University of Southern California Dissertations and Theses

The role of GRP78 in the regulation of apoptosis and prostate cancer progression

(USC Thesis Other)

The role of GRP78 in the regulation of apoptosis and prostate cancer progression

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
THE ROLE OF GRP78 IN THE REGULATION OF
APOPTOSIS AND PROSTATE CANCER PROGRESSION

by

Yong Fu

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY & MOLECULAR BIOLOGY)

May 2008

Copyright 2008 Yong Fu

ii

Dedication

To my dearest parents Chaofang Fu and Chunxiang Wang.

iii

Acknowledgments

I would like to thank my thesis advisor Dr. Amy S. Lee for her excellent mentorship and
great kindness in guiding my graduate at USC. I would also like to thank my guidance
committee members, Drs. Michael Stallcup, Robert Stellwagen, Neil Kaplowitz, and Ite
Laird, for their time, encouragement and advice. Their guidance and constructive advice
are an important integral part of this thesis and will certainly be beneficial to my future
academic development.
I thank Dr. Pradip Roy-Burman and Chun-Peng Liao at USC for the Pten
conditional knockout mice and generous technical support. I thank Dr. Gordon Shore
for the BIK expression plasmids and helpful discussions, and Dr. Ana M. Soto for the
MCF-7/BUS cells. I thank Dr. Martin Kast and Andrew Gray for the knockout mice. I
am grateful to Dr. Jianzi Li for the help in FACS analysis, Dr. Shengzhan Luo for the
construction of His-tagged GRP78, Gene Hung for the construction of adenoviral
expression vectors, and Brenda Lee for the construction of GST-GRP78. We thank Dr.
Cathie Chung for helpful discussions.
I would like to express my appreciation to present and former lab members of the
Lee lab. They are Drs. Dezheng Dong, Min Hong, Jianze Li, Shengzhan Luo, Changhui
Mao, Tylor Parr, Yi Zhang, and Peter Baumeister, Brenda Lee, Min Ni, Minal Patel,
Wei-Cheng Tai, Miao Wang, Shiuan Wey, and Risheng Ye.
I gratefully appreciate the West America Chapter of the Phi Tau Phi Scholastic
Honor Society and the President Paul L. Lee for the support with Phi Tau Phi Scholastic

iv
Award. I am also thankful to the California Breast Cancer Research Program (CBCRP)
for the generous support with the CBCRP Training Grant Award.

v
Table of Contents
Dedication ii
Acknowledgments iii
List of Figures vii
Abstract ix
Chapter 1. Introduction 1
Chapter 2. GRP78 associates with BIK and inhibits its apoptotic activity 8
2. 1 Introduction 8
2. 2 Materials and Methods 11
2. 2. 1 Cell culture, drug treatment and reagents 11
2. 2. 2 Plasmids 11
2. 2. 3 Transient transfections 12
2. 2. 4 Western blots 13
2. 2. 5 Co-immunoprecipitation assays 14
2. 2. 6 GST pull-down assays 15
2. 2. 7 Cell death and apoptotic assays 16
2. 3 Results 16
2. 3. 1 BIK protein is induced by etoposide treatment 16
2. 2. 2 Endogenous BIK selectively forms complex with GRP78 17
2. 2. 3 GRP78 associates with the endoplasmic-reticulum targeted BIK 20
2. 2. 4 GRP78 overexpression inhibits apoptotic activity of ER-targeted
BIK 21
2. 3 Discussion 22
Chapter 3. GRP78 protects human breast cancer cells against estrogen-starvation
induced apoptosis by inhibiting BIK 26
3. 1 Introduction 26
3. 2 Materials and methods 30
3. 2. 1 Cell culture and reagents 30
3. 2. 2 Transient transfections and adenovirus infections 30
3. 2. 3 Western blot and quantitation 31
3. 2. 4 Small interfering RNA 31
3. 2. 5 Apoptotic assay 32
3. 2. 6 Flow cytometric analysis of BAX-associated immunofluorescence 32
3. 3 Results 33
3. 3. 1 Estrogen starvation induces BIK protein and BAX activation in
MCF-7/BUS cells 33
3. 3. 2 GRP78 overexpression inhibits estrogen-starvation induced BAX
activation 34

vi
3. 3. 3 GRP78 overexpression inhibits estrogen-starvation induced
apoptosis 36
3. 3. 4 Knockdown of GRP78 by siRNA sensitizes human breast cancer
cells to estrogen-starvation induced apoptosis 38
3. 3. 5 Knockdown of BIK decreases the enhancement of
estrogen-starvation induced apoptosis resulted from GRP78 knockdown 40
3. 4 Discussion 42
Chapter 4. GRP78 protects cells from apoptosis induced by HDAC inhibitor 46
4. 1 Introduction 46
4. 2 Materials and methods 47
4. 2. 1 Cell culture and reagents 47
4. 2. 2 Expression vectors and transient transfections 47
4. 2. 3 Western blot 48
4. 2. 4 Cell death and apoptotic assays 48
4. 3 Results 48
4. 3. 1 GRP78 overexpression protects 293T cells from TSA-induced
apoptosis 48
4. 4 Discussion 51
Chapter 5. The role of GRP78 in the postnatal growth of prostate and progression of
prostate cancer 53
5. 1 Introduction 53
5. 2 Materials and Methods 55
5. 2. 1 Generation of prostate-specific Pten and Grp78 homozygous
deletion mice 55
5. 2. 2 Genotyping of mice 56
5. 2. 3 Autopsy and histopathology assessments 57
5. 2. 4 Immunohistochemistry analysis 57
5. 2. 5 Immunofluorescence analysis 58
5. 3 Results 59
5. 3. 1 Postnatal homozygous deletion of Grp78 does not affect the
development of prostate 59
5. 3. 2 The effect of Grp78 heterozygous deletion on prostate cancer 64
5. 3. 3 The effect of Grp78 homozygous deletion on prostate cancer 66
5. 3. 4 The AKT activity in the prostate of mouse model 68
5. 4 Discussion 69
Chapter 6. Summary and perspective 75
Bibliography 81

vii
List of Figures

Fig. 1.1. Schematic drawing of GRP78, BIK and their mutants. 2
Fig. 2.1. BIK protein is induced by etoposide treatment. 17
Fig. 2.2. Endogenous BIK selectively forms complex with GRP78. 19
Fig. 2.3. GRP78 associates with the endoplasmic-reticulum targeted BIK. 20
Fig. 2.4. GRP78 overexpression inhibits the apoptotic activity of ER-targeted
BIK. 22
Fig. 3.1. Estrogen starvation induces BIK protein and BAX activation in
MCF-7/BUS cells. 34
Fig. 3.2. GRP78 overexpression inhibits estrogen-starvation induced BAX
activation. 35
Fig. 3.3. Overexpression of GRP78 rescues MCF-7/BUS cells from estrogen-
starvation induced apoptosis. 38
Fig. 3.4. Knockdown of GRP78 by siRNA sensitizes human breast cancer cells
to estrogen-starvation induced apoptosis. 40
Fig. 3.5. Knockdown of BIK decreases the enhancement of estrogen-starvation
induced apoptosis resulting from GRP78 knockdown. 42
Fig. 4.1. Overexpression of GRP78 protects 293T cells from TSA induced
apoptosis. 50
Fig. 5.1. The breeding scheme of Grp78
F/F
; PB-Cre4 mouse. 60
Fig. 5.2. The breeding scheme of Pten
F/F
; Grp78
F/F
; PB-Cre4 mouse. 61
Fig. 5.3. The breeding scheme of Pten
F/F
; Grp78
F/-
; PB-Cre4 mouse. 62
Fig. 5.4. Postnatal homozygous deletion of Grp78 does not affect the
development of prostate. 63
Fig. 5.5. The immunofluorescent analysis of the PTEN, GRP78, and P-Akt
level in mouse prostate. 66
Fig. 5.6. The effect of Grp78 deletion on the prostate cancer formation and
progression initiated by homozygous Pten deletion. 68

viii
Fig. 6.1. The mechanism of the antiapoptotic activity of GRP78. 76

ix
Abstract

Glucose-regulated protein 78 (GRP78), a molecular chaperone at the endoplasmic
reticulum (ER), is a master regulator of ER stress and an important survival factor for
cell. GRP78 protein level is highly elevated in malignant tumors and correlates with
severe pathological grade and poor prognosis. It has been well established that GRP78
can affect apoptosis by regulating ER Ca
2+
signaling and unfolded protein response
pathway, but whether other mechanism exists remains unknown. Searching for novel
partners that interact with GRP78 at the ER, we discovered that BIK, an apoptotic
BH-3-only protein located principally at ER, selectively forms a complex with GRP78.
GRP78 overexpression decreases apoptosis of 293T cells induced by ER-targeted BIK.
For the MCF-7/BUS breast cancer cells that require BIK to mediate estrogen
starvation-induced apoptosis, overexpression of GRP78 inhibits estrogen-starvation
induced BAX activation, mitochondrial permeability transition, and consequent
apoptosis. Further, knockdown of endogenous GRP78 by siRNA sensitizes
MCF-7/BUS cells to estrogen-starvation induced apoptosis. This effect was
substantially reduced when the expression of BIK was also reduced by siRNA. In
addition to in vitro investigations, an in vivo study in a Pten conditional knockout mouse
model of prostate cancer reveals that homozygous deletion of Grp78 blocks prostate
cancer formation and progression initiated by Pten nullification. Our results provide
multiple lines of evidence that GRP78 is a critical player in the regulation of apoptosis
and the formation and progression of cancer. These results further support the concept

x
that GRP78 represents a novel marker for cancer progression and
chemo-responsiveness, as well as a novel target for cancer therapy.

1
Chapter 1. Introduction
GRP78 was originally discovered as the 78 KD glucose-regulated protein(Lee 2001)
and is also referred as BiP (immunoglobulin heavy-chain binding protein)(Haas and
Wabl 1983). As a member of the family of HSP70 molecular chaperones, GRP78 is an
ER-resident molecular chaperone. It has two domains, an N-terminal domain with an
A TPase activity site and a C-terminal client protein binding domain(Gething 1999) (Fig.
1.1). Although discovered as an ER luminal protein, a subpopulation of GRP78 exists
constitutively as a transmembrane protein(Reddy 2003). GRP78 binds to newly
synthesized proteins and misfolded proteins to assist their folding. In addition to
chaperoning, GRP78 participates in ER protein translocation, protein quality control,
ER-associated protein degradation, ER stress sensing and regulation, and ER calcium
binding(Hendershot 2004; Lee 2005; Yang and Li 2005). Under normal conditions,
GRP78 binds to ER transmembrane proteins PERK, IRE1, and ATF6 and keeps them
inactive. When unfolded proteins accumulate and compete for the binding to GRP78,
PERK, IRE1, and ATF6 are released; the corresponding pathways are activated to
initiate the UPR(Lee 2007).
GRP78 is constitutively expressed in various organs, but the basal level is low. In
contrast to its low basal level, GRP78 is strongly induced in tumors(Dong, Dubeau et al.
2004; Li and Lee 2006). In solid tumors, cells undergo glucose deprivation, acidosis,
and hypoxia due to the inadequate vascularization and rapid growth of solid tumors. All
these adverse factors result in the accumulation of underglycosylated and misfolded

2
proteins in ER and then trigger the unfolded protein response (UPR). UPR is a
cytoprotective response that tries to rescue cells from stress-induced death. One of the

Fig. 1.1. Schematic drawing of GRP78, BIK and their mutants. A, Schematic drawing of wild type
GRP78 and the mutants. The GRP78 protein has two major domains: the ATPase domain at the
N-terminal half and the peptide-binding domain at the C-terminal half. Topology analysis suggests that a
subpopulation of GRP78 could be transmembrane with part of the N-terminal portion being cytosolic and
the C-terminal half locating inside the ER lumen. In P45 mutant, the ER lumenal part, from a.a. 401-661,
was deleted. B, Schematic drawing of wild type BIK and the mutants. In BIK-b5TM, the C-terminal
transmembrane domain was replaced by the C-terminal transmembrane segment of cytochrome b5, a
sequence shown to selectively target fusion proteins to the ER.

principle protective effectors of UPR is GRP78. Recently, the role of GRP78 in cancer
growth and drug resistance has become a focus of research.(Misra, Gonzalez-Gronow et
al. 2002; Misra, Sharma et al. 2005; Koumenis 2006; Li and Lee 2006; Misra,
Deedwania et al. 2006; Lee 2007; Shani, Fischer et al. 2008)

3
In addition to the UPR pathway, GRP78 can be induced through UPR-independent
mechanisms. For instance, the human Grp78 promoter has a conserved c-Myb binding
site which acts independently of sequences associated with the UPR(Ramsay, Ciznadija
et al. 2005). C-Myb, an oncogenic transcription factor critical for colon cancer cell
growth, binds to this site and transactivates the Grp78 promoter, leading to induction of
endogenous GRP78(Ramsay, Ciznadija et al. 2005). Recently, it was discovered that
chromatin modification regulates Grp78 transcription(Baumeister, Luo et al. 2005).
These observations suggest that, in addition to ER stress, GRP78 may be upregulated
through other mechanisms during tumor growth and confer tumors survival advantage.
As early as in 1996, it was reported that GRP78 knockdown fibrosarcoma cells were
either unable to form tumors or they quickly regressed.(Jamora, Dennert et al. 1996)
This was the first clue that GRP78 is required for tumor growth. In vivo activation of the
Grp78 promoter in growing solid tumors was directly observed in mouse xenografts by
micro-Positron Emission Tomography.(Dong, Dubeau et al. 2004) Combined with the
cytoprotective function of GRP78, these observations suggest that GRP78 may be an
important factor for tumors to survive the adverse environment. This notion is
supported by the recent findings that cytoplasmic GRP78 level increases sequentially
with the progression from normal tissue to adenoma and to carcinoma.(Xing, Lai et al.
2006)
Protecting tumors against ER stress is not the only way that GRP78 promotes tumor
growth. Recent studies show that in some cancer cell types, cell surface GRP78 exists as
a receptor and transduces extracellular stimuli to intracellular signals to promote cancer
development. For example, in 1-LN prostate cancer cells, it is reported that cell surface

4
GRP78 acts as a receptor for the receptor-recognized forms of α2-Macroglobulin
( α2M*). Binding of α2M* to cell surface GRP78 activates Akt to promote cellular
proliferation(Misra, Gonzalez-Gronow et al. 2002; Misra, Gonzalez-Gronow et al. 2004;
Misra, Deedwania et al. 2005; Misra, Sharma et al. 2005; Misra, Deedwania et al.
2006).
Since GRP78 is upregulated in the presence of glucose starvation, acidosis, or
hypoxia, the efficiency of any treatment resulting in these conditions can be
compromised by GRP78. Antivascular therapy, a novel approach to block tumor growth,
shuts down the blood supply to the tumor and kills tumor cells by depriving them of
oxygen and nutrients. Therefore, tumors receiving antivascular therapy might acquire
resistance mediated through GRP78 upregulation. This notion is supported by a study
from our lab. Using xenograft models, this study showed that the antivascular agents,
combretastain-A4P and contortrostatin, induce Grp78 transcription and increase GRP78
expression. Thus, it is possible that tumor cells acquire resistance against this class of
anticancer drugs through induction of GRP78(Dong, Ko et al. 2005).
Because GRP78 shifts the balance between survival and death of ER stress toward
survival, it follows necessarily that any anticancer drug acting through ER stress must
be subjected to the influence of GRP78 level in tumor cells. For instance, some
non-steroidal anti-inflammatory drugs (NSAIDs), such as celecoxib, induce cancer cell
apoptosis through ER stress as well as inhibiting cyclooxygenase activity(Tsutsumi,
Gotoh et al. 2004). GRP78 overexpression reduces the apoptosis of human gastric
cancer cells induced by celecoxib and knockdown of GRP78 drastically enhances
apoptosis(Tsutsumi, Namba et al. 2006). Similarly, apoptosis caused by selenium,

5
which induces apoptosis through global thiol redox modification of proteins and the
consequent UPR, is also regulated by GRP78 level in cancer cells(Wu, Zhang et al.
2005; Zu, Bihani et al. 2006).
Drug resistance caused by GRP78 is mediated through not only its global effect on
ER stress but also its specific regulation of apoptosis. GRP78 specifically associates
with caspase-7, which is localized to the ER, and inhibits its activity(Rao,
Castro-Obregon et al. 2002; Rao, Peel et al. 2002; Reddy 2003). A GRP78 mutant
failing to bind to procaspase-7 loses its protective effect against etoposide-induced
apoptosis(Reddy, Mao et al. 2003). Since caspase-7 is a downstream effector caspase,
the action of various anticancer drugs goes through caspase-7. Inhibition of caspase-7
by GRP78 can confer general chemo-resistance to cancer cells.
GRP78 affects the action of apoptosis regulatory proteins as well as apoptosis
effectors. Recently, it was discovered that GRP78 is upregulated in dormant squamous
carcinoma cells, and through suppression of BAX activation, GRP78 protects these
cells from doxorubicin-induced cell death(Ranganathan, Zhang et al. 2006). This
discovery may partially explain how pre-existing dormant cancer cells contribute to the
relapses after chemotherapy. How can GRP78, a chaperone at the ER, affect the specific
apoptosis regulation of Bcl-2 family proteins? The present study demonstrates that
GRP78 associates with BIK, a BH3-only protein, and inhibits its apoptotic activity and
downstream targets, for example BAX.
Collectively, GRP78 is a pro-survival protein that has a variety of ways to inhibit
cell death. The drug resistance mediated through GRP78 is mainly determined by the
basal GRP78 level and its induction level. Thus, in cells with strong GRP78

6
overexpression, the degree of protection is substantially greater than in cells which
show minimal elevation of GRP78 level(Reddy, Mao et al. 2003; Gray, Mann et al. 2005;
Ranganathan, Zhang et al. 2006). To mimic the situation of GRP78-mediated drug
resistance, many experimental investigations focus on the overexpression of GRP78.
However, the overexpression approach has its limitations in probing the role of GRP78.
Such limitations necessitate investigations through specific knockdown of GRP78 by
siRNA. The present study combines both approaches to probe the role of GRP78 in the
regulation of apoptosis and the formation and progression of cancer.
In vitro experiments have limitations because the condition of in vitro study is very
different from the real situation of cancer. Avoiding these limitations, animal studies
confirm the conclusions drawn from in vitro experiments. Mice with heterozygous
deletion of GRP78 exhibit significantly retarded tumor progression, including a longer
latency period, reduced tumor size, and increased tumor apoptosis, as compared to
wildtype sibling mice(Dong, Ni et al. 2008). However, homozygous deletion of GRP78
causes early embryonic death of mice. Therefore, the conventional knockout technique
cannot probe the precise role of GRP78 in cancer formation and progression. Moreover,
the conventional knockout technique produces global nullification of GRP78. This
limitation constrains our research on the role of GRP78 in a specific physiological
process, a specific type of tissue, and a specific type of cancer. The site-specific
recombinase systems overcome the limitations of conventional knockout technique and
open new doors to study gene function in a complex background. Using the Cre/loxP
recombinase system to study the role of GRP78 in cancer formation and progression in
the mouse model is the focus of the present study.

7
In summary, the present study focuses on two points in view of the current status of
GRP78 research. The first focus is how GRP78 regulates apoptosis in a specific way,
like its regulation of ER stress through its specific interaction with PERK, IRE1, and
ATF6. The second focus is the role of GRP78 in cancer formation and progression in a
specific knockout mouse model. The in vivo mechanism of GRP78’s anticancer
function will be explored.

8
Chapter 2. GRP78 associates with BIK and inhibits its
apoptotic activity
2 . 1 Introduction
Apoptosis is regulated by diverse extrinsic and intrinsic signals. Mitochondria have
long been recognized as the major site of initiation and regulation of apoptosis.
However, more and more evidence suggest that other organelles, especially
endoplasmic reticulum (ER), are also very important for apoptosis. ER can sense local
stress through ER stress response, and send signals to mitochondria. The ER also
contains some Bcl-2 family proteins, such as Bcl-2, Bcl-XL, BIK/NBK, BAX, BAK,
etc. Mobilization of ER calcium stores can initiate the activation of cytoplasmic death
pathways as well as sensitize mitochondria to direct proapoptotic stimuli. Emerging
evidence suggests that the ER also regulates apoptosis both by sensitizing mitochondria
to a variety of extrinsic and intrinsic death stimuli and by initiating its own cell death
signals(Thomenius 2003; Thomenius and Distelhorst 2003).
Recently, it was found that overexpression of GRP78 can protect cells from
apoptosis induced by topoisomerase inhibitor, etoposide (Reddy 2003). GRP78 is a
broad specificity molecular chaperone at the ER. Its function in protein quality control
has already been established. However, how GRP78 gets involved in the regulation of
the apoptosis induced by etoposide remains largely obscure.
Etoposide treatment can activate p53 and result in the up-regulation of
BAX(Karpinich, Tafani et al. 2002). Activation of p53 can induce BIK mRNA and
protein, and that results in apoptosis (Mathai, Germain et al. 2002). BIK mediated
apoptosis is BAX dependent. BIK can induce a conformational switch in BAX, which is

9
believed to lead to the insertion of cytosolic BAX into the outer mitochondrial
membrane and cytochrome c release. However, BIK does not directly interact with
BAX (Gillissen, Essmann et al. 2003). BIK can interact with Bcl-2 and
Bcl-XL(Elangovan 1997). Although the functional role of these interactions has not
been determined, the interaction between BIK and Bcl-2 is very important in apoptosis
induction. Thus, Bcl-2 might participate in the mediation of BIK function(Thomenius
2003).
Among these factors, only BIK is primarily located at the ER(Breckenridge 2003).
Moreover, ER-targeted BIK can induce secondary mitochondria activation and
cytochrome c release(Germain, Mathai et al. 2002). This observation suggests that BIK
might be involved in a crosstalk of the ER with the mitochondrial apoptosis pathway.
GRP78 not only functions in protein folding and degradation of misfolded protein,
but also acts as a sensor and regulator of stress response. GRP78 binds to various
protein factors at the ER membrane, such as ATF6, PERK, and IRE1 and locks them at
the ER. For instance, GRP78 binds to the lumenal domain of ATF6 and inhibits the
function of the Golgi localization signal of ATF6. In ER stress, misfolded proteins
compete for binding to GRP78 with A TF6, which frees A TF6 molecules and leads to the
activation of ATF6(Shen, Chen et al. 2002).
It’s possible that GRP78 binds to BIK and affects the action of BIK. Although the
mechanism of this action is not clear, interaction of BIK with other proteins, for instance,
Bcl-2 and Bcl-XL, is very important for its function. GRP78 binding may mask the
interacting sites by steric interference. Alternatively, GRP78 binding may cause a
conformational change of BIK such that BIK is not recognized by its partners.

10
There is a major difference between BIK and ATF6. ATF6 has an ER lumenal
domain to which GRP78 binds, while BIK has no such ER lumenal domain. BIK has a
transmembrane domain at its C-terminus. Like other membrane proteins of the Bcl-2
family, BIK is tail-anchored to the mitochondrial outer membrane or the ER membrane,
and the N-terminal bulk of the protein faces the cytosol. Therefore, lumenal GRP78
cannot directly bind to the transmembrane BIK. Of course, GRP78 could bind to
transmembrane cofactors that form a complex with BIK, but there is a more direct way.
Reddy et al’s research showed that a subpopulation of GRP78 could exist as an ER
transmembrane protein(Reddy 2003), and that is consistent with the results of previous
studies that GRP78 is expressed on the cell surface(Misra, Gonzalez-Gronow et al. 2002;
Arap, Lahdenranta et al. 2004; Misra, Gonzalez-Gronow et al. 2004; Misra, Deedwania
et al. 2005; Misra, Sharma et al. 2005; Misra, Deedwania et al. 2006). Therefore,
transmembrane GRP78 could bind to and lock the BIK at the ER without the
requirement of other cofactors. Thus, it is reasonable to consider the possibility that
GRP78 regulates etoposide-induced apoptosis by interacting with BIK.
The present study demonstrates that GRP78, but not other ER chaperones, forms a
complex with endogenous BIK and ER-targeted BIK and blocks their apoptotic activity.
This pathway represents a novel mechanism in which GRP78 regulates apoptosis in a
specific manner.

11
2 . 2 Materials and Methods
2. 2. 1 Cell culture, drug treatment and reagents
The human embryonic kidney 293T cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum. For etoposide treatment,
the cells were incubated with 50 µmol/L etoposide for 6 hours and cultured for another
24 hours before harvest.
Phosphate-buffered saline: 11.5 g Di-sodium hydrogen orthophosphate anhydrous,
2.96 g sodium dihydrogen orthophosphate, and 5.84 g sodium chloride, diluted to 1000
ml with distilled water. Adjust pH to 7.5 and autoclave.
Cell lysis buffer: 20 mM Tris-HCl with pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mg
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM DTT, and 1 mM
Na
3
VO
4
; add one EDTA-free protease inhibitor cocktail tablet (Roche) per 10 ml lysis
buffer before using.
TBS-Tween (TBS-T): Dilute required volume of Tween 20 in TBS to give a 0.1%
(v/v) solution.
2. 2. 2 Plasmids
The plasmids pcDNA3-Flag-BIK-b5TM and pcDNA3-Flag-BIK were provided by G .C.
Shore (McGill University, Montreal, Canada) and their construction has been
described(Germain, Mathai et al. 2002). In pcDNA3-Flag-BIK-b5TM, the
COOH-terminal transmembrane domain (amino acids 135 – 160) of BIK was replaced
by the transmembrane domain of cytochrome b5 (amino acids 107 – 134), which targets
the protein to the ER. The construction of pcDNA3-His-GRP78 has been

12
described(Zeng, Lu et al. 2004). Briefly, For pcDNA-BiP/grp78, six copies of the
His-tag and two copies of Ser that serve as spacer were inserted directly after the ER
signal peptide of 18 amino acids. The full-length 6 x His-BiP/grp78 cDNA, assembled
by multiple subcloning and religation steps, was subcloned into the pKS vector. It was
excised as a BamH1 fragment and subcloned into the BamH1 site of the expression
vector pcDNA3 (Invitrogen, Carlsbad, CA) (Zeng, Lu et al. 2004).
2. 2. 3 Transient transfections
The day before transfection, seed 1.2 x 10
6
293T cells per 60 mm dish in 5 ml of DMEM
containing 10% fetal bovine serum. Incubate the cells at 37°C and 5% CO
2
in an
incubator. 293T cells were grown to 60% to 80% confluence. Two micrograms of
pcDNA3-Flag-BIK-b5TM plasmid with 2 µg of His-GRP78 or empty vector were
diluted with DMEM without serum or antibiotics to total volume of 150 µl. The green
fluorescent protein (GFP) gene driven by cytomegalovirus promoter was added to
monitor for transfection efficiency. Empty vector was added to adjust the total amount
of plasmids to be the same. Mix and spin down the solution for a few seconds to remove
drops from the top of the tube. Add 40 µl of PolyFect transfection reagent (Qiagen) to
the DNA solution. Mix by vortexing for 10 -20 seconds. Incubate samples for 5–10 min
at room temperature to allow complex formation. While complex formation takes place,
gently aspirate the growth medium from the dish, and add 3 ml of fresh cell growth
medium with serum and antibiotics. Add 1 ml of cell growth medium with serum and
antibiotics to the reaction tube containing the transfection complexes. Mix by pipetting
up and down twice, and immediately transfer the total volume to the cells in the 60 mm

13
dish. Gently swirl the dish to ensure uniform distribution of the complexes. Incubate
cells with the transfection complexes for 10 – 12 hours at 37°C and 5% CO
2
. Remove
medium containing the transfection complexes from the cells by gentle aspiration. Add
fresh growth medium with serum and antibiotics. Forty-eight hours later, the transfected
cells were subjected to cell death assays, Western blot, or coimmunoprecipitation.
2. 2. 4 Western blots
The Western blots were done as described(Lee 2005). Aspirate media from the culture
vessel. Wash cells once with PBS. Add the Trypsin–EDTA to the side of the vessel
opposite the cells (approximately 3 ml/ 25ml flask) and gently swirl the vessel to cover
the monolayer completely. Aspirate Trypsin–EDTA gently and incubate cells in 37°C
for 2 – 10 min with careful monitoring until the cells begin to round up. Add PBS
(approximately 0.15 ml/cm
2
) and disperse cells into suspension by pipetting in and out
repeatedly. Centrifuge cells at 3500 rpm for 10 min at 4°C. Remove the remaining
supernatant with pipette.
After lysing cells in the cell lysis buffer, clear the cell lysate by centrifugation at
12,000g for 10min at 4°C. Dilute the proteins with Laemmli sample buffer and incubate
for 5 min at 95°C. The proteins are separated by SDS–polyacrylamide gel
electrophoresis (SDS– PAGE) and transferred to nitrocellulose membranes according to
standard protocol. Load prestained protein molecular marker to the same
polyacrylamide gel.
Rinse the nitrocellulose membrane briefly with distilled water. Non-specific
binding sites are blocked by immersing the membrane in 5% (w/v) fat-free milk in

14
TBST for 30 min at room temperature on an orbital shaker. Incubate the membrane in
diluted primary antibody for 2 hours at room temperature or overnight at 4°C. Briefly
rinse the membrane using two changes of TBS-T and then wash once for 10 min and
twice for 5 min, with fresh changes of TBS-T at room temperature. Incubate the
membrane in the diluted secondary antibody for 1 h at room temperature. Wash the
membrane as detailed before. Take the ECL detection reagents (Amersham) supplied
and mix an equal volume of detection solution 1 with detection solution 2 to give
sufficient liquid to cover membrane and incubate for 1 min at room temperature. Drain
the excess ECL. Place the blot with the protein side facing up on a sheet of plastic wrap.
Wrap blots and expose to x-ray film.
The primary antibodies were goat anti-BIK (N-19, Santa Cruz Biotechnology,
Santa Cruz, CA), rat anti-GRP78 (76-E6, Santa Cruz Biotechnology), rat anti-GRP94,
rabbit anti-calnexin, rabbit anti-calreticulin (Stressgen), mouse anti-Flag M2, mouse
anti–poly(ADP-ribose) polymerase (PARP; F-2, Santa Cruz Biotechnology), and mouse
anti–ß-actin (Sigma-Aldrich). Anti–ß-actin was diluted at 1:2,000; anti-BIK at 1:500;
and other antibodies at 1:1,000. Respective horseradish peroxidase–conjugated
secondary antibodies (Santa Cruz Biotechnology) at 1:1,000 dilution were used.
2. 2. 5 Co-immunoprecipitation assays
Cells were lysed in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5%
Nonidet P-40, and 0.5% deoxycholate, with protease inhibitor tablet (Roche Molecular
Biochemicals)) and frozen and thawed three times. 500 µg of total protein extract from
each sample was pretreated with 50 µl of protein G-Sepharose beads (Upstate) for 1h at

15
4°C prior to incubation with 5 µg of either goat anti-BIK antibody (N-19, Santa Cruz
Biotechnology) or mouse anti-Flag M2 antibody (Sigma-Aldrich) for 2 hours. For
negative controls, the respective goat or mouse immunoglobulin G (IgG; Santa Cruz
Biotechnology) was used. Following the incubation period, 50 µl of protein
G-Sepharose beads was added, and the mixtures were rotated at 4°C overnight. The
beads were then washed five times with the extraction buffer. The immunoprecipitate
was released from the washed beads by the addition of 30 µl of 1 x SDS-PAGE sample
loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1%
bromphenol blue, 10% glycerol), followed by heating at 100°C for 5 min. The
supernatant obtained after centrifugation was resolved by SDS-PAGE and subjected to
Western blot analysis to detect the co-immunoprecipitated proteins.
2. 2. 6 GST pull-down assays
Glutathione S-transferase (GST)-GRP78, GST-GRP78/P45, and GST-BIK were
constructed by subcloning full-length hamster Grp78 cDNA, GRP78/P45, and human
BIK into the BamH1/XhoI and BamH1/Sal1 sites of pGEX 4T1, respectively
(Pharmacia Biotech). Following expression of GST-proteins in Escherichia coli and
IPTG induction, GST-proteins, prepared by sonication in PBS plus 1% Triton X-100,
were purified by affinity chromatography with glutathione-Sepharose beads
(Sigma-Aldrich), and the protein yields were verified by Coomassie Blue staining.
Five micrograms of bacterially expressed GST-BIK, GST-GRP78, and GST bound to
glutathione-Sepharose beads (Sigma-Aldrich) were incubated with 500 µg of total
protein extract on a rotating shaker at 4°C for 16 h. The beads were collected by

16
centrifugation at 2,000 rpm for 5 min and washed thrice with extraction buffer (20 mM
Tris–HCl, pH 8, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). The bound proteins on
beads were eluted in SDS-PAGE sample loading buffer and subjected to 10%
SDS-PAGE and Western blotting.
2. 2. 7 Cell death and apoptotic assays
Cell death was assessed by the trypan blue exclusion assay. Briefly, 20 μl of 0.4% trypan
blue (Sigma-Aldrich) was added into 20 μl of harvested cells resuspended in media.
After 5 minutes of equilibration, the cells were counted under the microscope.
For mitochondrial membrane potential staining, the Mitochondrial Permeability
Transition Detection Kit (Immunochemistry, Bloomington, MN) was used following
the manufacturer's protocol. Briefly, remove the media from monolayer cell cultures
and wash the cultures with PBS. Add enough 1 x MitoPT solution to cover the cells.
Incubate the cells with the MitoPT solution at 37°C for 15 minutes in a CO
2
incubator.
Carefully remove and discard staining media and wash the monolayer cultures with 1 to
2 ml of 1 x assay buffer of 37°C. Discard the assay buffer and add DMEM with serum
and antibiotics. The cell cultures were then examined under a fluorescence microscope.
Each assay was done in triplicate.
2 . 3 Results
2. 3. 1 BIK protein is induced by etoposide treatment
To determine the inducibility of BIK protein by etoposide treatment, we examined the
BIK protein level in the human embryonic kidney cell line 293T. In 293T cells, BIK

17
protein was present at a low basal level under normal culture conditions. On 6-hour
treatment with 50 μmol/L etoposide, a topoisomerase I inhibitor, the level of BIK protein
was substantially elevated (Fig. 2.1). In contrast, ER stress inducers such as
thapsigargin or tunicamycin do not induce BIK (Fig. 2.1). Thus, the induction of BIK
occurs under specific condition in 293T cells.

Fig. 2.1. BIK protein is induced by etoposide treatment. 293T cells were either non-treated or treated
with 50 µM etoposide (Etop) for 6 hours, 300 nM thapsigargin (TG) or 1.5 μg/ml tunicamycin (TN) for 16
h. Cells were harvested 24 hours later. Total proteins lysates were made from these cells and were
resolved in 10% SDS-PAGE gel. The levels of BIK and β-actin protein were determined by
immunoblotting with antibodies against BIK and β-actin.

2. 3. 2 Endogenous BIK selectively forms complex with GRP78
In order to understand how BIK is regulated at the ER, we need to know what protein
interacts with BIK at the ER. Thus, we searched for the interactive partners of BIK by
co-immunoprecipitation followed by Western blot with known ER proteins. We

18
discovered that BIK selectively interacts with GRP78. In co-immunoprecipitation
assays, BIK formed a complex with GRP78 in both the untreated cells and the cells
whose BIK level was upregulated by etoposide treatment (Fig. 2.2A). The complex
formation between endogenous GRP78 and BIK is specific because this complex was
not observed using control IgG as the precipitating antibody. Moreover, other major ER
proteins such as GRP94, calnexin, and calreticulin were not detected in the BIK
co-immunoprecipitate (Fig. 2.2A). GST pulldown assay was performed to confirm the
physical interaction between GRP78 and BIK. GRP78 and BIK were both expressed as
bacterial GST-fusion proteins. The yield and purity of the GST-proteins were confirmed
by Coomassie blue staining (Fig. 2.2B). In pull-down assays, GST-GRP78, but not the
control GST protein, was able to bind BIK from total cell extract, and reversely,
GST-BIK, but not the control GST protein, was able to bind GRP78 (Fig. 2.2C).
GST-GRP78/P45, the cytosolic part of surface GRP78, was able to bind to GST-BIK,
and that suggest that surface GRP78 can form complex with BIK(Fig. 2.2C). Thus, BIK
and GRP78 form a complex both in vivo and in vitro.

19

Fig. 2.2. Endogenous BIK selectively forms complex with GRP78. A, Cell lysates prepared from
control and Etop-treated 293T cells were immunoprecipitated with anti-BIK or normal IgG. The
immunoprecipitates were applied in parallel with input lysates to SDS-PAGE and Western blotted with
antibodies against GRP78, GRP94, calnexin, calreticulin and BIK. B, Coomassie blue staining of
GST-GRP78, GST-BIK, and GST resolved by SDS-PAGE. C, GST pulldown analysis of BIK interaction
with GRP78. Lysates of 293T cell were incubated with GST-GRP78, GST-GRP78/P45, GST-BIK, or
GST linked beads. Beads were collected, washed, and mixed with equal amount of 2× SDS-PAGE
loading buffer. Samples were boiled and resolved by SDS-PAGE and the presence of the GRP78 or BIK
was detected by immunoblotting.

20
2. 3. 3 GRP78 associates with the endoplasmic-reticulum targeted BIK
Although BIK is primarily located at the ER, it cannot be excluded that a part of BIK
proteins locates outside ER. To determine the functional interaction between GRP78
and BIK specifically at the ER, 293T cells were transfected with a vector expressing
Flag-tagged BIK, which is selectively targeted to the ER by the cytochrome b5
transmembrane domain (b5TM). First, the expression level of the Flag-tagged
BIK-b5TM in the transfected cells was confirmed by Western blot analysis. Then,
co-immunoprecipitation using anti-Flag antibody showed the complex formation
between GRP78 and the ER-targeted BIK in vivo (Fig. 2.3).

Fig. 2.3. GRP78 associates with the endoplasmic-reticulum targeted BIK. Cell lysates prepared from
293T cells transfected with either empty vector pcDNA3 (-) or vector expressing Flag-BIK-b5TM (+)
were immunoprecipitated with either anti-Flag antibody or normal IgG as a control. The
immunoprecipitates were resolved by SDS-PAGE and Western blotted with anti-GRP78 and anti-Flag
antibodies.

21
2. 3. 4 GRP78 overexpression inhibits apoptotic activity of ER-targeted BIK
GRP78 forms complex with endogenous BIK. What is the function significance of the
association between GRP78 and BIK? To investigate the effects of GRP78 on BIK
activity, the expression vector for ER-targeted BIK was cotransfected into 293T cells
with either the expression vector for His-tagged GRP78 or the empty vector pcDNA3.
Coexpression of the His-tagged GRP78 and Flag-tagged BIK in the transfected cells
was confirmed by Western blot (Fig. 2.4A). Cell death determined by trypan blue
exclusion reveals that cells expressing ER-targeted BIK exhibited a 5-fold increase in
the percent of cell death compared with cells transfected with pcDNA3 (Fig. 2.4B). This
increase was reduced by half in cells overexpressing His-GRP78, providing the
evidence that GRP78 is able to inhibit the cell death induced by BIK overexpression. To
determine whether the cell death observed was apoptosis, identical transfection
experiments were done and the extent of apoptosis was determined by lipophilic cation
fluorescent staining that detects changes in mitochondrial membrane potential. As
summarized in Fig. 2.4C, ER-targeted BIK expression induced apoptosis in the
transfected cells and GRP78 overexpression reduced ER-targeted BIK–induced
apoptosis by 3-fold.

22

Fig. 2.4. GRP78 overexpression inhibits the apoptotic activity of ER-targeted BIK. 293T cells were
transfected with empty vector (-), pcDNA3-Flag-BIK-b5TM, or pcDNA3-His-GRP78, alone or in
combination as indicated. A, the expression level of each protein was determined by Western blot; B, the
percent cell death in each transfection was assessed by trypan blue exclusion assay; and C, the percent of
apoptotic cells was assessed by mitochondrial membrane potential staining. The columns in figures 3B
and 3C are mean values from three experiments, each of which assayed at least 400 cells for every group;
bars represent standard errors; * denotes a P value < 0.05; ** denotes a P value < 0.01.
2 . 4 Discussion
Different from other BH3-only proteins, BIK is primarily localized at the ER(Germain,
Mathai et al. 2002; Mathai, Germain et al. 2005). This characteristic of BIK suggests
that ER also plays an important role in apoptosis regulation. Moreover, BIK targeted to
the ER can activate BAX indirectly and trigger cytochrome c release from the

23
mitochondria (Germain, Mathai et al. 2002). While the mitochondria have been
established as a major regulator of apoptosis, the ER has emerged as another key site for
the regulation of apoptosis and initiates independent apoptotic pathways in response to a
variety of stress conditions (Breckenridge 2003; Scorrano, Oakes et al. 2003; Zong, Li
et al. 2003). Moreover, there is crosstalk between the mitochondria and the ER. BIK
represents a promising novel link because it can send signal from ER to initiate the
cytochrome c release from mitochondria (Germain, Mathai et al. 2002; Germain,
Mathai et al. 2005).
Looking for the ER proteins that interact with BIK is critical in understanding how
BIK regulates apoptosis at the ER. In searching for partners interacting with BIK at the
ER, we discovered that BIK selectively forms complexes with GRP78, but not with
other major ER chaperones, such as GRP94, calnexin, and calreticulin. GRP78 is a
pivotal regulator of ER function due to its role in protein folding and assembly,
ER-associated degradation (ERAD), ER calcium binding, and controlling the activation
of transmembrane ER stress inducers (Little, Ramakrishnan et al. 1994; Lee 2001;
Hendershot 2004; Li and Lee 2006). In various experimental systems, the
cytoprotective function of GRP78 is firmly established (Rao, Peel et al. 2002; Reddy,
Mao et al. 2003; Dong, Ko et al. 2005; Lee 2005; Ermakova, Kang et al. 2006; Li and
Lee 2006; Ranganathan, Zhang et al. 2006). Although GRP78 was discovered as an ER
lumenal protein, it was revealed recently that a subpopulation of GRP78 exists as a
transmembrane protein (Rao, Peel et al. 2002; Reddy, Mao et al. 2003). Therefore,
GRP78 can potentially interact directly with the cytosolic components of the apoptotic
pathway and regulate their activity. In addition to the case presented in this study,

24
GRP78 has been reported to form complexes with procaspases, such as caspase-7 and
mouse caspase-12, both of which associate with the outer ER membrane. GRP78
overexpression blocks cleavage of procaspase-7 to its active form (Reddy, Mao et al.
2003). Conversely, inhibition of the formation of the GRP78-caspase-7 complex results
in caspase-7 activation, resulting in increase in apoptosis (Davidson, Haskell et al. 2005;
Ermakova, Kang et al. 2006).
Since GRP78 can interact with the downstream effectors of the apoptotic pathways,
it is reasonable to propose that GRP78 may also regulate the activity of key upstream
regulators of apoptosis in the same way. Here, we show that GRP78 forms a complex
with BIK and overexpression of GRP78 inhibits the apoptotic activity of BIK. A recent
report shows that in epidermoid carcinoma cells, knockdown of GRP78 by siRNA leads
to BAX activation, cytochrome c release and increased sensitivity to doxorubicin,
however the mechanism whereby GRP78 suppresses BAX activation is not known
(Ranganathan, Zhang et al. 2006). Since both etoposide and doxorubicin are strong
inducers of BIK and BAX is a downstream target of BIK(Gillissen, Essmann et al. 2003;
Mathai, Germain et al. 2005), our discovery that GRP78 is an interactive partner of BIK
and that GRP78 can block BIK activity explains why GRP78 suppression sensitizes
cells to BAX activation and apoptosis induced by doxorubicin as well as etoposide.
How can GRP78 suppress BIK activity? Binding to client proteins is a general
feature of molecular chaperones, which enables them to serve as buffering agents by
masking the functional domain or altering the conformation of the client protein
(Mitchell-Olds and Knight 2002). One scenario is that GRP78 binding to BIK may alter
its conformation or interfere with its heterodimerization with other interactive partners

25
essential for its proapoptotic activity. For example, it has been reported that BIK can
cooperate with the weak BH3-only protein NOXA to activate BAX, resulting in rapid
cytochrome c and caspase activation (Germain, Mathai et al. 2005). Interfering with this
interaction by GRP78 may impair the ability of BIK to induce apoptosis. Future studies
mapping the interactive domains of BIK and GRP78 will provide further insight and
resolve these issues.
The regulation of BIK activity by GRP78 has been demonstrated in this study.
However, this does not necessarily mean that GRP78 plays a role in apoptosis
regulation through BIK under physiological conditions. Therefore, it is important to
investigate the functional interaction between GRP78 and BIK in a physiological
system where BIK plays an essential role. Estrogen-dependent human breast cancer cell
line, MCF-7/BUS, which requires BIK to mediate its apoptosis induced by estrogen
starvation(Hur, Chesnes et al. 2004), is such a model system. The following chapter
presents a study on the effect of GRP78 on the BIK-mediated apoptosis in MCF-7/BUS
cells.

26
Chapter 3. GRP78 protects human breast cancer cells
against estrogen-starvation induced apoptosis by inhibiting
BIK
3 . 1 Introduction
Antiestrogen therapy is the treatment of choice for postmenopausal women with
estrogen-receptor-positive breast cancer. The first antiestrogens were generated in the
1950s. Their effects on breast cancer were soon found. Since the 1970s, tamoxifen has
been widely used as an adjuvant therapy. Tamoxifen remains the treatment of choice for
most women with estrogen-receptor-positive breast carcinoma because of its
effectiveness and low toxicities compared with other chemotherapy drugs (Clarke
2001). Fulvestrant (Faslodex), a newer estrogen receptor antagonist in clinical use in
metastatic hormone receptor positive breast cancer, has no agonist activity and causes
degradation of the estrogen receptor, thus eliminating estrogen-sensitive gene
transcription (Dowsett, Nicholson et al. 2005). In addition, third generation aromatase
inhibitors (e.g., anastozole, letrozole and exemestane), which block the conversion of
adrenally derived androgens to estrogen in postmenopausal women, provide even better
efficacy and tolerability (Baum, Buzdar et al. 2003).
However, most responsive cancers become resistant despite the fact that the initial
response rate can be up to 70%. The mechanisms for the resistance of
estrogen-receptor-positive cancers are unclear. Most cancers may acquire resistance
through multiple mechanisms, such as host immunity, endocrinology, or
pharmacokinetics. Significant changes occur within the cancer cells in response to
antiestrogen therapy, such as the switch of estrogen expression pattern from estrogen

27
Receptor α to estrogen receptor β and other changes in the downstream factors. These
may lead to the change in cellular response to antiestrogen treatment (Clarke 2001).
Understanding the molecular mechanisms responsible for endocrine resistance is of
primary importance for improving the treatment of breast cancer.
It is widely accepted that estrogen is required for the growth and proliferation of
breast cancer cells. Many lines of evidences show that estrogen is also essential for the
survival of breast cancer cells(Thiantanawat 2003). When subjected to estrogen
starvation, which mimics the effect of aromatase inhibitors, or exposed to antiestrogens,
significant apoptosis of breast cancer cells is observed. The BCL-2 family proteins are
key regulators of apoptosis. The antiapoptotic members of the BCL-2 family, such as
BCL-2, share three or four conserved domains known as BCL-2 homology (BH)
regions. The proapopotic members such as BAX share two or three BH domains.
Whereas the proapoptotic members facilitate the release of cytochrome c from the
mitochondria, resulting in Apaf-1 activation and subsequent caspase activation, the
antiapoptotic members suppress this pathway (Cheng, Wei et al. 2001). A third group of
apoptosis regulators, referred to as BH3-only proteins, only share the 9 amino acid BH3
region. In their active conformation, BH3-only BCL-2 members regulate the ability of
BAX and BAK to oligomerize in the mitochondrial outer membrane and release
intermediate proteins, including cytochrome c, to the cytosol (Wei, Zong et al. 2001).
BH3-only proteins can also bind directly to the antiapoptotic members of the BCL-2
family through the BH3 domain and inhibit their activity. Previous studies showed that
antiestrogens have no effect on the expression of proapoptotic protein BAX but
suppress antiapoptotic BCL-2 expression, correlating with induction of apoptosis (Diel,

28
Smolnikar et al. 1999). Nonetheless, the molecular mechanisms whereby the BCL-2
protein family members regulate estrogen-starvation mediated apoptosis are not well
understood.
Recently, Hur et al discovered that estrogen starvation or antiestrogen treatment
could induce apoptosis in MCF-7/BUS, a breast cancer cell line (Hur 2004). MCF-7
human breast cancer cell line is often used as an in vitro model of breast cancer.
However, many derivative lines of MCF-7 cells have changed during subculture with
respect to their estrogen responsiveness, degree of oncogene amplification, and
karyotypes. MCF-7/BUS has been characterized as growing in an
estrogen-dose-dependent manner(Coser 2003). Therefore, this cell line is an excellent
model for estrogen-receptor-positive breast cancer. In addition,
estrogen-receptor-negative cell lines, such as MDA-MB-435, are available as models
for estrogen-receptor-negative breast cancer. An apoptotic BH3-only protein, BIK,
mediates apoptosis. This is concluded because BIK mRNA and protein were strongly
induced by estrogen starvation or antiestrogen treatment, and knockdown of BIK by
siRNA significantly inhibited apoptosis caused by antiestrogen treatment. In contrast, in
estrogen-independent cell lines, for instance, T47D, ZR75, or SKBR3, a significant
amount of BIK mRNA was expressed without any detectable BIK protein.
BIK induction has been reported in human cells in response to p53 overexpression
and genotoxic agents such as doxorubicin. Interestingly, BIK contains a single
transmembrane segment at its extreme C-terminus, but in contrast to most BH3-only
proteins, which target primarily the mitochondria with some also localizing in the
endoplasmic reticulum (ER), BIK is integrated almost exclusively in the membrane of

29
the ER (Germain, Mathai et al. 2002). Immunofluorescence confocal microscopy shows
that BIK co-localizes with calnexin, an ER transmembrane protein, and subcellular
fractionation demonstrates that BIK co-distributes with ER proteins calnexin and
GRP78/BiP (Germain, Mathai et al. 2002; Mathai, Germain et al. 2005). Although BIK
does not interact directly with proapoptotic BAX and BAK, it regulates a BAX/BAK
dependent release of Ca2+ from the ER stores, and operates with other BH3-only
proteins to cause rapid release of cytochrome c from the mitochondria and the activation
of caspases (Germain, Mathai et al. 2002; Mathai, Germain et al. 2005). The discovery
that BIK is a key mediator for estrogen-starvation and antiestrogen-induced apoptosis
implies that inhibition of BIK expression or activity at the ER site may represent a novel
molecular mechanism for endocrine resistance in human breast cancer.
The previous work of our lab showed that overexpression of GRP78 can protect
cells from apoptosis induced by topoisomerase inhibitor, etoposide (Reddy, Mao et al.
2003). Etoposide treatment can activate p53 (Karpinich 2002), which can induce BIK
mRNA and protein (Germain, Mathai et al. 2002). The work presented in chapter 2
demonstrates that GRP78 interacts with BIK and overexpression of GRP78 inhibits the
apoptotic activity of BIK. Here it is further demonstrated that GRP78 overexpression
inhibits estrogen-starvation induced BIK upregulation, BAX activation and apoptosis in
an estrogen-dependent human breast cancer cell line, MCF-7/BUS; suppression of
endogenous GRP78 by siRNA sensitizes MCF-7/BUS cells to estrogen-starvation
induced apoptosis. This study presents a new mechanism that contributes to the
resistance of breast cancer to antiestrogen therapy, and provides a novel target for the
therapy to overcome the resistance of antiestrogen treatment of breast cancer.

30
3 . 2 Materials and methods
3. 2. 1 Cell culture and reagents
The estrogen-dependent breast cancer cell line MCF-7/BUS was provided by A.M. Soto
(Tufts University, Medford, MA). The characteristic has been described(Soto,
Sonnenschein et al. 1995). The MCF-7/BUS cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. For estrogen
starvation, MCF-7/BUS cells were washed three times with phenol red-free DMEM and
incubated in washing medium at 37°C for 60 minutes. The MCF-7/BUS cells were then
cultured in phenol red-free DMEM supplemented with 5% charcoal/dextran-stripped
fetal bovine serum for 24 to 72 hours as indicated(Hur, Chesnes et al. 2004).
3. 2. 2 Transient transfections and adenovirus infections
For construction of the adenovirus expression vectors, either GFP or a His-tagged
full-length hamster Grp78 cDNA was subcloned into an adenoviral vector and its
expression was driven by the cytomegalovirus promoter. The sequence in the final
construct was confirmed by DNA sequencing. MCF-7/BUS cells were infected at 100
plaque-forming units/cell with adenovirus vectors expressing GFP or GRP78. For
mitochondrial membrane potential staining, because GFP interferes with the green
fluorescence of this assay, the adenovirus empty vector was used as the negative control.
After 24 h, the infected cells were subjected to estrogen starvation for 48 h. Each
transfection or infection was done in duplicate and was repeated two to three times.

31
3. 2. 3 Western blot and quantitation
The Western blots were performed as described in chapter 2. The primary antibodies
were goat anti-BIK (N-19, Santa Cruz Biotechnology), rat anti-GRP78 (76-E6, Santa
Cruz Biotechnology), rat anti-GRP94, mouse anti-PARP (F-2, Santa Cruz
Biotechnology), and mouse anti- β -actin (Sigma-Aldrich). Anti- β -actin was diluted at
1:2000, anti-BIK at 1:500, and other antibodies at 1:1000. Respective horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at 1:1000
dilution were used. The Western blots were quantitated by Fluor-STM MultiImager
(BIO-RAD, Hercules, CA) according to manufacturer’s instructions. All quantitations
were normalized against β-actin.
3. 2. 4 Small interfering RNA
The siRNA against Grp78 is 5'-ggagcgcauugauacuagadTdT-3' as described(Tsutsumi,
Namba et al. 2006). The siRNA against Bik is 5'-aagaccccucuccagagacau-3'(Hur 2004).
The control siRNA is Silencer Negative Control #3 siRNA (Ambion) composed of a
19-bp scrambled sequence without significant homology to any known gene sequences
from mouse, rat, or human. MCF-7/BUS cells were grown to 50% confluence and
transfected with control siRNA or siRNA against Grp78 or Bik using Lipofectamine
2000 transfection reagent (Invitrogen).
Briefly, the day before transfection, plate MCF-7/BUS cells in the DMEM without
antibiotics so that the cells are about 50% confluent at the time of transfection. Dilute
the appropriate amount of control siRNA, siGrp78, or siBik alone or in combination as
indicated in 50 μl of Opti-MEM I Reduced Serum Medium without serum (Invitrogen)

32
and mix it gently. The total amount of siRNA in each condition was adjusted to be the
same by addition of control siRNA. Mix Lipofectamine 2000 gently before use, then
dilute the appropriate amount in 50 μl of Opti-MEM I Medium. Mix gently and incubate
for 5 minutes at room temperature. Then, combine the diluted siRNA with the diluted
Lipofectamine 2000 (total volume is 100 μl). Mix gently and incubate for 20 minutes at
room temperature to allow the siRNA and Lipofectamine 2000 to form complexes. Add
the 100 μl of siRNA-Lipofectamine 2000 complexes to each well. Mix gently by
rocking the plate back and forth. Incubate the cells at 37°C in a CO
2
incubator for 24
hours until they are ready for estrogen starvation. The experiments were repeated two to
three times.
3. 2. 5 Apoptotic assay
For mitochondrial membrane potential staining, the Mitochondrial Permeability
Transition Detection Kit (Immunochemistry, Bloomington, MN) was used as described
in chapter 2. The cell cultures were then washed with PBS and examined under a
fluorescence microscope. Each assay was done in triplicate.
3. 2. 6 Flow cytometric analysis of BAX-associated immunofluorescence
On initiation of apoptosis, BAX undergoes conformational change that exposes an
otherwise inaccessible NH2-terminal epitope(Mandic, Viktorsson et al. 2001). A mouse
monoclonal antibody against amino acids 12 to 24 (clone 6A7, PharMingen) was used
to detect the BAX with proapoptotic conformational change. MCF-7/BUS cells were
harvested and washed in ice-cold PBS. Cells were then fixed in 0.25%

33
paraformaldehyde in PBS for 5 min, washed three times in PBS, and incubated for 30
min with the mouse monoclonal antibody against amino acids 12 to 24 of Bax (clone
6A7; PharMingen) or anti-mouse IgG1 (BD Biosciences). Antibodies were diluted 1:50
in PBS containing digitonin (100 μg/ml). After three washes in PBS, cells were
incubated with phycoerythrin-labeled anti-mouse antibody for 30 min, washed twice in
PBS, and resuspended in PBS. The cells were analyzed on a FACstar flow cytometer
(BD Biosciences).
3 . 3 Results
3. 3. 1 Estrogen starvation induces BIK protein and BAX activation in
MCF-7/BUS cells
In the human breast cancer MCF-7/BUS cells, the level of BIK protein was dramatically
induced by estrogen starvation (Fig. 3.1 & Fig. 3.3). Because BIK is an upstream
regulator of BAX, estrogen starvation should activate BAX in MCF-7/BUS cells. As
shown in Fig. 3.2, Estrogen starvation resulted in fluorescent histogram curve shift with
the mean fluorescence value increased from 77 to 313 when compared with the
non-treated cells, indicating an increase of the active form of BAX as recognized by the
BAX conformation specific antibody.

34

Fig. 3.1. Estrogen starvation induces BIK protein and BAX activation in MCF-7/BUS cells.
MCF-7/BUS cells were cultured either in regular DMEM or in estrogen-free DMEM for 48 hours.
Western blots of total protein lysates from these cells were performed with antibodies against BIK and
β-actin.

3. 3. 2 GRP78 overexpression inhibits estrogen-starvation induced BAX
activation
Because GRP78 inhibits BIK activity, the downstream BAX activation should be
suppressed by GRP78 overexpression. To test this in the context of estrogen-dependent
human cancer cells, MCF-7/BUS cells were infected with adenovirus vectors
expressing either GRP78 (Ad-GRP78) or GFP (Ad-GFP) as a control. Overexpression
of GRP78 in the Ad-GRP78–infected cells was confirmed by Western blot (Fig. 3.3).
On estrogen starvation, BIK was induced with BAX activation (Fig. 3.2 & Fig. 3.3). As
shown in Fig. 3.2, estrogen starvation resulted in fluorescent histogram curve shift with
the mean fluorescence value increased from 77 to 313 when compared with the

35
non-treated cells. This result indicates an increase of the active form of BAX as
recognized by the BAX conformation specific antibody (Fig. 3.2). Consistent with
GRP78 counteracting BIK activity, the activation of BAX by estrogen starvation was
inhibited by GRP78 overexpression as compared with cells expressing GFP, with the
mean fluorescence value decreased from 183 for cells expressing GFP to 70 for cells
overexpressing GRP78 (~48% suppression; Fig. 3.2).

Fig. 3.2. GRP78 overexpression inhibits estrogen-starvation induced BAX activation. MCF-7/BUS
cells were infected with adenoviral vector expressing GFP (Ad-GFP) or GRP78 (Ad-GRP78). Cells were
then cultured either in regular medium or in estrogen-free medium for 48 hours and subjected to FACS
analysis using mouse anti-BAX and phycoerythrin-labeled anti-mouse antibodies. ES: estrogen
starvation.

36
3. 3. 3 GRP78 overexpression inhibits estrogen-starvation induced apoptosis
To test independently the protective effect of GRP78 in estrogen starvation–induced
apoptosis, MCF-7/BUS cells were infected with adenovirus vectors expressing either
GRP78 (Ad-GRP78) or GFP (Ad-GFP) as a control. To MCF-7/BUS cells subjected to
the mitochondrial permeability transition assay, empty adenovirus vectors were used as
a control to avoid the interference due to the green fluorescence of GFP. In the
mitochondrial permeability transition assay, the lipophilic MitoPT reagent penetrates
the healthy mitochondria in nonapoptotic cells, aggregates, and fluoresces red in the
negatively charged mitochondria. In early apoptotic cells, on collapse of the
mitochondrial membrane potential, the MitoPT reagent distributes throughout the cell
and fluoresces green. As shown in Fig. 3.3A, MCF-7/BUS cells overexpressing GRP78
showed substantial reduction in mitochondrial membrane potential change on 48 h of
estrogen starvation, as compared with cells infected with the empty vector.
Mitochondrial permeability transition assay detects the early stage of apoptosis. To
confirm the execution of apoptosis, a terminal marker of apoptosis should be checked.
Because MCF-7/BUS cells lack caspase-3, a useful indicator of apoptosis in these cells
is estrogen starvation–induced cleavage of endogenous PARP(Hur 2004). In
nonapoptotic cells, PARP exists in its uncleaved form (116 kDa), whereas in apoptotic
cells, PARP is cleaved by activated caspases into an 85-kDa fragment. As shown in Fig.
3.3B, the cleaved form of PARP was evident in estrogen-starved cells infected with
Ad-GFP but was not detected in cells infected with Ad-GRP78. In cells infected
Ad-GRP78, the level of BIK protein was higher than that in cells infected Ad-GFP (Fig.

37
3.3B). This may result from the apoptotic property of BIK: it cannot accumulate to high
level while provoking apoptosis. In cells overexpressing GRP78, BIK is inhibited by
GRP78 and able to accumulate to a higher level without disrupting host cells. This
phenomenon is very similar to the E1A-induced BIK accumulation in the cells
overexpressing BCL-2(Mathai, Germain et al. 2002).
Finally, as shown by light microscopy, cells transfected with Ad-GFP gradually lost
viability on estrogen starvation treatment, and by 72 h, most cells exhibited rounded
morphology, whereas ~50% the GRP78 overexpressing cells were still viable (Fig.
3.3C). Collectively, these results provide several lines of evidence that GRP78 protects
human breast cancer against estrogen starvation–induced apoptosis.

38

Fig. 3.3. Overexpression of GRP78 rescues MCF-7/BUS cells from estrogen- starvation induced
apoptosis. A, mitochondrial membrane potential staining of MCF-7/BUS cells culture either in regular
medium or in estrogen-free medium after infection of adenovirus empty vector (Ad-Vector) or
Ad-GRP78. Red fluorescence indicates normal mitochondrial membrane potential, and green
fluorescence indicates collapsed mitochondrial membrane potential and early apoptosis. B, cell lysates
from MCF-7/BUS cells infected with adenoviral vector expressing GFP (Ad-GFP) or GRP78
(Ad-GRP78) cultured either in regular medium or in estrogen-free medium for 48 hours were subjected to
SDS-PAGE and Western blots. The levels of GRP78, BIK, β-actin, the cleaved form of PARP (a signature
of apoptosis) and the uncleaved form are indicated. C, general morphology under light microscope of
MCF-7/BUS cells at 0, 48 and 72 hours after estrogen starvation.
3. 3. 4 Knockdown of GRP78 by siRNA sensitizes human breast cancer cells to
estrogen-starvation induced apoptosis
Overexpression method has limitations in probing the physiological function of a
protein. Knockdown method is a better way to understand the function of a protein. To

39
test directly whether the down-regulation of endogenous GRP78 protein level will
sensitize human breast cancer to estrogen starvation–induced apoptosis, we used siRNA
to knockdown GRP78 in MCF-7/BUS cells. As shown in Fig. 3.4A, transient
transfection of a Grp78-suppressing siRNA substantially reduced the level of GRP78 as
compared with control siRNA. The siRNA against Grp78 is specific because it has no
effect on the expression of another major ER chaperone protein, GRP94, or on the
expression of β-actin. In cells growing in normal culture medium, siRNA against Grp78
and control siRNAs had little effect on the mitochondrial membrane potential (Fig.
3.4B). In contrast, in cells undergoing estrogen starvation for 24 h, there was a marked
increase in apoptosis in cells transfected with the siRNA against GRP78 as compared
with cells transfected with the control siRNA (Fig. 3.4B). Thus, GRP78 protects human
breast cancer cells against estrogen starvation–induced apoptosis.

40

Fig. 3.4. Knockdown of GRP78 by siRNA sensitizes human breast cancer cells to
estrogen-starvation induced apoptosis. A, cell lysates from MCF-7/BUS cells transfected with siGrp78
oligomers or control siRNA (siCtrl) for 24 hours and subsequently cultured in regular or estrogen-free
medium (ES) for 24 hours were subjected to SDS-PAGE and Western blotting to probe for levels of
GRP78, GRP94 and β-actin. B, MCF-7/BUS cells were cultured either in regular or in estrogen-free (ES)
medium for 24 hours after transfection of siGrp78 or siCtrl as indicated. The percent of apoptotic cells
was assessed by mitochondrial membrane potential staining. Columns are mean values from two
experiments; bars represent standard errors; * denotes a P value < 0.05, ** denotes a P value < 0.01.

3. 3. 5 Knockdown of BIK decreases the enhancement of estrogen-starvation
induced apoptosis resulted from GRP78 knockdown
Although GRP78 overexpression can inhibit estrogen-starvation-induced apoptosis, it
cannot be excluded that GRP78 acts through a pathway other than BIK. To test further
whether this protective effect acts through BIK directly, we used siRNA to knock down
GRP78 and BIK, either alone or in combination, in MCF-7/BUS cells subjected to

41
estrogen starvation. To complement the measurement of apoptotic cells, the amount of
apoptosis induced by estrogen starvation was determined by quantitation of PARP
cleavage. As shown in Fig. 3.2C, the expression of GRP78 and BIK protein was
substantially reduced by their specific siRNA as compared with control siRNA.
Knockdown of BIK by siRNA decreased PARP cleavage as compared with cells
transfected with control siRNA whereas knockdown of GRP78 increased PARP
cleavage (Fig. 4D). Further, knockdown of BIK substantially reduced the enhanced
PARP cleavage mediated by knockdown of GRP78 (Fig. 4D). The level of PARP in the
double knockdown cells was more than that of BIK knockdown alone, and that means
there is overlap between the anti-apoptosis effect of GRP78 and the BIK pathway.
These results confirmed that BIK mediates estrogen starvation–induced apoptosis in
MCF-7/BUS cells and further showed that GRP78 inhibits apoptosis in
estrogen-starved breast cancer cells, in part, through suppression of BIK.

42

Fig. 3.5. Knockdown of BIK decreases the enhancement of estrogen-starvation induced apoptosis
resulting from GRP78 knockdown. A, MCF-7/BUS cells were transfected with control siRNA,
siGrp78, or siBik, alone or in combination as indicated for 24 hours and then cultured in ES medium for
24 hours. The total amount of siRNA in each condition was adjusted to be the same by addition of siCtrl.
Cell lysates were collected and subjected to SDS-PAGE and probed for levels of GRP78, BIK and β-actin
by Western blotting. B, cell lysates from A were subjected to SDS-PAGE and Western blotted with
anti-PARP antibody. The Western signal of full-length PARP and apoptosis-signature fragment were
quantitated by Fluor-STM MultiImager. The relative PARP cleavages are shown with the PARP cleavage
in cells transfected with control siRNA set as 1. Columns are mean values from two experiments; bars
represent standard errors; * denotes a P value < 0.05.

3 . 4 Discussion
Many breast cancers require estrogen for their continued growth. Blocking the action of
estrogen is an established method for treating breast cancer. Antiestrogen therapy, alone
or in combination with other drugs, has been proven effective for
estrogen-receptor-positive patients. For instance, tamoxifen can reduce the odds of

43
recurrence and death by 47% and 26% respectively(Early Breast Cancer Trialists'
Collaborative Group 1998). However, many initially responsive breast cancers acquire
resistance. It is believed that not a single mechanism or single gene confers antiestrogen
resistance. Several mechanisms have been proposed, but none is fully understood. In
this study, we explored the relationship between BIK, a proapoptotic BH3-only protein
recently discovered to be a critical contributor to estrogen-starvation and
antiestrogen-induced apoptosis (Hur, Chesnes et al. 2004), and GRP78, a major ER
chaperone with antiapoptotic properties naturally induced in the tumor
microenvironment. Our results support a new role for GRP78 as an inhibitor of
BIK-mediated apoptosis via physical and functional interactions, and that GRP78
confers resistance to estrogen-starvation induced apoptosis in human breast cancer cells.
Since both BIK and GRP78 are localized to the ER, this study also provides direct
evidence that the ER is a novel regulatory site for estrogen-starvation induced apoptosis
as well as resistance, and establishes GRP78 as an upstream regulator of the apoptosis
signaling cascade through targeting BIK.
BIK was first discovered by DNA microarray analysis as the only BH3-only protein,
among the thirteen other protein members being evaluated, that is strongly induced by
the presence or absence of estrogens or antiestrogens in human breast cancer cells (Hur,
Chesnes et al. 2004). BIK is also unique in that unlike the other BH3-only proteins, it is
primarily localized to the ER (Germain, Mathai et al. 2002; Mathai, Germain et al.
2005). Importantly, BIK targeted to the ER is capable of activating BAX indirectly and
provokes cytochrome c release from the mitochondria (Germain, Mathai et al. 2002).
While the mitochondria have been well established as a major player in apoptosis, the

44
ER has emerged as another key site for the regulation of apoptosis and initiates parallel
apoptotic pathways in response to a variety of stress conditions (Breckenridge 2003;
Scorrano, Oakes et al. 2003; Zong, Li et al. 2003). Further, there is crosstalk between the
mitochondria and the ER, and BIK represents an exciting new link whereby a protein
localized in the ER can initiate cytochrome c release from the mitochondria (Germain,
Mathai et al. 2002; Germain, Mathai et al. 2005). There are reports that the BIK gene
contains missense mutations and alterations within the intronic regions in human
peripheral B-cell lymphomas (Arena, Martini et al. 2003). This could potentially give
rise to isoforms with altered structure and/or function. However, in MCF-7/BUS cell
and other breast cancer cell lines either sensitive or insensitive to estrogen-starvation
induced apoptosis, the nucleotide sequence of the Bik cDNA did not show any changes
in the open reading frame or around the translation initiation site (Hur, Chesnes et al.
2004). Thus, it is unlikely that the fraction of BIK binding to GRP78 is structurally or
functionally different from wild-type BIK in the MCF-7/BUS cells.
It is generally believed that the mechanism of resistance to antiestrogen therapy is
multifactorial. Many specific mechanisms, for instance, immunity, host endocrinology,
drug pharmacokinetics, changes in estrogen receptor, and altered gene network
signaling may contribute to the resistance. However, the cytoprotective property of
GRP78 is a general mechanism that can protect all types of solid tumors. Hypoxic,
acidic, and low glucose tumor environments increase the expression of GRP78. The
level of GRP78 is highly elevated in many cancer cell lines, solid tumors and human
cancer biopsies, and that is correlated with malignancy and metastasis (Mintz, Kim et al.
2003; Reddy, Mao et al. 2003; Arap 2004). Breast cancer is also a solid tumor.

45
Therefore, it is reasonable that GRP78 also mediates the resistance to antiestrogen
treatment. We propose that resistance to antiestrogen therapy is partly due to GRP78
overexpression in human breast cancer cells.
Therefore, while the development of resistance against estrogen starvation will
probably be complex and multifactorial, our results establish the ER as a key cellular
organelle for apoptosis mediated by estrogen starvation. The two major factors are BIK,
a prodeath BH3-only protein, and GRP78, a prosurvival protein. Since about two thirds
of breast cancer patients showed elevated level of GRP78 (Fernandez, Tabbara et al.
2000; Lee, Nichols et al. 2006), our results predict that in this subset of human breast
cancer, high elevation of GRP78 will block the ability of BIK to cause cell death
resulting from estrogen starvation. If our hypothesis is correct, our findings may lead to
the development of two novel clinical applications of GRP78. First, for patients with
hormone receptor positive breast cancer, GRP78 overexpression may be a prognostic
marker for resistance to hormonal therapy based on estrogen starvation. Breast cancer
treatment can be individualized and refined based on the expression pattern of particular
breast cancers. Second, GRP78 might be used as a novel therapeutic target to overcome
resistance of hormone receptor positive breast cancers to hormonal therapy based on
estrogen starvation. Concerning the method to downregulate GRP78, drugs targeting
GRP78 have been developed and show promise in preclinical studies where they have
been shown to prevent tumor progression and sensitize tumors to chemotherapy
treatment (Zhou and Lee 1998; Arap 2004; Park, Tomida et al. 2004; Davidson, Haskell
et al. 2005; Ermakova, Kang et al. 2006).

46
Chapter 4. GRP78 protects cells from apoptosis induced by
HDAC inhibitor
4 . 1 Introduction
Histone acetyl transferases (HTAs) and histone deacetylases (HDACs) are opposing
enzymes that tightly regulate gene expression through acetylation and deacetylation of
the histones in nucleosomes. HDAC inhibitors (HDACi) are novel anticancer drugs that
selectively induce cancer cell death. Selective induction of apoptosis in cancer cells is
one of the major biological effects that make HDACi promising drugs for cancer
treatment. Transformed cells are at least tenfold more sensitive to HDACi compared to
normal cells. It is hypothesized that HDACi induces global changes in gene expression
that alter the balance between pro- and anti-apoptotic genes toward apoptosis.
However, preliminary studies of our lab indicate that GRP78, an anti-apoptotic
protein, is induced by HDAC inhibitors Trichostatin A (TSA) and Suberic
bishydroxamic acid (SAHA). In vitro treatment of a variety of cells, K12, NIH3T3, and
293, with TSA elevates the basal and ER stress level of GRP78 mRNA and protein. TSA
also induces GRP78 in human breast cancer xenografts. These results are consistent
with the clinical investigation of breast cancer patients subjected to SAHA treatment.
Breast cancer patients were subjected to SAHA treatment regimen at 200 mg orally
twice a day for 14 days. Compared with the tumor biopsy before treatment, the
immunostaining on the sections of tumor biopsy after treatment showed much stronger
staining of GRP78. Luciferase assay on successive deletions of Grp78 promoter
indicated a sequence inducible by TSA in the first ER stress response element (ERSE1)
of Grp78 promoter.

47
Since GRP78 is induced by HDACi, GRP78 might mediate the resistance to
HDACi treatment, because GRP78 is a powerful anti-apoptotic protein. Whether
GRP78 affects the HDACi-induced apoptosis is the key. In this study, we investigated
the effect of GRP78 overexpression on the apoptosis of 293T cells induced by TSA
treatment. We found that GRP78 overexpression conferred 293T cells resistance against
TSA-induced apoptosis in a dosage-dependent manner.
Although it is widely accepted that HDACi can kill cancer cells, the specific
molecular mechanism that is involved in HDACi mediated cell death largely remains
unknown.
4 . 2 Materials and methods
4. 2. 1 Cell culture and reagents
The human embryonic kidney 293T cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. For TSA
treatment, the cells were incubated with 0.5 µM TSA for 48 h.
4. 2. 2 Expression vectors and transient transfections
The construction of pcDNA3-His-GRP78 has been described in chapter 2. Empty
vector pcDNA3 or increasing amounts of the plasmid expressing His-GRP78 (0.25, 0.5
and 1.0 µg) as indicated were transfected to 293T cells. Empty vector was added to
adjust the total amount of plasmids to be the same. Twenty-four hours after transient
transfection, the cells were treated with 0.5 µM TSA for 48 hr and then subjected to
western blot and mitochondrial membrane potential staining.

48
4. 2. 3 Western blot
Cell lysates prepared from 293T cells transfected with either 1.0 µg of a plasmid
expressing His-tagged GRP78 or same amount of empty vector pcDNA3 were applied
to SDS-PAGE and Western blotted with anti-KDEL, anti-His, and anti- β-actin
antibodies. The anti-KDEL antibody recognized the C-terminal KDEL motif of GRP78,
GRP94 and protein disulphide isomeras (PDI). Cell lysates prepared from the 293T
cells transfected with plasmids and treated with 0.5 µM TSA for 48 hr were applied to
SDS-PAGE and Western blotted with anti-KDEL antibody.
4. 2. 4 Cell death and apoptotic assays
For mitochondrial membrane potential staining, the Mitochondrial Permeability
Transition Detection Kit (Immunochemistry, Bloomington, MN) was used fas described
in chapter 3. The cell cultures were then washed with PBS and examined under a
fluorescence microscope. The percent of apoptotic cells under each condition in was
quantitated and plotted against the transfected amount of GRP78. Each assay was done
in triplicate.
4 . 3 Results
4. 3. 1 GRP78 overexpression protects 293T cells from TSA-induced apoptosis
First, we examined the protein level of GRP78 and other major ER chaperons, GRP94
and protein disulfide isomerase (PDI) 24 hours after transfection. As shown Fig. 4.1A,
GRP78 protein level increased significantly compared to the vector control; the level of

49
GRP94 and PDL did not show any significant change (Fig. 4.1A). The result showed
that overexpression of GRP78 did not affect the expression of other ER chaperons.
To test the protective effect of GRP78 on the TSA-induced apoptosis, the same
293T cells transfected with His-Grp78 plasmid or vector control were subjected to
mitochondrial permeability transition assay. In normal cells, MitoPT agent aggregates
in mitochondria and fluoresces red. In early apoptotic cells, the mitochondrial potential
collapses and MitoPT diffuses to the whole cell and emits green fluorescence. As shown
in Fig. 4.1B, in 293T cells overexpressing GRP78, the mitochondrial potential change
after 48-hour 0.5 µM TSA treatment was reduced substantially compared to the cells
transfected with vector control. Moreover, increasing amount of the plasmids
expressing His-GRP78 (0.25, 0.5, and 1.0 µg) resulted in decreasing amount of cells
that showed positive green fluorescence (Fig. 4.1B). As quantitated in Fig. 4.1C,
positive green staining decreased from 95% in cells transfected with vector control, to
85% in cells transfected with 0.25 µg His-Grp78 plasmid, to 28% and 11% in cells
transfected with 0.5 and 1.0 µg His-Grp78 plasmid respectively. The protein levels of
GRP78 in these transfected cells were confirmed by western blot, as shown in the insert
figure in Fig. 4.1C. Collectively, these results provide evidence that overexpression of
GRP78 confers resistance to 293T cells against TSA-induced apoptosis in a dose
dependent manner.

50

Fig. 4.1. Overexpression of GRP78 protects 293T cells from TSA induced apoptosis. A, Cell lysates
prepared from 293T cells transfected with either 1.0 µg plasmid expressing His-GRP78 or same amount
of empty vector pcDNA3 were applied to SDS-PAGE and Western blotted with anti-KDEL, anti-His, and
anti- β-actin antibodies. B, Empty vector pcDNA3 or various amount of plasmid expressing His-GRP78
as indicated were transfected to 293T cells. Empty vector was added to adjust the total amount of
plasmids to be the same. Twenty four hours after transient transfection, cells were treated with 0.5 µM
TSA for 48 hours and then subject to mitochondrial membrane potential staining, which detects cells at
early stage of apoptosis. C, The quantitative result of C and the corresponding GRP78 levels detected by
Western blot.

51
4 . 4 Discussion
HDACi represents a novel promising class of anticancer agents. However, most cancers
become resistant to specific agents during treatment via various mechanisms. HDACi
are not the exception. To date, besides the general multiple drug resistance mediated by
P-glycoprotein(Yamada, Arakawa et al. 2006), several specific mechanisms have been
found responsible for the resistance to HDACi.
First, HDAC enzymes that are insensitive to HDACi may explain the resistance to
HDACi. For example, S. pombe phd1
+
, a gene highly homologous to human HDAC
genes, encodes an active but redundant HDAC enzyme. Although null mutation results
in a decrease in the total HDAC activity, the phd1
+
disruptant is viable but shows
supersensitivity to TSA treatment, as compared to the wildtype(Kim, Honda et al. 1998).
Selective pressure due to the treatment with HDACi may select the mutations that
produce HDACi insensitive HDAC. It has been reported that TSA treatment of FM3A
cell, a mouse mammary gland tumor cell line, generates a mutant cell line that is
resistant to TSA; this mutant cell line was found to possess a TSA-resistant
HDAC(Yoshida, Kijima et al. 1990). Second, the survival pathways are enhanced in
response to HDACi treatment. It has been reported that the autophagy pathway is
augmented when chronic myelogenous leukemia (CML) cell lines and primary cells.
Drugs that disrupt the autophagy pathway dramatically increase the antineoplastic
effects of SAHA in CML cell lines and primary cells(Carew, Nawrocki et al. 2007).
This study suggests a novel mechanism that the upregulation of GRP78 in response
to HDACi treatment contributes to the resistance of cancers to HDACi because GRP78
upregulation can confer cells resistance to HDACi treatment. GRP78 provides a novel

52
therapeutic target to overcome the resistance of cancers to HDACi. Further, GRP78
could be used as a prognostic marker to predict the effectiveness of HDACi treatment to
specific cancer patients.

53
Chapter 5. The role of GRP78 in the postnatal growth of
prostate and progression of prostate cancer
5 . 1 Introduction
As demonstrated in previous chapters, GRP78 is important for tumor growth.
Anti-sense downregulation of GRP78 in fibrosarcoma cells inhibits xenograft
progression when injected into syngenic mice(Jamora, Dennert et al. 1996). GRP78
may promote tumor growth through three ways. Cancer cells exhibit elevated energy
metabolism, which includes enhanced glycolytic activity and oxygen consumption.
This unique property of cancer cells, coupled with the poor vascularization of solid
tumor, leads to glucose starvation, low pH, and severe hypoxia in solid tumor. All these
conditions induce ER stress. Therefore, in solid tumors, GRP78 are upregulated to
alleviate ER stress and promote survival of tumor cells(DONG, DUBEAU et al. 2004;
Dong, Ko et al. 2005). Second, GRP78 may inhibit the apoptosis of cancer cells through
inhibiting the activity of BIK, as demonstrated in above chapters. Third, surface GRP78
can inhibit apoptosis and promote proliferation through activating Akt(Misra,
Deedwania et al. 2006). However, above cell culture experiments and xenograft studies
are insufficient to establish the role of GRP78 in tumor formation and progression. An
animal model study is required to understand the effect of GRP78 on the cancer
formation and progression in in situ cancer development. In the present study, a mouse
model of prostate cancer is utilized to investigate the effect of GRP78 on cancer
formation and progression.
Prostate cancer is the most common malignant noncutaneous neoplasm of men.
The major precancerous lesion of human prostate cancer is the prostatic intraepithelial

54
neoplasia (PIN). PIN is divided as low grade or high grade according to its
histopathological manifestation. High grade PIN is considered as a precursor of
prostatic adenocarcinoma(McNeal, Villers et al. 1991; Haggman, Macoska et al. 1997;
Roy-Burman, Wu et al. 2004). The molecular pathways that contribute to the genesis of
human prostate cancer remain largely unknown. One of the most common genes
involved in prostate cancer is PTEN (phosphatase and tensin homologue deleted on
chromosome 10). It has been reported that 30% of primary prostate cancers(Dahia 2000;
Sellers and Sawyers 2002) and 63% of metastatic prostate cancers(Suzuki, Freije et al.
1998) have PTEN deletions and/or mutations.
PTEN is a phosphatase that facilitates the removal of phosphate groups from a wide
range of macromolecules. Its major targets are highly specialized plasma membrane
lipids. PTEN dephosphorylates phosphatidylinositol-3, 4, 5-triphosphate (PIP3) to
produce phosphatidylinositol-3, 4-bisphosphate (PIP2), while phosphoinotitide
3-kinase (PI3K), a lipid kinase, phosphorylates PIP2 to generate PIP3. Therefore, PTEN
is an off switch of PIP3, which in turn activates Akt kinase. The substrates of Akt play
key roles in regulating cell cycle, apoptosis, metabolism, and translation. Loss of PTEN
function in human cancer cell lines and mouse knockout models results in constitutive
activation of Akt/PI3K pathway. As a result, phosphorylation of downstream signaling
molecules, such as glycogen synthase kinase-3 (GSK3), Caspase 9, MDM2 and I κB,
leads to enhanced cell growth and survival(Sellers 2004). PTEN homozygous deletion
in mice causes early embryonic death. Pten
+/-
mice exhibit hyperplastic-dysplastic
changes in the prostate, skin, and colon and spontaneously develop germ cell,
gonadostromal, thyroid, and colon tumors(Cristofano, Pesce et al. 1998). Pten
+/-
male

55
mice develop PIN with near 100% penetrance, but the latency is as long as 10 months.
Moreover, these PINs never progress to adenocarcinoma. Conditional homozygous
deletion of Pten in mouse prostate not only significantly shortens the latency of PINs
but also promotes the progression of PINs to metastatic cancer. Characterization of the
Pten model has indicated that the Pten model is similar to human prostate cancer in gene
expression profile(Wang, Gao et al. 2003).
In the present study, PTEN and GRP78 genes are specifically knocked out in mouse
prostate epithelium. Such conditional knockout is achieved through the Cre-loxP
site-specific recombination system. Both alleles of PTEN and GRP78 are floxed. The
expression of Cre recombinase is driven by PB-Cre4 promoter construct(Wu, Wu et al.
2001), which is an engineered derivative of rat probasin promoter. PB-Cre4 is a
post-natal, androgen regulated, and prostate epithelium-specific high efficient promoter.
In this chapter, we demonstrate that specific knockout of GRP78 in prostate epithelium
significantly delays the formation and progression of prostate cancer initiated by Pten
homozygous deletion. The potential underlying mechanism is discussed.
5 . 2 Materials and Methods
5. 2. 1 Generation of prostate-specific Pten and Grp78 homozygous deletion mice
To generate mice with biallelic deletion of Pten and Grp78, male Pten
F/F
; PB-Cre4
mice(Wang, Gao et al. 2003; Zhong, Saribekyan et al. 2006) (Pradip Roy-Burman,
University of Southern California Keck School of Medicine) on C57BL/6xDBA2
background were crossed with female Grp78
F/F
mice on C57BL6 background (Fig.
5.4A)(Luo, Mao et al. 2006). The Pten
F/F
; Grp78
F/F
; PB-Cre4, Pten
F/F
; Grp78
F/+
;

56
PB-Cre4, and Pten
F/F
Grp78
+/+
PB-Cre4 male mice were generated according to the
breeding scheme showed in Fig. 5.2. To generate Pten
F/F
; Grp78
F/-
; PB-Cre4 male mice,
male Pten
F/F
; PB-Cre4 mice were crossed with female Grp78
F/-
mice on C57BL6
background according to the breeding scheme (Fig. 5.3). The Pten
F/+
Grp78
F/+
PB-Cre4
male offspring were then crossed with the Pten
F/+
Grp78
+/-
female offspring to generate
the Pten
F/F
; Grp78
F/-
; PB-Cre4 male mice. In order to examine the effect of Grp78
homozygous deletion on prostate development, male Grp78
F/F
; PB-Cre4 mice were
generated according to the breeding scheme in Fig. 5.1.
5. 2. 2 Genotyping of mice
Mouse tail biopsies of no longer than one centimeter were lysed in 0.5 ml lysis buffer
(100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 μg Proteinase
K/ml). The lyses of tail biopsies were then spun and the supernatants were mixed with
the same volume of isopropanol until the precipitation of DNA is complete. The
aggregated precipitates of DNA were lifted with a disposable tip and dissolved in
double distilled water(Laird, Zijderveld et al. 1991). The DNA extracts were then
subjected to PCR analysis.
The PCR primers used for genotyping Pten wildtype and floxed alleles were P1
(5’-AAGCAAGCACTCTGCGAAACTGA-3’) and P2 (5’-GATTGTCATCT
TCACTTAGCCATTGGT-3’); the PCR conditions were 42 cycles (30 s at 94 , ℃ 60 s at
60℃, and 70 s at 72 ℃). The primers for PB-Cre4 were C1 (5’-GATCCTGG
CAATTTCGGCTAT-3’) and C2 (5’-TTGCCTGCATTACCGGTCGAT-3’); the PCR
conditions were 36 cycles (30 s at 94 , ℃ 40 s at 58 ℃, and 40 s at 72 ℃). The primers for

57
Grp78 wildtype and floxed alleles were PF3 (5’-GATTTGAACTCA
GGACCTTCGGAAGAGCAG-3’) and PR3 (5’-GCAATAGCAGCTGCT
GTACTGTGAGGA TGA-3’); the primers for Grp78 knockout allele were PF3 and PTR
(5’-TTGTTAGGGGTCGTTCACCTAGA-3’) (Fig. 5.4A); the PCR conditions were 40
cycles (30 s at 95 , 30 ℃ s at 60 ℃, and 30 s at 72 ℃)(Luo, Mao et al. 2006).
5. 2. 3 Autopsy and histopathology assessments
All mice were autopsied and all lobes of prostate were examined. All animal protocols
were conducted with the approval of the University of Southern California Animal Care
and Use Committee. Prostate samples were fixed in 10% buffered formalin and
embedded in paraffin. The paraffin-embedded tissues were sectioned (4-5 μm) and
stained with haematoxylin and eosin (H&E).
5. 2. 4 Immunohistochemistry analysis
The paraffin sections were first deparaffinized and rehydrated, and antigens were
retrieved via incubation of slides in Retrievagen A Solution (BD Pharmingen) at
95-100 ℃ for 30 minutes. Slides were then allowed to cool at room temperature for 1
hour in Retrievagen A Solution. After washing with double distilled water and PBS, the
slides were washed twice in 3% H
2
O
2
in PBS for 5 minutes to eliminate endogenous
peroxidase activity. The slides were subsequently blocked: for monoclonal primary
antibodies, sections were blocked with the horse serum (ABC Elite kit, Vector
Laboratories); for polyclonal primary antibodies, sections were blocked with the goat
serum of ABC Elite kit. After blocking, sections were incubated overnight with

58
polyclonal rabbit anti-GRP78 (H-129, Santa Cruz Biotechnology) diluted at 1:200 in
blocking solution (ABC Elite Kit, Vector Laboratories) at 4 ℃. After three 5-minute
washings in PBS, sections were then incubated with biotinylated secondary antibody
(ABC Elite Kit, Vector Laboratories) for 30 minutes at room temperature. Following
PBS washings, sections were developed with the ABC Elite kit according to
manufacturer’s protocol. Slides were then counterstained with haematoxylin,
dehydrated, and coverslipped. Negative control slides were processed without primary
antibody.
5. 2. 5 Immunofluorescence analysis
The paraffin sections were first deparaffinized and rehydrated, and antigens were
retrieved via incubation of slides in Retrievagen A Solution (BD Pharmingen) at
95-100 ℃ for 30 minutes. Slides were then allowed to cool at room temperature for 1
hour in Retrievagen A Solution. After washing in double distilled water and PBS, the
slides were blocked with 1% BSA in PBS. After removing excessive blocking solution,
sections were incubated with primary antibodies at 4 ℃ overnight. After three 5-minute
washings in PBS, sections were then incubated with FITC-conjugated anti-mouse or
rhodamine-conjugated anti-rabbit antibody for 60 minutes at room temperature. Slides
were then dehydrated and coverslipped. Negative control slides were processed without
primary antibody. Primary antibodies were polyclonal rabbit anti-PTEN (26H9, #9556,
Cell Signaling Technology), monoclonal mouse anti-P-AKT (Ser473, #9271S, Cell
Signaling Technology), monoclonal mouse anti-GRP78 (#610978, BD Pharmingen).
All primary antibodies were diluted at 1:200 in 1% BSA in PBS.

59
5 . 3 Results
5. 3. 1 Postnatal homozygous deletion of Grp78 does not affect the development of
prostate
Before investigating the role of GRP78 in prostate cancer formation and development,
we need to know whether GRP78 nullification affects prostate development and growth.
GRP78 plays an important role in development. GRP78 is expressed as early as at the
two-cell stage of embryonic development, and is essential for both proliferation and
survival of the embryonic cell mass. Homozygous knockout of Grp78 causes early
embryonic death. Considering that homozygous deletion of GRP78 may affect the
development of prostate, we first examined the morphology of prostate of Grp78
F/F
;
PB-Cre4 mice. The mice were generated according to the scheme in Fig. 5.1 and were
genotyped at 20 weeks and 30 weeks through PCR and agarose gel electrophoresis(Fig.
5.4A&B). At 20 weeks, mice were sacrificed and dissected; the gross anatomy of
Grp78
F/F
; PB-Cre4 mouse prostate was basically the same as that of wildtype and
Grp78
F/F
mouse prostate. The size and morphology of prostates, the number of ductal
tips, and the diameter of ducts did not show any significant difference between Grp78
F/F
;
PB-Cre4 and wildtype mice (Fig. 5.4C). At 30 weeks, the mice were genotyped, and the
immunohistochemistry analysis confirmed that the GRP78 protein level of Grp78
F/F
;
PB-Cre4 was significantly lower than that of wildtype. There was still no significant
difference in either gross anatomy or microscopic histology: the acini were variably
sized with various amount of epithelial infolding; all lobes had simple columnar
epithelium with basophilic granular cytoplasm and centrally placed nuclei; the stroma is

60
thin and there was no inflammatory cell infiltration(Fig. 5.4D). Further, Grp78
F/F
;
PB-Cre4 mice were as fertile as wildtype and Grp78
F/F
mice. All these observations
indicate that postnatal homozygous deletion of Grp78 driven by PB-Cre4 does not
affect the development and growth of prostate, or the fertility of mouse.

Fig. 5.1. The breeding scheme of Grp78
F/F
; PB-Cre4 mouse. Cre represents PB-Cre4.

61

Fig. 5.2. The breeding scheme of Pten
F/F
; Grp78
F/F
; PB-Cre4 mouse. Cre represents PB-Cre4.

62

Fig. 5.3. The breeding scheme of Pten
F/F
; Grp78
F/-
; PB-Cre4 mouse. Cre represents PB-Cre4.

63

Fig. 5.4. Postnatal homozygous deletion of Grp78 does not affect the development of prostate. A,
Schematic drawings for the Grp78 cDNA, the WT allele, the floxed allele, and the conventional knockout
allele. The exons encoding the ATPase domain and peptide-binding domain of GRP78 and the loxP sites
(arrow head) are indicated. The location of the primers used in the PCR genotyping are also indicated
(arrow). B, The DNA extracts of Grp78
F/F
; PB-Cre4 mouse, Grp78
F/F
mouse, and wildtype mouse were
subjected to PCR analysis. C, The gross anatomy of the prostates of Grp78
F/F
; PB-Cre4 mouse and
wildtype mouse of 20 weeks age. D. The gross anatomy of the prostates of wildtype mouse and Grp78
F/F
;
PB-Cre4 mouse of 30 weeks age. The immunohistochemistry analyses indicate the significantly lower
level of GRP78 in Grp78
F/F
; PB-Cre4 mouse than that of wildtype mouse. The H&E staining does not
show any difference in prostate histology between Grp78
F/F
; PB-Cre4 mouse and wildtype mouse of 30
weeks age.

64
5. 3. 2 The effect of Grp78 heterozygous deletion on prostate cancer
To confirm prostate-specific deletion of Pten and Grp78, the prostates, seminal vesicles,
and urinary bladder were dissected at the age of 20 and 30 weeks. The status of Pten
deletion and Grp78 deletion were examined by PCR analysis (Fig. 5.5A) and
immunofluorescence staining (Fig. 5.5B). PTEN immunofluorescence staining was
significantly reduced in the prostate of Pten
F/F
; Grp78
F/+
; PB-Cre4, Pten
F/F
; Grp78
F/F
;
PB-Cre4, and Pten
F/F
; Grp78
F/-
; PB-Cre4 mice when compared with wildtype mice. In
the prostate of Pten
F/F
; Grp78
F/+
; PB-Cre4 mice, the immunofluorescence staining of
GRP78 was moderately reduced when compared to the wildtype mice, but is still
significantly higher than that of Pten
F/F
; Grp78
F/F
; PB-Cre4, and Pten
F/F
; Grp78
F/-
;
PB-Cre4 mice, which have very low GRP78 immunofluorescence(Fig. 5.5B).
At the age of from 3 to 6 months, 21 of 21 Pten
F/F
; PB-Cre4 mice acquired prostate
cancer. The prostate lobes, including anterior prostate (AP), dorsolateral prostate (DLP),
and ventral prostate (VP), formed a solid mass and completely lost the clear fern-like
appearance of prostate (Fig. 5.6). Histological analysis indicated that 21 of 21 Pten
F/F
;
PB-Cre4 mice developed invasive adenocarcinoma in all prostate lobes. Cells exhibit
nuclear enlargement, nuclear contour irregularity, hyperchromatism, and prominent
nucleoli accompanied by the inversion of the nuclear to cytoplasmic ration; malignant
cells invaded through the basement membrane and into the adjacent stroma; this in turn
induced both an inflammatory and a desmoplastic response, which manifests as the
growth of fibrous or connective tissue around the tumor. In most of the area, the

65
invasion was so extensive that the glandular structure was completely lost and only the
trace of basement membrane was left (Fig. 5.6).
In contrast, in 3 of 3 20-week old Pten
F/F
; Grp78
F/+
; PB-Cre4 mice, only anterior
prostate developed a solid mass of tumor. Other lobes still kept the fern-like ductal
appearance, but some ducts enlarged and lost the clear and gelatinous appearance. In
agreement with this, histological analysis showed that AP had extensive invasive
adenocarcinoma and the glandular structure was lost, as in the prostate of Pten
F/F
;
PB-Cre4 mice at the same age. In DLP and VP, diffusive prostate intraepithelial
neoplasia (PIN) developed: the number of epithelial cells and the size of gland increased
significantly; the epithelial cells were cytologically atypic; the glands were
disorganized but the basement membrane was still intact (Fig. 5.6A). At the age of 30
weeks, the pathological change of was Pten
F/F
; Grp78
F/+
; PB-Cre4 mice very similar to
that of Pten
F/F
; PB-Cre4 mice at 20 weeks (Fig. 5.6B). The heterozygous deletion of
Grp78 moderately delayed the formation and progression of prostate cancer incurred by
Pten deletion.

66

Fig. 5.5. The immunofluorescent analysis of the PTEN, GRP78, and P-Akt level in mouse prostate.
A, The Pten
F/F
; Grp78
F/+
; PB-Cre4, Pten
F/F
; Grp78
F/F
; PB-Cre4, and Pten
F/F
; Grp78
F/-
; PB-Cre4 male
mice at the age of 20 weeks were genotyped through PCR analysis. B, The immunofluorescent analyses
of the prostates from the mice in A show the changes in the level of PTEN, GRP78, and P-Akt as
compared to Pten
F/F
; Grp78
F/F
mice of the same age.

5. 3. 3 The effect of Grp78 homozygous deletion on prostate cancer
The homozygous deletion of Grp78 was confirmed by PCR analysis(Fig. 5.5A) and
immunofluorescence staining (Fig. 5.5B). At 20 weeks, the prostate of Pten
F/F
; Grp78
F/F
;
PB-Cre4 (n=3 of 3) and Pten
F/F
; Grp78
F/-
; PB-Cre4 (n=3 of 3) mice developed neither

67
cancerous nor precancerous change. The fern-like prostate had clear and gelatinous
ducts. The number of ductal tips and the diameter of ducts did not show any obvious
difference compared to the Pten
F/F
; Grp78
F/F
mice. Moreover, the histology of the
prostate was normal: the number of epithelial cells, the morphology of cells, and the size
and organization of gland did not exhibit any abnormality (Fig. 5.6). The homozygous
deletion of Grp78 blocks the oncogenesis initiated by the homozygous deletion of Pten.

68

Fig. 5.6. The effect of Grp78 deletion on the prostate cancer formation and progression initiated by
homozygous Pten deletion. A, The gross anatomy of the prostate of the mice at the age of 20 weeks with
indicated genotype. All lobes of Pten
F/F
; PB-Cre4 mice formed a solid mass and completely lost the clear
fern-like appearance. In Pten
F/F
; Grp78
F/+
; PB-Cre4 mice, only AP developed a solid mass of tumor. In
mice with homozygous deletion of Grp78, prostates did not show any difference in gross anatomy when
compared with Pten
F/F
; Grp78
F/F
mice. B. A pathological comparison of the prostate from the mice in A.
Pten
F/F
; PB-Cre4 mice developed invasive adenocarcinoma in all prostate lobes. In Pten
F/F
; Grp78
F/+
;
PB-Cre4 mice, AP had extensive invasive adenocarcinoma, as in the prostate of Pten
F/F
; PB-Cre4 mice at
the same age, while DLP and VP only developed diffusive prostate intraepithelial neoplasia (PIN). In
mice with homozygous deletion of Grp78, prostates did not show any significant difference in histology
when compared with Pten
F/F
; Grp78
F/F
mice.

5. 3. 4 The AKT activity in the prostate of mouse model
Homozygous deletion of Pten results in the upregulation of AKT serine/threonine
kinase(Wang, Gao et al. 2003), the primary target of the PTEN signaling pathway.

69
Recently, it has been reported that surface GRP78 can activate AKT pathway(Misra,
Deedwania et al. 2006). To investigate the influence of Grp78 deletion on AKT
activation, we examined the phosphorylation of AKT through the immunofluorescence
staining using anti-phosphorylated-AKT (p-AKT) antibody. Consistent with the
previous study(Wang, Gao et al. 2003), p-AKT was detected in the prostate of one
20-week Pten
F/F
; Grp78
F/+
;PB-Cre4 mouse; the positive staining was mainly in the
epithelium rather than the stroma. In the prostate of Pten
F/F
; Grp78
F/F
; PB-Cre4 and
Pten
F/F
; Grp78
F/-
; PB-Cre4 mice, the p-AKT was not detected, as in the prostate of
wildtype mice. AKT phosphorylation was significantly decreased in Pten
F/F
; Grp78
F/F
;
PB-Cre4 and Pten
F/F
; Grp78
F/-
; PB-Cre4 mice, as compared to Pten
F/F
; Grp78
F/F
;
PB-Cre4 mouse (Fig. 5.5). Although the number of mouse sample stained at this time is
insufficient to draw a conclusion, it suggests that Grp78 homozygous deletion resulted
in the inhibition of AKT pathway.
5 . 4 Discussion
The animal model of cancer is critical for the in vivo investigation of cancer. A good
animal model has to meet two requirements: first, the formation and progression of
cancer in animal model should mimic the pathological and clinical course of
corresponding human cancer; second, the genetic changes underlying the model should
be similar to the genetic changes in corresponding human cancer. Mouse models of
cancer, generated through transgenic or knockout technique, are preferred over canine
or rat models because of their clearly defined genetics and molecular pathways.

70
Concerning prostate cancer, most currently available mouse models have only
hyperplasia-dysplasia phenotype in aged mouse, for example the constitutively
activated AKT model(Majumder, Yeh et al. 2003) and the conditional NKX3.1knockout
model(Abdulkadir, Magee et al. 2002; Bethel, Faith et al. 2006). To date, the transgenic
adenocarcinoma mouse prostate (TRAMP) is the only mouse model that goes beyond
the PIN lesion. However, this SV40 T antigen transgenic model is characterized with its
high neuroendocrine differentiation potential, which is morphologically and
biologically different from the adenocarcinoma seen in most human prostate
cancers(Powell, Cardiff et al. 2003).
The mouse model of prostate cancer through tissue-specific Pten deletion
resembles the course of formation and progression of human prostate cancer: from
hyperplasia to PIN, invasive adenocarcinoma, and finally metastasis. Moreover, the
Pten null cancer originates in secretory epithelial cells, same as the origin of most
human prostate cancers (Wang, Gao et al. 2003; Roy-Burman, Wu et al. 2004). Biallelic
deletion of Pten leads to the gene expression changes that are common in human
prostate cancer and other human cancer with metastatic potentials(Wang, Gao et al.
2003; Roy-Burman, Wu et al. 2004). Therefore, the Pten prostate-specific knockout
model is a very good model for studying the molecular mechanism underlying the
formation and progression of prostate cancer as well as other cancers.
In this study, we found that prostate-specific knockout of Grp78 can prevent the
formation of prostate cancer initiated by the Pten deletion in prostate secretory epithelial
cells. GRP78 can protect cells against a wide variety of stress and promote the survival

71
of cells. Several mechanisms may be responsible for the inhibitory effect of GRP78 on
cancer formation and progression.
First, as shown in the previous chapters, GRP78 can associate with BIK and inhibit
its apoptotic activity. Knockdown of GRP78 sensitizes cells to the apoptosis induced by
various stresses, such as anticancer chemotherapy and estrogen starvation. It has been
reported that BIK is lost in human renal cell carcinoma(Sturm, Stephan et al. 2006) and
mutated in human peripheral B-cell lymphoma(Arena, Martini et al. 2003). Expression
of BIK suppresses tumor formation and restores sensitivity to anti-cancer drugs in
resistant tumor cells(Tong, Yang et al. 2001; Radetzki, Kohne et al. 2002; Zou, Peng et
al. 2002). In oncogenesis and various stresses, the cell’s decision to die or survive is
mainly determined by the balance between pro-apoptotic BH3-only proteins and
pro-survival Bcl-2 proteins. Such balance plays an important role in cancer
development: apoptosis can eliminate cells whose normal physiology, such as cell cycle
control, is disturbed by oncogenic mutations. Impaired regulation of apoptosis would
preserve these precancerous cells and allow them to develop to cancer(Thomenius and
Distelhorst 2003). While overexpression of pro-survival Bcl-2 proteins can promote
oncogenesis, inhibition of pro-apoptotic BIK by GRP78 may have the same promoting
effect on cancer formation. Knockout of Grp78 enhances apoptosis and may block
oncogenesis by eliminating precancerous cells. Although Pten deletion provides an
initiating force, the enhanced apoptosis through Grp78 deletion inhibits the steps
following initiation. This mechanism can explain why and how Grp78 deletion blocks
the cancer formation incurred by Pten deletion.

72
Second, in addition to its protection against cell death, GRP78 can directly promote
cancer cell proliferation. GRP78 is ER-resident protein. A subpopulation of GRP78 can
exist as transmembrane protein(Reddy 2003). These transmembrane GRP78 proteins
serve as functional chaperones on tumor cell surface. Recent evidence suggests that in
some cancer cell types, GRP78 can exist as a cell surface protein and transmit
extracellular stimuli to intracellular signaling pathways to promote cancer development.
For example, in 1-LN prostate cancer cells, cell surface GRP78 acts as a receptor for
ligand-forms of α2-macroglobulin. Binding of α2-macroglobulin ligand to cell surface
GRP78 activates AKT to promote cellular proliferation(Misra, Deedwania et al. 2006).
There is a positive correlation between the amount of cell surface GRP78 and the
aggressive behavior of prostate cancer(Mintz, Kim et al. 2003; Arap, Lahdenranta et al.
2004). Because the major function of PTEN relies on the downstream AKT pathway, it
is reasonable to hypothesize that homozygous deletion of Grp78 abolishes the
oncogenesis initiated by Pten deletion partially through the AKT pathway. This is
consistent with our observation that p-AKT is downregulated in Pten
F/F
; Grp78
F/F
;
PB-Cre4 and Pten
F/F
; Grp78
F/-
; PB-Cre4 mice. However, AKT pathway alone cannot
account for the oncogenesis by Pten deletion, because transgenic mice expressing
constitutively activated form of AKT in prostate only acquire hyperplasia-dysplasia
instead of adenocarcinoma(Majumder, Yeh et al. 2003; Roy-Burman, Wu et al. 2004).
This suggests that inhibiting AKT pathway might be only one of many effects of GRP78
deletion.
As a master regulator of ER stress, GRP78 can relieve ER stress and thus promote
survival. Under normal conditions, GRP78 expression is at low basal level in adult

73
organs. However, GRP78 is strongly induced in tumors. One cause is the enhanced
glucose metabolism of cancer cells with elevated glycolytic activity. Another cause is
the abnormal microenvironment of hypovascularized solid tumor, which is
characterized by glucose deprivation, acidosis, and hypoxia(Lee 2007) (Fu and Lee
2006). In other words, cancer cells undergo chronic ER stress. As a pro-survival factor
in ER stress, GRP78 relieves ER stress of cancer cells and thus promotes the
progression of cancer. Another mechanism contributing to the effect of GRP78 on
cancer progression is its inhibition of angiogenesis in solid tumors(Dong, Ni et al. 2008).
Although inhibition of angiogenesis does not affect the initiation of oncogenesis, it can
contribute to the inhibitory effect of GRP78 on tumor growth.
Consistent with this study, a previous study from our lab showed that Grp78
heterozygous deletion in a transgenic mouse model of mammary tumor does not affect
organ development and growth, but prolongs the latency period, impedes tumor growth,
increases apoptosis, and inhibits tumor angiogenesis(Dong, Ni et al. 2008). In our study,
heterozygous deletion of Grp78 results in mild to moderate delay of cancer progression
in DLP and VP: the pathological changes of DLP and VP were mainly PIN, compared to
the extensive invasive adenocarcinoma in Pten null positive control of the same age,
while the AP did not exhibit any significant difference. The effect of Grp78
heterozygous deletion in the mouse model of mammary tumor is more pronounced than
that in the mouse model of prostate cancer. The reason could be that the heterozygous
deletion in the former is universal while it is tissue specific in the latter; Grp78
downregulation in the non-cancerous cells in the tumor microenvironment, for example

74
blood vessel, and the host immune system may inhibit the formation and progression of
cancer.

75
Chapter 6. Summary and perspective
The study presented in this paper demonstrates that GRP78, but not other ER
chaperones, forms a complex with endogenous BIK and ER-targeted BIK and blocks
their apoptotic activity. This functional interaction between GRP78 and BIK is further
confirmed in an estrogen-dependent human breast cancer cell line, MCF-7/BUS.
GRP78 overexpression inhibits estrogen-starvation induced BIK upregulation, BAX
activation and apoptosis in MCF-7/BUS cells; suppression of endogenous GRP78 by
siRNA sensitizes MCF-7/BUS cells to estrogen-starvation induced apoptosis. Finally,
the whole effect of GRP78 on cancer formation and progression, not limited in its
interaction with BIK, is examined collectively in a mouse model of prostate cancer:
specific knockout of GRP78 in prostate epithelium significantly delays the formation
and progression of prostate cancer initiated by Pten heterozygous deletion. It can be
concluded that GRP78 is an influential player in the genesis and development of cancer.
The pathways regulated by GRP78 are summarized in Fig. 6.1.

76

Fig. 6.1. The mechanism of the antiapoptotic activity of GRP78. Etoposide treatment can activate p53
via DNA double strand break. Activation of p53 up-regulates BAX, which inserts into the outer
mitochondrial membrane and results in mitochondrial permeability transition and cytochrome c release.
Activated p53 also induces BIK, which stimulates cell death. Estrogen starvation can induce apoptosis
with up-regulation of BIK mRNA and protein and down-regulation of GRP78 in an estrogen-dependent
breast cancer cell line. Knockdown of BIK by SiRNA can protect cells from apoptosis induced by
estrogen starvation or antiestrogen treatment. Although it is reported that surface GRP78 activates AKT
pathway, such activation in vivo is to be confirmed.

In the context of physiology of whole body, why and how can a chaperone at the ER
modulate pathways not involving protein folding? GRP78 is not the only case that a
chaperone specifically regulates a signaling pathway. There are many reports that
Hsp90 can buffer against genetic variations by interacting with the protein constituents

77
of signal transduction pathways (Rutherford/Lindquist, Nature, 1998;
Queitsch/Lindquist, Nature, 2002). Since binding to client proteins is a general feature
of all chaperones, any chaperone could potentially exhibit such buffering activity
(Sangster/Queitsch, Bioessays, 2004; Mitchell-Olds/Knight, Science, 2002). It is quite
natural if GRP78 acts as a buffering agent in apoptosis by masking the functional
domain or altering the conformation of BIK and other relevant factors. This scenario is
consistent with the mode of action that GRP78 binds to and inactivates PERK, IRE1,
and A TF6.
In view of multiple lines of evidence, it is reasonable to propose that GRP levels
may be exploited as a prognostic as well as diagnostic marker for malignancy and
chemo-responsiveness. Previous work using transgenic mouse models showed that
transgene expression driven by the Grp78 or the Grp94 promoter is highly elevated in
malignant tumors, but is relatively quiescent in major adult organs such as the heart,
lung, brain and liver(Reddy, Dubeau et al. 2002; Dong, Dubeau et al. 2004). This
observation is confirmed in human cancers and higher GRP78 level generally correlates
with higher pathological grade and aggressive phenotype in breast, liver, colon and
prostate carcinoma(Fernandez, Tabbara et al. 2000; Shuda, Kondoh et al. 2003; Arap
2004; Luk, Lam et al. 2006; Xing, Lai et al. 2006). While this implies that GRP78 might
predict poor prognosis for major solid tumors, there could be exceptions. For lung
cancer, there are conflicting reports on the correlation between GRP78 level and
pathological grade and one study suggests positive GRP78 is indicative of favorable
prognosis(Uramoto, Sugio et al. 2005; Wang, He et al. 2005). It has also been proposed
that GRP78 may promote neuroblastoma differentiation, thereby halting tumor

78
progression, leading to longer survival of patients with positive GRP78 expression(Hsu,
Hsieh et al. 2005). The essential role of GRP78 in embryonic development has recently
been established by genetic knockout(Luo, Mao et al. 2006). Future investigations are
required to define the role of GRP78 in differentiation and its prognostic value in
neuroblastoma.
Based on its cytoprotective function, GRP78 represents a prime target for solid
tumor cancer therapy. A macrocyclic compound, versipelostatin (VST), inhibits
glucose-starvation induced GRP78 expression and significantly inhibits tumor growth
of stomach cancer xenograft in combination with cisplatin therapy(Park, Tomida et al.
2004). The discovery that GRP78 is present on the cell surface of cancer cells, but not
normal tissues, provides novel therapeutic intervention strategies that can specifically
target tumor cell death. Systemic administration of synthetic chimeric peptides with
GRP78 binding motifs fused to a programmed cell-death inducing sequence suppresses
tumor growth in xenograft models of breast and prostate(Arap 2004). It is recently
reported that cell surface GRP78 serves as a receptor for angiogenesis inhibitor Kringle
5 (K5). The antiangiogenic and proapoptotic activity of recombinant K5 depends on a
high-affinity binding interaction with GRP78 on the surface of stimulated endothelial
cells and hypoxic and cytotoxic stressed tumor cells(Davidson, Haskell et al. 2005).
Thus, drugs mimicking the high-affinity binding of K5 with GRP78 may have
antiangiogenic and anticancer activities. Similarly, blocking α2M* association with
GRP78 may inhibit cancer proliferation and motility, thereby suppressing
metastasis(Misra, Deedwania et al. 2005; Misra, Deedwania et al. 2006).

79
To further investigate the mechanism of GRP78’s action, we expect several lines of
experiment might be beneficial. First, isolate a panel of MCF-7/BUS clones that are
resistant to estrogen starvation-induced apoptosis and determine whether knockdown of
GRP78 will overcome the resistance. This experiment will further elucidate the role of
GRP78 in the resistance to estrogen starvation-induced apoptosis.
Second, determine the interactive domain(s) between GRP78 and BIK. BIK protein
is known to have three functional regions: BH3 domain, phosphorylation sites, and
C-terminal transmembrane region (Fig. 1.1). BH3 is required for its apoptotic function
and interacting with anti-apoptotic proteins (Elangovan 1997). BIK has two
phosphorylation sites, Thr-33 and Ser-35. Mutation of these sites reduces apoptotic
activity of BIK without significantly affecting its ability to heterodimerize with Bcl-2.
BIK mutants, in which the phosphorylation sites are changed to aspartic acid to mimic
the phosphorylation, enhance their binding affinity with the antiapoptotic proteins
Bcl-XL and Bcl-2 and are more potent than wild-type BIK in inducing apoptosis and
inhibiting cell proliferation in various human cancer cells. The transmembrane domain
anchors the BIK protein at the ER membrane. GRP78 binding to any one of the
above-mentioned regions could possibly affect the activity of BIK. GRP78 is an ER
lumenal protein, but a subpopulation of GRP78 could exist as an ER transmembrane
protein(Reddy 2003). The ATPase domain locates in the cytosolic part, and the
protein-binding domain in the lumenal part. Because each region has its specific role,
knowing which region is required for the GRP78-BIK interaction is very helpful for the
understanding the underlying mechanism. However, it is probable that GRP78-BIK
interaction requires a region without any previously known function. Therefore,

80
construction of serial deletion mutant is as important as construction of mutants with
deletion of known domains.
Third, for the mouse model of prostate cancer, molecular markers associated with
apoptosis, differentiation, and metastasis, etc., not limited in AKT, should be assessed
by immunohistological analyses. Cell culture study may assist and complement mouse
model investigation.
In summary, in vitro systems in combination with a mouse model strongly suggest
that GRP78 plays an important role in tumor growth and survival, drug resistance, and
dormancy. The next challenge is to identify and weigh the molecular pathways involved
in the action of GRP78 on cancer formation and progression. Since this process is
complex and involves many interacting factors, such as tumor, stroma, endothelial cell
types, as well as immune system, an integration of in vitro studies in cell culture and in
vivo studies in a specific knockout animal model is mandatory in dissecting the
functional output of GRP78.

81
Bibliography
Abdulkadir, S. A., J. A. Magee, et al. (2002). "Conditional Loss of Nkx3.1 in Adult
Mice Induces Prostatic Intraepithelial Neoplasia." Mol. Cell. Biol. 22(5):
1495-1503.
Arap, M., Lahdenranta J, Mintz PJ, Hajitou A, Sarkis AS, Arap W, Pasqualini R (2004).
"Cell surface expression of the stress response chaperone GRP78 enables tumor
targeting by circulating ligands." Cancer Cell 6: 275-284.
Arap, M. A., J. Lahdenranta, et al. (2004). "Cell surface expression of the stress
response chaperone GRP78 enables tumor targeting by circulating ligands."
Cancer Cell 6(3): 275-84.
Arena, V., M. Martini, et al. (2003). "Mutations of the BIK gene in human peripheral
B-cell lymphomas." Genes Chromosomes Cancer 38(1): 91-6.
Baum, M., A. Buzdar, et al. (2003). "Anastrozole alone or in combination with
tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal
women with early-stage breast cancer: results of the ATAC (Arimidex,
Tamoxifen Alone or in Combination) trial efficacy and safety update analyses."
Cancer 98(9): 1802-10.
Baumeister, P., S. Luo, et al. (2005). "Endoplasmic reticulum stress induction of the
Grp78/BiP promoter: activating mechanisms mediated by YY1 and its
interactive chromatin modifiers." Mol Cell Biol 25: 4529-4540.
Bethel, C. R., D. Faith, et al. (2006). "Decreased NKX3.1 Protein Expression in Focal
Prostatic Atrophy, Prostatic Intraepithelial Neoplasia, and Adenocarcinoma:
Association with Gleason Score and Chromosome 8p Deletion." Cancer Res
66(22): 10683-10690.
Breckenridge, D., Germain M, Mathai JP, Nguyen M, and Shore GC (2003).
"Regulation of apoptosis by endoplasmic reticulum pathways." Oncogene 22:
8608-8618.
Carew, J. S., S. T. Nawrocki, et al. (2007). "Targeting autophagy augments the
anticancer activity of the histone deacetylase inhibitor SAHAto overcome
Bcr-Abl–mediated drug resistance." Blood 110(1): 313-322.
Cheng, E. H., M. C. Wei, et al. (2001). "BCL-2, BCL-X(L) sequester BH3 domain-only
molecules preventing BAX- and BAK-mediated mitochondrial apoptosis." Mol
Cell 8(3): 705-11.

82
Clarke, R., Leonessa F, Welch JN, and Skaar TC (2001). "Cellular and Molecular
Pharmacology of Antiestrogen of Antiestrogen Action and Resistance."
Pharmacological Reviews 2001(53): 25-71.
Coser, K., Chesnes J, Hur J, Ray S, Isselbacher KJ, and Shioda T (2003). "Global
analysis of ligand sensitivity of estrogen inducible and suppressible genes in
MCF-7/BUS breast cancer cells by DNA microarray." PNAS. 100(24):
13994-13999.
Cristofano, A. D., B. Pesce, et al. (1998). "Pten is essential for embryonic development
and tumour suppression." Nature Genetics 19: 348 - 355.
Dahia, P. (2000). "PTEN, a unique tumor suppressor gene." Endocrine-Related Cancer
7: 115 - 129.
Davidson, D. J., C. Haskell, et al. (2005). "Kringle 5 of human plasminogen induces
apoptosis of endothelial and tumor cells through surface-expressed
glucose-regulated protein 78." Cancer Res 65(11): 4663-72.
Diel, P., K. Smolnikar, et al. (1999). "The pure antiestrogen ICI 182780 is more
effective in the induction of apoptosis and down regulation of BCL-2 than
tamoxifen in MCF-7 cells." Breast Cancer Res Treat 58(2): 87-97.
Dong, D., L. Dubeau, et al. (2004). "Spontaneous and controllable activation of suicide
gene expression driven by the stress-inducible grp78 promoter resulting in
eradication of sizable human tumors." Hum Gene Ther 15: 553-561.
DONG, D., L. DUBEAU, et al. (2004). "Spontaneous and Controllable Activation of
Suicide Gene Expression Driven by the Stress-Inducible Grp78 Promoter
Resulting in Eradication of Sizable Human Tumors." HUMAN GENE
THERAPY 15: 553 - 561.
Dong, D., B. Ko, et al. (2005). "Vascular targeting and antiangiogenesis agents induce
drug resistance effector GRP78 within the tumor microenvironment." Cancer
Res. 65: 5785 - 5791.
Dong, D., B. Ko, et al. (2005). "Vascular targeting and antiangiogenesis agents induce
drug resistance effector GRP78 within the tumor microenvironment." Cancer
Res 65(13): 5785-91.
Dong, D., M. Ni, et al. (2008). "Critical role of the stress chaperone GRP78/BiP in
tumor proliferation, survival and tumor angiogenesis in transgene-induced
mammary tumor development." Cancer Res 68: 498-505.

83
Dowsett, M., R. I. Nicholson, et al. (2005). "Biological characteristics of the pure
antiestrogen fulvestrant: overcoming endocrine resistance." Breast Cancer Res
Treat 93: S11-8.
Early Breast Cancer Trialists' Collaborative Group (1998). "Tamoxifen for early breast
cancer: an overview of the randomised trials." Lancet 351: 1451-1467.
Elangovan, B., Chinnadurai G (1997). "Functional Dissection of the Pro-apoptotic
Protein BIK." J Biol. Chem. 39: 24494-24498.
Ermakova, S. P., B. S. Kang, et al. (2006). "(-)-Epigallocatechin gallate overcomes
resistance to etoposide-induced cell death by targeting the molecular chaperone
glucose-regulated protein 78." Cancer Res 66(18): 9260-9.
Fernandez, P . M., S. O. Tabbara, et al. (2000). "Overexpression of the glucose-regulated
stress gene GRP78 in malignant but not benign human breast lesions." Breast
Cancer Res Treat 59: 15-26.
Fu, Y. and A. S. Lee (2006). "Glucose regulated proteins in cancer progression, drug
resistance and immunotherapy." Cancer Biol Ther 5(7): 741-744.
Germain, M., J. P. Mathai, et al. (2005). "Endoplasmic reticulum BIK initiates
DRP1-regulated remodelling of mitochondrial cristae during apoptosis." EMBO
J 24((8)): 1546-1556.
Germain, M., J. P. Mathai, et al. (2002). "BH-3-only BIK functions at the endoplasmic
reticulum to stimulate cytochrome c release from mitochondria." J Biol Chem
277(20): 18053-18060.
Gething, M.-J. (1999). "Role and regulation of the ER chaperone BiP ." Seminars in Cell
& Development Biology 10: 465-472.
Gillissen, B., F. Essmann, et al. (2003). "Induction of cell death by the BH3-only Bcl-2
homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial
pathway." EMBO J 22(14): 3580-90.
Gray, M. D., M. Mann, et al. (2005). "Activation of the unfolded protein response is
necessary and sufficient for reducing topoisomerase IIalpha protein levels and
decreasing sensitivity to topoisomerase-targeted drugs." Mol Pharmacol 68(6):
1699-707.
Haas, I. G . and M. Wabl (1983). "Immunoglobulin heavy chain binding protein." Nature
306(5941): 387-9.

84
Haggman, M. J., J. A. Macoska, et al. (1997). "The Relationship Between Prostatic
Intraepithelial Neoplasia and Prostate Cancer: Critical Issues." Journal of
Urology 158(1): 12 - 22.
Hendershot, L. M. (2004). "The ER function BiP is a master regulator of ER function."
Mt Sinai J Med 71(5): 289-97.
Hsu, W. M., F. J. Hsieh, et al. (2005). "GRP78 expression correlates with histologic
differentiation and favorable prognosis in neuroblastic tumors." Int J Cancer
113(6): 920-7.
Hur, J., J. Chesnes, et al. (2004). "The Bik BH3-only protein is induced in
estrogen-starved and antiestrogen-exposed breast cancer cells and provokes
apoptosis." Proc Natl Acad Sci USA 101(8): 2351-6.
Hur, J., Shioda T, et al. (2004). "The Bik BH3-only protein is induced in
estrogen-starved and antiestrogen-exposed breast cancer cells and provokes
apoptosis." PNAS 101(8): 2351-2356.
Jamora, C., G . Dennert, et al. (1996). "Inhibition of tumor progression by suppression of
stress protein GRP78/BiP induction in fibrosarcoma B/C10ME." PNAS 93:
7690 - 7694.
Jamora, C., G . Dennert, et al. (1996). "Inhibition of tumor progression by suppression of
stress protein GRP78/BiP induction in fibrosarcoma B/C10ME." Proc Natl
Acad Sci USA 93: 7690-7694.
Karpinich, N., Tafani M, Rothman RJ, Russo MA, and Farber JL (2002). "The course of
etoposide-induced apoptosis from damage to DNA and p53 activation to
mitochondrial release of cytochrome c." J Biol. Chem. 277(19): 16547-16552.
Karpinich, N. O., M. Tafani, et al. (2002). "The course of etoposide-induced apoptosis
from damage to DNA and p53 activation to mitochondrial release of cytochrome
c." J Biol Chem 277(19): 16547-52.
Kim, Y. B., A. Honda, et al. (1998). "phd1+, a histone deacetylase gene of
Schizosaccharomyces pombe, is required for the meiotic cell cycle and
resistance to trichostatin A." FEBS Letters 436: 193-196.
Koumenis, C. (2006). "ER stress, hypoxia tolerance and tumor progression." Curr Mol
Med 6(1): 55-69.
Laird, P. W., A. Zijderveld, et al. (1991). "Simplified mammalian DNA isolation
procedure." Nucleic Acids Research 19(15): 4923.

85
Lee, A. S. (2001). "The glucose-regulated proteins: stress induction and clinical
applications." Trends Biochem Sci 26: 504-510.
Lee, A. S. (2005). "The ER chaperone and signaling regulator GRP78/BiP as a monitor
of endoplasmic reticulum stress." Methods 35(4): 373-81.
Lee, A. S. (2007). "GRP78 induction in cancer: therapeutic and prognostic
implications." Cancer Res 67: 3496-3499.
Lee, E., P. Nichols, et al. (2006). "GRP78 as a novel predictor of responsiveness to
chemotherapy in breast cancer." Cancer Res 66: 7849-7853.
Li, J. and A. S. Lee (2006). "Stress induction of GRP78/BiP and its role in cancer." Curr
Mol Med 6(1): 45-54.
Little, E., M. Ramakrishnan, et al. (1994). "The glucose-regulated proteins (GRP78 and
GRP94): functions, gene regulation, and applications." Crit Rev Eukaryot Gene
Expr 4: 1-18.
Luk, J. M., C. T. Lam, et al. (2006). "Proteomic profiling of hepatocellular carcinoma in
Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78)
up-regulation and their associated prognostic values." Proteomics 6(3):
1049-57.
Luo, S., C. Mao, et al. (2006). "GRP78/BiP is required for cell proliferation and
protecting the inner cell mass from apoptosis during early mouse embryonic
development." Mol Cell Biol 26(15): 5688-97.
Luo, S., C. Mao, et al. (2006). "GRP78/BiP Is Required for Cell Proliferation and
Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic
Development." Molecular and Cellular Biology 26(15): 5688-5697.
Majumder, P. K., J. J. Yeh, et al. (2003). "Prostate intraepithelial neoplasia induced by
prostate restricted Akt activation: The MPAKT model." Proceedings of the
National Academy of Sciences 100(13): 7841-7846.
Mandic, A., K. Viktorsson, et al. (2001). "Cisplatin Induces the Proapoptotic
Conformation of Bak in a deltaMEKK1-Dependent Manner." Molecular and
Cellular Biology 21(11): 3684 - 3691.
Mathai, J. P., M. Germain, et al. (2002). "Induction and endoplasmic reticulum location
of BIK/NBK in response to apoptotic signaling by E1A and p53." Oncogene
21(16): 2534-44.

86
Mathai, J. P., M. Germain, et al. (2005). "BH3-only BIK regulates
BAX,BAK-dependent release of Ca2+ from endoplasmic reticulum stores and
mitochondrial apoptosis during stress-induced cell death." J Biol Chem 280(25):
23829-36.
McNeal, J., A. Villers, et al. (1991). "Microcarcinoma in the prostate: its association
with duct-acinar dysplasia." Human Pathology 22(7): 644 - 652.
Mintz, P . J., J. Kim, et al. (2003). "Fingerprinting the circulating repertoire of antibodies
from cancer patients." Nat Biotechnol 21(1): 57-63.
Misra, U. K., R. Deedwania, et al. (2005). "Binding of activated alpha2-macroglobulin
to its cell surface receptor GRP78 in 1-LN prostate cancer cells regulates
PAK-2-dependent activation of LIMK." J Biol Chem 280(28): 26278-86.
Misra, U. K., R. Deedwania, et al. (2006). "Activation and Cross-talk between Akt,
NF- κB, and Unfolded Protein Response Signaling in 1-LN Prostate Cancer
Cells Consequent to Ligation of Cell Surface-associated GRP78." THE
JOURNAL OF BIOLOGICAL CHEMISTRY 281(19): 13694-13707.
Misra, U. K., M. Gonzalez-Gronow, et al. (2002). "The role of Grp78 in alpha
2-macroglobulin-induced signal transduction. Evidence from RNA interference
that the low density lipoprotein receptor-related protein is associated with, but
not necessary for, GRP 78-mediated signal transduction." J Biol Chem 277(44):
42082-7.
Misra, U. K., M. Gonzalez-Gronow, et al. (2004). "A novel receptor function for the
heat shock protein Grp78: silencing of Grp78 gene expression attenuates
alpha2M*-induced signalling." Cell Signal 16(8): 929-38.
Misra, U. K., T. Sharma, et al. (2005). "Ligation of cell surface-associated
glucose-regulated protein 78 by receptor-recognized forms of
alpha2-macroglobulin: activation of p21-activated protein kinase-2-dependent
signaling in murine peritoneal macrophages." J Immunol 175(4): 2525-33.
Mitchell-Olds, T. and C. A. Knight (2002). "Evolution. Chaperones as buffering
agents?" Science 296(5577): 2348-9.
Park, H. R., A. Tomida, et al. (2004). "Effect on tumor cells of blocking survival
response to glucose deprivation." J Natl Cancer Inst 96(17): 1300-10.
Powell, W. C., R. D. Cardiff, et al. (2003). "Mouse Strains for Prostate Tumorigenesis
Based on Genes Altered in Human Prostate Cancer." Current Drug Targets 4:
263-279.

87
Radetzki, S., C.-H. Kohne, et al. (2002). "The apoptosis promoting Bcl-2 homologues
Bak and Nbk/Bik overcome drug resistance in Mdr-1-negative and
Mdr-1-overexpressing breast cancer cell lines." Oncogene 21: 227-238.
Ramsay, R. G., D. Ciznadija, et al. (2005). "Expression of stress response protein
glucose regulated protein-78 mediated by c-Myb." Int J Biochem Cell Biol
37(6): 1254-68.
Ranganathan, A. C., L. Zhang, et al. (2006). "Functional coupling of p38-induced
up-regulation of BiP and activation of RNA-dependent protein kinase-like
endoplasmic reticulum kinase to drug resistance of dormant carcinoma cells."
Cancer Res 66(3): 1702-11.
Rao, R. V ., S. Castro-Obregon, et al. (2002). "Coupling endoplasmic reticulum stress to
the cell death program. An Apaf-1-independent intrinsic pathway." J Biol Chem
277: 21836-21842.
Rao, R. V., A. Peel, et al. (2002). "Coupling endoplasmic reticulum stress to the cell
death program: role of the ER chaperone GRP78." FEBS Lett 514: 122-128.
Reddy, R., Mao C, Baumeister P, Austin RC, Kaufman RJ, Lee AS. (2003).
"Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis
induced by topoisomerase inhibitors: role of ATP binding site in suppression of
caspase-7 activation." J Biol. Chem. 278: 20915-20924.
Reddy, R. K., L. Dubeau, et al. (2002). "Cancer-inducible transgene expression by the
Grp94 promoter: spontaneous activation in tumors of various origins and
cancer-associated macrophages." Cancer Res 62(24): 7207-7212.
Reddy, R. K., C. Mao, et al. (2003). "Endoplasmic reticulum chaperone protein GRP78
protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP
binding site in suppression of caspase-7 activation." J Biol Chem 278:
20915-20924.
Roy-Burman, P., H. Wu, et al. (2004). "Genetically defined mouse models that mimic
natural aspects of human prostate cancer development." Endocrine-Related
Cancer 11: 225 - 254.
Scorrano, L., S. A. Oakes, et al. (2003). "BAX and BAK regulation of endoplasmic
reticulum Ca2+: a control point for apoptosis." Science 300(5616): 135-139.
Sellers, I. S. a. W. R. (2004). "The Biology and Clinical Relevance of the PTEN Tumor
Suppressor Pathway." JOURNAL OF CLINICAL ONCOLOGY 22(14): 2954 -
2963.

88
Sellers, W. and C. Sawyers (2002). Somatic Genetics of Prostate Cancer: Oncogenes
and Tumore Suppressors. Philadelphia, Lippincott Williams & Wilkins.
Shani, G., W. Fischer, et al. (2008). "GRP78 and Cripto form a complex at the cell
surface and collaborate to inhibit TGF-{beta} signaling and enhance cell
growth." Mol Cell Biol 28(2): 666-667.
Shen, J., X. Chen, et al. (2002). "ER stress regulation of ATF6 localization by
dissociation of BiP/GRP78 binding and unmasking of golgi localization
signals." Dev Cell 3: 99-111.
Shuda, M., N. Kondoh, et al. (2003). "Activation of the A TF6, XBP1 and grp78 genes in
human hepatocellular carcinoma: a possible involvement of the ER stress
pathway in hepatocarcinogenesis." J Hepatol 38(5): 605-14.
Soto, A., C. Sonnenschein, et al. (1995). "The E-SCREEN assay as a tool to identify
estrogens: an update on estrogenic environmental pollutants." Environ. Health
Perspect. 103: 113 - 122.
Sturm, I., C. Stephan, et al. (2006). "Loss of the tissue-specific proapoptotic BH3-only
protein Nbk/Bik is a unifying feature of renal cell carcinoma." Cell Death and
Differentiation 13: 619-627.
Suzuki, H., D. Freije, et al. (1998). "Interfocal heterogeneity of PTEN/MMAC1 gene
alterations in multiple metastatic prostate cancer tissues." Cancer Res. 58: 204 -
209.
Thiantanawat, A., Long BJ, Brodie AM. (2003). "Signaling pathways of apoptosis
activated by aromatase inhibitors and antiestrogens." Cancer Res. 63:
8037-8050.
Thomenius, M., Wang NS, Reineks EZ, Wang Z, and Distelhorst CW (2003). "Bcl-2 on
the Endoplasmic Reticulum Regulates Bax Activity by Binding to BH3-only
Proteins." J Biol. Chem. 278(8): 6243-6250.
Thomenius, M. J. and C. W. Distelhorst (2003). "Bcl-2 on the endoplasmic reticulum:
protecting the mitochondria from a distance." J Cell Sci 116(Pt 22): 4493-9.
Tong, Y., Q. Yang, et al. (2001). "The Pro-apoptotic Protein, Bik, Exhibits Potent
Antitumor Activity That Is Dependent on Its BH3 Domain." Molecular Cancer
Therapeutics 1: 95-102.
Tsutsumi, S., T. Gotoh, et al. (2004). "Endoplasmic reticulum stress response is
involved in nonsteroidal anti-inflammatory drug-induced apoptosis." Cell Death
Differ 11(9): 1009-16.

89
Tsutsumi, S., T. Namba, et al. (2006). "Celecoxib upregulates endoplasmic reticulum
chaperones that inhibit celecoxib-induced apoptosis in human gastric cells."
Oncogene 25(7): 1018 - 1029.
Uramoto, H., K. Sugio, et al. (2005). "Expression of endoplasmic reticulum molecular
chaperone Grp78 in human lung cancer and its clinical significance." Lung
Cancer 49(1): 55-62.
Wang, Q., Z. He, et al. (2005). "Overexpression of endoplasmic reticulum molecular
chaperone GRP94 and GRP78 in human lung cancer tissues and its
significance." Cancer Detect Prev 29(6): 544-51.
Wang, S., J. Gao, et al. (2003). "Prostate-specific deletion of the murine Pten tumor
suppressor gene leads to metastatic prostate cancer." Cancer Cell 4(3): 209-21.
Wei, M. C., W. X. Zong, et al. (2001). "Proapoptotic BAX and BAK: a requisite
gateway to mitochondrial dysfunction and death." Science 292(5517): 727-30.
Wu, X., J. Wu, et al. (2001). "Generation of a prostate epithelial cell-specific Cre
transgenic mouse model for tissue-specific gene ablation." Mechanisms of
Development 101: 61-69.
Wu, Y., H. Zhang, et al. (2005). "Endoplasmic reticulum stress signal mediators are
targets of selenium action." Cancer Res 65(19): 9073-9.
Xing, X., M. Lai, et al. (2006). "Overexpression of glucose-regulated protein 78 in
colon cancer." Clin Chim Acta 364(1-2): 308-15.
Yamada, H., Y. Arakawa, et al. (2006). "Depsipeptide-resistant KU812 cells show
reversible P-glycoprotein expression, hyper-acetylated histones, and modulated
gene expression profile." Leukemia Research 30: 723-734.
Yang, Y . and Z. Li (2005). "Roles of heat shock protein gp96 in the ER quality control:
redundant or unique function?" Mol Cells 20(2): 173-82.
Yoshida, M., M. Kijima, et al. (1990). "Potent and Specific Inhibition of Mammalian
Histone Deacetylase Both in Vivo and in Vitro by Trichostatin A." Journal of
Biological Chemistry 265(28): 17174-17179.
Zeng, L., M. Lu, et al. (2004). "ATF6 modulates SREBP2-meidated lipogenesis."
EMBO J 23: 950 - 958.
Zhong, C., G. Saribekyan, et al. (2006). "Cooperation between FGF8b Overexpression
and PTEN Deficiency in Prostate Tumorigenesis." Cancer Res. 66(4):
2188-2194.

90
Zhou, Y. and A. S. Lee (1998). "Mechanism for the suppression of the mammalian
stress response by genistein, an anticancer phytoestrogen from soy." J Natl
Cancer Inst 90: 381-388.
Zong, W. X., C. Li, et al. (2003). "Bax and Bak can localize to the endoplasmic
reticulum to initiate apoptosis." J Cell Biol 162(1): 59-69.
Zou, Y ., H. Peng, et al. (2002). "Systemic Tumor Suppression by the Proapoptotic Gene
bik." Cancer Res 62: 8-12.
Zu, K., T. Bihani, et al. (2006). "Enhanced selenium effect on growth arrest by
BiP/GRP78 knockdown in p53-null human prostate cancer cells." Oncogene
25(4): 546-54.

Abstract (if available)

Abstract Glucose-regulated protein 78 (GRP78), a molecular chaperone at the endoplasmic reticulum (ER), is a master regulator of ER stress and an important survival factor for cell. GRP78 protein level is highly elevated in malignant tumors and correlates with severe pathological grade and poor prognosis. It has been well established that GRP78 can affect apoptosis by regulating ER Ca2+ signaling and unfolded protein response pathway, but whether other mechanism exists remains unknown. Searching for novel partners that interact with GRP78 at the ER, we discovered that BIK, an apoptotic BH-3-only protein located principally at ER, selectively forms a complex with GRP78. GRP78 overexpression decreases apoptosis of 293T cells induced by ER-targeted BIK. For the MCF-7/BUS breast cancer cells that require BIK to mediate estrogen starvation-induced apoptosis, overexpression of GRP78 inhibits estrogen-starvation induced BAX activation, mitochondrial permeability transition, and consequent apoptosis. Further, knockdown of endogenous GRP78 by siRNA sensitizes MCF-7/BUS cells to estrogen-starvation induced apoptosis. This effect was substantially reduced when the expression of BIK was also reduced by siRNA. In addition to in vitro investigations, an in vivo study in a Pten conditional knockout mouse model of prostate cancer reveals that homozygous deletion of Grp78 blocks prostate cancer formation and progression initiated by Pten nullification. Our results provide multiple lines of evidence that GRP78 is a critical player in the regulation of apoptosis and the formation and progression of cancer. These results further support the concept that GRP78 represents a novel marker for cancer progression and chemo-responsiveness, as well as a novel target for cancer therapy.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The role of endoplasmic reticulum chaperone protein GRP78 in breast cancer

PDF

The role of endoplasmic reticulum protein GRP78 in normal hematopoeises and PTEN-null leukemogenesis

PDF

Role of GRP78 and its novel cytosolic isoform GRP78va in regulating the unfolded protein response in cancer

PDF

The role of endoplasmic reticulum proteins GRP78 and IP3R1 in regulation of glucose homeostasis and acute pancreatitis

PDF

Utilizing novel small molecules to modulate GRP78 expression and activity in cancer

PDF

The role of nuclear GRP78 in regulation of EGFR expression in lung cancer

PDF

Molecular mechanisms of chemoresistance in breast cancer

PDF

Creating a multiple micrornia expression vector to target GRP78, an ER chaperone and signaling regulator in cancer

PDF

Role of endoplasmic reticulum chaperones GRP78 and GRP94 in regulating unfolded protein response and cell survival

PDF

The roles of endoplasmic reticulum chaperones in regulating liver homeostasis and tumorigenesis

PDF

The role of endoplasmic reticulum chaperone glucose-regulated 78-kilodalton (GRP78) in lung cancer

PDF

The role of glucose-regulated proteins in endometrial and pancreatic cancers

PDF

Cell surface translocation and therapeutic targeting of GRP78

PDF

Cell surface translocation mechanism of stress-inducible GRP78 in human cancer

PDF

The role of endoplasmic reticulum chaperones in adipogenesis, liver cancer and mammary gland development

PDF

The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies

PDF

The transcriptional regulation of the pro-survival protein Grp78 by activating transcription factors and chromatin-modifying enzymes: its upregulation in response to HDAC-inhibitor treatment and ...

PDF

The molecular mechanism of GRP78 translocation from the endoplasmic reticulum to the cell surface

PDF

Study of the role of bone morphogenetic proteins in prostate cancer progression

PDF

Design of novel anticancer agents targeting cellular stress response pathways

Asset Metadata

Creator Fu, Yong (author)

Core Title The role of GRP78 in the regulation of apoptosis and prostate cancer progression

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-05

Publication Date 04/17/2010

Defense Date 03/19/2008

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

Tag apoptosis,BIK,BiP,GRP78,OAI-PMH Harvest,prostate cancer,PTEN

Language English

Advisor Lee, Amy S. (committee chair), Kaplowitz, Neil (committee member), Stallcup, Michael R. (committee member), Stellwagen, Robert H. (committee member)

Creator Email yongfu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1145

Unique identifier UC161569

Identifier etd-Fu-20080417 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-58415 (legacy record id),usctheses-m1145 (legacy record id)

Legacy Identifier etd-Fu-20080417.pdf

Dmrecord 58415

Document Type Dissertation

Rights Fu, Yong

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu