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Study of the mechanism by which hypoxia-inducible-factor-1 alpha (HIF-1α) regulates heat shock protein-90 alpha (Hsp90α) secretion in breast cancer

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Study of the mechanism by which hypoxia-inducible-factor-1 alpha (HIF-1α) regulates heat shock protein-90 alpha (Hsp90α) secretion in breast cancer

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Content STUDY OF THE MECHANISM BY WHICH HYPOXIA-INDUCIBLE-FACTOR-1
ALPHA (HIF-1α) REGULATES HEAT SHOCK PROTEIN-90 ALPHA (HSP90α)
SECRETION IN BREAST CANCER

By
Archana Ashok

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2014

ii

ACKNOWLEDGEMENTS
I express my gratitude to Dr. Wei Li for his guidance and for giving me an opportunity to
carry out the project at his lab. I thank him for his constant support and suggestions. I thank
my committee members Dr. James Ou and Dr. Chengyu Liang for being a part of my
committee and finding the time for me.
I would like to thank my lab members Priyamvada Jayaprakash, Ayesha Bhatia and Divya
Sahu for constantly guiding and supporting me throughout my project work.
I thank my parents for their constant motivation and encouragement.
On the onset, I would like to take this opportunity to express my gratitude to all those great
minds and hearts that have touched this project in the path of its success.

iii

TABLE OF CONTENTS
Acknowledgements…………………………………………………………………………ii
List of Figures………………………………………………………………………………v
Abstract…………………………………………………………………………………...viii
1.0 Introduction……………………………………………………………………………..1
1.1 Hypoxia- Inducible Factor 1 Alpha (HIF-1α)…………………………………1
1.2 Heat Shock Protein (Hsp90)…………………………………………………...3
1.3 Upstream Regulators of Hsp90 Secretion in Normal and Tumor Cells……….4
1.4 Exosomes Pathway for Hsp90 Secretion……………………………………...5
1.5 Relationship between HIF-1α and Secretion of Hsp90α in cancer……………6
2.0 Materials and Methods………………………………………………………………….7
2.1 Construction of HIF-1α genes into inducible lentiviral vector, pSLIK……….7
2.2 Cell culture and reagents……………………………………………………..13
2.3 Lentivirus production………………………………………………………...14
2.4 Lentivirus infection of MDA-MB-231 cells and HDFs..................................14
2.5 Hygromycin-B selection..................................................................................14
2.6 Analysis of drug-inducible expression………………………………………15
iv

3.0 Results…………………………………………………………………………………16
3.1 MDA-MB-231 cells constitutively secrete Hsp90α………………………….16
3.2 HIF-1α is responsible for Hsp90α secretion…………………………………17
3.3 Evidence for specific control of Hsp90α secretion by HIF-1α………………18
3.4 Dose Curve showing HIF-1αCA induction by Doxycycline in
MDA-MB-231 cells……………………………………………………………...19
3.5 Time Course showing HIF-1α CA induction by Doxycycline and
secretion of Hsp90α in MDA-MB-231 cells……………………………………..20
3.6 Dose Curve showing HIF-1αCA induction by Doxycycline in HDFcells ..…23
3.7 Dose Curve showing HIF-1αDN induction by Doxycycline in
MDA-MB-231 cells……………………………………………………………...24
3.8 Time Course showing HIF-1α DN induction by Doxycycline and
secretion of Hsp90α in MDA-MB-231 cells……………………………………..25
3.9 Dose Curve showing HIF-1αCA-719 induction by Doxycycline in
MDA-MB-231 cells……………………………………………………………...27
4.0 Discussion……………………………………………………………………………..29
Bibliography……………………………………………………………………………….32

v

LIST OF FIGURES
Figure 1: HIF-1α regulation by proline hydroxylation……………………………………..1
Figure 2: Hsp90α secretion occurs in normal cells only under stress, but constitutively in
certain tumor cells…………………………………………………………………………..4
Figure 3: Generation of entry clone by restriction enzyme digestion and ligation………...8
Figure 4: A schematic representation of the Gateway recombination cloning technology.
Entry clone with attL recombines with a destination vector with attR to form a new
expression clone with attB and a byproduct with attP……………………………………...9
Figure 5: Restriction enzyme digestion of HIF-1αCA/pSLIK with XhoI and sequencing
results showing 5’ SacII site and start codon……………………………………………...10
Figure 6: Restriction enzyme digestion of HIF-1αCA-719/pSLIK with XhoI and
sequencing results showing 5’ SacII site and start codon…………………………………11
Figure 7: Restriction enzyme digestion of HIF-1αDN/pSLIK with XhoI and sequencing
results showing 5’ SacII site and start codon……………………………………………...12
Figure 8: Serum free CM of HBL-100 and MDA-MB-231 cells incubated under normoxia
(N) or hypoxia (H) for 14h analyzed for the presence of Hsp90α proteins by Western
Blotting…………………………………………………………………………………….16
Figure 9: Analysis of CM for Hsp90α secretion from HIF-1α or HIF-1β down-regulated
MDA-MB-231 cells by Western Blotting…………………………………………………17
vi

Figure 10: Reintroduction of WT-HIF-1α and CA HIF-1α but not DN HIF-1α rescued
Hsp90α secretion in HIF-1α downregulated MDA-MB-231 cells………………………...18
Figure 11: Dose curve kinetics for HIF-1α CA induction by doxycycline in MDA-MB-231
cells………………………………………………………………………………………...19
Figure 12: Time course kinetics showing HIF-1α CA induction by doxycycline and
secretion of Hsp90α in MDA-MB-231 cells………………………………………………20
Figure 13: Time course kinetics showing absence of HIF-1α CA induction in the absence
of doxycycline in MDA-MB-231 cells…………………………………………………….21
Figure 14: Time course kinetics showing endogenous HIF-1α in MDA-MB-231 cells in
the presence of doxycycline……………………………………………………………….22
Figure 15: Time course kinetics showing endogenous HIF-1α in MDA-MB-231 cells in
the absence of doxycycline………………………………………………………………...23
Figure 16: Dose curve kinetics for HIF-1α CA induction by doxycycline in HDF cells…24
Figure 17: Dose curve kinetics for HIF-1α DN induction by doxycycline in MDA-MB-231
cells………………………………………………………………………………………...25
Figure 18: Time course kinetics showing HIF-1α DN induction by doxycycline and
secretion of Hsp90α in MDA-MB-231 cells………………………………………………26
Figure 19: Time course kinetics showing absence of HIF-1αDN induction in the absence
of doxycycline in MDA-MB-231 cells…………………………………………………….27
vii

Figure 20: Dose curve kinetics for HIF-1α CA-719 induction by doxycycline in MDA-
MB-231 cells………………………………………………………………………………28

viii

ABSTRACT
HIF-1α protein levels in the case of normal cells under normoxic conditions are usually
very low or undetected. Unlike normal cells, in the case of tumor cells, studies have shown
that there is a constitutive level of HIF-1α due to intratumoral hypoxia and also due to
some mutations in genes encoding for oncoproteins and tumor suppressors. . Normal cells
do not secrete Hsp90α unless they are under environmental stress, but tumor cells have
been shown to constitutively secrete Hsp90α under both normoxia and hypoxia.
Knockdown of HIF-1α by shRNA in MDA-MB-231 cells was shown to block Hsp90α
secretion in a previous study. Their results indicated that HIF-1α is a direct upstream
regulator of Hsp90α secretion. However, the exact mechanism by which HIF-1α triggers
Hsp90α secretion is still unknown to date. In the present study, we have investigated if
HIF-1α regulated secretion of Hsp90α requires HIF-1α to dimerize with HIF-1β in the
cytosol, translocate into the nucleus and initiate transcription of downstream genes before
triggering Hsp90α secretion, or does HIF-1α trigger Hsp90α secretion without performing
any of the nuclear function; by the use of a doxycyline controlled (Tet-ON) single
lentiviral vector platform for inducible overexpression of HIF-1α mutants namely HIF-1α
CA, HIF-1α DN and HIF-1α CA719 in MDA-MB-231 breast cancer cells and human
dermal fibroblasts

1

1.0 INTRODUCTION
1.1 Hypoxia- Inducible Factor 1 Alpha (HIF-1α)
In normal cells, under normoxic conditions (approximately 8% oxygen level in tissues), the
hypoxia-inducible factor-1α (HIF-1α) protein is synthesized constantly and subjected to an
oxygen dependent prolyl hydroxylation. This modification then targets the HIF-1α to
degradation by the ubiquitination-proteosome machinery (Semenza, 2003). Hence, the
overall steady state level of HIF-1α is kept low. However, under hypoxia (less than 2%
oxygen level in tissues), HIF-1α hydroxylation and degradation are suppressed and HIF-1α
levels start to rise. The increased HIF-1α then dimerizes with the constitutively active HIF-
1β, resulting in a functional heterodimer called HIF-1 which is a master transcriptional
complex. HIF-1 can then translocate into the nucleus and regulate the expression of
hypoxia response element (HRE) containing genes in a p300/CBP-dependent manner
(Arany et al., 1996).

2

Figure 1: HIF-1α regulation by proline hydroxylation
In contrast to the normal cells, in many tumors, HIF-1α proteins are kept at a constitutive
level due to the tissue hypoxia generated by outgrowth of the rapidly proliferating tumor
cells, creating a distance which is longer than the reach of the oxygen supply from the
nearest blood circulation (Bertout et al., 2008). Due to constant ischemia, these tumor cells
then undergo genetic changes to adapt alternative and self supporting mechanisms for their
continued survival, expansion, and progression, and start to neovascularize around them.
Thus the action of oncogenes, deactivation of enzymes involved in the HIF-1α
ubiquitination and degradation, and inhibition of tumor suppressor genes could all
contribute to deregulated expression of HIF-1α in tumor cells. Majmundar et al (2010)
showed that the downregulation of deregulated HIF-1α expression or inhibition of HIF-1α
action could slow tumor growth and render them to be more susceptible to killing by
chemotherapy and radiotherapy.
In humans, approximately 50% of all solid tumors show upregulated HIF-1α. For instance,
Dales et al (2005) carried out anti-HIF-1α immunohistochemical assays on frozen sections
of 745 breast cancer samples and found that the levels of HIF-1α expression correlated to
poor prognosis, lower overall survival, and high risk of metastasis among both node-
negative and node-positive patients. It was estimated that 25-40% of all invasive breast
cancer samples are HIF-1α positive. The constitutively expressed HIF-1α has become a
marker to predict possible outcomes in patients with tumor metastasis. In theory,
destroying the deregulated HIF-1α in tumor cells could prevent tumor progression, but
directly targeting the intracellularly located HIF-1α or the enzymes that regulate the
stability of HIF-1α is challenging (Poon et al., 2009).
3

1.2 Heat Shock Protein (Hsp90)
The human heat shock protein-90 (Hsp90) chaperone family consists of four confirmed
members namely the cytosolic Hsp90α and Hsp90β which share 86% amino acid identity
and are expressed ubiquitously in all nucleated cells; the endoplasmic reticulum GRP94
and the mitochondrial TRAP1, all encoded by distinct genes (Chen et al., 2005).
Overexpression or accumulation of HIF-1α in tumor cells was accompanied by Hsp90α
overexpression quantitatively or qualitatively overactivated in a variety of tumors (Kamal
et al., 2003). These overactive Hsp90α proteins are believed to bind and protect the
stability of oncogene products inside the cell (Welch and Feramisco, 1982; Grenert et al.,
1997; Neckers and Neckers, 2002).
Recent studies by our group have demonstrated that extracellular Hsp90α (eHsp90α) plays
a critical role in skin wound healing and tumor progression (Li et al, 2012). Normal cells
do not secrete Hsp90α until and unless they are under stress, such as reactive oxygen
species (Liao et al., 2000), heat (Hightower and Guidon, 1989; Clayton et al., 2005), γ-
radiation (Yu et al., 2006), injury-released growth factors (Cheng et al., 2008), hypoxia (Li
et al., 2007; Woodley et al., 2009), serum starvation (Chen et al., 2010) and virus infection
(Hung et al., 2011). But the mechanisms by which the stress signals cause Hsp90α
secretion remain to be further studied. The main function of eHsp90α in normal cells is to
help tissue repair by promoting the cells at the edge of the damaged tissue to migrate into
the damaged area.
But in the case of tumor cells, constitutively active oncogenes such as HIF-1α trigger
Hsp90α secretion even in the absence of environmental stress cues. Tumor-secreted
4

Hsp90α promotes both tumor and tumor stroma cell migration during invasion and
metastasis (Li et al., 2013). A schematic illustration of how normal cells and tumor cells
secrete Hsp90α is shown in the figure below.

Figure 2: Hsp90α secretion occurs in normal cells only under stress, but constitutively in
certain tumor cells (Li et al., 2013).
1.3 Upstream Regulators of Hsp90 Secretion in Normal and Tumor Cells
MDA-MB-231 breast cancer cells have been shown to constitutively secrete Hsp90
(Eustace et al., 2004; Wang et al., 2009; McCready et al., 2010). Li et al (2007) and
Woodley et al (2009) reported that HIF-1α mediates hypoxia-triggered Hsp90α secretion in
primary human keratinocytes and dermal fibroblasts. They showed that a dominant
negative mutant of HIF-1α (DN-HIF-1α) blocked Hsp90α secretion, but a constitutively
active mutant of HIF-1α (CA-HIF-1α) made the cells to secrete Hsp90α even under
5

normoxia. The same mechanism was observed in tumor cells also. Depletion of HIF-1α or
HIF-1β from MDA-MB-231 cells by RNAi completely blocked the constitutive secretion
of Hsp90α by these cells. This inhibition of Hsp90α secretion could be rescued by
exogenously reintroducing CA-HIF-1α, but not DN-HIF-1α gene into the endogenous HIF-
1α downregulated cells (Sahu et al., 2012). Since approximately 50% of all invasive human
tumors express higher levels of HIF-1α (Semenza, 2007; Semenza, 2012), extracellular
Hsp90α could be used as a new diagnostic and / or therapeutic target for the “HIF-1α
positive” tumors.
1.4 Exosomes Pathway for Hsp90 Secretion
Exosomes or intraluminal vesicles are non-plasma membrane derived vesicles that are 30-
90 nm in diameter and are contained within multivesicular bodies (MVBs) and performs
functions such as fusion with lysosomes. MVBs can also fuse with the plasma membrane
to release their cargo proteins, such as Hsp90 which lacks signal sequence at the amino
terminus of the protein and hence cannot be secreted via the classical endoplasmic
reticulum (ER)/ Golgi peptide transport pathway. MVBs secrete Hsp90 into the
extracellular space via the following steps: sorting into smaller vesicles, fusion with the
cell’s surface membrane and release of small vesicles into the extracellular space (Fevrier
and Raposo, 2004; Stoorvogel et al., 2002). Whether eHsp90 stays inside exoxomes all
time or is spilled out to the environment after the exosomes get to the cell surface or
outside the cells is still unclear.
Hsp90 is secreted from cells via the exosome protein trafficking pathway (Yang and
Robbins, 2011; Li et al., 2012) and this mechanism was supported by evidence from
6

studies with chemical inhibitors, proteomic analysis and electron microscopic (EM)
visualization of Hsp90-containing exosomes. Dimethyl amiloride (DMA) which is a
chemical inhibitor that selectively blocks the exosome mediated protein secretion pathway
was used to show that it inhibited membrane translocation and secretion of Hsp90α,
Hsp90β and / or Hsp70 in various cell types (Li et al., 2012); but not by Brefeldin A (BFA)
which is a selective chemical inhibitor against the classical ER/ Golgi protein transport
pathway (Lancaster and Febbrio, 2005; Savina et al., 2003). Clayton et al (2005) used
proteomic methods to analyze the peptide contents of B cell secreted exosomes under
either physiological temperature (37°C) or heat shock (42°C for 3 hours), and found that
heat shock increased Hsp90α presence in secreted exosomes isolated by
ultracentrifugation. Yu et al (2006) provided EM evidence that Hsp90β is located outside
the cells in response to γ-irradiation.
1.5 Relationship between HIF-1α and Secretion of Hsp90α in cancer
The key upstream regulator of Hsp90α secretion is HIF-1α (Li et al., 2007; Woodley et al.,
2009). Because constitutive accumulation of HIF-1α occurs in more than 40% of human
tumors (Dales et al., 2005; Poon et al., 2009), secreted Hsp90α could be a new and
effective target for treatment of these HIF-1α positive tumors. Previous studies by Sahu et
al (2012), proved the importance of the HIF-1α and Hsp90α secretion axis in control of
cancer cell migration and invasion. In the present study, we have tested the possibility that
nuclear translocation and transcription activation might be required for HIF-1α regulated
Hsp90α secretion in triple negative MDA-MB-231 cells and primary human dermal
fibroblast cells, by the use of a tightly regulated Tet-on inducible overexpression system.
7

2.0 MATERIALS AND METHODS
2.1 Construction of HIF-1α genes into inducible lentiviral vector, pSLIK
The cDNAs that encode HIF-1αCA5 (constitutively active), HIF-1αCA5-719
(constitutively active nuclear localization signal mutant) and HIF-1αDN (dominant
negative) were amplified by Polymerase Chain Reaction (PCR) from gene storage with
gene specific primers introducing a 5’ SacII site and a 3’ XbaI site. The primers used for
amplification are shown below in table 1.
OLIGO NAME SEQUENCE 5’-3’
HIF-1α CA5 fwd GAT CAC CCG CGG TTC ACC ATG GAG GGC
HIF-1α CA5 rev GTA CAG TCT AGA TCA GTT AAC TTG ATC
HIF-1α CA5-719 fwd GAT CAC CCG CGG TTC ACC ATG GAG GGC
HIF-1α CA5-719 rev GTA CAG TCT AGA TCA GTT AAC TTG ATC
HIF-1α DN fwd ACC GAC CCG CGG ACC ATG TTT TAC CCA TAC
GAT GTT
HIF-1α DN rev GTA CAG TCT AGA TCA AAG TTT GTC AAA GAG
GCT ACT
Table 1: Oligonucleotides used for PCR amplification of the cDNAs
The PCR products were then ligated into the pCR2.1-TOPO vector using the Topo-TA
cloning system purchased from Invitrogen. A recombinational cloning method called the
8

Gateway cloning was then adapted in subsequent steps. The gateway cloning is a 2 step
cloning in which the gene of interest is first cloned into a pEN_TmiRc3 entry vector
(American Type Tissue Culture no. MBA-248) between 2 attachment (att) sites called
attL1 and attL2. This is done by the standard restriction enzyme digestion and ligation,
followed by transformation with XL-10 Gold Ultra Competent cells purchased from
Agilent Technologies.

Figure 3: Generation of entry clone by restriction enzyme digestion and ligation.
Then a recombination reaction is employed to clone the gene of interest into the final
destination vector pSLIK-Hygro (American Type Tissue Culture no. MBA-237) between
attR1 and attR2 sites from the pEN_TmiRc3 entry vector by the use of a Gateway LR
Clonase enzyme purchased from Invitrogen. It was then followed by a transformation with
TOP10 One Shot Chemically competent E.coli purchased from Invitrogen. Figure 4 shown
below illustrates the LR reaction.

9

Figure 4: A schematic representation of the Gateway recombination cloning technology.
Entry clone with attL recombines with a destination vector with attR to form a new
expression clone with attB and a byproduct with attP.
The presence of ccdB gene allows negative selection of the destination vector in E.coli
following recombination and transformation. When recombination occurs between an entry
vector and a destination vector, the ccdB gene is replaced by the gene of interest. Cells that
take up unreacted vectors carrying the ccdB gene or by-product molecules retaining the
ccdB gene will fail to grow, because the ccdB protein interferes with E.coli DNA gyrase
and inhibits growth of most E.coli strains. And this allows high efficiency recovery of the
desired clones.
10

Figure 5: Restriction enzyme digestion of HIF-1αCA/pSLIK with XhoI and sequencing
results showing 5’ SacII site and start codon
11

Figure 6: Restriction enzyme digestion of HIF-1αCA-719/pSLIK with XhoI and
sequencing results showing 5’ SacII site and start codon
12

Figure 7: Restriction enzyme digestion of HIF-1αDN/pSLIK with XhoI and sequencing
results showing 5’ SacII site and start codon.
13

As shown in Figures 5, 6 and 7, the cloned HIF-1αCA/pSLIK, HIF-1αCA-719/pSLIK and
HIF-1αDN/pSLIK respectively were verified using restriction enzyme digestion and also
by sequencing which shows the 5’ SacII site followed by the Kozak sequence and start
codon. Since pSLIK vector is very large (13918bp), restriction digest with SacII and XbaI
resulted in several bands as the vector itself had these sites. Hence XhoI was chosen in the
case of HIF-1αCA/pSLIK and HIF-1αCA-719/pSLIK and EcoRI was used to verify HIF-
1αDN/pSLIK.
2.2 Cell culture and reagents
MDA-MB-231, which is a triple negative breast cancer cell line and primary human
dermal fibroblasts (HDFs) were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with low glucose concentration of 1g/L supplemented with 10% Fetal Bovine
Serum (FBS), 100U/ml penicillin and 0.1mg/ml streptomycin. 293T cells were maintained
in DMEM with high glucose concentration of 4.5g/L supplemented with 10% FBS,
100U/ml penicillin and 0.1mg/ml streptomycin. The cells were cultured in an incubator
with a humidified atmosphere of 5% CO
2
at 37°C.
Anti-HIF-1α antibody (610958) was purchased from BD Transduction Laboratories
(Lexington, KY). Anti-Hsp90α antibody (CA1023) was from Calbiochem and antibody
against GAPDH was from Genetex. Anti-HA-tag antibody (11867423001) was purchased
from Roche Applied Science. Hygromycin-B was purchased from Invitrogen.

14

2.3 Lentivirus production
293T cells were cultured to a 90% confluence in 10cm culture dishes. 15µg of HIF-1α wild
type or mutants DNA in pSLIK-Hygro vector was then transfected along with 15µg of
packaging plasmid pCMVΔR8.2 and 5µg of fusion protein vesicular stomatitis virus
(VSV) G envelope plasmid, using the Calcium Phophate Transfection kit from Promega.
After 16 hours, the media containing the plasmids were removed from the transfected cells
and replaced with fresh culture media. Conditioned media were collected 48 hours post-
transfection and filtered through a 0.45µm filter to remove any cell debris, before being
used to infect MDA-MB-231 cells and HDFs.
2.4 Lentivirus infection of MDA-MB-231 cells and HDFs
MDA-MB-231 cells and HDFs were plated to 30% confluence in 6cm culture dishes 24
hours prior to infection. 1.5ml of the collected virus containing medium was added along
with 1.5µl of polybrene. The plates were washed with fresh culture medium 8 hours post
infection and replaced with DMEM media supplemented with 10% FBS, 100U/ml
penicillin and 0.1mg/ml streptomycin.
2.5 Hygromycin-B selection
The infected MDA-MB-231 cells and HDFs were then passaged to 10cm culture dishes 48
to 72 hours post infection and subjected to Hygromycin-B selection for 2 weeks at a
concentration of 700µg/ml and 150µg/ml respectively. The selected cells were then used
for dose course and time course experiments.

15

2.6 Analysis of drug-inducible expression
Cells were grown to log phase on 10cm tissue culture dishes and then treated with
Doxycycline to induce HIF-1α, along with a control non-treatment group for the desired
time of treatment. After the desired treatment time, the cells were harvested for protein
extraction for analysis by Western blotting. The cells were washed thrice with cold
Phosphate Buffered Saline (PBS) and then lysis buffer containing protease inhibitor was
added to it. The cells were scraped off the plate using a cell scraper and vortexed at high
speed for 20 seconds, following which it was centrifuged at 14,000 rpm for 10 minutes in a
cold room centrifuge. The clear lysate was then transferred to fresh tubes on ice and the
pellet was discarded. The protein concentration was then estimated by the Bio-Rad protein
assay kit.
The protein samples were subjected to SDS-PAGE in 10% polyacrlyamide gels in Tris
Glycine buffer. It was then transferred to 0.45µm nitrocellulose membranes. The
membranes were then subjected to Ponceau-S stain to test transfer adequacy. It was then
washed twice with Tris-buffered Saline containing tween (TBS-T) followed by one wash
of Tris-buffered Saline (TBS). Each wash was for 3 minutes in a shaker at speed 4 at room
temperature. The membranes were then blocked for one hour in blocking buffer containing
5% Bovine Serum Albumin (BSA) in TBS and incubated with primary antibody (1:500
dilution of antibody in 5% BSA in TBS) overnight. Membranes were washed four times
and then incubated for an hour in Horse-radish peroxidase (HRP) conjugated secondary
antibody (1:1000 dilution of antibody in 5% skimmed milk in TBS). The membranes were
then washed with TBS once followed by three washes in TBS-T, and then developed by
Enhanced Chemiluminescence (ECL) kit purchased from Amersham.
16

3.0 RESULTS
3.1 MDA-MB-231 cells constitutively secrete Hsp90α
A study by Sahu et al in 2012 showed that secreted Hsp90α was detected from the
conditioned media (CM) of MDA-MB-231 cells cultured under either normoxia or
hypoxia. They cultured HBL-100 and MDA-MB-231 cells under either normoxia or
hypoxia and collected their serum free CM after 14h and analyzed it for the presence of
Hsp90α. As shown in Figure 8, secreted Hsp90α was detected from the CM of HBL-100
cells incubated under hypoxia (lane 2), but not under normoxia (lane 1). In contrast, an
equal amount of secreted Hsp90α was detected from the CM of MDA-MB-231 cells under
both normoxia and hypoxia.

Figure 8: Serum free CM of HBL-100 (lanes 1 and 2) and MDA-MB-231 cells (lanes 3
and 4) incubated under normoxia (N) or hypoxia (H) for 14h analyzed for the presence of
Hsp90α proteins by Western Blotting. (Used with permission from Sahu et al, Molecular
Biology of the Cell, 2012)

17

3.2 HIF-1α is responsible for Hsp90α secretion
They then went on to show that the secretion of Hsp90α in MDA-MB-231 cells under both
normoxia and hypoxia was due to the deregulated HIF-1α in these cells. They used a
lentiviral system FG-12 to deliver a U6 promoter-driven short hairpin RNA (shRNA)
against human HIF-1α or HIF-1β into MDA-MB-231 cells. As shown in Figure 9, secreted
Hsp90α was undetectable from the CM of either HIF-1α or HIF-1β downregulated MDA-
MB-231 cells (lanes 2a and 3a), whereas Hsp90α secretion was unaffected in control
RNAi-infected MDA-MB-231 cells (lanes 1a and 1b). This inhibition appeared to be
specific, given that under identical conditions, secretion of matrix metalloproteinase 9
(MMP9) by the cells was present (lanes 2b and 3b).

Figure 9: Analysis of CM for Hsp90α secretion from HIF-1α or HIF-1β down-regulated
MDA-MB-231 cells by Western Blotting. (Used with permission from Sahu et al,
Molecular Biology of the Cell, 2012)

18

3.3 Evidence for specific control of Hsp90α secretion by HIF-1α
To validate the specific control of Hsp90α secretion by HIF-1α, Sahu et al in 2012 then
carried out HIF-1α gene rescue experiments. They exogenously reintroduced wild type
(WT), constitutively active (CA) HIF-1α and dominant negative (DN) HIF-1α into
endogenous HIF-1α depleted MDA-MB-231 cells. As shown in Figure 10, they used anti-
HIF-1α antibody to detect wt-HIF-1α (lane 2a) and CA-HIF-1α (lane 3a). Because DN-
HIF-1α has a large C-terminal truncation, most of the commercial antibodies do not
recognize it, and hence they used an anti-hemagglutinin (HA) tag antibody to detect HA-
DN-HIF-1α (lane 4b). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
an internal loading control. From the CM of these cells, they found that only wt-HIF-1α
(lane 2c) and CA-HIF-1α (lane 3c) were able to rescue Hsp90α secretion, but not DN-HIF-
1α (lane 4c). These results indicated that secreted Hsp90α was a direct downstream target
for deregulated HIF-1α.

19

Figure 10: Reintroduction of WT-HIF-1α and CA HIF-1α (lanes 2c and 3c), but not DN
HIF-1α (lane 4c) rescued Hsp90α secretion in HIF-1α downregulated MDA-MB-231 cells.
(Used with permission from Sahu et al, Molecular Biology of the Cell, 2012)
3.4 Dose Curve showing HIF-1αCA induction by Doxycycline in MDA-MB-231 cells
In order to determine the minimum doxycycline concentration required to induce HIF-
1αCA, a dose curve experiment was performed. MDA-MB-231 cells transduced with HIF-
1αCA/pSLIK were plated to 85-90% confluence in six 10cm dishes, post Hygromycin-B
selection. The cells were allowed to attach overnight and the following day, media was
replaced with serum-free media.

Figure 11: Dose curve kinetics for HIF-1α CA induction by doxycycline in MDA-MB-231
cells
The cells were then treated with varying concentrations of doxycycline (0, 0.03, 0.1, 0.3, 1
and 3μg/ml) for a constant time of 24 hours. As shown in Figure 11, we observed a gradual
induction of HIF-1αCA with increasing concentrations of doxycycline. GAPDH was used
as an internal loading control. The induction of HIF-1αCA started to stabilize from
20

0.3μg/ml of doxycycline onwards. Hence this concentration was chosen and a time course
experiment was performed as described below.
3.5 Time Course showing HIF-1α CA induction by Doxycycline and secretion of
Hsp90α in MDA-MB-231 cells
In order to determine the minimum time required to induce HIF-1αCA, a time course
experiment was then performed. As previously described, MDA-MB-231 cells transduced
with HIF-1αCA/pSLIK were plated to 85-90% confluence in six 10cm dishes, post
Hygromycin-B selection. The cells were allowed to attach overnight and the following day,
media was replaced with serum-free media.

Figure 12: Time course kinetics showing HIF-1α CA induction by doxycycline and
secretion of Hsp90α in MDA-MB-231 cells
Based on the dose curve experiment result shown in Figure 11, doxycyline concentration
of 0.3μg/ml was chosen and HIF-1αCA induction at various time points (0, 20min, 1h, 3h,
21

9h, and 24h) was determined by immunoblotting. As shown in Figure 12, HIF-1αCA
induction started as early as 1 hour and stabilized from 3 hours.
We then looked for secretion of Hsp90α from the conditioned medium of these cells. The
serum free conditioned media was collected at the various time points of doxycycline
induction and concentrated by using 50,000kDa molecular weight cut-off concentration
columns purchased from Millipore. As shown in Figure 12, secreted Hsp90α was detected
at 24h, but not at other time points. This delay in the secretion of Hsp90α compared to
HIF-1α induction at 1h, suggests that nuclear translocation of HIF-1α might be necessary
for Hsp90α secretion.
However, unlike intracellular proteins (like glyceraldehyde-3-phosphate dehydrogenase or
β-actin for intracellular protein standards), there are few reliable loading control markers
for secreted proteins. Hence the equal loadings of CM were justified by loading equal
volumes of CM from the same number of cells cultured under identical conditions.

Figure 13: Time course kinetics showing absence of HIF-1α CA induction in the absence
of doxycycline in MDA-MB-231 cells.
22

In the case of control experiment, when there was no addition of doxycycline there was no
induction of HIF-1α CA, as shown in Figure 13. However we still observed secretion of
Hsp90α at 24h and this could be because of the constitutive secretion of Hsp90α by MDA-
MB-231 cells.

Figure 14: Time course kinetics showing endogenous HIF-1α in MDA-MB-231 cells in
the presence of doxycycline.
In another set of control experiments performed on uninfected MDA-MB-231 cells in the
presence and absence of doxycycline, we observed the basal levels of HIF-1α and a basal
secretion of Hsp90α at 24h as shown in Figures 14 and 15.
23

Figure 15: Time course kinetics showing endogenous HIF-1α in MDA-MB-231 cells in
the absence of doxycycline.
3.6 Dose Curve showing HIF-1αCA induction by Doxycycline in HDFcells
Since MDA-MB-231 cells already have an endogenous HIF-1α and constitutively secrete
Hsp90α, we wanted to establish this inducible overexpression system in a cell line which
does not contain endogenous HIF-1α and no basal level secretion of Hsp90α. Hence we
chose HDF cells and infected them with HIF-1αCA/pSLIK. Once the infected cells were
selected with Hygromycin-B, they were subcultured to six 10cm dishes to a confluence of
85-90% The cells were then used to perform dose curve experiment as shown in Figure 16.
24

Figure 16: Dose curve kinetics for HIF-1α CA induction by doxycycline in HDF cells.
The cells were treated with various doses of doxycycline concentration (0, 0.03, 0.1, 0.3, 1
and 3μg/ml) for a constant time of 24 hours. As shown in Figure 16, a gradual induction of
HIF-1αCA with increasing concentrations of doxycycline was observed and the induction
started to stabilize from 0.3μg/ml of doxycycline onwards. Hence this concentration has
been chosen and a time course experiment is to be performed.
3.7 Dose Curve showing HIF-1αDN induction by Doxycycline in MDA-MB-231 cells
A dose curve experiment was performed on MDA-MB-231 cells infected with HIF-
1αDN/pSLIK. Post Hygromycin-B selection, the cells were plated to 85-90% confluence in
six 10cm dishes and allowed to attach overnight. The next morning, media was replaced by
serum-free media.
25

Figure 17: Dose curve kinetics for HIF-1α DN induction by doxycycline in MDA-MB-231
cells.
The cells were then treated with various doses of doxycycline concentration (0, 0.03, 0.1,
0.3, 1 and 3μg/ml) for a constant time of 24 hours. Since HIF-1αDN has a C-terminal
truncation, most of the commercial antibodies did not recognize it. Hence we used a HA
tag antibody to detect HA-DN-HIF-1α. As shown in Figure 17, a gradual induction of HIF-
1αDN with increasing concentrations of doxycycline was observed and the induction
started to stabilize from 1 μg/ml of doxycycline onwards. Hence this concentration was
chosen and a time course experiment was performed.
3.8 Time Course showing HIF-1α DN induction by Doxycycline and secretion of
Hsp90α in MDA-MB-231 cells
A time course experiment was performed in order to determine the minimum time required
to induce HIF-1αDN. As previously described, MDA-MB-231 cells infected with HIF-
1αDN/pSLIK were plated to 85-90% confluence in six 10cm dishes, post Hygromycin-B
selection. The cells were allowed to attach overnight and the following day, media was
replaced with serum-free media.
26

Figure 18: Time course kinetics showing HIF-1α DN induction by doxycycline and
secretion of Hsp90α in MDA-MB-231 cells.
Based on the dose curve experiment result shown in Figure 17, doxycyline concentration
of 1 μg/ml was chosen and HIF-1αDN induction at various time points (0, 20min, 1h, 3h,
9h, and 24h) was determined by Western blotting. As shown in Figure 18, HIF-1αDN
induction started from 3h onwards.
When the CM of these cells was concentrated for secreted Hsp90α, we observed secretion
of Hsp90α at 24h, but not at other time points.
27

Figure 19: Time course kinetics showing absence of HIF-1αDN induction in the absence
of doxycycline in MDA-MB-231 cells.
However in the case of control experiment, MDA-MB-231 cells transduced with HIF-
1αDN/ pSLIK were not treated with doxycycline. Hence there was no induction of HIF-
1αDN due to absence of doxycycline as shown in Figure 19. However we still observed
secretion of Hsp90α at 24h. This experiment needs better design, since a dominant
negative mutation has been reported to block Hsp90α secretion previously. Hence we must
either knockdown endogenous HIF-1α in the case of MDA-MB-231 cells or use a cell line
which does not have endogenous HIF-1α to study the role of this DN mutant in regulating
Hsp90α secretion.
3.9 Dose Curve showing HIF-1αCA-719 induction by Doxycycline in MDA-MB-231
cells
28

After Hygromycin-B selection, MDA-MB-231 cells infected with HIF-1αCA-719/pSLIK
were plated to 85-90% confluence in six 10cm dishes and allowed to attach overnight.
Media was replaced by serum-free media the next morning.

Figure 20 : Dose curve kinetics for HIF-1α CA-719 induction by doxycycline in MDA-
MB-231 cells.
The cells were then treated with various doxycycline concentrations (0, 0.03, 0.1, 0.3, 1
and 3μg/ml) for a constant time of 24 hours. As shown in Figure 20, an induction of HIF-
1αCA-719 was observed at 0.03μg/ml, and was highest at 0.1 and 0.3μg/ml of
doxycycline.

29

4.0 DISCUSSION
HIF-1α protein levels in the case of normal cells under normoxic conditions are usually
very low or undetected. This is because of the constant oxygen dependant hydroxylation
and degradation of HIF-1α by the ubiquitination-proteasome pathway. However, in the
case of hypoxia, HIF-1α starts to accumulate in the cytosol and can dimerize with the
constitutively present HIF-1β in the cytosol, translocate into the nucleus and initiate
transcription of several downstream genes.
Unlike normal cells, in the case of tumor cells, studies have shown that there is a
constitutive level of HIF-1α due to intratumoral hypoxia and also due to some mutations in
genes encoding for oncoproteins and tumor suppressors. When Dales et al screened
approximately 745 breast cancer patient samples in 2005; he found approximately 25 to
40% of them to be HIF-1α positive. The constitutively expressed HIF-1α in tumors has
often been linked to large tumor size and tumor metastasis.
Although Hsp90α and Hsp90β share 86% amino acid identity, only secreted Hsp90α has
been shown to play an important role in promoting tumor and tumor stroma cell migration
during invasion and metastasis and also in skin wound healing. Normal cells do not secrete
Hsp90α unless they are under environmental stress, but tumor cells have been shown to
constitutively secrete Hsp90α under both normoxia and hypoxia by Sahu et al in 2012.
MDA-MB-231 is one such breast cancer cell line to constitutively secrete Hsp90α. Studies
has shown that HIF-1α mediates hypoxia-triggered Hsp90α secretion in MDA-MB-231
cells. A dominant negative mutant of HIF-1α was shown to block Hsp90α secretion, but
30

not the constitutively active mutant of HIF-1α. Similar results were shown in primary
human dermal fibroblasts and in keratinocytes as well.
Knockdown of HIF-1α by shRNA in MDA-MB-231 cells was shown to block Hsp90α
secretion, and this was so specific to Hsp90α alone, in that secretion of other molecules for
instance MMP9 was not blocked. When Sahu et al in 2012, then performed a rescue
experiment by exogenously reintroducing HIF-1α into HIF-1α depleted 231 cells, they
found that HIF-1α WT and HIF-1α CA were able to rescue secretion of Hsp90α, but not in
the case of HIF-1α DN. These results indicated that HIF-1α is a direct upstream regulator
of Hsp90α secretion. However, the exact mechanism by which HIF-1α triggers Hsp90α
secretion is still unknown to date.
In the present study, we have investigated if HIF-1α regulated secretion of Hsp90α requires
HIF-1α to dimerize with HIF-1β in the cytosol, translocate into the nucleus and initiate
transcription of downstream genes before triggering Hsp90α secretion, or does HIF-1α
trigger Hsp90α secretion without performing any of the nuclear function.
In order to investigate HIF-1α role in Hsp90α secretion, we wanted to construct a tightly
regulated inducible overexpression system. Hence we constructed a doxycyline controlled
(Tet-ON) single lentiviral vector platform for inducible overexpression of HIF-1α mutants
namely HIF-1α CA, HIF-1α DN and HIF-1α CA719 (Nuclear Localisation Signal mutant).
The advantage of this system over normal overexpression systems is that, expression of
HIF-1α is under the control of the researcher and HIF-1α is induced only by the addition of
doxycycline.
31

Dose curve experiment in MDA-MB-231 cells infected with HIF-1α CA/pSLIK showed
that HIF-1αCA induction stabilized from 0.3μg/ml of doxycycline concentration onwards.
Hence this concentration was chosen for time course experiment. At a constant
doxycycline concentration of 0.3μg/ml, HIF-1α CA was induced at 1h and started to
saturate from 3h. However, when the CM of these cells was used to detect secreted
Hsp90α, Hsp90α secretion occurred only at 24h. This delay in secretion might probably be
because of the nuclear translocation of HIF-1αCA once it dimerizes with HIF-1β in the
cytosol. Further studies need to be conducted to verify the same.
In the case of HIF-1αDN, at a constant doxycycline concentration of 1μg/ml, HIF-1αDN
induction was observed from 3h onwards. However, secretion of Hsp90α was observed at
24h. This secretion could be because of the endogenous HIF-1α present in the MDA-MB-
231 cells. Further studies of the HIF-1α DN/pSLIK in human dermal fibroblasts would be
a possible solution to check if secretion is indeed due to the endogenous HIF-1α or not,
since HDFs lack an endogenous HIF-1α.
Preliminary dose curve kinetics of HIF-1αCA-719 has shown induction of HIF-1αCA-719
to be highest at 0.1 and 0.3μg/ml of doxycycline. Since this mutant of HIF-1α is a Nuclear
Localisation Signal mutant, we expect not to see secretion of Hsp90α. Time course kinetics
need to be carried out to verify if the same is true.

32

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Abstract (if available)

Abstract HIF-1α protein levels in the case of normal cells under normoxic conditions are usually very low or undetected. Unlike normal cells, in the case of tumor cells, studies have shown that there is a constitutive level of HIF-1α due to intratumoral hypoxia and also due to some mutations in genes encoding for oncoproteins and tumor suppressors. Normal cells do not secrete Hsp90α unless they are under environmental stress, but tumor cells have been shown to constitutively secrete Hsp90α under both normoxia and hypoxia. Knockdown of HIF-1α by shRNA in MDA-MB-231 cells was shown to block Hsp90α secretion in a previous study. Their results indicated that HIF-1α is a direct upstream regulator of Hsp90α secretion. However, the exact mechanism by which HIF-1α triggers Hsp90α secretion is still unknown to date. In the present study, we have investigated if HIF-1α regulated secretion of Hsp90α requires HIF-1α to dimerize with HIF-1β in the cytosol, translocate into the nucleus and initiate transcription of downstream genes before triggering Hsp90α secretion, or does HIF-1α trigger Hsp90α secretion without performing any of the nuclear function

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Asset Metadata

Creator Ashok, Archana (author)

Core Title Study of the mechanism by which hypoxia-inducible-factor-1 alpha (HIF-1α) regulates heat shock protein-90 alpha (Hsp90α) secretion in breast cancer

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 04/22/2014

Defense Date 03/13/2014

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

Tag breast cancer,HiF-1α,OAI-PMH Harvest,secreted Hsp90α

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Advisor Li, Wei (committee chair), Liang, Chengyu (committee member), Ou, J.-H. James (committee member)

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