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Molecular mechanisms of chemoresistance in breast cancer
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Molecular mechanisms of chemoresistance in breast cancer
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Content
MOLECULAR MECHANISMS OF CHEMORESISTANCE IN
BREAST CANCER
by
Minal Chandravadan Patel
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2008
Copyright 2008 Minal Chandravadan Patel
ii
Dedication
To my dearest parents Chandravadan and Aarti Patel,
my grandparents,
and Shobha Aunty and Smita Aunty.
iii
Acknowledgements
I would like to thank the members of my committee for their kind support. Their guidance has
been most appreciated. I would like to thank Dr. Amy Lee for giving me the opportunity to
participate in her lab and learn so much in my time there. I would like to thank Dr. Cote for his
support and confidence in me. I would also like to thank Dr. Zoltan Tokes for guiding me
through this process with kindness and good humor.
I would like to express my appreciation to members of the Lee lab for their helpfulness: Dezheng
Dong, Jianze Li, Yong Fu, Peter Baumeister, Min Ni, Risheng Ye, Miao Wang, Yi Zhang and
Shiuan Wey. I would like to especially thank Yong Fu for his guidance and patience as well as
Peter Baumeister for his encouragement.
I would like to express thanks to members of the Cote lab for their help and encouragement: Dr.
Debra Hawes, Dr. Nancy Barr, Dr. Ram Datar, Dr. Shan-rong Shi, Dr. Mohammad Alavi, Lillian
Young, William Win, Carmela Villajin-Busque, Anirban Mitra, Llana Pootrakul, Henry Lin and
Anthony Williams. I would especially like to thank Dr. Debra Hawes for her guidance and Win
for his support throughout.
iv
Table of Contents
Dedication ii
Acknowledgements iii
List of Figures vi
Abstract vii
Chapter 1: Introduction 1
Chapter 2: Interaction of GRP78 and BIK and the effect of BIK mutagenesis 4
2.1 Introduction 4
2.2 Materials and Methods 6
2.2.1 Cell Culture 6
2.2.2 Plasmids 6
2.2.3 Site-directed mutagenesis 6
2.2.4 Plasmid MiniPrep and MaxiPrep 7
2.2.5 Transient Transfection 9
2.2.6 Co-immunoprecipitation 9
2.2.7 Western blots 10
2.3 Results 12
2.3.1 Deletion of BH3-only domain in pcDNA3-Flag-BIK plasmid 12
2.3.2 Association between BIK, GRP78 and BCL-2 15
2.4 Discussion 18
Chapter 3: Characterization of Estrogen Starvation Resistant MCF-7/BUS Clones 19
3.1 Introduction 19
3.2 Materials and Methods 20
3.2.1 Cell culture and treatments 20
3.2.2 Western Blots 20
3.2.3 Si94 in MCF-7/BUS Resistant Clones 21
3.2.4 Trypan Blue Exclusion Cell Death Assay 22
3.3 Results 23
3.3.1 Elevated levels of GRP94 in resistant MCF-7/BUS clones. 23
3.3.2 Higher levels of cell death in si94-treated resistant MCF-7/BUS clones. 26
3.4 Discussion 30
Chapter 4: Putative breast cancer stem cell population in lymph node occult
micrometastases of breast cancer patients 32
4.1 Introduction 32
4.2 Materials and Methods 34
4.2.1 Double-staining immunohistochemistry 34
4.2.2 Light microscopy 36
4.2.3 Spectral imaging 36
v
4.3 Results 37
4.3.1 Lymph node occult micrometastases in lymph node negative breast cancer
patients possess a minor putative breast cancer stem cell population. 37
4.4 Discussion 41
Chapter 5: Summary 43
Bibliography 47
vi
List of Figures
Fig. 2.1 Schematic of BCL-2 family members: BH3-only proteins 5
Fig. 2.2 Successful deletion of BH3-only domain in pcDNA3-Flag-BIK
plasmid yielded mutant pcDNA3-FLAG-BIK 13
Fig. 2.3 Sequence results indicated successful BH3-only domain deletion 14
Fig. 2.4 Deletion of the BH3-only domain in BIK results in altered protein
interactions in 293T cells. 17
Fig. 3.1 Elevated levels of GRP94 in estrogen-starvation resistant MCF-7/BUS
breast cancer clones. 24
Fig. 3.2 Elevated levels of GRP94 in estrogen-starvation resistant MCF-7/BUS
breast cancer clones. 25
Fig. 3.3 Higher levels of cell death in si94-treated MCF-7/BUS resistant clones. 28
Fig. 3.4 Higher percent of cell death in si94-treated MCF-7/BUS resistant clones. 29
Fig. 4.1 Double immunohistochemical staining reveals a population of putative
breast cancer stem cells. 39
Fig. 4.2 Lymph node occult micrometastases in lymph node histologic-negative
breast cancer patients possess a minor putative breast cancer stem cell population. 40
vii
Abstract
Resistance to chemotherapy represents a major obstacle in breast cancer treatment. GRP78 is a
protein upregulated in malignant but not benign human breast tumors, and is associated with
resistance to chemotherapy in breast cancer patients. While it has been shown that this resistance
depends on the BH3-only protein BIK, the mechanism of interaction between GRP78 and BIK
still remains to be elucidated. Investigating the nature of this interaction, we discovered that
deletion of the BH3-only domain in BIK results in altered protein interactions in 293T cells.
Characterization of MCF-7/BUS human breast cancer cell clones that were resistant to estrogen-
starvation therapy showed markedly elevated levels of GRP94. Knockdown of GRP94 by small
interfering RNA in these resistant clones led to an increase in cell death. Resistance to
chemotherapy is also a characteristic of cancer stem cells so we characterized molecular markers
of putative breast cancer stem cells. Our results showed a minor putative breast cancer stem cell
population in lymph node occult micrometastases. Overall these results help elucidate molecular
characteristics that contribute to chemoresistance and that may serve as valuable targets for
therapy, as well as help delineate molecular mechanisms contributing to estrogen-starvation
therapy resistance in breast cancer.
1
Chapter 1: Introduction
Breast cancer treatment involves cytotoxic chemotherapy to kill rapidly dividing cancer cells.
Amongst other physiological effects, chemotherapy can reduce estrogen production by the
ovaries, which has been shown to increase patient survival rates and decrease relapse incidence in
women under the age of 70 (Cochrane collaboration, 2001). Estrogen is important in estrogen
receptor-positive breast cancer cell proliferation as well as in breast cancer cell survival (Lee,
2005) and blocking the estrogen receptor signaling pathway is important in breast cancer
treatment. Aromatase inhibitors can also block estrogen production by blocking the enzyme
aromatase from converting androgens to estrogens (Mokbel, 2002). Estrogen can cause breast
cancer cell growth, and estrogen-starvation therapy is thought to be the reason adjuvant treatment
is so effective in premenopausal women.
But what remains one of the biggest problems with any chemotherapy in patients of all
ages is the development of resistance. Tumor cells can acquire characteristics that render them
resistant to further treatment, so tumor cells will continue to grow despite therapy. The question
arises as to what confers this resistance. GRP78 is overexpressed
in malignant but not benign
human breast lesions, and associates
with resistance to chemotherapy in breast cancer patients
(Fernandez, 2000). Furthermore, GRP78 knockdown results in abatement or regression of
tumors. GRP78 confers chemoresistance to cell against drugs such as doxorubicin and other
topoisomerase inhibitors (Zhang, 2006). Based on these findings questions arise as to the
mechanism of resistance conferred by GRP78.
GRP78/BIP, or glucose-regulated protein 78, is a molecular chaperone protein found in
the endoplasmic reticulum (ER). The functions of GRP78 in the ER include protein folding,
prevention of protein aggregation, calcium binding, maintenance of ER homeostasis, regulation
of the unfolded protein response and activation of ER stress pathway components (Ni, 2007).
2
GRP78 is prosurvival in function and suppresses ER stress-induced apoptosis as part of the
unfolded protein response (Kaufman, 2002). The unfolded protein response (UPR) is a response
to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Upon ER stress,
GRP78 releases and thereby activates PERK, IRE1, and ATF6 pathways, which activate nuclear
signals to induce the UPR. GRP78 allows for cell survival in stressful environments such as the
tumor microenvironment where cells may be exposed to conditions such as glucose starvation or
hypoxia.
BIK, previously known as Bip1, is a proapoptotic protein that possesses a BH3-only
domain. Like most other proapoptotic BH3-only proteins, when BIK is activated it binds BCL-2
and other antiapoptotic proteins to neutralize their prosurvival activities. Unlike many other
BH3-only proteins, BIK is localized in the ER (Germain and Mathai, 2002). It is known to play a
vital role in estrogen-starvation induced apoptosis in breast cancer (Hur, 2004). In MCF-7/BUS
human breast cancer cells, estrogen-starvation therapy causes an induction of BIK mRNA and
protein, and BIK knockdown results in inhibition of estrogen-starvation induced apoptosis.
Drugs such as doxorubicin also induce BIK.
The lab of my mentor, Amy Lee, found that BIK selectively complexes with
the glucose-regulated
protein GRP78/BiP (Fu, 2007). GRP78 confers resistance to
estrogen starvation–induced
apoptosis in human breast cancer cells via a novel
mechanism
mediated by BIK. They found that, GRP78 overexpression inhibited estrogen
starvation–induced
mitochondrial permeability transition and apoptosis in estrogen
receptor-positive MCF-7/BUS human breast cancer cells.
Knockdown of endogenous
GRP78 by small interfering RNA (siRNA) sensitized MCF-7/BUS cells to estrogen
starvation–induced
apoptosis, but the effect was markedly reduced when BIK expression
3
was also reduced by siRNA. Whereas these relationships have been characterized, the
exact mechanism of interaction between GRP78 and BIK still remain to be elucidated.
While GRP78 overexpression inhibits response to estrogen-starvation therapy, there are
also other characteristics of cancer cells that render them less responsive to therapy in general.
Cancer cells that are more likely to become invasive and metastasize are those with putative stem
cell-like characteristics (Dick, 1997). Cancer stem cells and stem cells both have the shared
properties of self-renewal and differentiation. The ability to continually perpetuate drives
tumorigenesis. Even miniscule quantity of these cancer stem cells transmitted to a new animal
can cause tumor growth (J. Dick, 1997; Marx, 2007). Cancer stem cells have been found in a
variety of cancers, including breast cancer, leukemia, melanoma, lung, pancreatic and prostate
cancer and have been implicated in invasion, metastasis, and angiogenesis. It is often this
population of tumor cells that is most resistant to chemotherapy or drug treatment and it is very
important to characterize these putative stem cells for therapeutic and diagnostic purposes. This
study will examine molecular mechanisms of GRP78-mediated resistance to estrogen-starvation
therapy in breast cancer, as well as investigate molecular characteristics of putative breast cancer
stem cells in breast cancer metastases. The three sections will examine: the interaction of GRP78
and BIK and implications in resistance to estrogen-starvation therapy, characterization of protein
levels in estrogen-starvation resistant MCF-7/BUS breast carcinoma clones and characterization
of the putative breast cancer stem cell population in lymph node occult micrometastases.
4
Chapter 2: Interaction of GRP78 and BIK and effect of BIK
mutagenesis
2.1 Introduction
Treatment for estrogen-receptor positive breast cancer has significantly improved with the advent
of aromatase inhibitor drugs that block estrogen production (Mokbel, 2002). However, breast
cancers that are responsive to treatment at first often become refractory because they acquire
adaptations that lead to resistance. Breast cancer cell apoptosis induced by estrogen-starvation
requires BIK, a pro-apoptotic BH3-only protein located predominantly at the ER (Hur, 2004).
GRP78 is a resident ER protein that belongs to the family of HSP70 heat shock proteins
and functions in preventing ER stress-induced apoptosis (Rauschert, 2008). In the stressful
environment in many tumors GRP78 is found to be upregulated.
The lab of Amy Lee, my mentor, discovered that endogenous BIK complexes with
glucose-regulated protein GRP78/BiP, a highly conserved ER chaperone that is upregulated in
many tumors and in approximately 70% of breast tumors (Fu, 2007). In human cell cultures,
GRP78 overexpression reduces BIK-induced apoptosis. In estrogen-dependent MCF-7/BUS
breast cancer cells, GRP78 overexpression inhibits estrogen-starvation induced mitochondrial
permeability transition, and subsequent apoptosis. Furthermore, siRNA knockdown of
endogenous GRP78 sensitizes MCF-7/BUS cells to estrogen-starvation induced apoptosis. This is
the first evidence that GRP78 confers resistance to estrogen-starvation induced apoptosis in
human breast cancer cells through inhibition of the action of the BH3-only protein BIK.
However, the exact mechanism of resistance and the interaction between GRP78 and Bik remains
to be elucidated.
5
Fig. 2.1 Schematic of BCL-2 family members: BH3-only proteins. This schematic depicts members
of the BCL-2 family including prosurvival BCL-2-like members as well as proapoptotic BH3-only
members such as BIK (Inohara, 2001). BIK’s BH3-only domain is responsible for protein dimerization.
BIK’s BH3-only domain is required for interaction with anti-apoptotic proteins (Elangovan,
1997). Structural studies have shown that proapoptotic BH3-only proteins possess an
amphipathic helix whose hydrophobic face inserts into the hydrophobic grooves of the BH1, BH2
and BH3 domains in antiapoptotic proteins (Petros, 2000; Liu, 2003). It has been shown that
transfection of BIK/Bip1 BH3-only domain deletion mutants resulted in lower rates of cell death
as well as an inability to interact with Bcl-2 (Chittenden, 1995). Based on this information, we
hypothesized that mutation of the BH3-only domain of BIK may interfere with GRP78-BIK
interaction.
6
2.2 Materials and Methods
2.2.1 Cell Culture
293T human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium
(DMEM)
supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (PS).
2.2.2 Plasmids
The wildtype pcDNA3-Flag-BIK plasmid was obtained from G.C. Shore (McGill University,
Montreal, Canada). Plasmid construction has been previously described.
2.2.3 Site-directed mutagenesis
Site-directed mutagenesis was used to delete the BH3-only domain of the pcDNA3-Flag-BIK
plasmid. The oligonucleotide primer sequences utilized for site-directed deletion of the BH3-
only domain in BIK were: forward primer 5'-agtgacgcactcagggccccgcgcctggcccagc-3' and reverse
primer 5'-ggccctgagtgcgtcactgccctccatgcattcc-3'. The BIK protein sequence deleted out was
ALRLACIGDEMDVSL.
The QuikChange
Site-Directed Mutagenesis Kit protocol was used to mutate a template
wild type pcDNA3-Flag-BIK plasmid. Two complimentary oligonucleotide primers were
designed as described above. The sample reactions were then prepared using 125 ng of each
oligonucleotide primer, varying levels of double-stranded DNA template (5 ng, 20 ng, 50 ng), 1
μl dNTP mix, 5 μl 10x reaction buffer (composition: 100 mM KCl, 100 mM (NH
4
)
2
SO
4,
200 mM
Tris-HCl ph 8.8, 20 mM MgSO
4
, l% Triton
X-100, 1 mg/ml nuclease-free bovine serum
albumin or BSA ), 1 μl PfuTurbo
DNA polymerase, and distilled H
2
O to a final volume of 50
7
μl. The reaction was cycled through the Mastercycler gradient Thermal Cycler (Eppendorf) using
the following cycling parameters: first cycle for 30 seconds at 95
o
C and the remaining 18 cycles
cycle between 30 seconds each at 95
o
C, one minute at 55
o
C, and 5 minutes at 68
o
C. After
thermal cycling, the reaction was placed on ice for two minutes to cool it to 37
o
C. The sample
reaction underwent Dpn-1 restriction enzyme digestion in which 1 μl of Dpn-1 was added to the
sample reaction and incubated for one hour at 37
o
C to digest any non-mutated supercoiled
double-stranded DNA. Then, 1 μl of Dpn-1 treated DNA was transformed into 50 μl DH-5α
Supercompetent E. Coli Cells, gently swirled, and incubated on ice for 30 minutes. The
transformation reaction was then heat pulsed for 45 seconds at 42
o
C and placed on ice for 2
minutes to optimize transformation efficiency. 0.5 ml of NZY broth preheated to 42
o
C was
added to the transformation reactions, and this mixture was incubated for one hour with shaking
at 225-250 rpm. 250 μl of sample reaction was put on each of two LB-Ampicillin Agar plates
and incubated at 37
o
C for at least 16 hours or overnight.
Smaller colonies were selected 24 hours later, expanded in 2-5 ml of LB-Ampicillin
media in an incubator-shaker at 37
o
C overnight, and then expanded in 150 ml LB-Ampicillin
media for another 12-16 hours. Mutant pcDNA3-Flag-BIK plasmid was isolated from these
cultures using QIAPrep
Spin Miniprep Kit
and Qiagen Plasmid Purification Maxi Kit. Glycerol
stocks of the mutant pcDNA3-Flag-BIK plasmid were made by adding 800 μl of 50% glycerol to
200 μl of bacterial cells from the expanded cultures. Mutations were confirmed by forward and
reverse sequencing (DNA Core, University of Southern California, Los Angeles, CA).
2.2.4 Plasmid Mini Prep and MaxiPrep
Mutant pcDNA3-Flag-BIK plasmid was isolated from the above cultures using the QIAPrep
Spin Miniprep Kit protocol. This protocol should be used for plasmid DNA grown up in 1-5 ml
overnight cultures of E. Coli in Luria-Bertani (LB) medium. Briefly, 250 μl Buffer P1 with
8
Rnase A added was used to resuspend the pellet of bacterial cells and then the entire volume
transferred to a microcentrifuge tube. 250 μl of Buffer P2 was added and the tube inverted 4-6
times until the solution became slightly clear and viscous. 350 μl Buffer N3 was then added and
the tube inverted 4-6 times until the solution becomes cloudy. The tube was centrifuged for 10
minutes at 13000 rpm in a table-top microcentrifuge. The supernatant was applied to a QIAprep
spin column by decanting, this was centrifuged for 60 seconds and the flow-through discarded.
0.75 ml Buffer PE was used to wash the QIAprep spin column and this was centrifuged for 30-60
seconds. The flow-through was discarded and centrifuged for another one minute to remove any
residual wash buffer. The QIAprep column was placed in a new 1.5 ml microcentrifuge tube and
50 μl Buffer EB (10 mM Tris-HCl, pH 8.5) added to elute the DNA.
Larger preparations of mutant pcDNA3-Flag-BIK plasmid were isolated from 150 ml
bacterial cultures using the Qiagen Plasmid Purification Maxi Kit protocol, with some minor
procedural modifications. Briefly, after addition of 10.5 ml isopropanol and centrifugation at
15000g for 30 minutes at 4
o
C, the isopropanol was carefully decanted so as to not disturb the
pellet. To preserve as much plasmid DNA as possible, 1-1.5 ml of 70% ethanol was then added
to dissolve the pellet and the mixture was pipetted up and down 10 times. This solution was
transferred into a 1.7 ml Eppendorf tube and spun down in a microcentrifuge. The ethanol was
carefully removed and the pellet allowed to air-dry for 5-10 minutes. An appropriate volume of
TE Buffer (composition: 10mM Tris-HCl pH 8.0, 1 mM EDTA) was added to dissolve the pellet.
If a more concentrated plasmid preparation is necessary then alcohol precipitation can be utilized.
Briefly, 1/10 volume of 3 M sodium acetate was added to a maximum volume of 450 μl of DNA
in water and briefly inverted. Two volumes of 100% ethanol were added and inverted many
times to mix well. DNA was precipitated by placing the sample in dry ice for 5 minutes. The
sample was centrifuged at high speed for 15-30 minutes at 4
o
C. The supernatant was decanted
9
and the pellet located and labeled. The tube was drained by inversion, the pellet washed with
cold 70% ethanol, and the pellet dried again. The DNA was resuspended in TE (10 mM Tris-
HCl, 0.1 mM EDTA; pH 8.0) and stored at 4
o
C. This, in effect, allows the DNA to be re-
precipitated and resuspended in a smaller volume, thereby concentrating the DNA.
2.2.5 Transient Transfection
The day before transfection 6 x 10
5
293T human embryonic kidney cells were seeded in a 6-well
plate in 3 mL growth media without antibiotics (DMEM+10% FBS only) and incubated at 37
o
C
and 5% CO
2
. They were grown to 40% to 60% confluence. Two μg of mutant pcDNA3-Flag-
BIK plasmid was diluted to a final volume of 100 μl in media lacking serum, protein or
antibiotics. Empty vector as well as wild type pcDNA3-Flag-BIK plasmid were used as
transfection controls. 20 μl of Qiagen PolyFect Transfection Reagent was added and mixed into
the solution by pipeting for 10 seconds, and the DNA solution was incubated for 5-10 minutes at
20-25
o
C to allow complex formation. While this was incubating, growth medium from the plate
was carefully aspirated and 1.5 ml of fresh media with antibiotics and serum was added to the
cells. 0.6 ml of growth media with antibiotics and serum were then added to the transfection
complexes, mixed briefly by pipetting, and transferred entirely to the cells in the 6-well plate.
The plate was briefly swirled to evenly disperse complexes. The plate was incubated at 37
o
C and
5% CO
2
for 6-8 hours, then the media was gently aspirated and replaced with fresh growth media
containing antibiotics and serum. This was incubated for 48 hours, and cells were then utilized in
Western Blot and co-immunoprecipitation.
2.2.6 Co-immunoprecipitation
Immunoprecipitation (IP) extraction buffer was prepared as follows: 50mM Tris (pH 7.5), 150
mM NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, with a protease inhibitor cocktail tablet
(Roche Applied Science) added for every 10 ml buffer. 293T cells were collected and lysed in IP
10
extraction buffer. Briefly, cells were washed twice and dissolved in extraction buffer. Lysates
were then frozen in liquid nitrogen and thawed in a 37
o
C water bath three times. Cell lysates
were centrifuged at 14000 rpm at 4
o
C for 20 minutes and the clear supernatant was collected. 1
mg of protein extract from each sample was pretreated by rotation with 50 μl Protein G Agarose
beads (Pierce Biotechnology, Rockford, IL) for one hour at 4
o
C. Samples were then incubated
for two hours with 5 μg of either goat anti-BIK N19 antibody (Santa Cruz Biotechnology) or
mouse anti-Flag M2 antibody (Sigma-Aldrich). Goat and mouse immunoglobulin G (IgG)
antibodies were used as controls (Santa Cruz Biotechnology). After incubation, 50 μl of Protein
G Agarose beads was added and the mixture was rotated overnight at 4
o
C. The following day,
the mixture was spun down and the pellet was washed with IP extraction buffer five times. 50 μl
SDS-PAGE protein loading buffer was added to release product from the beads and this mixture
was boiled for five minutes.
The samples were centrifuged and the supernatants resolved by SDS-PAGE and analyzed
by Western Blot using anti-GRP78, anti-BCL2, anti-BIK, and anti-FLAG antibodies to identify
any proteins that were coimmunoprecipitated.
2.2.7 Western Blots
Resolving gels with the following compositions were prepared for Tris-glycine sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE): 10% separating gel (5.9 ml distilled
H
2
O, 5.0 ml of 30% acrylamide mix, 3.8 ml of 1.5 M Tris (pH 8.8), 0.15 ml of 10% SDS, 0.15 ml
of 10% ammonium persulfate, 0.006 ml TEMED and 5% stacking gel (5.5 ml distilled H
2
O, 1.3
ml of 30% acrylamide mix, 1.0 ml of 1.0 M Tris (pH 6.8), 0.08 ml of 10% SDS, 0.08 ml of 10%
ammonium persulfate, 0.008 ml TEMED). Samples to be loaded into the gel were mixed with 4x
SDS-PAGE loading buffer, boiled for five minutes, and spun down at 3000 rpm for five minutes.
Samples, controls, and a protein-loading marker were loaded into the gel and proteins were
11
separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). Wet transfer to a
nitrocellulose membrane was then performed overnight (or for at least 16 hours). After washing
the membrane with distilled H
2
O, the membrane was incubated in 5% Blotting Grade Blocker
Non-Fat Dry Milk (BioRad, Hercules, CA) in TBS-T (TBS-T composition: 0.1% solution of
Tween 20 in TBS) on an Rotomix orbital shaker (Thermolyne; Dubuque, Iowa) at 25
o
C for one
hour in order to block non-specific binding sites. The membrane was then incubated in diluted
primary antibody on an orbital shaker overnight (16-24 hours) at 4
o
C. The next day fresh TBS-T
was added to the membrane and washed on an orbital shaker at room temperature for 15 minutes;
this procedure was repeated three times. The membrane was then incubated in diluted secondary
antibody for two hours at room temperature. The membrane was washed again as described
above. The membrane was visualized using enhanced chemiluminescence (ECL) signal detection
reagents (consisting of SuperSignal West Femto Stable Peroxide Buffer and Luminol/Enhancer
Solution). 0.5 ml of solution 1 was mixed with 0.5 ml solution 2 and used to cover entire
membrane surface. After incubation for two minutes the excess ECL was drained off and the
membrane, placed in plastic wrap so that the solution could not escape, was exposed using
autoradiography film.
The primary antibodies utilized were: goat anti-GRP78 C20 (1:1000 dilution; Santa Cruz
Biotechnology), goat anti-GRP94 C-19, mouse anti-BCL2 C-2 (1:1000 dilution; Santa Cruz
Biotechnology), goat anti-BIK N-19 (1:500 dilution; Santa Cruz Biotechnology), and mouse anti-
Flag M2 (1:1000 dilution; Sigma-Aldrich). The appropriate horseradish peroxidase-conjugated
secondary antibodies (IgG-HRP) were utilized at a 1:1000 dilution (Santa Cruz Biotechnology).
Mouse anti-CHOP/GADD153 B-3, mouse anti-beta-actin C-2, rabbit anti-calnexin C-20, rabbit
anti-ATF4 C-20, goat anti-BIK N-19 (1:500), rabbit anti-calreticulin/calregulin H-170, rabbit
anti-PDI H-160 (1:200), mouse anti-CHOP B-3 (Santa Cruz Biotechnology).
12
2.3 Results
2.3.1 Deletion of BH3-only domain in pcDNA3-Flag-BIK plasmid
Wildtype pcDNA3-Flag-BIK plasmid underwent site-directed mutagenesis using the
QuikChange
Site-Directed Mutagenesis Kit. The forward primer had nucleotide sequence
5'-agtgacgcactcagggccccgcgcctggcccagc-3' and the reverse primer had nucleotide sequence
5'-ggccctgagtgcgtcactgccctccatgcattcc-3'.
When the resulting plasmid was run on a gel (Fig. 2.4) the purity of the sample was
confirmed. It was then forward- and reverse-sequenced (Fig. 2.5) and results indicated successful
site-directed mutagenesis which involved BH3-only domain deletion with the rest of the
pcDNA3-Flag-BIK plasmid still intact.
13
Fig. 2.2 Successful deletion of BH3-only domain in pcDNA3-Flag-BIK plasmid yielded mutant
pcDNA3-FLAG-BIK (mutant FLAG-BIK). Wildtype pcDNA3-Flag-BIK plasmid underwent site-
directed mutagenesis with forward primer with nucleotide sequence 5'-agtgacgcactcagggccccgcgcctggcccagc-3'
and reverse primer 5'-ggccctgagtgcgtcactgccctccatgcattcc-3'.
14
Fig. 2.3 Sequence results indicated successful BH3-only domain deletion (with BIK’s BH3-only
domain in bold). The sequence comparison of wildtype pcDNA3-Flag-BIK plasmid (WtFlagBIK) versus
mutant pcDNA3-Flag-BIK plasmid (MuFlagBIK). Sequence matches are indicated with an asterisk and
regions of sequence variation are indicated with dashed line.
WtFlagBIK ATGGACTACAAAGACGATGACGATAAAGCCTCTGAAGTAAGACCCCTCTCCAGAGACATC
MuFlagBIK ATGGACTACAAAGACGATGACGATAAAGCCTCTGAAGTAAGACCCCTCTCCAGAGACATC
************************************************************
WtFlagBIK TTGATGGAGACCCTCCTGTATGAGCAGCTCCTGGAACCCCCGACCATGGAGGTTCTTGGC
MuFlagBIK TTGATGGAGACCCTCCTGTATGAGCAGCTCCTGGAACCCCCGACCATGGAGGTTCTTGGC
************************************************************
WtFlagBIK ATGACTGACTCTGAAGAGGACCTGGACCCTATGGAGGACTTCGATTCTTTGGAATGCATG
MuFlagBIK ATGACTGACTCTGAAGAGGACCTGGACCCTATGGAGGACTTCGATTCTTTGGAATGCATG
************************************************************
WtFlagBIK GAGGGCAGTGACGCATTGGCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGACGTGAGC
MuFlagBIK GAGGGCAGTGACGCA---------------------------------------------
***************
WtFlagBIK CTCAGGGCCCCGCGCCTGGCCCAGCTCTCCGAGGTGGCCATGCACAGCCTGGGTCTGGCT
MuFlagBik CTCAGGGCCCCGCGCCTGGCCCAGCTCTCCGAGGTGGCCATGCACAGCCTGGGTCTGGCT
************************************************************
WtFlagBIK TTCATCTACGACCAGACTGAGGACATCAGGGATGTTCTTAGAAGTTTCATGGACGGTTTC
MuFlagBIK TTCATCTACGACCAGACTGAGGACATCAGGGATGTTCTTAGAAGTTTCATGGACGGTTTC
************************************************************
WtFlagBIK ACCACACTTAAGGAGAACATAATGAGGTTCTGGAGATCCCCGAACCCCGGGTCCTGGGTG
MuFlagBIK ACCACACTTAAGGAGAACATAATGAGGTTCTGGAGATCCCCGAACCCCGGGTCCTGGGTG
************************************************************
WtFlagBIK TCCTGCGAACAGGTGCTGCTGGCGCTGCTGCTGCTGCTGGCGCTGCTGCTGCCGCTGCTC
WtFlagBIK TCCTGCGAACAGGTGCTGCTGGCGCTGCTGCTGCTGCTGGCGCTGCTGCTGCCGCTGCTC
************************************************************
WtFlagBIK AGCGGGGGCCTGCACCTGCTGCTCAAGTGA
WtFlagBIK AGCGGGGGCCTGCACCTGCTGCTCAAGTGA
******************************
15
2.3.2 Association between BIK, GRP78 and BCL-2
To examine the association between GRP78, BCL-2, and mutant pcDNA3-FLAG-BIK with the
BH3-only domain deletion, 293T cells were transfected with mutant pcDNA3-FLAG-BIK
plasmid, wildtype pcDNA3-FLAG-BIK plasmid with intact BH3-only domain, and empty buffer
transfection control. Interactions were examined via co-immunoprecipitation (co-IP) assays.
Results for IP using anti-BIK antibody (Fig 2.6) showed that in the cell lysates, similar
levels of GRP78 were seen in the wildtype (wt) FLAGBIK, mutant (mut) FLAGBIK and empty
buffer control samples. BCL-2 levels were also similar in the cell lysates in all three cases. BIK
levels in the cell lysates were higher in mut FLAGBIK input than in wt FLAGBIK input. BIK
levels represent both endogenous BIK and transfected FLAGBIK levels. The control lane
showed very low levels of BIK, which is expected as the basal level of BIK in 293T cells is low
under normal cell culture conditions (Fu, 2007). FLAG levels in cell lysates were very high in
mut FLAGBIK input versus wt FLAGBIK, which was unexpected because equal amounts were
transfected. Control showed no FLAG as expected for the empty buffer control, which was not
transfected with FLAGBIK. These levels reflect only transfected FLAGBIK as there is no
endogenous FLAG in the cells.
In the immunoprecipitated samples, similar levels of GRP78 were seen in the wildtype
FLAGBIK and mut FLAGBIK samples, while the control lane showed lower amounts of GRP78.
Very low levels of BIK-GRP78 binding have been reported in immunoprecipitates of unstressed
MCF-7/BUS cells (Fu, 2007). BCL2 levels were similar in all three lanes, with mut FLAGBIK
levels slightly higher than wt FLAGBIK. For the immunoprecipitated samples, similar BIK
levels were seen for all lanes with mut FLAGBIK showing slightly higher amounts. FLAG levels
were significantly higher for mutant FLAGBIK versus wt FLAGBIK, and absent in control lanes,
which was expected.
16
Considering that there is a much higher level of mut FLAGBIK, equal amounts of both
GRP78 and BCL2 are pulled down by mut FLAGBIK and wt FLAGBIK. This could be that
without the BH3-only domain there is more protein stability and less protein turnover so there is
more mut FLAGBIK expression. Similar levels of interactions for BCL2 and mut FLAGBIK or
wt FLAGBIK could also be due, for example, to BCL2 saturation. This means that for whatever
level of FLAGBIK, samples that immunoprecipitate with BCL2 will be similar because of the
limited amount of BCL2. These results are preliminary and the experiment should be repeated so
that IgG immunoprecipitation controls can be used to separate specific binding from non-specific
binding and the results interpreted in light of this information.
Since the antibody light chain runs at 25 kilodaltons this can obscure Western Blots of
BIK, FLAGBIK and BCL-2, which run at similar levels. This may also explain why a band
appears in the control lane, especially for BIK. Next time, 3X FLAG peptide should be run to
separate the protein of interest from the FLAG antibody so that Western Blots will only display
proteins and not the FLAG antibody itself. Then, results will reflect the separate proteins without
concern for antibody light chains obscuring physiological protein levels.
17
293 T cells
lysate_____ Co-IP: anti-BIK__ _
wt ctrl wt1a wt ctrl
GRP78
BCL2
BIK
FLAG
Fig. 2.4 Deletion of the BH3-only domain in BIK results in altered protein interactions in 293T cells.
293T cells were transfected with 8 μg of wild type pcDNA3-FLAG-BIK per dish, 8 μg of mutant pcDNA3-
FLAG-BIK plasmid (5.5 kb) per dish, or empty buffer control, and cells were harvested after 48 hours.
These were immunoprecipitated with 5 μg anti-Bik antibody, resolved by SDS-PAGE and Western Blotted
with anti-GRP78, anti-BCL2, anti-BIK, and anti-FLAG antibodies.
18
2.4 Discussion
The cell lysates represent the amount of total protein in the sample after transfection , while
immunoprecipitated proteins depict protein that is complexed with BIK or FLAG within these
transfected samples. These experiments are inconclusive and should be repeated with a few
adjustments. There was too much FLAG antibody used in the anti-FLAG immunoprecipitation
itself because even after stripping the blot the heavy chain and light chain of the FLAG antibody
still remained. Since the light chain runs at 25 kilodaltons this will obscure Western Blots of
BIK, FLAGBIK and BCL-2, which run at similar levels. Next time, 3X FLAG peptide should be
run to separate the protein of interest from the FLAG antibody so that Western Blots will only
display proteins and not the FLAG antibody itself. The blot should also be stripped in between
probes for different proteins as some of the proteins are of similar size and will therefore run at
similar positions. Perhaps it would be informative to Western Blot for beta-actin to see if there
are discrepancies in transfection levels or in protein loading. Next time IgG control should be
used for immunoprecipitation to control for non-specific binding.
19
Chapter 3: Characterization of Estrogen Starvation Resistant MCF-
7/BUS Breast Cancer Cell Clones
3.1 Introduction
Estrogen-starvation therapy targets estrogen receptor-positive breast cancer cells and leads to
apoptosis of tumors. However, tumor cells often develop resistance to such therapy. MCF-7/BUS
human breast cancer cell clones that are resistant to estrogen-starvation therapy were previously
generated (Fu, 2007). For selection of resistant cell clones from MCF-7/BUS, the cells were
cultured in estrogen-free medium containing 5% charcoal-dextran stripped fetal calf serum for 5
days and surviving cells were cultured in regular medium for one week to allow expansion. This
selection procedure was repeated 6 times to generate resistant clones capable of growth in
estrogen-free medium. Estrogen-starvation resistant cells were maintained in estrogen-free
medium for 2 months to stabilize the resistant property.
There is a plethora of ER chaperones, co-chaperones and isomerases in the ER. It would
be informative to look at changes in basal levels of protein expression in these resistant clones.
GRP94 is a resident ER chaperone and stress protein that binds calcium, plays a role in protein
folding and assembly, and is antiapoptotic in function. Protein disulfide isomerase (PDI) is a
protein isomerase as well as a chaperone. It is a diverse catalyst of reduction, oxidation and
isomerization of protein disulfides and is also involved in protein folding that is independent of
disulfide bond formation (Song & Wang 1995, Yao et al. 1997). Calnexin is an ER integral
protein that functions as a chaperone and aids in glycoprotein folding. ATF4 is a transcription
factor that induces CHOP and ultimately leads to ER-stress induced apoptosis (Oyadomari, 2004).
20
3.2 Materials and Methods
3.2.1 Cell culture and treatment
The estrogen-dependent MCF-7/BUS human breast cancer cell line was obtained from A.M. Soto
(Tufts University, Medford, MA). Cells were maintained in Dulbecco's modified Eagle's medium
(DMEM)
supplemented with 10% fetal bovine serum (FBS). The estrogen-starvation resistant
MCF-7/BUS clones were previously generated as described (Fu, 2007).
Estrogen starvation therapy was completed as previously described. Briefly, phenol-red
free DMEM was used to wash MCF-7/BUS cells three times and cells were then incubated in
washing medium for 60 minutes at 37 o C. Cells were then cultured in phenol-red free DMEM
supplemented with 5% charcoal/dextran-stripped fetal bovine serum for 48 hours.
3.2.2 Western Blots
Western Blots were carried out as previously indicated. The primary antibodies utilized were:
goat anti-GRP78 C-20, goat anti-GRP94 C-19, rabbit anti-calnexin C-20, rabbit anti-ATF4 C-20,
rabbit anti-PDI H-160 (1:200), and mouse anti-beta-actin C-2. All primary antibodies were used
at a 1:1000 dilution except for PDI which was diluted 1:200. The appropriate horseradish
peroxidase-conjugated secondary antibodies were added at a 1:1000 dilution and incubated on an
orbital shaker at room temperature for two hours (Santa Cruz Biotechnology). Western Blots
were quantitated using the Bio-Rad Fluor-S Max MultiImager and SuperSignal ECL detection
solution and were normalized using beta-actin.
21
3.2.3 Si94 in MCF-7/BUS Resistant Clones
The siRNA against GRP94 was sense 5’-AUC UGG GAC AAG CGA GUU UUU-3’ and
antisense AAA AAC UCG CUU GUC CCA GAU (Biquan Luo and Miao Wang, Los Angeles,
CA). The control siRNA was Silencer Negative Control #3 siRNA (Ambion) and is comprised of
a 19 base pair sequence that is scrambled and lacks any significant homology to any known
mouse, rat or human gene sequences. The day before transfection, MCF-7/BUS estrogen
starvation-resistant clones were plated in a 12-well plate in 1 ml DMEM without antibiotics such
that they would be 30-40% confluent on the day of transfection. For each transfection sample,
oligomer-Lipofectamine
TM
2000 complexes were prepared as follows: 40 pmol of siRNA (or 40
pmol of control siRNA) was diluted in 100 μl Opti-MEM
®
Reduced Serum Medium without
antibiotics (OMEM) and mixed gently; in another set of Eppendorfs 2 μl Lipofectamine
TM
2000
was diluted in 100 μl OMEM and incubated at room temperature for 5 minutes; siRNA oligomer
solution was then mixed with the Lipofectamine
TM
2000 solution and kept at room temperature
for 20 minutes; the Lipofectamine
TM
2000-oligomer complexes were added to each well and
mixed carefully by rocking the plate side to side; 4-6 hours later the media was changed and
regular DMEM with 10% FBS (no PS) was added. Cells were incubated for 24 hours at 37 o C.
After this 24 hour period, estrogen-starvation therapy was carried out for 48 hours as described
above. Briefly, phenol-red free DMEM was added to sample reactions while regular DMEM was
added to control reactions and incubated for 48 hours. After this procedure cells were collected
for the Trypan Blue Exclusion Assay.
22
3.2.4 Trypan Blue Exclusion Cell Death Assay
The Trypan Blue Exclusion Assay was used to ascertain relative cell death in each of the groups.
This is a dye exclusion stain in which viable cells with intact membranes can exclude the dye
whereas dead cells will not be able to exclude the dye and will take up the trypan blue color.
Briefly, a 1:1 dilution of cell suspension in 0.4% trypan blue was loaded into a hemacytometer
counting chamber and allowed to sit at room temperature for 2-5 minutes. The number of stained
cells, unstained cells, and total cells were counted and the number of unstained cells over the total
number of cells represented the percentage of viable cells.
23
3.3 Results
3.3.1 Elevated levels of GRP94 in estrogen-starvation resistant MCF-7/BUS
breast cancer clones.
To investigate the protein expression profiles of estrogen-starvation resistant MCF-7/BUS
breast cancer clones, MCF-7/BUS breast cancer cell clones that were resistant to estrogen-
starvation therapy were characterized using Western Blot. These experiments were repeated 4-6
times on 13 different clones.
In MCF-7/BUS human breast cancer cell clones resistant to estrogen-starvation therapy,
the levels of GRP94 were markedly elevated in 12 of 13 clones (Fig. 3.1, Fig. 3.2). 8 of these 12
clones exhibited at least a three-fold increase in GRP94 levels. Levels of calnexin (CNX),
GRP78 and protein disulfide isomerase (PDI) were slightly increased whereas levels of ATF4
were slightly decreased. These levels were ascertained as compared to control parental MCF-
7/BUS clones that were not estrogen-resistant, and were normalized to beta-actin levels.
GRP78 is already present at relatively high basal levels whereas GRP94 is present at a
relatively low basal level, so a dramatic increase in GRP94 is possible whereas a similar increase
would not be expected in GRP78. Since marked elevation of GRP94 was exhibited in 11 of 13
clones that were resistant to estrogen-starvation therapy, the next step was to knockdown GRP94
expression to see if sensitivity to estrogen-starvation therapy could be regained. The knockdown
was carried out in one of the resistant clone lines, resistant clone 10.
24
8 9 10 11 12 13 14 Ctrl
Fig. 3.1 Elevated levels of GRP94 in estrogen-starvation resistant MCF-7/BUS
breast cancer clones. MCF-7/BUS breast cancer cell clones that were resistant to estrogen-starvation
therapy were characterized using Western Blot. The primary antibodies utilized were: goat anti-GRP78
C-20, goat anti-GRP94 C-19, rabbit anti-calnexin C-20, rabbit anti-ATF4 C-20, rabbit anti-PDI H-160
(1:200), and mouse anti-beta-actin C-2. All primary antibodies were used at a 1:1000 dilution except for
PDI which was diluted 1:200. The appropriate horseradish peroxidase-conjugated secondary antibodies
were added at a 1:1000 dilution and incubated on an orbital shaker at room temperature for two hours
(Santa Cruz Biotechnology).
◄GRP94
◄GRP
◄CNX
◄PDI
◄ATF4
◄B-actin
25
Protein Fold Change in Resistant Clones
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Ctrl 1 3 4 5 6 7 Ctrl 8 9 10 11 12 13 14
Fold Change
Relative GRP78
Relative CNX
Relative ATF4
Relative GRP94
Relative PDI
Fig. 3.2 Elevated levels of GRP94 in estrogen-starvation resistant MCF-7/BUS breast cancer clones.
To generate MCF-7/BUS resistant cell clones, the cells were cultured in estrogen-free medium containing
5% charcoal-dextran stripped fetal calf serum for 5 days. 10% of cells survived and were cultured in
regular medium for one week to allow expansion of remaining cells. This selection process was repeated 4
to 6 times to get resistant clones that could grow and divide in estrogen-free medium. Estrogen-starvation
resistant cells were maintained in estrogen-free medium for about 2 months to stabilize the resistant
property. Briefly, phenol-red free DMEM was used to wash MCF-7/BUS cells three times and cells were
then incubated in washing medium for 60 minutes at 37
o
C. Cells were then cultured in phenol-red free
DMEM supplemented with 5% charcoal/dextran-stripped fetal bovine serum for 48 hours. Western Blot
was used to characterize the protein levels in the resistant clones. The primary antibodies utilized were:
goat anti-GRP78 C-20, goat anti-GRP94 C-19, rabbit anti-calnexin C-20, rabbit anti-ATF4 C-20, rabbit
anti-PDI H-160, and mouse anti-beta-actin C-2. Western Blots were quantitated using the Bio-Rad Fluor-S
Max MultiImager and SuperSignal ECL detection solution and were normalized using beta-actin.
26
3.3.2 Higher levels of cell death in si94-treated resistant MCF-7/BUS clones.
Upon siRNA knockdown of GRP94 in resistant clone 10, differing levels of cell death were seen
in both siCtrl and si94-treated clones grown in regular DMEM medium versus phenol red-free
medium (Fig. 3.3, Fig. 3.4). The Trypan Blue Exclusion Assay was used to ascertain relative cell
death in each of the groups. This is a dye exclusion stain in which viable cells with intact
membranes can exclude the dye whereas dead cells will not be able to exclude the dye and will
take up the trypan blue color. Briefly, a 1:1 dilution of cell suspension in 0.4% trypan blue was
loaded into a hemacytometer counting chamber and the number of blue-stained cells were
counted out of 400 cells total. The number of stained cells over the total number of cells
represented the percentage of cell death.
The average percentages of cell death for each group was: 13.4% for siCtrl-transfected
cells in regular medium, 32.4% for siCtrl-transfected cells in estrogen-free media, 14.4% for si94-
transfected cells in regular medium, and 52.1% for si94-transfected cells in estrogen-free media.
Higher levels of cell death were seen in clones transfected with si94 than in clones transfected
with siControl. Also, slightly more cell death was seen in resistant clones treated with si94 that
were grown in estrogen-free medium versus those treated with si94 and grown in regular medium
that contained estrogen.
A t-test was completed to check for significance between the siCtrl regular medium and
phenol-red free treatment group (p=0.0129), between si94 regular medium and phenol-red free
treatment group (p=0.00006), and between the siCtrl phenol-red free treatment group and the si94
regular medium and phenol-red free treatment group (p=0.013). All the p-values were found to
be significant (p<0.05). The experiment should be repeated again on more samples to further
characterize this trend.
27
This study also suggests that GRP94 may be a protein that is upregulated in resistant
MCF-7/BUS human breast cancer clones. GRP94 expression is generally upregulated under
conditions of stress, such as heat shock or glucose starvation. GRP94 was found to be
upregulated in esophageal adenocarcinomas in a rat surgical esophageal adenocarcinoma model
(Chen, 2002) and overexpression of GRP94 has also been shown to correlate with human lung
cancer progression (Wang, 2005). Another interesting future experiment could involve using
Western Blot to characterize chaperone levels in clones after knockdown of GRP94 to see if there
are any compensatory changes in the levels of other chaperones such as GRP78 or calnexin.
The next step would be to confirm knockdown of GRP94 utilizing Western Blot. Any
behaviors seen here cannot be attributed to GRP94 knockdown until further experiments are
conducted to confirm levels of GRP94. Parental clones, which are not resistant to estrogen-
starvation therapy, should also be included in future experiments to see their behavior in
estrogen-free medium versus regular medium, which can then be compared to the behavior of
resistant clones as a control. In this way there can also be a comparison to see if resistant
properties of the resistant clones are in fact intact. Since parental clones were not included here
the levels of cell death cannot be compared to non-estrogen starvation resistant MCF-7/BUS
cells. So any comparisons of cell death in the si94 group can only be made relative to siCtrl
levels, and cannot be attributed to GRP94 knockdown until Western Blots are run to confirm
GRP94 knockdown.
This experiment shows promise for the potential role of GRP94 upregulation in resistance
to estrogen starvation therapy, but should be repeated on more clones including resistant clone 10,
should include parental clone controls, and should confirm GRP94 knockdown. Whereas GRP78
levels have been correlated with resistance to cancer therapy this could provide clues to the role
GRP94, another ER resident chaperone, may play in resistance to therapy.
28
siCtrl si94
Regular Phenol-red Regular Phenol-red
Medium free medium medium free medium
Fig. 3.3 Higher levels of cell death in si94-treated MCF-7/BUS resistant clones.
MCF-7/BUS estrogen therapy-resistant cells grown to 50% confluence in regular DMEM medium were
transfected with si94 or siControl, and after 24 hours select samples were treated with phenol-red free
(estrogen-free) medium. 48 hours later, cells were harvested and the Trypan Blue Exclusion Assay was
carried out and number of stained (dead) cells were calculated out of 400 cells total.
66
42
48
140
97
152
53
64
55
194
210
241
52 (13%) 129.7 (32.4%) 57.3 (14.3%) 215(53.8%)
Mean
cell
death=
29
Fig. 3.4 Higher percent of cell death in si94-treated MCF-7/BUS resistant clones.
MCF-7/BUS estrogen therapy-resistant cells grown to 50% confluence in regular DMEM medium were
transfected with si94 or siControl and after 24 hours, select samples were treated with phenol-red free
(estrogen-free) medium. 48 hours later, cells were harvested and the Trypan Blue Exclusion Assay was
carried out and percentage of cell death calculated out of 400 cells total.
0
10
20
30
40
50
60
siCtrl si94
P
e
r
c
e
n
t
C
e
l
l
D
e
a
t
h
Regular Medium Phenol-red Free Medium
*
** *
30
3.4 Discussion
Proteins located in the endoplasmic reticulum include GRP94, GRP78, ATF4, PDI, and
calnexin. These chaperones and isomerases play critical roles in the endoplasmic reticulum.
Since marked elevation of GRP94 was exhibited in 11 of 13 clones that were resistant to
estrogen-starvation therapy, the next step was to knockdown GRP94 expression to see if
sensitivity to estrogen-starvation therapy could be regained.
Slightly more cell death was seen in resistant clones treated with si94 that were grown in
estrogen-free medium versus those treated with si94 and grown in regular medium that contained
estrogen. Also, slightly more cell death was seen in resistant clones treated with siCtrl that were
grown in estrogen-free medium versus those treated with siCtrl and grown in regular medium that
contained estrogen. The experiment should be repeated again on more samples to further
characterize this trend. Since such high relative levels of cell death were seen in clones grown in
phenol-red free medium, treated with either siCtrl of si94, the question arises whether the
resistant clones have maintained their resistant properties or if resistance has diminished due to
time or prolonged cell culture in regular medium.
Another good step would be to utilize Western Blot to check GRP94 levels to look at
levels of si94 knockdown efficiencies and perhaps blot for cleaved PARP as another measure of
cell death. Future experiments can also involve knockdown of GRP78 in MCF-7/BUS resistant
clones to see whether the resistance can be reversed and sensitivity to estrogen-starvation therapy
regained. Parental clones, which are not resistant to estrogen-starvation therapy, should also be
included in future experiments to see their behavior in estrogen-free medium versus regular
medium, which can then be compared to the behavior of resistant clones. In this way there can be
a comparison to see if resistant properties of the resistant clones are in fact intact.
A t-test was completed to check for significance between the siCtrl regular medium and
31
phenol-red free treatment group (p=0.0129), between si94 regular medium and phenol-red free
treatment group (.00006), and between the siCtrl phenol-red free treatment group and the si94
regular medium and phenol-red free treatment group (p=0.013). All the p-values were found to
be significant (p<0.05). The experiment should be repeated again on more samples to further
characterize this trend.
32
Chapter 4: Putative breast cancer stem cell population in lymph node
occult micrometastases of breast cancer patients
4.1 Introduction
It was postulated over 150 years ago that cancer may arise from an isolated population of cells
within the tumor cell population that possess stem-cell like properties (Durante, 1874). Cancer
stem cells and stem cells both have the shared properties of self-renewal and differentiation. Self-
renewal drives tumorigenesis and multipotency allows tumor cells to differentiate (Wicha, 2006).
Cancer stem cells have been implicated in processes of invasion, metastasis, angiogenesis and
resistance to apoptosis and cancer therapy.
Even before lymph node metastases can be identified through standard visualization
techniques, micrometastases can be identified in breast cancer patients. Breast cancer patients
with occult lymph node micrometastases are at increased risk of cancer recurrence so
characterization of these micrometastases has implications on earlier diagnosis as well as
potential therapeutics. Studies have shown that even minor populations of tumor cells can cause
disease relapse (Braun, 2005; Coombes, 1986; Cote, 1991; Lugo, 2003). While some patients
with residual tumor cells progress to disease relapse, others do not. This tumorigenic potential
could be caused by a difference in biologic properties of the disseminated tumor cells. Cancer
stem cells may represent a tumor subpopulation with these properties.
One of these cell populations could be the putative stem cell phenotype CD44
+
/CD24
-/low
that has been found in hematologic malignancies (Huntly, 2005), brain tumors (Singh, 2004), and
breast cancer (Al-Hajj, 2003). These cells have been shown to cause tumor growth and may also
33
be a cause of failure of therapy in mouse xenograph models (Al-Hajj, 2004). In primary breast
cancers, cells with this phenotype represent a minority of the tumor cell population, about 10-
20% (Abraham, 2005). A study by the lab of my mentor, Dr. Richard Cote, found over 71%
CD44
+
/CD24
-/low
subpopulation in occult bone marrow micrometastases of breast cancer patients
(Balic, 2006).
CD44 is a plasma membrane glycoprotein that functions as a receptor for hyaluronic acid,
an extracellular glycosaminoglycan. It interacts with osteopontin, collagens, and matrix
metalloproteinases and has known functions in cell migration, cell adhesion, and tumor metastasis
(Draffin, 2004). It is also found in normal breast tissue stem cells (Hebbard, 2000). CD24 is
involved in cell adhesion and is an independent prognostic marker in breast cancer. High levels
of CD24 inhibit the metastatic potential of tumors as evinced by studies in CXCR4 breast cancer
cell lines where cells with higher CD24 expression levels had decreased stromal cell-derived
factor-1 mediated migration and signaling. CD24
-/low
cells appear to have increased metastatic
potential (Schabath, 2006). Cytokeratin (CK) is an intermediate filament keratin that is an
epithelial tissue marker. The cytokeratin profile of epithelial tissue remains fairly consistent even
after malignant transformation, so immunohistochemistry can be utilized to diagnose and typify a
tumor with epithelial origins.
70% of primary breast tumors have less than 10% of cells with stem cell phenotype-
CD44
+
/CD24
-/low
cells (Abraham, 2005). While it is known that 71% of breast cancer
micrometastases to bone marrow are CD44
+
/CD24
-/low
, the cell surface marker profile of
micrometastases to lymph nodes is unknown and is the subject which Dr. Debra Hawes and I
investigated. This study was carried out to investigate the molecular characteristics of breast
cancer occult micrometastases to the lymph node.
34
4.2 Materials and Methods
Double-staining immunohistochemistry was utilized to investigate 50 sentinel lymph node-
positive, occult micrometastases-positive specimens obtained from early-stage breast cancer
patients who were previously classified as having cytokeratin-positive tumor cells. These
samples were evaluated for the presence of CD24 and CD44 via light microscopy and spectral
imaging.
4.2.1 Double-staining Immunohistochemistry
The general staining procedure was carried out on formalin-fixed paraffin-blocked tissue. Patient
sentinel lymph node samples previously characterized as cytokeratin positive were utilized, as
well as a positive control consisting of breast cancer tissue that was CD44
+
/CD24
+
. The negative
control consisted of patient samples without primary antibody. Slides with paraffin-blocked
tissue were placed in a slide holder and immersed in Histoclear for 10 minutes, 100% ethanol for
6 minutes, 95% ethanol for 6 minutes, and finally in a 3% hydrogen peroxide-methanol solution
for 20 minutes. To regain immunoreactivity by uncrosslinking nonspecific proteins from target
antigens, Antigen Retrieval was performed (Immunohistochemical Dako Staining Methods).
Briefly, slides were washed in distilled water and placed inside a pressure cooker containing 10x
Antigen Retrieval Citra buffer (Biogenex, San Ramon, CA). The cooker was microwaved for 30
minutes and then slides were removed and washed with distilled water. Slides were blocked with
non-immune horse serum. Slides were then placed in a humidity control chamber (to prevent
evaporation and drying of tissue sections) and primary antibody was then added to the samples as
appropriate. Phosphate-buffered saline (PBS) was added to the samples to wash off primary
antibody and slides were soaked in PBS for 10 minutes. Secondary antibody was then added as
appropriate. PBS was then added to the slides for another 10 minutes. The appropriate
35
chromagen was then added the slides and incubated for the appropriate amount of time. Slides
were then rinsed with water, placed in Mayer’s Hematoxylin (Sigma Aldrich) to serve as a
nuclear stain and rinsed with water once again. Slides were then mounted.
CD24/CD44 slides were stained as follows:
Slides were incubated with the first primary antibody (CD24 mouse monoclonal antibody,
Neomarkers, 1:50 dilution) overnight, then incubated with secondary antibody (goat anti-mouse
Mach2 Poly-HRP Polymer, Santa Cruz) for 30 minutes followed by staining with Betazoid DAB
(Biocare Medical) for 10 minutes. Slides were then incubated with the second primary antibody
CD44 (CD44 mouse monoclonal antibody, Neomarkers, 1:300) for 2 hours, followed by wash
with tris buffer TBS (not PBS, as phosphates act as a competitive inhibitor to alkaline
phosphatase enzymes) and incubation in alkaline phosphatase secondary antibody (goat anti-
mouse Mach2 Polymer-ALP conjugate, Biocare Medical) for 30 minutes and then stained with
Fast Red chromagen (Vulcan Fast Red Chromogen, Biocare Medical) for 15 minutes.
To confirm CD44 staining or absence of staining for some patient cases, slides were double-
stained for CD44/CK with Betazoid DAB and Fast Red, respectively, as described above.
CD44/CK slides were stained as follows:
Slides were stained for CD44 primary antibody for 2 hours (Santa Cruz Labs, 1:300 dilution),
then incubated with secondary antibody (Mach2 Mouse HRP Polymer, Santa Cruz) for 30
minutes followed by staining with Betazoid DAB (Santa Cruz Labs) for 10 minutes. Slides were
then incubated with the second primary antibody (CK, Santa Cruz, cocktail of AE-1 1:200 and
Cam5.2 1:100) for 30 minutes, followed by incubation in secondary antibody (alkaline
phosphatase, Santa Cruz Laboratories) for 30 minutes and then staining with Fast Red (Santa
Cruz Labs) for 15 minutes.
36
4.2.2 Light Microscopy
An OlympusBH-2 light microscope set at 40X and 100X magnification was used to visually
identify CD24 staining (brown) as well as CD44 and CK staining (red) on respective slides. A
small subset of patient cases was double-stained for CD44/CK and evaluated using a light
microscope (40X magnification and 100x magnification) to further validate results.
4.2.3 Spectral Imaging
The Olympus BX-51 microscope (Olympus, Center Valley, PA) and Nuance™FLEX Imaging
System (Cambridge Research Instruments, Woburn, WA) were utilized to resolve staining in
cases where staining appeared to overlap as well as to validate results from light microscopy
quantification.
37
4.3 Results
4.3.1 Lymph node occult micrometastases in lymph node negative breast cancer patients
possess a minor putative breast cancer stem cell population.
In this study, we analyzed the sentinel lymph node (SLN) specimens from patient with early-
stage breast cancer who were known to have LNOM from the American College of Surgeons
Oncology Group (ACOSOG) Z0010 study for the presence of putative BCSC CD44
+
/CD24
-/low
.
Fifty LNOM positive nodes from the American College of Surgeons Oncology Group
(ACOSOG) Z0010 clinical trial were assessed using immunohistochemistry (IHC) double marker
analysis for the presence of CD44/CD24 (Fig. 4.1 and Fig. 4.2). The purpose was to investigate
the proportion of tumor cells that showed the breast cancer stem cell phenotype (BCSC). It is
known that less than 10% of primary tumors show this putative phenotype and a study by Balic
showed that 71% of occult micrometastases to bone marrow showed the BCSC. Our purpose
here was to investigate the percentage of breast cancer occult micrometastases that showed the
putative BCSC phenotype.
Sufficient tumor cells were identified on 44 of the 50 cases for analysis. All 50 cases
were stained three times to validate results. Of these 44 cases 23 (52%) had no putative BCSC
(CD24>CD44,12 cases; CD44
-
/CD24
+
, 2 cases; CD44=CD24, 7 cases; CD44
-
/CD24
-/low
, 2 cases).
Eleven (25%) had 100% of tumor cells showing the putative BCSC phenotype (CD44
+
/CD24
-/low
)
and 10 (23%) had a subpopulation of stem cells with the BCSC phenotype (range7-84%).
Of the 44 cases analyzed, 23(52%) were negative for putative BCSC. Of the remaining
21 cases 11 showed all of the tumor cells present to be of the putative BCSC phenotype. The
remaining 10 cases had at least some of the tumor cells showing the putative BCSC phenotype.
The number of cells with the BCSC phenotype was variable ranging from 7% to 84%. Overall
less than 48% of the cases were positive for putative BCSC.
38
There was considerable variation in the number of tumor cells found in the lymph nodes.
In 7 of the cases that were positive, there was less than 5 tumor cells present, 15 had 6-20 cells
present and 22 had more than 21 cells present. In lymph nodes in which there were 5 or fewer
cells were far more likely to express the BCSC phenotype, 5 of the 7 (71%) had 100% of the cells
positive, 1 had 17% and 1 no tumor cells showing the BCSC phenotype. A concern is that tumor
deposits with few tumor cells may not be representative of other possible occult tumor deposits
not present on the slide. A study on a group larger than this small pilot sample must be carried
out before any conclusions can be reached. Issues such as patient variation or antigenic
heterogeneity could be larger issues in smaller sample sizes.
Bone marrow is the most common site of breast cancer metastases and up to 80% of
patients with tumor recurrence have metastases to the bone marrow. The highest risk of
recurrence is for patients with bone marrow occult micrometastases (BMOM) whereas there is an
intermediate risk of recurrence for patients with lymph node occult micrometastases as compared
to patients with no occult micrometastases in either the lymph node or bone marrow (Hawes,
Cote, 2005). The results presented in this study lend support to these trends. Occult
micrometastases to the lymph node (LNOM), which represent a lower risk of recurrence than
BMOM, also show a lower percentage of breast cancer stem cell phenotype (less than 48%) than
do BMOM. BMOM which represent a higher risk of tumor recurrence show a higher (71%)
BCSC phenotype. It is possible that occult micrometastases to the bone marrow versus the lymph
node represent separate pathways of metastasis and that the stem cell phenotype makeup of the
separate occult micrometastases may be indicators of recurrence.
39
Fig. 4.1 Double immunohistochemical staining reveals a population of putative breast cancer stem
cells. Only cells that were CK positive were assessed for CD44 and CD24 status. The Olympus BX-51
microscope (Olympus, Center Valley, PA) and Nuance™FLEX Imaging System (Cambridge Research
Instruments, Woburn, WA) was used to resolve staining in cases where staining appeared to overlap as
well as to validate results from light microscopy quantification.
40
1-5 tumor cells 6-20 tumor cells 21-500+ tumor
cells
0%
BCSC positive
cells
.023 .205 .295
7-84%
BCSC positive
cells
.023 .091 .114
100%
BCSC positive
cells
.114 .045 .091
Fig. 4.2 Lymph node occult micrometastases in lymph node histologic-negative breast cancer patients
possess a minor putative breast cancer stem cell population. This table (Debra Hawes, Los Angeles,
CA) summarizes the proportion of tumor cells expressing the putative breast cancer stem cell phenotype
out of 44 cases.
41
4.4 Discussion
Sentinel lymph node status has been designated one of the most critical prognostic factors in
breast cancer. Sentinel lymph node status is also one of the factors that determines appropriate
adjuvant therapy. With a more minute description of the tumorigenic, stem-cell like population
that contributes to tumor metastasis, the cells that are actually able to re-create the tumor can be
specifically and systematically targeted for elimination.
A study on a group larger than this small pilot sample must be carried out before any
conclusions can be reached. However, the pattern seen here may suggest that cells with the breast
cancer stem cell phenotype are the first to metastasize to the lymph nodes. This may also show
that as the tumor cells multiply many of them also differentiate as in primary tumors, and the
stem cells population may become diluted. These results are in dramatic contrast to findings in
the bone marrow in which the CD44
+
/CD24
-/low
phenotype was seen in 71% of all early detected
tumor cells had this phenotype (Balic, 2006), and more than half (52%) of the sentinel lymph
nodes investigated here were negative for the putative stem cell phenotype. Approximately one
third (36%) of the cases were negative due to CD44 (4 cases) negativity or CD44 less than CD24
(12 cases) and that approximately one third (16%) were negative due to the CD44
+
/CD24
+
phenotype. The significance, if any, of the different combinations of phenotypes is not known.
The ability to identify and isolate this cancer stem cell subpopulation may not only allow a more
detailed identification of occult micrometastases at an earlier stage, but also a potential target for
therapeutics. Further insights into the independent roles of both CD44 and CD24 may shed light
on the subject, though more investigation is needed to clarify which, if any, patterns are relevant.
CD24 has been studied as an independent marker of disease prognostication. One study
(Kristiansen, 2003) examined 201 primary breast carcinomas. They found that in invasive
disease, CD24 expression was observed in 85% of cases, and that there was a significant
association of CD24 expression with shortened overall survival (p=0.031) and disease-free
42
survival (p=0.0008). While there is a report of CD24 expression being an indicator of a worse
prognosis (Kristiansen, 2003), CD24 has also been shown to suppress the metastatic potential of
tumor cells as evidenced by studies in breast cancer cell lines (CXCR4) in which cells with
enhanced CD24 expression were found to decrease stromal cell-derived factor-1 mediated
migration and signaling.
CD44 functions in cell adhesion largely through its ability to bind hyaluronic acid
(Herrera-Gayol, 1999). Because the extracellular matrix contains high levels of HA, differing
levels of CD44 expression may affect tumor-matrix and tumor metastasis. While there is
evidence suggesting that CD44 promotes tumor metastasis, there has recently been evidence to
the contrary. There is evidence that CD44 may indeed antagonize breast cancer metastasis
(Lopez, 2005). Using a mouse model involving spontaneously metastasizing breast cancer, Lopez
et al showed that CD44 loss augments lung metastasis. While CD44 overexpression has been
associated with tumor growth and spread, in neuroblastoma, advanced tumors often have
decreased CD44 expression, especially in advanced / MYCN amplified tumors (Gross, 1997).
A study by Schneider et al. showed that CD44 negativity is a statistically significant
predictor of lymph node invasion (Schneider, 1999). In this case, loss of CD44 expression can be
a marker for lymph node metastasis. Lopez et al found that invasion
of CD44-positive tumor
cells was inhibited in hyaluronan (HA)-containing
matrices, suggesting that epithelial-stromal
interactions such as CD44-hyaluronan may be factors in preventing metastasis.
Overall the study of breast cancer micrometastases to the lymph node may be informed
by investigation of factors such as cell type, tumor progression, CD44 variant, HA content of the
extracellular matrix, estrogen receptor status, or whether HA is produced by epithelia or stroma
(Lopez, 2005). Investigating how the nature or expression of these factors relates to CD44
upregulation or downregulation in tumors, and the resulting implications on metastatic potential,
could further clarify the outcomes that we have seen in this study.
43
Chapter 5: Summary
Resistance to chemotherapy represents a major obstacle in breast cancer treatment. Investigating
the nature of this interaction, we discovered that deletion of the BH3-only domain in BIK results
in altered protein interactions in 293T cells. Characterization of MCF-7/BUS human breast
cancer cell clones that were resistant to estrogen-starvation therapy showed markedly elevated
levels of GRP94. Knockdown of GRP94 by small interfering RNA in these resistant clones led to
an increase in cell death. Resistance to chemotherapy is also a characteristic of cancer stem cells
so we characterized molecular markers of putative breast cancer stem cells. Our results showed a
minor putative breast cancer stem cell population in lymph node occult micrometastases. Overall
these results help characterize molecular characteristics that contribute to chemoresistance and
that may serve as valuable targets for therapy, as well as help delineate molecular mechanisms
contributing to estrogen-starvation therapy resistance in breast cancer.
Follow-up studies for the GRP78-BIK interaction studies could include other potential
deletion mutations to check for points of interaction between GRP78 and BIK. It has been
postulated that BIK activity is regulated by phosphorylation. BIK’s threonine-33 and serine 35
phosphorylation sites were mutated to aspartic acid to mimic phosphorylation, and mutants were
found to have increased affinity for BCL-2 and higher apoptotic activity in vitro and greater
antitumor activity in vivo (Yan, 2003). When these sites were mutated to alanine residues, the
apoptotic activity of BIK was decreased (Verma, 2001). This suggests that posttranslational
phosphorylation of BIK may be required for efficient apoptotic activity.
Other studies have shown that the BH3-only domain’s heterodimerization activity alone
may be not sufficient to induce cell death (Elangovan, 1997; Holmgreen, 1999). For several
BH3-only proteins, optimal binding to prosurvival members may require not only their BH3
44
domain but also a membrane targeting function (Cory, 2003). This is usually mediated by the
hydrophobic C-terminal domain. By ensuring colocalization of the two proteins, the tail
enhances their interaction. With Bim, for example, that domain is required both for
mitochondrial targeting and proapoptotic activity (Yamaguchi and Wang, 2002). Therefore, a
future experiment could use a mutant pcDNA3-FLAG-BIK plasmid with the same BH3-only
deletion but with the addition of a C-terminus transmembrane cytochrome B5 segment (b5TM)
domain which would target the mutant specifically to the endoplasmic reticulum.
Resistance to chemotherapy represents a major obstacle in breast cancer treatment.
Clarification of the molecular mechanisms that contribute to chemoresistance may provide
valuable targets for cancer therapy as well as markers for earlier diagnosis. It is known that
doxorubicin induces BIK, but also that GRP78 overexpression causes resistance to this drug.
GRP78 is also required for angiogenesis. GRP78 is primarily a lumenal protein in the ER, but in
its role as a surface receptor it promotes tumor proliferation and metastasis (Misra, 2005). Anti-
GRP78 therapies, especially against tumor-specific cell-surface variants of GRP78 such as the
fully human IgG monoclonal antibody SAM-6, may provide effective antitumor activity
(Rauschert, 2008).
BIK, when ectopically expressed, was found to resensitize resistant tumor cells to cancer
drugs and delay tumor progression in a mouse xenograft model (Daniel et al., 1999; Radetzki et
al., 2002). Clarifying the interaction between these molecules could provide specific targets for
therapy, and moreover further characterization of this interaction in normal versus malignant cells
could provide tumor-specific targets for therapy and diagnostic purposes.
This study also suggests that GRP94 may be a protein that is upregulated in resistant
MCF-7/BUS human breast cancer clones. GRP94 expression is generally upregulated under
conditions of stress, such as heat shock or glucose starvation. GRP94 was found to be
upregulated in esophageal adenocarcinomas in a rat surgical esophageal adenocarcinoma model
45
(Chen, 2002) and overexpression of GRP94 has also been shown to correlate with human lung
cancer progression (Wang, 2005). Another interesting future experiment could involve using
Western Blot to characterize chaperone levels in clones after knockdown of GRP94 to see if there
are any compensatory changes in the levels of other chaperones such as GRP78 or calnexin.
Cancer cells can also possess putative stem cell subpopulations that may contribute to
chemoresistance and may play a large part in tumor growth and metastasis. The ability to
identify and isolate this cancer stem cell subpopulation may not only allow a more detailed
identification of occult micrometastases, but also a potential target for therapeutics. Previously it
has been shown that as little as 100 CD44
+
/CD24
-/low
/Lineage
-
cells can reconstitute a tumor in
NOD/SCID mice, whereas thousands of cells with alternate cell-surface markers failed to do so
(Al-Hajj, 2003). CD44
+
/CD24
-/low
/Lineage
-
cells, which constitute only 1-10% of the total tumor
cell population, could also recreate the heterogeneity seen in the original tumor. If these cancer
stem cells could be targeted in a specific manner, there may be ways to identify and eradicate the
cells that may lead to a future relapse in patients. There is evidence from cell culture and mouse
models that the drug parthenolide kills AML progenitor and stem cells specifically without
harming normal cells (Jordan, 2002).
In addition to drugs, antibodies against cancer stem cell markers are also promising
avenues for investigation. Dick et al. utilized an antibody to CD44 that is highly expressed on the
surface of AML stem cells. When human AML stem cells were transplanted into mice and then
treated with CD44 antibody, the tumor stem cells driving the leukemia were destroyed (Jin and
Dick, 1997). This antibody interferes with trafficking of leukemic stem cells to niches that
support their stem cell properties.
The ability to identify and isolate this cancer stem cell subpopulation may not only allow
a more detailed identification of occult micrometastases, but also a potential target for
therapeutics. Obliterating the bulk of tumor cells may be less effective if the more resilient
46
cancer stem cell population remains largely intact (Rich, 2006). A clearer description of stem cell
markers could widen the repertoire of tumor markers available for diagnosis. Furthermore, it
could lead to more effective early diagnosis because many current tumor markers widely in use
are from differentiated tumor cells. This also has implications for earlier, more targeted
interventions as newer tumor markers emerge.
Overall these results help characterize molecular characteristics that contribute to
chemoresistance and that may serve as valuable targets for therapy, as well as help delineate
molecular mechanisms contributing to estrogen-starvation therapy resistance in breast cancer.
Findings could run the gamut from leading to earlier identification of cancer as well as
elucidating targeted therapies that may even extend therapeutic advantage to patients with
advanced stages of cancer.
47
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Abstract (if available)
Abstract
Resistance to chemotherapy represents a major obstacle in breast cancer treatment. GRP78 is a protein upregulated in malignant but not benign human breast tumors, and is associated with resistance to chemotherapy in breast cancer patients. While it has been shown that this resistance depends on the BH3-only protein BIK, the mechanism of interaction between GRP78 and BIK still remains to be elucidated. Investigating the nature of this interaction, we discovered that deletion of the BH3-only domain in BIK results in altered protein interactions in 293T cells. Characterization of MCF-7/BUS human breast cancer cell clones that were resistant to estrogen-starvation therapy showed markedly elevated levels of GRP94. Knockdown of GRP94 by small interfering RNA in these resistant clones led to an increase in cell death. Resistance to chemotherapy is also a characteristic of cancer stem cells so we characterized molecular markers of putative breast cancer stem cells. Our results showed a minor putative breast cancer stem cell population in lymph node occult micrometastases. Overall these results help elucidate molecular characteristics that contribute to chemoresistance and that may serve as valuable targets for therapy, as well as help delineate molecular mechanisms contributing to estrogen-starvation therapy resistance in breast cancer.
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Asset Metadata
Creator
Patel, Minal Chandravadan
(author)
Core Title
Molecular mechanisms of chemoresistance in breast cancer
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2008-12
Publication Date
10/15/2008
Defense Date
08/20/2008
Publisher
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Tag
breast cancer,drug resistance,GRP78,GRP94,OAI-PMH Harvest,stem cells,stress
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), Tokes, Zoltan A. (
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