the buffer mentioned above with 10% glycerol at -80ºC
degrees. Prior to injecting in the mice, the glycerol was removed by gel filtration and the
peptide was re-concentrated (Fig 4.7B).

Screening the tail bleeds from F-5 immunized mice
The monoclonal antibody production was outsourced to the Caltech Monoclonal
antibody facility and all the screening procedures were performed by me. Three female
Balb/c mice were immunized with 150µg F-5 antigen/injection/mice. After 3 injections,
we started testing the serum from tail bleeds for reactivity against Hsp90α. Since we wish
to develop a neutralizing antibody against secreted Hsp90α, the full length protein was

126

Figure 4.7 Preparation of F-5 antigen for immunization. (A) Ni-NTA purified F-5
was subjected to thrombin cleavage to remove the His-tag with different incubation times
and 24hr time point was chosen to achieve 100% cleavage. A representative SDS-PAGE
gel stained with Coomassie blue is shown (3μg / lane) (B) Before injecting into mice,
glycerol is removed by gel filtration. A representative SDS-PAGE gel stained with
Coomassie blue is shown. Further the eluted fractions 2 and 3 containing F-5 are
combined and concentrated before use.

Time (hr) in 37
o
C 0 1 2 4 6 24 1 2 3
BSA (ug)
1 2 3
BSA (ug)
1 2
BSA (ug)
A B

127
chosen as antigen for screening. By testing different amounts of the antigen, 300ng/well
was found to be saturating and was used in all subsequent assays. Using indirect ELISA
method, we found that serum from all 3 mice had high titers against Hsp90α, showing
reactivity in a concentration dependent manner, compared to the pre-immune serum, with
Mouse #1 having highest titer (Fig 4.8). A commercially available mouse monoclonal
antibody against Hsp90α served as the positive control while whole molecule mouse IgG
was used as the negative control. We also subjected the serum for IgG v/s IgM isotyping
using specific secondary antibodies and found that antibodies present in the serum from
all 3 mice were primarily of IgG isotype. We then asked if there was cross-reactivity
against Hsp90β. Using same amounts of Hsp90α and Hsp90β on the same plate, we
found that serum from all 3 mice gave high signal for both the proteins (Fig 4.9). This is
not unexpected since the corresponding F-5 regions of both the Hsp90 isoforms have
81% identity (Li et al., 2012) and the serum is of polyclonal nature. Further, all 3 serum
recognized F-5 antigen by western blot, but Mouse #2 serum had lesser affinity than the
other two (Fig 4.10).
With promising results using immunoassays, we next wanted to test if the serum
had neutralizing activity using a functional assay. We used colloidal gold migration assay
to test if the F-5 immunized mice serum could inhibit hypoxia driven human dermal
fibroblast secretion, which is known to be mediated through secreted Hsp90α. As shown
in Fig 4.11, serum from all 3 mice inhibited hypoxia induced migration while pre-
immune serum had no effect. Mouse #1 had highest effect.

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Figure 4.8 F-5 immunized mice serum has primarily IgG antibodies. The sera from 3
Balb/c mice injected atleast 3 times with F-5 were subjected to an indirect ELISA assay
on Hsp90α coated plates with secondary antibodies specific to IgG and IgM. A
commercially available mouse monoclonal antibody against Hsp90α served as the
positive control while whole molecule mouse IgG was used as the negative control. Each
condition was carried out in duplicate. OD readings at 405nm were taken in an Odyssey
Microwin reader.

Sec Ab - IgG Sec Ab - IgM
OD 405nm

129

Figure 4.9 F-5 immunized mice serum has Hsp90β cross-reactivity . The sera from 3
Balb/c mice injected atleast 3 times with F-5 were subjected to an indirect ELISA assay
using same amounts of Hsp90α and Hsp90β coated on the same plate. A commercially
available mouse monoclonal antibody against Hsp90α served as the positive control
while whole molecule mouse IgG was used as the negative control. Each condition was
carried out in duplicate. OD readings at 405nm were taken in an Odeyssey Microwin
reader.
Antigen – Hsp90α Antigen – Hsp90β
OD 405nm

130

Figure 4.10 F-5 immunized mice serum recognizes F- 5 by Western Blot. Increasing
amounts of F-5 were run in quadruplicate on 2 gels and the sera from 3 Balb/c mice
injected atleast 3 times with F-5 and the pre-immune serum were used to detect it by
western blotting. Running the gel, transfer, washings, secondary antibody incubations,
ECL and developing was done in same containers for same time duration.

F-5 (ng) 50 150 450 50 150 450 50 150 450 50 150 450
Pre-Imm Mouse # 1 Mouse # 2 Mouse # 3

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Figure 4.11 F-5 immunized mice serum inhibits hypoxia induced migration. (A)
Primary HDFs were serum-starved overnight and subjected to colloidal gold salt assay
either under normoxia (21% O2) or hypoxia (1%O2), either untreated or treated with sera
from 3 Balb/c mice injected atleast 3 times with F-5 and the pre-immune serum. Images
of cell migration from 1 representative experiment are shown. Dotted circles point out the
averaged migration tracks under indicated conditions. (B) Migration index of the tracks is
shown.
serum free FBS PDGF-bb Hyp Hyp+NPGA
Hyp+Pre -imm Hyp+Mouse1 Hyp+Mouse2 Hyp+Mouse3
A
serum FBS PDGF minus NPGA Pre-imm Mouse#1 Mouse#2 Mouse#3
free
Normoxia Hypoxia
Migration Index
14
12
10
8
6
4
2
0
B

132
DISCUSSION
Our lab previously reported secretion of Hsp90α by primary skin cells under
hypoxia. We discovered that in contrast to earlier thinking, the driving force for cell
migration during wound healing was a secreted molecule - Hsp90α. Considering the
potential of Hsp90α based therapies, we wanted to further validate the role of secreted
Hsp90α in the process of wound healing. We took advantage of a small molecule non-
permeable inhibitor of Hsp90α, DMAG-N-oxide (NPGA), reported by Tsutsumi and
colleagues. Like all derivatives of GA, NPGA binds to the ATP binding cleft of Hsp90α
and inhibits its ATPase activity, suggested to be responsible for its inhibition of Hsp90α
(Tsutsumi et al., 2008). However our lab has shown that ATPase defective mutants of
Hsp90α retain their pro-motility function (Cheng et al., 2008) and that the minimum pro-
motility fragment of Hsp90αt, F-5, lacks the N-terminal (Cheng et al., 2011). These
contrasting findings are right now up to speculation. GA binding to Hsp90α is known to
cause conformational changes in Hsp90α; it is likely that NPGA binding causes a
conformational change which prevents F-5 from being surface exposed in the full-length
Hsp90α and thus hinders its binding to its receptor LRP1. However, structural studies of
Hsp90α bound to NPGA would be required to conclusively prove this.
We have shown in this study that NPGA is indeed cell impermeable as it does not
affect levels of Hsp90α intracellular client proteins and that as a secreted Hsp90α
antagonist, it inhibits Hsp90α induced HDF migration. To confirm its activity in a
physiological setting, we have shown that NPGA inhibits hypoxia induced migration,
which is known to be driven by secretion of Hsp90α. We also show that NPGA’s

133
inhibition of cell migration is rescued by F-5 possibly because F-5 lacks the ATPase
carrying N-terminal domain which is the binding site for NPGA. This is consistent with
the findings of Gopal and colleagues who reported that NPGA could inhibit cancer cell
migration and invasion driven by full length Hsp90α but not driven by a N-terminal
truncated fragment (Gopal et al., 2011). To investigate if secreted Hsp90α drives wound
healing in vivo, we topically applied NPGA on mice wounds. In agreement with our
hypothesis, NPGA delayed wound healing through retardation of re-epithelialization of
the wound. Taken together, these data provide another line of evidence for the
importance of secreted Hsp90α in the process of wound healing and validate F-5 as a a
promising candidate for future wound healing therapies, thus, shifting the paradigm away
from the array of growth factor based therapies.
Apart from wound healing, cancer is another process where cell motility plays an
essential role in tumor formation and metastasis. Our work has shown that secreted
Hsp90α is essential for cancer cell migration and invasion in Hif-1α positive breast
cancer. This finding has greater implications if we take into account that more than 40%
of all solid tumors have increased expression of Hif-1α (Semenza et al., 2007). Several
groups have reported an essential role of Hsp90α, constitutively secreted by cancer cells,
in promoting their invasiveness (Li et al., 2012). Therefore, we were interested in
targeting extracellular Hsp90α with an aim to develop anti-cancer therapeutics.
Previously, NPGA was used by Tsutsumi et al. to inhibit cancer migration and invasion
in vitro but it was found to be structurally unstable for potential use in vivo. Therefore
they treated the cells ex vivo before injecting them into mice and reported reduced lung

134
colonization. Similar results were reported by the Patsaovadi group who developed a
monoclonal antibody 4C5 against Hsp90 and showed that melanoma cells treated with
the antibody and then injected into mice had decreased lung colonization (Stellas et al,
2007, Cancer therapy preclinical). However, how a single treatment by drug/antibody
could neutralize constitutive secretion of Hsp90α by cancer cells in vivo over the duration
of a multi-week experiment is beyond reason. In addition, this antibody neutralizes both
Hsp90α and Hsp90β. This calls for the need to develop better cell impermeable Hsp90α
specific antagonists. Monoclonal antibody based drugs are a good option because they
are stable inside the body and thus many are already in clinical use, such as Trastuzumab,
which targets ErbB2 and is used in HER-2 positive breast cancer patients. We
hypothesize that an anti-F-5 antibody would specifically bind to the pro-motility region
of Hsp90α and would neutralize its function. The F-5 region within Hsp90α is also
known to be surface exposed and is hence a good choice as an epitope. Our data show
that F-5 injected into mice is indeed immunogenic and serum from these mice showed
high titer of IgG antibody which recognized Hsp90α. We also confirmed functional
neutralization of Hsp90α function through inhibition of hypoxia induced migration by the
serum obtained from mice injected with F-5. With these promising results, we now plan
to proceed to hybridoma production and finally derive monoclonal antibody secreting
clones. These would then be tested for anti tumor activity in vivo using an orthotopic
breast cancer model, whereby we will inject invasive breast cancer cells in the mammary
fat pad of SCID mice and will then systemically administer the monoclonal antibody.
This study might provide the first direct evidence of the F-5 epitope in Hsp90α mediating

135
tumor cell invasion and validate the idea of developing anti-cancer drugs targeting
secreted Hsp90α. If we do obtain positive results, antibodies against F-5 would then have
to be humanized for future therapeutic use in humans to avoid immunogenic reactions
against a mouse antibody. Moreover, since extracellular Hsp90α is not required by
normal cells for any physiological process and is only required during a stress response,
an anti-F-5 antibody should not give rise to toxicity issues that the inhibitors targeting the
intracellular Hsp90α chaperone have been previously associated with, rendering it as a
safer therapeutic option for cancer therapy.

136
CHAPTER 5
SUMMARY AND CONCLUSIONS

Hsp90α is the most abundant protein inside the cell, constituting 1-2% of cellular
proteins. It has traditionally been known as a cytoplasmic molecular chaperone protein
which helps in folding, stabilization and intracellular targeting of its more than 200 client
proteins which are involved in various cellular pathways. In addition to its intracellular
role, Hsp90α had also been detected on the cell surface as early as 1970s, involved in
antigen presentation, but was ignored to be an artifact of leaked protein from lysed cells
(Basu S et al, Cell stress chaperones, 2000). In 2000, Liao et al showed Hsp90α secretion
from rat vascular smooth muscle cells upon oxidative stress (Liao et al, 2000 JBC).
Subsequently in the last decade, many other groups reported Hsp90α in the extracellular
space as a result of various stimuli such as heat shock (Hightower et al, 1989, Clayton et
al, JCS 2005, Sidera et al, JBC 2004), hypoxia ( Li et al, Embo 2007, Woodley et al, JCS
2009), cytokines (Cheng et al, MCB 2008, Song et al, BBRC 2010), and gamma radiation
( Yu et al, Can Res 2006).
Secreted Hsp90α has been shown to function as a pro-motility agent. Sidera et al
showed that neuronal cell migration is driven by cell surface Hsp90α (Sidera et al, JBC,
2004). Our lab has shown that secreted Hsp90α drives hypoxia mediated migration in
skin cells (Li Embo 2007, Woodley JCS 2009). Recently extracellular Hsp90α has been
shown to be important for migration of primary mouse cranial mesenchymal cells (Sarkar
and Zohn, JCB, 2012). Our group has shown that secreted Hsp90α binds to the cell

137
surface receptor LRP-1 in an autocrine or paracrine fashion and reorganizes the actin
cytoskeleton to cause cell movement. Chen et al reported integrin α
v
binding to LRP-1 to
be important in cancer invasion (Chen et al, JBC, 2010). Work by Tsutsumi and
colleagues imply the role of Hsp90α in focal adhesion complexes involving Src and
integrin β1 (Tsutsumi et al, Oncogene, 2008).
Cell migration is known to be a rate limiting step in the process of wound healing.
Upon wounding, the epidermal keratinocytes at the wound edge need to move in to close
the wound first and the dermal fibroblasts and endothelial cells then need to move in to
deposit ECM and build new blood vessels, respectively. Our lab has shown that Hsp90α
is secreted by skin cells upon stimulation and promotes cell migration. This led to the
possibility of Hsp90α being a wound healing agent and we found that recombinant
Hsp90α accelerates closure of wounds in mice. Next, by sequential mutagenesis, our lab
narrowed down the pro-motility region to be at the boundary of the linker region and
middle domain, a 115 amino acid fragment, F-5. We have shown that F-5 causes wound
closure more efficiently than the current lone FDA approved therapeutic agent, PDGF-
BB or Beceplermin, in mice. Any fragment shorter than F-5 showed much lesser efficacy,
albeit comparable to that of Beceplermin. The wounds which pose a greater challenge
clinically are chronic wounds, such as venous, pressure and diabetic ulcers. Only the
latter has an established model for research use, i.e. the db/db mouse, which lacks the
leptin gene and is characterized by hyperglycemia. We showed that F-5 heals diabetic
wounds better than Beceplermin. Mice are loose-skinned animals compared to humans,
and heal largely due to contraction while human skin heals largely due to re-

138
epithelialization. We found that the F-5 treated wounds show increased re-
epithelialization compared to placebo control. Even though we did not observe an
increase in number of endothelial cells in F-5 treated wounds compared to placebo, Song
et al reported increased blood vessel densities in Hsp90α treated wounds (Song et al,
BBRC, 2010). These differences may be due to differences in treatment regimen (one-
time v/s everyday) or day of analysis (day 14 v/s day 5). Wound healing being such a
complex process, further analyses is needed to understand these results.
We then asked what makes F-5 much superior to PDGF-BB. We found that the
receptor for PDGF-BB, PDGFR, is present only on dermal fibroblasts, but not on
keratinocytes or endothelial cells. On the other hand, the receptor for Hsp90α F-5 is LRP-
1, which is present on all the three aforementioned skin cell types. Secondly, a negative
regulator of migration is the cytokine TGFβ found abundantly in the wound bed and is
known to selectively inhibit dermal cell migration. We found that in contrast to PDGF-
BB stimulated migration which is sensitive to TGFβ, F-5 is able to override the inhibitory
effect of TGFβ. Thirdly, in chronic diabetic wounds, hyperglycemia causes a delay in
wound healing by destabilizing Hif-1α, the regulator of Hsp90α secretion (Botusan et al,
PNAS, 2009). We found that F-5 is able to rescue hypoxia induced migration in the
presence of hyperglycemia. These three key properties make it a better wound healing
agent than any growth factor based therapy.
To validate the role of secreted Hsp90α in the process of intrinsic wound healing,
we took advantage of a cell impermeable inhibitor of Hsp90α, DMAG-N-oxide (NPGA),
which is a derivative of GA (Tsutsumi et al, Oncogene, 2008). We showed that NPGA

139
while being impermeable, could inhibit the activity of secreted Hsp90α i.e. Hsp90α
stimulated migration of dermal fibroblasts. We observed delay in wound healing on
topical application of NPGA, through decreased re-epithelialization. This data shows that
secreted Hsp90α is required for the process of wound healing.
Tumors are non-healing wounds, sharing key characteristics, such as hypoxia
(Dvorak et al, N Eng J Med, 1986). The uncontrolled growth of cells and haphazard
formation of blood vessels cause intra-tumoral hypoxia. More than 40% of solid tumors
have elevated levels of Hif-1α. Hif-1 is a transcription factor which is a heterodimer of
two isoforms, the inducible Hif-1α and the constitutively present Hif-1β. It has been
studied extensively for its role in regulating several pathways for survival of cancer cells
such as angiogenesis, glucose metabolism etc. Many reports have shown the key role of
Hifs in cancer cell invasion and metastasis (Semenza et al, Trends Mol Med, 2012).
However, not a single drug targeting Hif has been approved so far. This has been
attributed to the lack of specific inhibitors. Recently, a topoisomerase inhibitor,
Topotecan has been reported to inhibit Hif-1α activity. The first multihistology pilot trial
of Topotecan reported reduced expression of Hif-1α and Hif target genes in patient
samples (Kummar et al, Clin Can Res, 2011). However it has a short half life and was
involved with several toxic side effects which actually led to dose reduction during the
course of the trial. Even though this study provides proof of principle of importance of
Hif-1α in a clinical setting, Topotecan is not suitable for clinical use. Moreover topotecan
was found to be inactive in renal cell cancer, a disease where Hif plays a major role (Law
et al, Invest New Drugs, 1994). Thus targeting Hif-1α is not a feasible option, atleast for

140
now. Just recently, Hif-1 has been reported to promote breast cancer metastasis through
LOX, LOXLs, ANGPL and LICAM (Wong et al, PNAS, 2011, Zhang et al, Oncogene,
2012). However, there is no comparison of invasive properties of parental, Hif
knockdown and double infected rescued cells to provide conclusive evidence to show that
any of these candidates can fully mediate the effects of Hif-1 on invasion.
Our lab has shown that Hif-1α is key upstream regulator of Hsp90α secretion in
skin cells. Also, Hsp90α is found on the cell surface of several cancers (Li et al, BBA,
2011). This drew our attention to the possibility of Hif-1α regulated Hsp90α secretion
and signaling through LRP-1 affecting cancer cell migration. >50% of breast cancers are
reported to have elevated Hif-1α levels which correlate with tumor stage and is predictive
of mortality (Semenza, Trends mol med, 2012). We decided to focus on triple negative
breast cancer i.e. lacking the ER, PR and Her/Neu receptor, because it is the most
aggressive form of breast cancer and has no current therapies. Therapy targeting the Hif
 secreted Hsp90α pathway would be independent of receptor status and thus potentially
can be used to treat larger number of patients. We utilized lentiviral knockdown strategy
to down-regulate Hif-1α and observed decrease in cancer migration and invasion, along
with decrease in Hsp90α secretion. Rescue experiments with a wild type or constitutive
active form of Hif-1α, but not a dominant negative form, could restore Hsp90α secretion
and invasiveness in vitro. To establish secreted Hsp90α as the effector of Hif-1α
mediated cancer cell invasiveness, we took two approaches. Firstly, addition of an anti
Hsp90α antibody could inhibit cancer migration and invasion in a dose dependent
manner, the effect of which was reversed on addition of excess recombinant Hsp90α.

141
Secondly, recombinant Hsp90α is able to rescue invasiveness of Hif-1α knockdown cells.
Consistent with the activity of different Hsp90α fragments, F-5 was the shortest peptide
that was able to rescue invasion in both Hif-1α and Hif-1β knockdown cells. To further
establish the importance of Hsp90α signaling in cancer cell invasion, we knocked down
LRP-1, the cell surface receptor for Hsp90α. The LRP-1 down-regulated cells showed
reduced invasion, which could not be rescued by F-5. We wanted to test our hypothesis
of Hif-1α  Hsp90α  LRP-1 signaling playing a role in tumor invasion in vivo.
Specific inhibitors of Hif-1α are still not available, making it an unsuitable target. The
current inhibitors of Hsp90α are cell permeable and hence would target intracellular pool.
The non-permeable derivative of Geldanamycin, DMAG-N-oxide has been reported to be
unstable in vivo. The use of large quantities of commercially available Hsp90α antibodies
was not feasible. Thus targeting extracellular Hsp90α was also challenging. We therefore
decided to target LRP-1 for this experiment. We injected LRP-1 knock-down MDA-MB-
231 breast cancer cells via tail vein in immunodeficient mice and observed them for lung
colonization. LRP-1 null cells showed much less tumor formation than control cells. We
got similar results using a LRP-1 null cell line, MDA-MB-468. Though these are
promising results, this is not conclusive evidence as LRP-1 binds to more than 50
structurally unrelated ligands and is known as a scavenger receptor, so these results
cannot entirely be attributed to interrupted Hsp90α – LRP-1 signaling. The direct
evidence would be attained if we could specifically neutralize secreted Hsp90α. We are
addressing this issue by developing a monoclonal antibody against F-5 (discussed later).
Though our model is that of breast cancer, hypoxia and elevated Hif-1α levels are

142
characteristic of many cancers and hence our results can be extrapolated to all Hif-1α
positive tumors.
Hsp90α has been in focus as an anti cancer target for two decades. As a
chaperone, Hsp90α was believed to preferentially have a protective effect on the
oncoproteins in tumor cells (Kamal et al) and this was the premise on which several
Hsp90α inhibitors have been developed and tested. However all of them have faced the
challenge of achieving the balance between effectiveness and toxicity. Though the
biggest attraction of targeting Hsp90α was believed to be the simultaneous targeting of
several pathways, anti-climactically clinically the only instance of its effectiveness has
been in Her2 positive breast cancer, in combination with Transzumab, a monoclonal
antibody against Her2 receptor, presumably because Her2 is a very important client of
Hsp90α (Chiosis et al, 2012). Therefore we believe that extracellular is a much specific
and effective anti cancer target than intracellular Hsp90α.
Considering the importance of secreted Hsp90α in cancer cell invasiveness, there
is high therapeutic potential in inhibitors against secreted Hsp90α. High levels of Hsp90α
have been reported in the circulation of cancer patients (Wang et al, PNAS 09, Chen JS,
JBC 2010). Extracellular Hsp90α can therefore be used as a diagnostic and prognostic
marker and an anti cancer target. We were thus interested in developing a neutralizing
monoclonal antibody against Hsp90α, specifically F-5, which is responsible for the pro-
motility function of Hsp90α. For this purpose, we collaborated with the Caltech
Monoclonal Antibody Facility. His-tag free F-5 was used to immunize 3 Balb/c mice and
then serum from these mice were screened to test if they are producing antibodies. F-5 is

143
immunogenic and we observed very high titers of IgG antibodies against Hsp90α. We
would use one of these mice for hybridoma development and envisage getting a panel of
monoclonal antibody secreting clones which would be validated by functional assays and
used to study the role of secreted Hsp90α in both cancer and wound healing. We plan to
establish an orthotopic breast cancer model with MDA-MB-231 cells and systemically
administer anti F-5 antibody. This experiment would conclusively tell us if secreted
Hsp90α driven migration and invasion is required for tumor formation and metastasis. If
this antibody gives us anticipated results, it would be a very powerful anti cancer drug,
because it can potentially work against all Hif-1α positive tumors, across the board. It
would especially beneficial to triple negative breast cancer patients, who do not have any
treatment options. For future clinical use in humans, the monoclonal antibody would
have to be humanized to avoid possible anti- drug antibody (ADA) reactions.
Since we have demonstrated the critical role of Hsp90α in both cancer and wound
healing, as we go forward to use it in the clinic, the obvious concern would be its
contrasting roles in the two processes. While it can be a novel wound healing agent
because of its pro-motility effect, for the same reason it is undesirable for cancer patients.
Therefore before prescribing it to patients there has to be a case-by-case assessment of
their relative risks – for example, a cancer patient who has chronic wounds would not be
a good candidate to receive anti Hsp90α therapy just as a patient with chronic wounds
with cancer risk would not receive Hsp90α wound healing therapy.
In summary, this thesis work characterizes the minimum active fragment of
secreted Hsp90α that drives cell motility, a 115 amino acid peptide F-5, validates its role

144
in the processes of wound healing and cancer and demonstrates its potential as a
therapeutic agent in the clinic for the first time. We show that F-5 can stimulate
migration in vitro and accelerate acute and diabetic wounds healing in vivo, better than
the only current FDA approved therapy, Beceplermin. We demonstrated that
mechanistically this is attributed to three key properties that F-5 possesses which
Beceplermin doesn’t and prove that the molecule responsible for driving cell migration
post wounding is Hsp90α, not growth factors as was believed previously, bringing about
a paradigm shift in current growth factor based wound healing therapies. In the context of
cancer, we show that Hsp90α can drive cancer cell migration and invasion in a Hif-1α
regulated manner and that F-5 is the minimum fragment of Hsp90α that retains this
ability. We are developing a monoclonal antibody against F-5 which has immense
potential as an anti cancer therapy. We foresee its use as an anti cancer drug in the clinic
or serve and as proof of principle for an F-5 based vaccine which would induce anti F-5
antibodies in the cancer patients which would neutralize any Hsp90α in their circulation
and hence retard tumor progression and invasion. We also foresee its use as a diagnostic
or prognostic marker in the clinic. It would also be useful to demonstrate for the first time
that neutralizing the F-5 epitope of secreted Hsp90α delays the intrinsic wound healing
process.
Several questions still remain unanswered in this field. How Hif-1α regulates
Hsp90α secretion is still unknown. Hif regulates transcription of its targets through
binding to the Hypoxia response elements (HREs) in their promoter regions. Hsp90α
lacks any HRE element, so a direct transcriptional regulation is not likely. However, it is

145
possible that Hif regulates the expression of some other protein which regulates Hsp90α
secretion or increases exosomal secretion in general. Also, we have observed Hsp90α
secretion as little as 2 min after TGFα stimulation, so a transcriptional regulation theory
doesn’t fit with this piece of data. However, a combination of factors responsible for
Hsp90α secretion is also possible. On urgent need upon an acute stimulus such as
cytokines, cells secrete out Hsp90α immediately and then re-stock their reserves of
Hsp90α by transcription and translation.
Proteins secreted by the classical golgi-ER secretion pathway have a canonical
signal peptide at the animo terminus which targets it for secretion. Hsp90α lacks this
signaling sequence and is secreted by the exosomal pathway. DMA, an inhibitor of
exosome pathway, but not BFA an inhibitor of golgi pathway inhibits Hsp90α secretion.
Hsp90α has also been detected in tumor cell exosomes (Yu et al, 2006). However, how
Hif regulates this pathway is still unknown. We are currently studying the role of
different domains of Hif-1α to study if its dimerization, DNA binding or transactivating
properties play a role in Hsp90α secretion. It would be very interesting to study if a Hif-
1α canonical pathway is involved in this process or if a non-canonical pathway would be
elucidated.

146
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Abstract (if available)

Abstract Heat shock protein 90alpha (Hsp90α) is an abundant molecular chaperone. We have recently shown that normal cells under stresses such as hypoxia secrete Hsp90α which promotes migration, signaling through LDL receptor-related protein-1 (LRP-1). In this study, we investigated the mechanism behind role of secreted Hsp90a in wound healing and cancer. ❧ Using systematic mutagenesis, we identified the minimum functional element in Hsp90α, F-5, required for cell migration. Topical application of F-5 enhances closure of acute and diabetic wounds in mice more effectively than PDGF-BB, the only FDA-approved therapy. The superior efficacy of F-5 is due to its ability to 1) recruit both epidermal and dermal cells through universally expressed LRP-1 receptor, 2) override inhibition of dermal cell migration by TGFβ and 3) override anti-migration effects of hyperglycemia. Therefore, this study challenges the long-standing paradigm for developing growth factor-based therapies and identifies a novel wound-healing agent. ❧ Like wounds, hypoxia is common in cancer and ~40% of solid tumors overexpress hypoxia-inducible factor-1alpha (Hif-1α). We show that lentiviral knock-down of Hif-1α blocks Hsp90α secretion and invasion/migration of breast cancer cells while reintroducing active Hif-1α rescues these effects. Neutralization of secreted Hsp90α by inhibitor or antibody or knock-down of LRP-1 reduces tumor cell invasion in vitro and tumor formation in nude mice. Introduction of Hsp90α F-5 bypasses the Hif-1α depletion blockade and rescues cancer cell invasion. Since normal cells do not secrete Hsp90α under physiological conditions, our data suggest that drugs targeting secreted Hsp90α F-5 region should be more effective and less toxic for treatment of Hif-1α positive tumors. ❧ In light of the clinical relevance of extracellular Hsp90α, we conducted further target validation studies. We used a non permeable inhibitor of Hsp90α, DMAG-N-oxide, to show that secreted Hsp90α is important for the wound healing process. We are also developing a monoclonal antibody against the F-5 region of Hsp90α. It would be useful in validating the role of pro-motility effect of Hsp90α on wound healing and cancer. It can potentially be further developed into an anti cancer therapeutic agent.

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Asset Metadata

Creator Sahu, Divya (author)

Core Title Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 02/06/2014

Defense Date 10/24/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer,HiF-1α,Hsp90α,invasion,migration,OAI-PMH Harvest,wound healing

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stallcup, Michael R. (committee chair), Kobielak, Agnieszka (committee member), Li, Wei (committee member)

Creator Email divyasahu1@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-317686

Unique identifier UC11295471

Identifier etd-SahuDivya-1974.pdf (filename),usctheses-c3-317686 (legacy record id)

Legacy Identifier etd-SahuDivya-1974.pdf

Dmrecord 317686

Document Type Dissertation

Format application/pdf (imt)

Rights Sahu, Divya

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA