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The role of glucose-regulated proteins in endometrial and pancreatic cancers
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The role of glucose-regulated proteins in endometrial and pancreatic cancers
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Content
THE ROLE OF GLUCOSE-REGULATED PROTEINS IN
ENDOMETRIAL AND PANCREATIC CANCERS
by
Jieli Shen
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2016
Copyright 2016 Jieli Shen
ii
Dedication
To my parents, Changning Shen and Xiaojun Zhou for raising me
up and shaping me who I am today.
&
To my love, Yu Guo for her understanding and support.
Happiness is the way.
iii
ACKNOWLEDGEMENTS
This dissertation would not have been possible without all the guidance and support
from the following incredible individuals. First of all, I would like to start my
acknowledgements with my appreciation to my mentor Dr. Amy S. Lee for her insightful
guidance and persistent encouragement throughout my Ph.D. study at USC. Amy not only
provided guidance on research and science, but also supported me on exploring my
curiosity on fields outside research.
I am grateful to my wonderful committee members throughout the years. I really
appreciate the insightful discussions, and helpful consultations on histology and pathology
from Dr. Louis Dubeau. I would also like to thank Dr. Yvonne Lin and members from her
lab for the wonderful collaboration, helpful discussions and experimental assistance. Dr.
Agnieszka Kobielak and her lab members for the interactive collaboration, helpful
discussions and resource support on my projects. Dr. Young Hong for his insightful advice
and encouragement at the student seminar. Dr. Bangyan Stiles and Dr. Amir Goldkorn on
my projects for the helpful discussions.
I would like to thank all the former and current members of the Lee lab for creating
such a welcoming, friendly and interactive lab environment. Former lab members include
Yi Zhang, Kyle Pfaffenbach, Shiuan Wey, Biquan Luo, Wan-Ting Chen, Genyuan Zhu,
Michelle Pong, Kate Ott, Shan Wei and Emma Hadley. Current lab members include Chun-
Chih Tseng, Yuan-Li Tsai, Daisy Rangel, Dat Ha, He Zhao and John Johnson. I enjoyed
the enthusiastic and fun discussions about science and I am thankful for their company and
cordial help throughout the years.
iv
This dissertation would not have been possible without the technical support and
core facilities at USC: Lina He from USC School of Pharmacy Histology Lab, Michelle
Mac Veigh from the Cell and Tissue Imaging Core of the USC Research Center for Liver
Diseases, Denis Alexander Trana from Translational Pathology Core, and Nancy Wu from
the Transgenic Mouse Core at USC Norris Comprehensive Cancer Center. I am also
thankful to the PIBBS program, the program advisors, specifically Bami Andrada, and the
amazing classmates from the PIBBS program.
Lastly, I would like to take this opportunity to thank my parents who raised me up
and taught me right from wrong, and Yu Guo, who has always been there for me through
the rain and sunshine.
v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
Chapter 1: Overview and Introduction 1
1.1 Glucose-regulated protein 94
1.1.1 GRP94 and its functions
1.1.2 GRP94 in cancer
1
1
2
1.2 Glucose-regulated protein 78
1.2.1 GRP78 and its functions
1.2.2 GRP78 in cancer
5
5
7
1.3 Endometrial Cancer 9
1.4 Pancreatic Cancer 11
Chapter 2: GRP94 Deficiency Induces Squamous Cell Metaplasia and
Suppresses PTEN-null Driven Endometrial Epithelial Tumor Development
17
2.1 Introduction 17
2.2 Materials and methods 20
2.3 Results 23
2.3.1 GRP94 mRNA and protein levels are elevated in human
EAC and contribute to cell viability
23
2.3.2 Endometrial GRP94 ablation leads to spontaneous
squamous cell metaplasia and loss of active nuclear β-catenin
expression
26
2.3.3 Endometrial GRP94 deficiency suppresses PTEN-null
driven adenocarcinoma
30
2.3.4 cP
f/f
Grp94
f/f
uteri display early squamous metaplasia and
reduced β-catenin signaling
32
2.3.5 Attenuation of AKT/S6 activation and proliferation in
cP
f/f
Grp94
f/f
SCM
34
vi
2.3.6 cP
f/f
Grp94
f/f
uteri show no EAC myometrial invasion and
high E-cadherin expression
2.3.7 Prolonged depletion of GRP94 in PTEN-null uteri leads
to massive glandular expansion but no EAC formation
2.3.8 GRP78 deficiency inhibits AKT activation in PTEN-null
endometrium
36
39
41
2.4 Discussion 44
Chapter 3: Glucose-regulated protein 78 Haploinsufficiency Suppresses
Pancreatic Tumorigenesis Driven by K ras Mutation and p53
Heterozygosity
49
3.1 Introduction 49
3.2 Materials and methods 53
3.3 Results
3.3.1 Grp78 homozygous deletion induced fat infiltration in the
pancreas
3.3.2 Grp78 heterozygous deletion in the pancreas
3.3.3 PKC pancreata exhibit ER stress
3.3.4 Grp78 heterozygosity in the pancreas delayed PKC-driven
tumorigenesis and prolonged survival
3.3.5 Suppression of proliferation and oncogenic signaling in
PKC78
f/+
pancreata
3.3.6 Reduction of ADM in PKC78
f/+
pancreata and in c78
f/+
acinar cells induced by TGFα
3.4 Discussion
57
57
60
62
65
66
69
72
Chapter 4: Conclusions and Perspectives 76
BIBLIOGRAPHY
94
vii
LIST OF TABLES
Table 1.1: Association of elevated GRP protein level in patient samples with
cancer aggressiveness and poor survival
3
viii
LIST OF FIGURES
Figure 1.1: GRPs in the unfolded protein response and the stress response
6
Figure 2.1: Increased GRP94 expression in human EAC and its effect on EAC
cell viability.
25
Figure 2.2: Generation of PR-Cre-mediated GRP94 knockout mouse models
27
Figure 2.3: GRP78 deficient uteri showed no SCM
27
Figure 2.4: Induction of squamous metaplasia in developing c94
f/f
uteri
29
Figure 2.5: c94
f/f
uteri showed SCM but no EAC at 9 months
29
Figure 2.6: Reduced endometrial cancer in cP
f/f
94
f/f
uteri
31
Figure 2.7: Accelerated squamous metaplasia and reduced β-catenin signaling in
cP
f/f
94
f/f
uteri
33
Figure 2.8: Attenuated AKT activation and decreased proliferation in cP
f/f
94
f/f
SCM
35
Figure 2.9: cP
f/f
94
f/f
uteri showed no change on ERK activation
36
Figure 2.10: cP
f/f
94
f/f
uteri showed no increase in apoptosis
36
Figure 2.11: Characterization of cP
f/f
94
f/f
uteri on myometrial invasion and
prolonged stage
38
Figure 2.12: Characterization of uteri at prolonged stage
40
Figure 2.13: Summary model on the effect of GRP94 deficiency in mouse uteri
in the presence or absence of PTEN-deficiency
41
Figure 2.14: Pten-null mediated AKT activation was suppressed in the uteri of
cPten
f/f
78
f/f
mice
42
Figure 2.15: Analysis of unfolded protein response (UPR) markers in the mouse
uteri
44
Figure 3.1: Mating scheme
53
Figure 3.2: Generation of Pdx1-Cre-mediated Grp78 homozygous knockout
mouse model
59
ix
Figure 3.3: Generation of Pdx1-Cre-mediated Grp78 heterozygous knockout
mouse model
61
Figure 3.4: Analysis of glucose tolerance
62
Figure 3.5: Analysis of GRP78 and UPR markers in PKC and PKC78
f/+
pancreata
64
Figure 3.6: Reduced pancreatic tumorigenesis in PKC78
f/+
pancreata
66
Figure 3.7: Characterization of proliferation, apoptosis and signaling pathways
in PKC78
f/+
pancreata
68
Figure 3.8: Analysis of acinar-to-ductal metaplasia in WT, PKC and PKC78
f/+
pancreata
69
Figure 3.9: Acinar transdifferentiation of WT and c78
f/+
pancreata
71
Figure 4.1: IHC staining of PTEN
82
x
Abstract
The glucose-regulated proteins (GRPs), including GRP78 and GRP94, are stress-
inducible molecular chaperones belonging to the heat shock protein family. The GRPs
primarily reside in the ER, however, they are also detected in other compartments of the
cell and even secreted outside the cell. Apart from their traditional function as ER
chaperones, evidence are emerging that GRPs are elevated in various cancers associating
with aggressive properties.
Endometrial carcinoma is the most prevalent gynecologic cancer in the United
States. The tumor suppressor gene Pten (phosphatase and tensin homolog) is commonly
mutated in the more common type 1 (endometrioid) subtype. Here we report that
expression profiles from the Cancer Genome Atlas (TCGA) showed significantly increased
Grp94 mRNA levels in endometrial tumor versus normal tissues, correlating with highly
elevated GRP94 protein expression in patient samples and the requirement of GRP94 for
maintaining viability of human endometrioid adenocarcinoma (EAC) cell lines. Through
generation of uterus-specific knockout mouse models with deletion of Grp94 alone (c94
f/f
)
or in combination with Pten (cP
f/f
94
f/f
), we discovered that c94
f/f
uteri induced squamous
cell metaplasia (SCM) and reduced active nuclear β-catenin. The cP
f/f
94
f/f
uteri showed
accelerated SCM and suppression of PTEN-null driven EAC, with reduced cellular
proliferation, attenuated β-catenin signaling and decreased AKT/S6 activation in the SCM.
In contrast to single PTEN knockout uteri (cP
f/f
), cP
f/f
94
f/f
uteri showed no decrease in E-
cadherin level and no invasive lesion. Collectively, our study implies that GRP94
downregulation induces SCM in EAC and suppresses AKT/S6 signaling, providing a novel
mechanism for suppressing EAC progression.
xi
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest disease
with <10% overall 5-year survival rate. High GRP78 expression in PDAC was reported in
several studies. Here, we investigated the role of GRP78 in pancreatic cancer using the
established PKC model (Pdx1-Cre;Kras
G12D/+
;p53
f/+
) in the absence or presence of Grp78
heterozygosity (PKC78
f/+
mice). The PKC78
f/+
pancreata showed decreased tumor area,
associating with prolonged survival. In understanding the mechanism, we determined that
Grp78 heterozygous pancreas exhibited reduced proliferation, AKT/S6 and ERK activation.
Acinar-to-ductal metaplasia (ADM) is recently recognized as a key tumor initiation event
of PDAC. We discovered that Grp78 heterozygosity, while having no effect on ductal
formation under normal physiologic conditions, suppressed ADM in response to
stimulation by TGFα. In PKC78
f/+
mice, reduced ADM was also observed, supporting a
novel role of GRP78 in tumor initiation process of PDAC.
1
Chapter 1
Overview and Introduction
1.1 Glucose-regulated protein 94
1.1.1 GRP94 and its functions
The endoplasmic reticulum (ER) is a specialized perinuclear organelle where
secretory and membrane proteins, as well as lipids, are synthesized. Glucose-regulated
protein 94 (GRP94) is a member of the HSP90 family, that mainly localizes in the
endoplasmic reticulum (ER). It has 50% homology to its cytosolic paralog HSP90. The
domain organization is conserved within the HSP90 family: an N-terminal domain (NTD),
a charged linker domain, a middle domain (MD), and a C-terminal domain (CTD) (Marzec,
Eletto, & Argon, 2012). The 4 domains serve different roles in GRP94: NTD binds ATP,
peptide and small molecule inhibitors (Gidalevitz et al., 2004); linker domain mediates
conformational changes and binds to calcium (Biswas et al., 2007); MD possesses catalytic
loop, which is required for ATP hydrolysis (Dutta & Inouye, 2000) and CTD provides
dimerization of GRP94.
While GRP94 primarily resides in the ER, it has also been detected in other
compartments of the cell. GRP94 accumulates in the Golgi apparatus, when treated with
calcium ionophore (Booth & Koch, 1989; Welch, Garrels, Thomas, Lin, & Feramisco,
1983). It has been found to form a complex with bile salt-dependent lipase (BSDL), and
secreted into extracellular milieu of pancreatic cells (Bruneau, Lombardo, & Bendayan,
1998). Additionally, GRP94 is present on the cell surface of normal immature thymocytes
2
(Wiest et al., 1997) and tumor cells (Altmeyer et al., 1996; Hou et al., 2015; X. Li et al.,
2015).
The function of GRP94 has been determined in various organisms. In
Dictyostelium, down-regulating GRP94 resulted in delay in development and decreased
growth rate (Baviskar & Shields, 2011). In plant, ablation of SHEPEHRD (Arabidopsis
ortholog of GRP94) led to expanded shoot apical and floral meristems (Ishiguro &
Watanabe, 2002). In drosophila, Gp93 (ortholog of GRP94) mutant larvae showed
prominent defect in midgut epithelium (Maynard et al., 2010). In murine models, GRP94
is required for mesoderm and muscle development (Wanderling et al., 2008).
Scrutinizing the role of GRP94, the most prominent activity of GRP94 is
chaperoning unfolded proteins. Unlike other chaperones with a long list of clients, GPR94
has rather limited substrates, which are either proteins in the secretory pathways or cell
surface ligands and receptors (Y . Yang & Li, 2005). In addition to its chaperone function,
GRP94 participates in the calcium storage (Biswas et al., 2007), ER-associated degradation
of misfolded proteins (Christianson, Shaler, Tyler, & Kopito, 2008) and ER stress (Gass,
Gifford, & Brewer, 2002).
1.1.2 GRP94 in Cancer
Solid tumors harbor hypoxic, nutrient deprived environment and it is reasonable to
anticipate that this microenvironment would trigger glucose regulated proteins, which
sense the stress and facilitate the folding of nascent proteins (Ma & Hendershot, 2004).
Indeed, studies have demonstrated that GRP94 is elevated in a number of human cancers
including: esophageal adenocarcinomas, colorectal cancer, oral carcinogenesis, lung
3
cancer, gastric carcinomas, breast cancer, liver cancer and pancreatic cancer (Table 1.1)
(A. S. Lee, 2014). In addition, Grp94 promoter has been shown to be strongly activated in
spontaneous and chemically induced tumors in mice (Reddy et al., 2002), which lends
support to the notion that GRP94 is upregulated in cancerous tissue.
Table 1.1 Association of elevated GRP protein level in patient samples with cancer aggressiveness and poor
survival (A. S. Lee, 2014).
The above-mentioned works led to the question: what is the role of GRP94 in
cancer? One research found that ovarian cancer cells with highest GRP94 level, exhibited
lowest sensitivity to chemotherapy (Zheng et al., 2008). Another study indicated that
cervical cancer cells with high GRP94 level had low sensitivity to X-ray (Kubota et al.,
2005). A third study demonstrated that adopting antisense to reduce GRP94 level in
4
etoposide treated Jurkat cells resulted in decrease cell viability (Reddy, Lu, & Lee, 1999).
Genetically engineered mouse models have also been utilized to study the role of GRP94
in various cancers. Deletion of Grp94 in the liver rendered hyperproliferation of liver
progenitor cells, and deletion of Grp94 in combination with PTEN loss accelerated liver
tumorigenesis (Chen, Tseng, et al., 2014). Depletion of GRP94 in the B cell compartment
attenuated the development of multiple myeloma in mice (Hua et al., 2013). Thus, the role
of GRP94 in cancer may be context-dependent.
In addition, GRP94 has also been indicated in cancer cell migration and invasion.
Downregulation of GRP94 in breast cancer cells inhibited its migration and proliferation
(Dejeans et al., 2012). GRP94 knockdown prostate cancer cells changed their cell polarity
and consequently were defective in cell migration and adhesion (Ghosh, Shinogle, Galeva,
Dobrowsky, & Blagg, 2016). As the role of cell surface GRP94 emerging, cell membrane
form of GRP94 has been indicated to enhance invasion of liver cancer cell, and facilitate
HER2 dimerization and promote proliferation in breast cancer cells (Hou et al., 2015; X.
Li et al., 2015).
Since GRP94 ash been indicated to play such an important role in cancer, small
molecule inhibitors and monoclonal antibodies have been developed to target GRP94.
GRP94 selective small molecular inhibitor PU-WS13 was developed at Sloan-Kettering
Institute. It was reported to inhibit GRP94 level in breast cancer cells, leading to HER2
expression disruption (Patel et al., 2013). Another small molecule inhibitor was developed
at University of Kansas, which suppressed migration of breast cancer cells and reduced
proliferation in multiple myeloma cell (Muth et al., 2014).
5
1.2 Glucose-regulated protein 78
1.2.1 GRP78 and its functions
Glucose-regulated protein 78, also known as BiP and HSPA5, is another important
chaperone residing in the endoplasmic reticulum (ER). The accumulation of misfolded
proteins in the ER will induce ER stress, which will further trigger a quality control
mechanism, the unfolded protein response (UPR) (B Luo & Lee, 2012). The UPR signaling
pathway consists of three canonical branches, which are mediated by three ER stress
sensors: protein-kinase RNA-like ER kinase (PERK), inositol-requiring kinase 1 (IRE1α),
and the activating transcription factor 6 (ATF6) (Figure 1.1) (A. S. Lee, 2014). Upon ER
stress, GRP78 will be sequestered to fold malfolded proteins and dissociate from the three
sensors, which allow activation of the three pathways. PERK will phosphorylate eukaryotic
translation initiation factor 2α (eIF2α), which will suppress global protein synthesis. The
IRE1α pathways will activate its downstream X-box-binding protein 1 (XBP-1) splicing.
The ATF6 will be cleaved, and act in conjuction with spliced XBP-1 to translocate into the
nucleus and selectively induce transcription of gene encoding ER chaperones and enzymes.
6
Figure 1.1 GRPs in the unfolded protein response and the stress response. Endoplasmic reticulum (ER)
luminal glucose-regulated protein 78 (GRP78) functions as a unfolded protein response (UPR) signalling
regulator by binding to and maintaining the ER stress sensors PERK, ATF6 and IRE1 in inactive forms (A.
S. Lee, 2014).
GRP78 has been shown to assume novel functions beyond protein folding in the
ER. Cell surface GRP78 has been detected on tumor cells mediating oncogenic signaling,
on proliferating endothelial cells and also on host cells serving as a critical portal for viral
entry (Ni, Zhang, & Lee, 2011a). Cytoplasmic GRP78 has been reported to regulate UPR
signaling, viral protein assembly and suppress lead neurotoxicity. GRP78 has also been
indicated to regulate energy balance of mitochondria. Moreover, GRP78 was found in the
nucleus and might be important for DNA repair capacity (A. S. Lee, 2014). Lastly, GRP78
7
was also secreted outside the cell and potentially regulate a multitude of biological
processes. For instance, GRP78 secreted by tumor cells was reported to block the anti-
angiogenic activity of bortezomib (Kern et al., 2009).
1.2.2 GRP78 in cancer
Tremendous efforts have been made to explore the function of GRP78 in cancers.
GRP78 is reported to be elevated in numerous cancers, such as breast, colorectal,
endometrial, esophageal, head and neck, leukemia, liver, lung, pancreatic cancers etc
(Table 1.1) (A. S. Lee, 2014). It is reported to have an anti-apoptotic function through
blocking caspase 7 activation by etoposide (Reddy et al., 2003) and suppress apoptosis
induced by BCL-2-interacting killer (BIK) (Zhou, Zhang, Fu, Chan, & Lee, 2011). In
addition, suppression of GRP78 was reported to reduce proliferation of human
cholangiocarcinoma cells (Feng et al., 2014), endometrial cancer cells (Cali et al., 2014;
Gray et al., 2013). Apart from its direct effect on cancer cells, GRP78 also has an impact
on the tumor microenvironment. Reduction of GRP78 impaired immortalized human
endothelial cell proliferation, survival and migration, and heterozygous knockout of
GRP78 in the mouse endothelial cells showed substantial reduction of tumor angiogenesis
with minimal effect on normal microvessel density (Dong et al., 2011).
Furthermore, GRP78 was reported to affect cancer cell invasion and metastasis.
Cell surface GRP78 facilitated colorectal cancer cell migration and invasion partially
through its association with urokinase receptor protease system (Z. Li et al., 2013).
Suppression of GRP78 decreased invasion of human cholangiocarcinoma cells (Feng et al.,
2014). Interaction of cell surface GRP78 with α2-macroglobulin, a pan-proteinase inhibitor,
promoted the invasion and metastasis of hepatocellular carcinoma (Zhao et al., 2015).
8
Silencing Grp78 inhibited cell invasion and suppressed cancer stemness in head and neck
cancer cells (Chiu et al., 2013).
The role of GRP78 in tumorigenesis has been tested using genetically engineered
mouse models. Homozygous deletion of Grp78 in mouse prostate epithelium suppressed
prostate tumorigenesis and AKT activation driven by Pten null (Fu et al., 2008).
Conditional Grp78 deletion from the endometrium blocked endometrial cancer
development and inhibited AKT activation in Pten-null uterus (Lin et al., 2015). In addition
to solid tumors, in the hematopoietic system, heterozygous knockout of GRP78 inhibited
Pten-null leukemogenesis and AKT oncogenic signaling (Wey, Luo, Tseng, et al., 2012).
In the liver cancer, the case is more complicated. In hepatocytes, concomitant reduction of
PTEN and GRP78 led to increased hepatomegaly, steatosis, livery injury and progenitor
cell proliferation (Chen, Zhu, et al., 2014). Although accelerated liver cancer was evident
in GRP78 depleted PTEN-null liver, intense GRP78 re-expression was detected in those
cancer lesions. Thus, the effects of GRP78 depletion could be tissue-specific and complex.
With emerging role of GRP78 in cancer, it has been presented as a novel cancer
therapeutic target. GRP78 was discovered to be substrate of a bacterial cytotoxin SubAB
(Backer et al., 2009). Engineering the single A subunit to epidermal growth factor (EGF),
selectively targeted cancer cells expressing EGF receptor, and significantly inhibited breast,
prostate and glioblastoma xenograft tumors (Backer et al., 2009; Prabhu, Sarcar, Kahali,
Shan, & Chinnaiyan, 2012). Several plant compounds targeting ATPase domain of GRP78
was reported to inhibit its activity. Epigallocatechin gallate (EGCG), a flavonoid
component of Green Tea Camellia sinensis, and honokiol (HNK), a Magnolia grandiflora
derivative, has been shown to bind to unfolded conformation of the GRP78 ATPase domain
9
and induced death in six neuro-ectodermal tumor cell lines (Martin et al., 2013). Salicylic
acid, from which Aspirin is derived, is a natural occurring molecule that can activate plant
defense mechanism and is a pharmacological agent that can control the inflammatory
response in humans. Salicylic acid was reported to bind GRP78 and inhibit its ATPase
activity (Deng, Ruan, Du, Saunders, & Wu, 2001).
Since cell surface GRP78 is selectively enriched on cancer cells, targeting its cell
surface form allows specific tumor targeting with minimal harmful effects on normal cells
(A. S. Lee, 2014). Antibodies targeting cell surface GRP78 were developed. Mouse
monoclonal antibody targeting C-terminus of GRP78, C107 induced apoptosis in
melanoma cells and slowed tumor growth (de Ridder, Ray, & Pizzo, 2012). A fully human
monoclonal IgM antibody isolated from a gastric cancer patient, PAT-SM6 was found to
bind GRP78 and induce apoptosis in primary human multiple myeloma cells (Rasche et al.,
2013) and suppressed human melanoma growth (Hensel, Eckstein, Rosenwald, &
Brändlein, 2013). Furthermore, a high affinity GRP78-specific mouse monoclonal
antibody, MAb159 triggered GRP78 endocytosis and inhibited tumor cell proliferation,
activated apoptosis (R. Liu et al., 2013).
In summary, GRP78 is rising as a promising therapeutic target for cancer. Since its
function in cancer is context-dependent and complex, exploring its function in different
types of cancers is needed.
1.3 Endometrial Cancer
Endometrial cancer refers to malignancies that arise from the endometrium, which
is a complex tissue with epithelial component and stromal component. During the past
10
decade, incidence of endometrial cancer has increased more than 80,000 per year. In the
United States, it is the most common gynecological malignancy in women with over 10,470
estimated deaths each year (Siegel, Miller, & Jemal, 2016). Although endometrial cancer
exhibit high prevalence, it has received far less attention than ovarian and cervical cancer
from public health and research, possibly partially due to its high overall 5 year survival
rate (83%). In Western populations endometrial cancer is most commonly found in women
between the ages of 50 and 65 years, and is rare before the age of 40. The peak incidence
occurs in the post-menopausal age group (Purdie, 2003).
Endometrial cancer has been categorized into 3 types (Sorosky, 2008). Type 1
tumors are with endometrioid histology and account for approximately 85% of the cases,
whereas type 2 tumors often display serous or clear cell morphology (Di Cristofano &
Ellenson, 2007). In type 1 tumors, tumor suppressor gene Pten is altered in 30-80% of the
tumors, Tp53 mutation occurs in 10-20% of the tumors, oncogene Kras is mutated in 10-
30% of the cases and Ctnnb1 (β-catenin) mutations has been identified in 15-30% of the
cases. In contrast to type 1 tumors, 90% type 2 tumors predominantly bear Tp53 mutations,
but they rarely have Kras, Pten mutations. Finally, genetic disease consists of 10% of the
cases, of which 5% are Lynch II syndrome (Sorosky, 2008).
For treatment options, well-differentiated endometrioid carcinoma and atypical
hyperplasia are commonly treated with progestin in combination with surgery, since
estrogen receptor alpha and progesterone receptor are usually expressed in those lesions
(Di Cristofano & Ellenson, 2007). For moderately or poorly differentiated carcinoma,
surgery and adjuvant radiation therapy are required. If the carcinoma is confined in the
uterus, the long-term survival is excellent, but if it has spread beyond the uterus, neither
11
radiation nor chemotherapy can improve long-term prognosis. Therefore, early detection
and new approaches preventing the progression of endometrial cancer to metastasis are in
need.
To study endometrial cancer in vivo, genetically engineered mouse models have
been established. Type 1 endometrial cancer has been mimicked by using progesterone
receptor driven-Cre recombinase to conditionally knock out Pten in mouse uterus, which
resulted in hyperplasia around day 10 and endometrial cancer around 4 weeks (Daikoku et
al., 2008). In addition, type 2 endometrial cancer mouse model has also been developed,
where Trp53 and Cdh1 (E-cadherin) were deleted by PR-Cre recombinase and nuclear
atypia was observed at 6 months of age and highly aggressive tumor was observed at 12
months of age (Stodden et al., 2015).
Metaplasia is defined as the reversible replacement of one differentiated cell type
with another differentiated cell type and it is observed in both physiological and
pathological conditions. In the human uterus, squamous metaplasia is commonly
encountered in both adenocarcinoma cases and benign lesions, such as chronic
inflammation and hyperplasia of endometrium (Makishi et al., 2006). It likely represents a
reactive or reparative response to some chronic injury or irritation.
1.4. Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest disease
with overall 5-year survival rate <10% (Siegel et al., 2016). Risk factors for PDAC include
advanced age, smoking and long-standing chronic pancreatitis (Ying et al., 2016).
12
PDAC is believed to arise from three preneoplastic lesions, namely pancreatic
intraepithelial neoplasm (PanIN), the most common precursor lesions observed in humans,
as well as mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm
(IPMN) (Ying et al., 2016). PanINs are microscopic lesions and classified into PanIN-1,
PanIN-2 and PanIN-3 lesions according to their degree of cytologic and architectural atypia
(Maitra & Hruban, 2008). They can be papillary or flat, and are composed of columnar to
cuboidal cells with varying amount of mucin. The progression from minimal abnormal
epithelium (PanIN 1) to more severe (PanIN 2 and 3) and ultimately to PDAC, is in parallel
with the successive accumulation of gene mutations that include activation of Kras (90%
of tumors), inactivation of Cdkn2a (p16, >50% of tumors), mutation of p53 (60-70% of
tumors) and inactivation of Dpc4 (SMAD4, 50% of tumors) (Ryan, Hong, & Bardeesy,
2014). IPMN arise within the main pancreatic duct with papillary architecture and occurs
in approximately 2% of adults (Ryan et al., 2014). The frequency of Kras mutation in IPMN
is 40%-65%. MCN typically form large cystic masses at the tail of pancreas with ovarian-
type stroma. Interestingly, most MCN occur in female (Maitra & Hruban, 2008). Yet, the
underlying genetic mutations in MCN have not been well-characterized.
Currently, standard of care for resectable PDAC, is surgery followed by adjuvant
(post-operative) chemoradiotherapy with either gemcitabine alone or with fluorouracil
(Hidalgo, 2010). For locally advanced unresectable PDAC, treatment options are limited
to chemotherapy alone or combined forms of chemoradiation and chemotherapy. For
metastatic PDAC, single-agent gemcitabine is the treatment of choice in the past decade.
Although tremendous efforts have been made in testing multiple new agents in
combination with gemcitabine, little favorable outcome was observed. One agent that
13
showed a marginal 12-day improvement, was an EGFR small molecule inhibitor erlotinib
(Moore et al., 2007). Taking its toxicity and modest effect in survival into consideration,
the combination therapy of erlotinib and gemcitabine is not widely accepted in the clinic.
Two other finished clinical trials showed more promising regimen. In the first study,
combination of nab-paclitaxel and gemcitabine increased PDAC patient survival by 2
months (V on Hoff et al., 2013). In another study, FOLIRINOX (folinic acid, 5-fluorouracil,
irinotecan, and oxaliplatin) demonstrated 5 months longer median survival than
gemcitabine (Conroy et al., 2011).
PDAC is mostly lethal because it is usually diagnosed at an advanced stage and
resistant to therapy. In patients whose tumors are resectable and receive adjuvant
chemotherapy, the 5-year survival rate is up to 20% (Neoptolemos et al., 2004). Therefore,
effective screening tests that would potentially improve survival rate for PDAC are
urgently needed.
Another difficulty for treating PDAC is the dense stroma formed around the cancer
cells, called desmoplasia, which forms a natural barrier for drug delivery. The desmoplasia
is composed of cancer-associated fibroblasts, endothelial cells, immune cells, and
extracellular matrix (Hidalgo, 2010). The stroma may produce factors that favor the
survival of tumor cells and suppress the immune response (Ryan et al., 2014). However,
depletion of cancer-associated fibroblasts in mice accelerated PDAC and decreased
survival, which suggest the existence of cancer-associated fibroblasts surrounding cancer
cells restricts the progression of PDAC (Ozdemir et al., 2014; Rhim et al., 2014). Hedgehog
pathway is activated in the stroma. With the notion to enhance drug delivery by loosening
cancer stroma, combination of gemcitabine and small molecule inhibitors (SMO inhibitor
14
saridegib) targeting Hedgehog pathway was investigated in a clinical trial with
disappointing results (Garrido-Laguna & Hidalgo, 2015). Immunotherapies are also
actively tested to treat PDAC.
The cell of origin in PDAC has been a point of debate and has been studied
extensively. While the histological appearance of PDAC suggests a ductal cell of origin,
mutating Kras in ductal cells utilizing cytokeratin 19 promoter in the pancreas failed to
form mouse PanIN (mPanIN) or formed mPanIN at exceedingly low frequency (Brembeck
et al., 2003; Ray et al., 2011). Interestingly, mPanIN in these mice were located mainly in
large ducts. The possibility of a progenitor cell population giving rise to PDAC has been
tested. Mutation of Kras in Nestin-positive exocrine cells, which is expressed in embryonic
day 10.5-12.5 and later will give rise to acinar and ductal cells, was sufficient for mPanIN
to form (Carrière, Seeley, Goetze, Longnecker, & Korc, 2007). Pdx1 (pancreatic and
duodenal homeobox 1) and p48 are transcription factors first expressed at embryonic day
8.5-9.5, and required to commit cells to a pancreatic fate (Hingorani et al., 2003).
Oncogenic Kras directed to Pdx1/p48-positive progenitor cells resulted in mPanIN and
spontaneous progress to PDAC (Hingorani et al., 2003). These research indicate the
potential origin of PDAC in pancreatic progenitor cells.
The notion of existing adult cells in the pancreas transdifferentiate into duct-like
cells further leading to PDAC were tested in a number of studies. The pancreas consists of
exocrine part, namely ductal cells, centroacinar cells and acinar cells, and endocrine part.
Centroacinar cells were reported to expand and form malignant transformation driven by
PTEN loss in the pancreas (Stanger et al., 2005). Insulin-expressing endocrine cells were
reported to be refractory to Kras mutation, however, they formed exocrine neoplasia under
15
chronic pancreatic injury (Friedlander et al., 2009).
More importantly, mounting evidence are suggesting that PDAC precursor cells
primarily arise from duct-like state of acinar cells and such process is called, acinar-to-
ductal metaplasia (ADM) (Kopp et al., 2011). Targeting Kras
G12D
to open reading frame of
Mist1, a transcription factor required for acinar organization, resulted in mixed metastatic
pancreatic carcinoma (Tuveson et al., 2006). Elastase promoter enabled selective targeting
to acinar and centroacinar cells in the pancreas. Mutant Kras under the control of elastase
promoter induced acinar cell hyperplasia and preinvasive pancreatic neoplasia (Grippo,
Nowlin, Demeure, Longnecker, & Sandgren, 2003). To mimic human pancreatic cancer
where PDAC arise in adult pancreas, the susceptibility to oncogenic Kras in adult mice
pancreas were studied using inducible Cre recombinase. One study discovered that
Kras
G12V
activated in embryonic acinar/central acinar lineage led to PanIN and invasive
PDAC, however, they were only detected when mice were challenged with chronic
pancreatitis (Guerra et al., 2007). Another separate study reported similar findings
(Friedlander et al., 2009). However, two independent studies have shown that activation of
oncogenic Kras in acinar cells of the adult mouse led to ADM and efficient mPanIN
formation (De La O et al., 2008; Habbe et al., 2008). More recently, the identification of
the ductal cell determinant Sox9, allowed targeting ductal/centroacinar lineage cells with
Sox-9 promoter at an earlier stage than cytokeratin 19 promoter (Kopp et al., 2011). The
propensity of ductal/centroacinar cells and acinar cells to form PanIN were compared
directly and oncogenic Kras induced PanIN was reported to predominantly arise from
acinar but not ductal/centroacinar cells (Kopp et al., 2012). Taken together, the plastic
centroacinar, ductal, endocrine and acinar cells have the potential to initiate malignant
16
tumor in the pancreas given corresponding genetic alterations and environmental insults,
however, the PanIN primarily initiate from acinar cells transdifferentiating into ductal
morphology.
In summary, pancreatic cancer remains to be one of the deadliest disease for human
with minimal progress made in prevention, early diagnosis and treatment in the past few
decades. ADM has been identified as the tumor initiating event for PDAC and targeting
this process may potentially delay PDAC.
Since the role of GRP94 in cancer is context-dependent and depletion of GRP94 in
the liver cancer and multiple myeloma indicated opposite effects, we further explored the
function of GRP94 depletion in another cancer model - endometrial cancer driven by the
PTEN loss. Given the suppression of cancer growth by GRP78 haploinsufficiency were
mostly in Pten-null driven cancers, we investigated the function of GRP78 in a cancer
model driven by Kras mutations. Overall, in this dissertation, we uncovered the functions
of GRP94 and GRP78 in endometrial and pancreatic tumorigenesis.
17
Chapter 2
GRP94 Deficiency Induces Squamous Cell Metaplasia
and Suppresses PTEN-null Driven Endometrial
Epithelial Tumor Development
2.1 Introduction
Endometrioid adenocarcinoma (EAC), the most common gynecological malignancy in the
United States, arises from the inner lining of the uterus(Siegel et al., 2016). EAC has been
categorized into two distinct types. Type 1 tumors exhibit endometrioid differentiation and
account for approximately 85% of the cases, whereas type 2 tumors, non-endometrioid,
often display serous or clear cell histology (Di Cristofano & Ellenson, 2007). The most
common mutation in type 1 tumors is in the Pten tumor suppressor gene which is altered
in 40 to 80% of cases (Di Cristofano & Ellenson, 2007). An endometrial cancer mouse
model mimicking human EAC has been established using the progesterone receptor
promoter driven Cre-recombinase (PR-Cre) to conditionally knockout Pten in the
endometrium, resulting in rapid onset of EAC at 4 weeks of age, with infiltration into the
myometrium by 8 weeks (Daikoku et al., 2008). Thus, this model is useful for studying the
key determinants of EAC development and invasion.
Glucose-regulated protein 94 (GRP94) encoded in humans by HSP90B1, is a
member of the HSP90 family sharing 50% homology to cytosolic paralog HSP90 (A. S.
Lee, 2014; Ni et al., 2011a). It is a multifunctional protein playing important roles as a
molecular chaperone and a Ca
2+
-binding protein in the endoplasmic reticulum (ER) (B Luo
& Lee, 2012; Wanderling et al., 2008). Homozygous knockout of GRP94 results in
18
embryonic lethality (B Luo & Lee, 2012; Wanderling et al., 2008). As an ER chaperone,
GRP94 controls the maturation and secretion of insulin growth factors (IGFs) and the
processing of Toll-like receptors (B. Liu et al., 2010; Marzec et al., 2012). Recently, the
Wnt co-receptor, LRP6, has been identified as a new client of GRP94 (B. Liu et al., 2010).
When GRP94 was conditionally knocked out in the gut, LRP6 failed to export to the cell
surface, and nuclear translocation of β-catenin was compromised (B. Liu et al., 2010). As
the β-catenin pathway is a key regulator of development as well as tumorigenesis, this
suggests that loss of GRP94 could impact cellular differentiation and cancer progression.
Invasive cancer requires remodeling of the cell adhesion molecules that maintain cell-cell
contacts and intercellular junctions (Gupta & Massague, 2006). Another major function
attributed to GRP94 is in maintaining cell matrix integrity due to its chaperoning activities
for key cell surface adhesion proteins such as E-cadherin and integrins (Chen, Tseng, et al.,
2014; Hong, Liu, Chiosis, Gewirth, & Li, 2013; Biquan Luo et al., 2011).
GRP94 is commonly elevated in human cancers (A. S. Lee, 2014). The Grp94
promoter was strongly activated in spontaneous and chemically-induced tumors in mice
(Reddy et al., 2002). GRP94 loss in B cells attenuated multiple myeloma, and GRP94
deficiency in macrophages reduced colitis and inflammation-associated colon
tumorigenesis (Hua et al., 2013; Morales et al., 2014). However, in the bone marrow and
hepatocytes, GRP94 loss perturbed extracellular matrix proteins, oncogenic signaling
pathways and led to hyper-proliferation of progenitor/stem cells (Chen, Tseng, et al., 2014;
Biquan Luo et al., 2011). Interestingly, repopulation of GRP94-positive hepatocytes was
detected in GRP94-deficient livers, correlating with spontaneous hepatocellular carcinoma
development in aged mice and promotion of chemically-induced hepatocellular
19
carcinogenesis (Chen, Ha, Kanel, & Lee, 2014; Rachidi et al., 2015). Therefore, GRP94
could have a multifaceted effect on tumorigenesis that is context- and age-dependent. In
this study, we report that GRP94 expression is significantly elevated in human endometrial
cancer and required for human endometrial cancer cell viability. We further report the
creation of two endometrial-specific knockout mouse models with deletion of Grp94 alone
or in combination with Pten deletion. Our study reveals that loss of GRP94 in mouse
endometrium induces squamous cell metaplasia (SCM), attenuated PTEN-deficiency
mediated β-catenin and AKT/S6 activation in the SCM and suppressed EAC.
The role of another ER chaperone GRP78 in endometrial cancer has also been
studied. GRP78 overexpression is more frequently detected in EAC than in normal
endometrium (Bifulco et al., 2012; Gray et al., 2013). GRP78 has also been reported to
mediate cell growth and invasiveness in EAC cells (Cali et al., 2014). To determine whether
targeting GRP78 could suppress EAC development, a conditional knockout mouse model
using progesterone receptor-Cre-recombinase to achieve Pten and Grp78 (cPten
f/f
Grp78
f/f
)
deletion in the endometrial epithelium was created (Lin et al., 2015). Strikingly,
no cPten
f/f
Grp78
f/f
mice developed EAC, even after more than 8 months of observation.
Histologic examination of uteri from cPten
f/f
Grp78
f/f
mice also revealed no complex
atypical hyperplasia, a well-established EAC precursor (Lin et al., 2015). Thus, we
explored possible underlying mechanism and observed these histologic observations
among the cPten
f/f
Grp78
f/f
uteri corresponded to abrogation of AKT activation within the
endometrium. We further observed that GRP78 co-localized with activated AKT on the
surface of EAC, thus providing an opportunity for therapeutic targeting.
20
2.2 Materials and methods
Mice
PR-Cre;Pten
f/f
mice on a C57BL6/129SV background (Daikoku et al., 2008) were
crossed with Pten
f/f
;Grp94
f/f
mice (Chen, Tseng, et al., 2014) to generate the PR-
Cre;Pten
f/f
;Grp94
f/f
(cP
f/f
94
f/f
) mice. Grp94
f/f
mice on a C57BL/6;129/SV background (Mao
et al., 2010) were crossed with cP
f/f
94
f/f
to generate PR-Cre;Grp94
f/f
(c94
f/f
). Genotyping
was performed by PCR using mouse tail genomic DNA as previously described (Chen,
Tseng, et al., 2014). All protocols for animal use and euthanasia were reviewed and
approved by the University of Southern California Institutional Animal Care and Use
Committee.
Tissue processing and histology
Female mice were euthanized and uteri were isolated. Collected samples were
either frozen in liquid nitrogen for biochemical analysis or fixed in 10% zinc formalin
(Sigma-Aldrich, St. Louis, MO) or frozen in OCT compound (Tissue-Tek Sakura, Torrance,
CA) for tissue analysis. Both paraffin-embedded and OCT-embedded tissues were
sectioned at 7 μm.
Western blot analysis
Tissue lysates were subjected to SDS-PAGE and Western blot analysis as described
previously (Ye, Jung, et al., 2010). Primary antibodies used were GRP94 (1:5000, Enzo
Life Sciences, Farmingdale, NY), PTEN (1:1000, Cell Signaling, Danver, MA) and
GAPDH (1:5000, Santa Cruz Biotechnology, Dallas, TX).
21
Tissue section staining
Immunostaining on paraffin-embedded or frozen tissue sections was performed as
described previously (Chen, Tseng, et al., 2014; Fu et al., 2008). Tissue sections were
incubated at 4°C overnight with primary antibodies against GRP94 (1:250, Enzo Life
Sciences, Farmingdale, NY), cytokeratin 14 (1:400), p63 (1:200), Cyclin D1 (1:100), Ki67
(1:200) from Thermo Scientific (Fremont, CA), GRP78 (1:50), β-catenin (1:100) from
Santa Cruz Biotechnology, Dallas, TX, active β-catenin (1:50, Millipore, Billerica, MA),
phospho-S6 (1:200), S6 (1:50), phospho-AKT (Ser 473, 1:50), AKT (1:200), phospho-
Histone H3 (1:200), p-eIF2α (1:50), eIF2α (1:200) from Cell Signaling Technology
(Danver, MA), cytokeratin 8 (1:100, Developmental Studies Hybridoma Bank, Iowa City,
IA), α-Smooth Muscle Actin (1:2000, Sigma-Aldrich, St. Louis, MO), pan cytokeratin
(1:50, Abcam, Cambridge, MA) and E-cadherin (1:50, BD Biosciences, San Jose, CA).
Immunofluorescence was analyzed using a Zeiss LSM 510 confocal microscope with LSM
510 Version 4.2 SP1 acquisition software. Confocal images were acquired with 20X or 40X
oil lens. Images were then processed with LSM Image Browser R4.2 and Adobe Photoshop
CS5.
Staining for mucin by either mucicarmine or periodic acid-Schiff stain (PAS)
followed by diastase digestion was performed by the clinical histology laboratory at
University of Southern California using standard protocols.
Cell lines and culture conditions
Human endometrial cancer cell lines AN3CA and ECC-1 have been described and
authenticated by mitochondrial sequencing (7.1.11) (Gray et al., 2013). Only short term
22
cultures from the verified frozen stocks were used. The cells were maintained in
Dulbecco’s Modified Eagle media (Corning, Manassas, VA) supplemented with 10% fetal
bovine serum (Omega Scientific, Tarzana, CA), 1% penicillin and streptomycin at 37°C in
a humidified atmosphere of 5% CO2.
WST-1 viability assay
Cell viability was assessed with the WST-1 reagent (Roche, Indianapolis, IN).
Briefly, 10,000 cells per well were plated onto 96-well plates and the cell viability was
measured by incubating each plate with 10 μl/well of WST-1 substrate for 2 hours and
plates were red at a wavelength of 450 nm.
RNA-seq dataset analyses
TCGA Level 3 RNA-seq data were downloaded from TCGA data access
(https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/tumor/). The
version number of RNA-seq dataset is IlluminaHiSeq_RNASeqV2_3.1.12. The data were
all generated on Illumina HiSeq platform, mapped with the RSEM algorithm and
normalized so that the third quartile for each sample equals 1000. Entrez gene IDs were
used for mapping to genomic locations using GenomicRanges (Lawrence et al., 2013).
To compare the mRNA expression level of Grp94 and Pten between normal and
endometrial cancer samples, we log2 transformed the expression data [log2(RSEM+1)],
and then performed a Student’s t-test on gene expression between 24 normal and 177 tumor
samples. In order to investigate the gene expression correlation between Grp94 and Pten,
Pearson correlation test was used on log2 transformed expression data.
23
Statistical analysis
Statistical analysis was performed with the least significant difference (LSD)
method, the 2-tailed Fisher’s exact test or 2-tailed Student’s t-test as indicated.
2.3 Results
2.3.1 GRP94 mRNA and protein levels are elevated in human EAC and contribute to
cell viability
First, we performed Grp94 mRNA expression analyses using 24 normal and 177
endometrial cancer expression profiles from TCGA (Kandoth et al., 2013). We observed
significantly increased Grp94 mRNA expression in tumor versus normal samples (p value
= 4.7E-14), and comparable Grp94 mRNA levels in different grade and stage of
endometrial cancer (Figures 2.1A-C). As many of Pten mutations result in decreased
expression (Di Cristofano & Ellenson, 2007; Djordjevic et al., 2012), Pten mRNA is
downregulated in tumor samples compared to normal samples (p value = 5.6E-16) (Figure
2.1D). Furthermore, examination of GRP94 protein expression in human uterine tissue
samples by immunohistochemistry (IHC) showed basal GRP94 expression in epithelial
cells in the normal endometrium, and upon development of EAC which is populated by
epithelial cells, strong GRP94 expression was detected uniformly in Grade 1, 2 and 3
tumors (Figure 2.1E), in agreement with the RNA-seq dataset analysis. Furthermore,
consistent with GRP94 over-expression in endometrial cancer which is predominantly
mutated in Pten, in RNA-seq dataset analysis, an inverse correlation was detected between
Grp94 and Pten mRNA levels using both normal (green) and tumor samples (red) (p value
24
= 0.001) due to the opposite expression changes between Pten and Grp94 mRNA levels
from normal to tumor (Figure 2.1F).
To test the effect of GRP94 deficiency on the viability of human endometrial cancer
cells, two cell lines (AN3CA and ECC-1), both reported to have loss of PTEN (Gray et al.,
2013), were examined. Treatment with siRNA targeting Grp94 reduced its protein level in
AN3CA cells by 70% and in ECC-1 cells by 90% compared to control siRNA (siCtrl),
corresponding with a 35% and 60% decrease in viability of AN3CA and ECC-1 cells,
respectively, compared to siCtrl, as measured by the WST-1 assay (Figures 2.1G and H).
Thus, GRP94 contributes to human endometrial cancer cell viability.
25
Figure 2.1 Increased GRP94 expression in human EAC and its effect on EAC cell viability. Grp94
mRNA expression in A. normal uterine (n=24) and EAC tissues (n=177), B. different grade EAC, and C.
different stage EAC. D. Pten mRNA expression in normal uterine and EAC tissues. Student’s t-test p values
are indicated. E. Immunohistochemistry (IHC) analysis of GRP94 in human normal uterine and EAC tissues.
Scale bar, 50 μm. F. Scatterplot of Grp94 and Pten mRNA expression of human normal uterine (green) and
tumor samples (red). Pearson correlation p value is indicated. Western blot analysis of GRP94 knockdown
efficiency in G. AN3CA cells and H. ECC-1 cells. The level of GRP94 reduction after normalization to
GAPDH which served as loading control is shown below. WST-1 assay measuring cell viability in si-control
(siCtrl) and si-Grp94 (si94) treated G. AN3CA cells at day 4 and H. ECC-1 cells at day 5. Data are presented
as mean ± s.e., **p<0.01 (Student’s t-test).
26
2.3.2 Endometrial GRP94 ablation leads to spontaneous squamous cell metaplasia
and loss of active nuclear β-catenin expression
To study the role of GRP94 in endometrial cancer in vivo, we utilized genetically
engineered mouse models. First, we determined the effect of GRP94 ablation in the normal
mouse endometrium. Deletion of GRP94 in the post-natal mouse endometrium was
achieved through creation of mice bearing a PR-Cre transgene and Grp94 floxed alleles.
The PR promoter is activated in endometrial tissues around post-natal day 3 leading to gene
ablation in luminal and glandular epithelial cells at first and subsequently in endometrial
stroma and myometrium (Soyal et al., 2005). The PR-Cre;Grp94
f/f
mice, referred to below
as c94
f/f
, were analyzed in parallel with littermates lacking PR-Cre serving as WT controls.
The genotypes of the mice were determined by PCR of tail genomic DNA (Figure 2.2A).
At both 4 and 8 weeks, the c94
f/f
uteri were smaller (Figure 2.2B) and lighter (Figure 2.2C).
Histological examination of uterine sections stained with H&E revealed two major
differences. First, the c94
f/f
uteri exhibited reduced number of endometrial glands compared
to WT (Figure 2.2D, white arrows), resulting in a flat luminal surface. Second, while the
WT uteri were lined with a single layer of columnar cells (black bars), at 8 weeks the c94
f/f
uteri were lined by multi-layered squamous epithelium (Figure 2.2D, red bars). In contrast,
uteri deficient of another ER chaperone GRP78 did not exhibit SCM and were lined with
a single layer of columnar cells (Figure 2.3). As demonstrated by IHC, GRP94 expression
was largely depleted in the c94
f/f
uteri except in occasional stromal cells (Figure 2.2D).
27
Figure 2.2 Generation of PR - Cre -mediated GRP94 knockout mouse models. A. Representative mouse
tail PCR genotyping of the indicated alleles. B. Representative gross anatomy of uteri of the indicated
genotypes at 4 and 8 weeks. Scale bar, 1 cm. C. The ratio of uterine weight to body weight in WT and c94
f/f
mice at 4 weeks (n=16 and 5, respectively) and 8 weeks (n=4 and 11, respectively). The data are presented
as mean ± standard error (s.e.), *p<0.05, **p<0.01 (LSD Method, and data was log transformed prior to
analysis). D. H&E staining (upper panel) and IHC analysis of GRP94 (lower panel) of WT and c94
f/f
uteri
from 4 and 8 weeks. Scale bar, 50 μm. White arrows indicate glands and black and red bars denote columnar
luminal epithelial cells and SCM, respectively.
28
Figure 2.3 GRP78 deficient uteri showed no SCM. H&E staining of c78
f/f
uteri at 4 and 8 weeks. Black
bars denote a single layer of columnar epithelial cells. Scale bar, 100 μm.
To confirm that the multi-layered epithelium in c94
f/f
uteri represented squamous
metaplasia, the expression of p63 and cytokeratin 14 (K14), representing early and late
markers of squamous differentiation, respectively, was examined by IHC (Koster & Roop,
2007; A. Yang et al., 1998). At 4 weeks, p63 expression was detected in some c94
f/f
endometrial epithelial cells located within the luminal lining, indicating initiation of SCM;
by 8 weeks c94
f/f
endometrial epithelial cells showed robust p63 and K14 expression,
confirming their squamous nature (Figure 2.4A). As expected, both markers were absent
from WT endometrium.
We next tested whether SCM observed in the c94
f/f
endometrium could be due to β-
catenin dysfunction since ablation of endometrial β-catenin has been reported to induce
SCM (Jeong et al., 2009; Villacorte et al., 2012). IHC analysis revealed that at 4 weeks
both c94
f/f
and WT endometria showed strong active nuclear β-catenin expression in the
columnar epithelium (Figure 2.4B, box), indicating that GRP94 is not required for β-
catenin activation and nuclear translocation in columnar cells. However, 8 week old c94
f/f
endometrium, which had undergone SCM, showed minimal active nuclear β-catenin
expression compared to WT (Figure 2.4B, box). Total β-catenin levels were similar in c94
f/f
and WT endometria at 4 and 8 weeks (Figure 2.4B). No spontaneous endometrial cancer
was observed in the c94
f/f
uteri for at least 9 months (Figure 2.5).
29
Figure 2.4 Induction of squamous metaplasia in developing c9 4
f/f
uteri. A. IHC of p63 and cytokeratin
14 (K14). B. IHC of active β-catenin and β-catenin in WT and c94
f/f
uteri at 4 and 8 weeks. Enlarged views
of the boxed regions are shown. Scale bar, 50 μm.
30
Figure 2.5 c 9 4
f/f
uteri showed SCM but no EAC at 9 months. H&E staining of c94
f/f
uteri at 9 months.
Red bars denote SCM. Scale bar, 200 μm.
2.3.3 Endometrial GRP94 deficiency suppresses PTEN-null driven adenocarcinoma
To test the role of GRP94 in type 1 endometrial cancer, which can be induced by
PTEN depletion, PR-Cre;Pten
flox/flox
(cP
f/f
) mice were crossed with Pten
f/f
94
f/f
mice to
generate a biallelic deletion strain lacking both Pten and Grp94 (cP
f/f
94
f/f
). Littermates
lacking the PR-Cre transgene were used as WT controls. The mouse genotypes were
determined by PCR of tail genomic DNA (Figure 2.6A). At 4 weeks, the cP
f/f
uteri were
considerably larger than the WT and cP
f/f
94
f/f
uteri (Figure 2.6B), and the average weight
of cP
f/f
uteri normalized against body weight was 5.5 and 2.0 times that of the WT and
cP
f/f
94
f/f
uteri, respectively (Figure 2.6C). Western blot analysis of uterine tissue lysates
confirmed minimal expression of PTEN and GRP94 in the respective knockout mice, with
GAPDH serving as loading control (Figure 2.6D).
Histological examination showed that at 4 weeks all 9 cP
f/f
uteri examined
contained extensive and aggressive EAC which, in 8 of the 9 cases, replaced more than 70%
of the endometrium (Figure 2.6E). One out of 7 cP
f/f
94
f/f
uteri showed no evidence of
malignancy. The remaining 6 cP
f/f
94
f/f
uteri showed a mixture of normal and transformed
glands (Figure 2.7E, white and red arrows, respectively). Five out of 6 showed 30% or
less of the endometrium replaced by tumor, while only one uterus showed more extensive
tumor involvement. In addition, all tumors seen in cP
f/f
94
f/f
uteri were separated from the
myometrium by a layer of normal endometrium, as opposed to the tumors from cP
f/f
uteri
that typically involved the full thickness of the endometrium and abutted the myometrium.
All cP
f/f
94
f/f
uteri showed extensive squamous metaplasia of the endometrial luminal lining
31
(Figure 2.6E, red bars). At 8 weeks, cP
f/f
uteri continued to show aggressive EAC, and
SCM of the luminal epithelium was detected (Figure 2.6F). Strikingly, 8 week old cP
f/f
94
f/f
uteri only showed low grade lesions characterized by large well-differentiated glandular
structures with no morphological features of invasiveness such as ragged edges or stromal
reactions (Figure 2.6F). GRP94 depletion in both 4 and 8 week cP
f/f
94
f/f
uteri, including in
the well-differentiated glandular structures, was confirmed by IHC (Figures 2.6E and F).
Figure 2.6 Reduced endometrial cancer in cP
f/f
94
f/f
uteri. A. Representative mouse tail PCR genotyping
of the indicated alleles. B. Representative gross anatomy of uteri of the indicated genotype at 4 weeks. Scale
bar, 1 cm. C. The ratio of uterine weight to body weight in WT (n=16), cP
f/f
(n=10) and cP
f/f
94
f/f
(n=9) mice
at 4 weeks. The data are presented as mean ± s.e., **p<0.01 (LSD Method). D. Western blot analysis of
GRP94 and PTEN levels in uteri of indicated genotypes with GAPDH serving as the loading control. E. H&E
staining and IHC of GRP94 in uteri of the indicated genotype at 4 weeks and F. at 8 weeks. White and red
32
arrows indicate normal and transformed glands, respectively, the broken lines denote EAC and red bars
denote SCM. Scale bar, 200 μm.
2.3.4 cP
f / f
94
f / f
uteri display early squamous metaplasia and reduced β-catenin
signaling
Through IHC, we observed luminal epithelial cells were positive for both p63 and
K14 at 4 week old cP
f/f
94
f/f
uteri (Figure 2.7A), indicating accelerated onset of SCM
compared to age-matched c94
f/f
and cP
f/f
uteri. Active nuclear β-catenin expression was
readily detected in the WT and cP
f/f
but not the cP
f/f
94
f/f
uteri while total β-catenin was
robustly expressed in all three genotypes (Figure 2.7B). As a critical transcription factor,
nuclear β-catenin activates downstream targets such as cyclin D1 which induces
progression through the G1 phase of the cell cycle (Shtutman et al., 1999). Expression of
cyclin D1 was more prominent in the transformed glandular structures in cP
f/f
uteri
compared to WT epithelium attesting to increased cell cycle activity (Figure 2.7B).
Expression of cyclin D1 was low and confined to the basal layers in the metaplastic
epithelium of cP
f/f
94
f/f
uteri, indicating an ordered proliferation and a level of organization
characteristic of untransformed squamous epithelium (Figure 2.7B). At 8 weeks, IHC for
K14 confirmed that PTEN-null uteri showed SCM in some areas and, interestingly, those
same regions showed low GRP94 expression (Figure 2.7C, broken lines). Thus, both c94
f/f
and cP
f/f
uteri showed squamous differentiation, correlating with GRP94 deficiency. We
compared the distribution of metaplastic squamous cells (K14 positive) versus columnar
cells (K8 positive) in cP
f/f
94
f/f
uteri. Co-staining for K14 (green) and K8 (red) showed that
at 4 weeks, whereas endometrial glands were K8 positive (white arrow), most of the
endometrial luminal cells expressed the marker of squamous differentiation (K14) with
33
some residual cells located in the top layer (luminal surface) expressing K8 (Figure 2.7D).
By 8 weeks, both endometrial luminal and glandular cells expressed the squamous marker
(K14), with some cells expressing glandular marker (K8) located near the glandular lumen.
Occasional cells expressed both K14 and K8 markers (Figure 2.7D, yellow arrows and
box). Intra- and extra-cellular mucin, indicative of secretory activity, was present in 8 week
old cP
f/f
94
f/f
uteri, as confirmed by PAS-diastase (Figure 2.7D) and mucicarmine (not
shown) positivity.
Figure 2.7 Accelerated squamous metaplasia and reduced β-catenin signaling in cP
f/f
94
f/f
uteri. A. IHC
of p63, K14 and B. IHC of active β-catenin, β-catenin, cyclin D1 in uteri of indicated genotypes at 4 weeks.
The white arrows denote glands. Enlarged view of the boxed region is shown. C. IHC of K14, GRP94 in
uteri of indicated genotypes at 8 weeks. Scale bar, 200 μm. D. Merged images of immunofluorescence (IF)
of K8 (red) and K14 (green) with nuclei stained with DAPI (blue) in cP
f/f
94
f/f
uteri and PAS-diastase staining
of cP
f/f
94
f/f
uteri at the indicated age. Scale bar, 200 μm. White arrows indicate glands, white bars denote
34
SCM and yellow arrows indicate cells positive for both K8 and K14. Enlarged view of the boxed region is
shown.
2.3.5 Attenuation of AKT/S6 activation and proliferation in cP
f / f
94
f / f
SCM
Pten deletion leads to PI3K signaling with activation of downstream targets
including AKT and S6 via phosphorylation (Song, Salmena, & Pandolfi, 2012). As
expected, cP
f/f
uteri showed robust activation of S6 in the EAC (Figure 2.8A, white arrows)
and luminal epithelial cells (black bars), whereas in cP
f/f
94
f/f
uteri S6 activation was
confined to the glands but absent in the SCM (Figure 2.8A, red bars). Immunofluorescence
(IF) analysis for activated AKT showed a pattern similar to that of pS6 in the three
genotypes such that AKT phosphorylation was evident in the glands of cP
f/f
and cP
f/f
94
f/f
uteri (white arrows), but substantially reduced in the SCM of the cP
f/f
94
f/f
uteri (Figure
2.8B, red bars) compared to the columnar luminal epithelial cells of cP
f/f
uteri (white bars).
IHC analysis showed no ERK activation in uteri of all three genotypes (Figure 2.9).
Proliferation as measured by IHC markers expressed either throughout the cell cycle (Ki67)
or during the mitotic phase (phospho-histone H3), was substantially reduced in the cP
f/f
94
f/f
compared to cP
f/f
uteri and was confined to glandular cells and basal layer of SCM (Figures
2.8C and D). We investigated the potential contribution of apoptosis to the reduced size of
cP
f/f
94
f/f
uteri. IHC showed that whereas cP
f/f
uteri exhibited a few cells positive for cleaved
caspase-3 located towards the lumen, cP
f/f
94
f/f
uteri showed little if any cells positive for
cleaved caspase-3 (Figure 2.10). Collectively, these results indicate that suppression of
AKT/S6 activation and reduced proliferation, rather than leading to increased apoptosis,
likely contribute to the suppression of PTEN-null driven EAC in cP
f/f
94
f/f
uteri.
35
Figure 2.8 Attenuated AKT activation and decreased proliferation in cP
f/f
94
f/f
SCM. A. IHC of phospho-
S6 (pS6) and S6 in uteri of the indicated genotypes at 4 weeks. White arrows indicate glands, black and red
bars denote columnar luminal epithelial cells and SCM respectively. B. IF of pAKT (Ser 473) and IHC of
AKT. White arrows indicate glands, white and red bars denote columnar luminal epithelial cells and SCM,
respectively. C. IHC of Ki67 and phospho-Histone H3 (p-H3) in mouse uteri. Scale bar, 100 μm. D.
Quantification of the p-H3 positive epithelial cells in the uteri sections of WT (n=6), cP
f/f
(n=3) and cP
f/f
94
f/f
(n=4) uteri at 4 weeks. The data are presented as mean ± s.e., **p<0.01 (LSD Method).
36
Figure 2.9 cP
f/f
94
f/f
uteri showed no change on ERK activation. IHC of p-ERK and ERK in WT, cP
f/f
and
cP
f/f
94
f/f
mice uteri at 4 weeks with pancreas bearing Kras mutation as p-ERK positive control (arrow). The
p-ERK (1:400), ERK (1:100) antibodies were purchased from Cell Signaling Technology (Danver, MA).
Scale bar, 100 μm.
Figure 2.10 cP
f/f
94
f/f
uteri showed no increase in apoptosis. IHC of cleaved caspase-3 in WT, cP
f/f
and
cP
f/f
94
f/f
mice uteri at 4 weeks. The cleaved caspase-3 (1:250) antibody was purchased from Cell Signaling
Technology (Danver, MA). Scale bar, 100 μm.
2.3.6 cP
f / f
94
f / f
uteri show no EAC myometrial invasion and high E-cadherin
expression
We next compared myometrial invasion of cP
f/f
and cP
f/f
94
f/f
uteri through
histological examination of H&E stained tissue sections by a pathologist (L.D.), as well as
37
examination of IHC stains on consecutive microtome sections for a muscle layer marker,
α-smooth muscle actin (α-SMA) and an epithelial cell marker, pan-cytokeratin (panCK)
(Daikoku et al., 2008). Myometrial invasion was readily observed in cP
f/f
uteri at 8 weeks
(Figure 2.11A, arrows). In contrast, no myometrial invasion was detected in age-matched
cP
f/f
94
f/f
uteri (Figure 2.11A). Seven of 10 cP
f/f
uteri exhibited myometrial invasion at that
age compared to 0 out of 8 cP
f/f
94
f/f
uteri (Figure 2.11B). These differences were
statistically significant (p = 0.004). Consistent with the notion that invasive cancer cells
loosen their connection to neighboring cells and basement membrane via downregulation
of intercellular adhesion (Gupta & Massague, 2006), cP
f/f
uteri in which myometrial
invasion was present, exhibited very low level of E-cadherin (Figures 2.11C and D). In
contrast, both WT and cP
f/f
94
f/f
uteri showed high E-cadherin expression in epithelial cells.
38
Figure 2.11 Characterization of cP
f/f
94
f/f
uteri on myometrial invasion and prolonged stage. A. IHC of
pan-cytokeratin (panCK) and α-smooth muscle actin (α-SMA) on consecutive slides in uteri of the indicated
genotype at 8 weeks. The red broken lines denote the boundaries between the endometrium and the
myometrium. Arrows indicate EAC invasion into the myometrium. Scale bar, 400 μm. B. Frequency of
myometrial invasion in cP
f/f
and cP
f/f
94
f/f
uteri at 8-9 weeks. Each circle represented one mouse. p=0.004 (2-
sided Fisher’s exact test). C. IHC of E-cadherin at 8 weeks. Scale bar, 200 μm. D. IF of E-cadherin on frozen
tissue sections (red) at 8 weeks. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. E. H&E of WT,
cP
f/f
and cP
f/f
94
f/f
uteri at 12 weeks (upper panel) and 20 weeks (lower panel). Scale bar, 200 μm.
39
2.3.7 Prolonged depletion of GRP94 in PTEN-null uteri leads to massive glandular
expansion but no EAC formation
In following WT, cP
f/f
and cP
f/f
94
f/f
mice past 8 weeks, we noticed that the size of
the cP
f/f
94
f/f
uteri was considerably larger than the cP
f/f
uteri by 12 and 20 weeks (Figure
2.12A). For both 12 and 20 week uteri, histological examination showed aggressive EAC
in the cP
f/f
uteri, whereas the cP
f/f
94
f/f
uteri displayed massively expanded, but mature
glands with abundant secretions lacking any of the cytological and architectural features
typically associated with malignancy (Figure 2.11E). IHC further showed that GRP94
depletion had persisted in the cP
f/f
94
f/f
uteri at least through 20 weeks (Figure 2.12B). The
effects of GRP94 deficiency in the WT (c94
f/f
) and PTEN-null uteri (cP
f/f
94
f/f
) are
summarized in Figure 2.13, with the striking observation that while PTEN-null uteri with
WT GRP94 level all developed EAC by 4 weeks, no EAC was detected in the uteri of 12
to 20 week cP
f/f
94
f/f
mice.
40
Figure 2.12 Characterization of uteri at prolonged stage. A. Gross anatomy of WT, cP
f/f
and cP
f/f
94
f/f
mice
uteri at 12 weeks and 20 weeks. Scale bar, 0.5 cm. B. IHC of GRP94 in WT, cP
f/f
and cP
f/f
94
f/f
uteri at 12
weeks (upper panel) and 20 weeks (lower panel). Scale bar: 400 μm.
41
Figure 2.13 Summary model on the effect of GRP94 deficiency in mouse uteri in the presence or
absence of PTEN-deficiency. The development of 4 and 8 week uteri of the indicated genotypes is shown.
The morphological transitions of the glands, SCM replacement, EAC formation, alteration of the signaling
pathways and E-cadherin expression are summarized for 4 and 8 week uteri. EAC: endometrial cancer. SCM:
squamous cell metaplasia.
2.3.8 GRP78 deficiency inhibits AKT activation in PTEN-null endometrium
To compare GRP94 knockout with GRP78 knockout uteri in the context of PTEN
depletion, we analyzed the cPten
f/f
Grp78
f/f
uteri. AKT activation is an important
mechanism in the development and progression of many solid tumors, including this Pten-
null EAC model. Recent in vitro evidence showed that GRP78 was needed for optimal
AKT activation in EAC (Gray et al., 2013); therefore, we sought to determine whether
genetic deletion of Grp78 from the endometrial epithelia could block AKT activation in
the cPten
f/f
Grp78
f/f
uteri. As demonstrated by immunofluorescent staining followed by
confocal microscopy, endometrial tumors harvested from necropsy showed robust
42
expression of AKT and GRP78 in cPten
f/f
Grp78
+/+
endometrial epithelial cells (Figure
2.14A). Although AKT was activated in the cPten
f/f
Grp78
+/+
uteri as evidenced by serine
473 phosphorylation, this activation was markedly suppressed in the cPten
f/f
Grp78
f/f
uteri,
correlating with the lack of GRP78 expression (Figure 2.14A). Dual immunofluorescent
staining of pAKT and GRP78 in 4 week cPten
f/f
Grp78
+/+
uteri further demonstrated that
GRP78 colocalized with pAKT at the cell surface (Figure 2.14B).
43
Figure 2.14 P ten-null mediated AKT activation was suppressed in uteri of cP ten
f/f
Grp 7 8
f/f
mice. A.
Immunofluorescent staining for pAKT (Ser473) (red) and GRP78 (green) in cPten
f/f
Grp78
+/+
and
cPten
f/f
Grp78
f/f
mice uteri at 4 weeks. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. B. Co-
immunofluorescent staining for pAKT (red) and GRP78 (green) in 4-week cPten
f/f
Grp78
+/+
mice uteri.
Nuclei were stained with DAPI (blue). Yellow represents the colocalization of pAKT and GRP78. Negative
control (Neg ctrl) is without primary antibodies. Each row represents one individual mouse uterus. Scale bar,
20 μm.
UPR activation is often associated with tumorigenesis (B Luo & Lee, 2012). In the
Pten-null EAC model, eIF2α phosphorylation downstream of PERK signaling was
observed in the cancerous cPten
f/f
Grp78
+/+
uteri but not in the WT (Figure 2.15).
Phosphorylation of eIF2α was also detected in the cPten
f/f
Grp78
f/f
uteri (Figure 2.15),
consistent with previous reports of eIF2α activation in GRP78 knockdown in other cell
types (Wey, Luo, & Lee, 2012). In contrast, the expression of CHOP, a pro-apoptotic UPR
marker downstream of PERK signaling, was detected in only a few cells in the
cPten
f/f
Grp78
+/+
uteri and not detected in the WT or the cPten
f/f
Grp78
f/f
uteri (Figure 2.15).
44
Figure 2.15 Analysis of unfolded protein response (UPR) markers in the mouse uteri.
Immunohistochemical staining of p-eIF2α, eIF2α and CHOP on uteri of indicated genotypes at 4 weeks.
Examples of CHOP-positive cells are indicated by arrows and the insert showed an enlarged image of a
CHOP-positive cell. Scale bar, 100 μm.
2.4 Discussion
The glucose regulated proteins including GRP94 and GRP78 are stress-inducible
chaperones that mostly reside in the ER and are well-established as molecular chaperones
critical for the ER homeostasis (A. S. Lee, 2014). While GRP94 shares partial amino acid
homology with HSP90, its cellular localization and functions are distinct from HSP90 (B
Luo & Lee, 2012). At the same time while GRP94 and GRP78 are coordinately upregulated
by ER stress, knockout of either GRP94 or GRP78 in mouse models resulted in embryonic
lethality, implying distinct and non-compensatory functions in early mammalian
development (S. Luo, Mao, Lee, & Lee, 2006; Mao et al., 2010). Recently we have
45
examined the consequence of GRP78 knockout alone or in combination of PTEN in the
mouse uteri and discovered that while both GRP78 and GRP94 deficient uteri were smaller
in size and showed a decrease in the number of glands, unlike GRP94, GRP78 deficient
uteri were lined with a single layer of columnar cells. The uteri from the cP
f/f
78
f/f
uteri
showed decreased AKT activation and were devoid of atypical hyperplasia, a well-
established EAC precursor, throughout the experimental period of up to 8 months (Lin et
al., 2015).
These interesting observations raise the question whether other ER chaperones such
as GRP94 could also impact EAC development, and if so, do they utilize the same or
different mechanism? Results from the Cancer Genome Atlas analysis in this study
provided the first clue that Grp94 mRNA expression is significantly increased in human
EAC tissues across all grades of EAC, confirmed by IHC staining of human EAC samples.
Recent evidence also showed that GRP94 suppressed AKT activation in hepatocellular
carcinoma in vivo and in vitro (Chen, Tseng, et al., 2014; Huang et al., 2016), implying that
AKT activation in PTEN-null driven EAC may also be impaired by the loss of GRP94. In
this study, we investigated the effect of GRP94 deficiency in the mouse uterus in the
absence or presence of the tumor suppressor gene Pten, which is commonly altered in type
1 endometrial cancer. This study reveals new GRP94 function on uteri development and
oncogenic signaling which could offer novel mechanisms for curbing EAC.
First, compared to WT, GRP94 deficient uteri exhibited a decrease in the number
of endometrial glands, depriving adenocarcinoma of a substrate from which to develop.
This could explain in part the lower incidence of EAC in the cP
f/f
94
f/f
uteri, as well as the
smaller size of such uteri, since budding of nascent glands from the luminal epithelium is
46
critical for uterine development (Cooke, Spencer, Bartol, & Hayashi, 2013). Loss of
GRP94 might also lead to decreased uterine size through IGF1 deficiency, since GRP94 is
an essential chaperone for the production of IGFs (Barton et al., 2012; Marzec et al., 2012;
Wanderling et al., 2008), and IGF-1 deficient mice possess an infantile uterus with severe
hypoplasia especially in the myometrium (Baker et al., 1996).
Strikingly, uterine GRP94 deficiency resulted in spontaneous replacement of the
columnar luminal epithelial cells with squamous cells as early as 8 weeks. Such
replacement of one differentiated cell type with another, defined as metaplasia, while a rare
occurrence in the healthy endometrium, can be observed occasionally and under both
physiological and pathological conditions such as endometrial cancer, chronic irritation or
progestin effects (Anderson, Robboy, & Russell, 2002; Y . Lee, Kim, Ro, Kwak, & Song,
1983; Miranda & Mazur, 1995). The reduced number of glands and the presence of SCM
in GRP94-deficient uteri resembled the phenotypes associated with uterine β-catenin
ablation (Jeong et al., 2009). Indeed, depletion of uterine GRP94 resulted in loss of active
nuclear β-catenin, suggesting that this could contribute in part to spontaneous SCM in the
c94
f/f
uteri. SCM was accelerated when GRP94 deficiency was superimposed on a PTEN
deficiency, which independently induces endometrial SCM (van der Zee et al., 2013). In
support of the notion that GRP94 deficiency promotes SCM induction, we observed that
the SCM cells in the PTEN-null uteri also expressed low GRP94. Interestingly, uterine
depletion of GRP78, in the presence (Lin et al., 2015) or absence of PTEN-deficiency did
not induce SCM (Figure 2.3), underscoring the unique role of GRP94 in SCM.
Our work provides the first evidence that GRP94 deficiency can delay the
development of cancers driven by the absence of a functional PTEN. Interestingly, this
47
study revealed that the majority of cP
f/f
94
f/f
uteri at 4 weeks did develop endometrial cancer
albeit the tumors were less aggressive and much smaller in overall volume compared to the
EAC seen in the cP
f/f
uteri. However, by 8 weeks, the morphological appearance of the
cP
f/f
94
f/f
tumors had become substantially different than that of the tumors seen in the cP
f/f
uteri. The lesions were low-grade and less aggressive as evidenced by smooth acinar
contours and abundant secretions indicative of higher degree of differentiation. The
cP
f/f
94
f/f
uteri showed WT level of E-cadherin and no sign of myometrial invasion by tumor.
We further noted that in 8 week cP
f/f
94
f/f
uteri, the majority of the cells expressed the
squamous marker (K14). Cells expressing glandular marker (K8) were located in the
luminal layer, suggesting that the precursor cells normally giving rise to columnar
epithelial cells alternatively initiate a different differentiation pathway leading to squamous
cells. Beyond 8 weeks, the cP
f/f
94
f/f
uteri rapidly increased in size and the endometrium
showed expanded glands with abundant secretions. The accumulation of secretion could
be due either to an effect of GRP94 deficiency on secretory activity or to entrapment of
secretions within glandular lumina because of narrowing of secretory ducts due to
squamous metaplasia. In summary, loss of uterine GRP94 leads to several unique
phenotypes clearly distinguishable from those associated with suppression of EAC
development in conditional GRP78 knockout mice (Lin et al., 2015). We show that
replacing columnar epithelial cells with squamous epithelial cells induced by GRP94
deficiency may represent a novel mechanism for suppression of EAC.
In vitro and in vivo cancer models support the relationship between GRP78 and
AKT activation (A. S. Lee, 2014; R. Liu et al., 2013; Misra, Deedwania, & Pizzo, 2006;
Shin et al., 2003; Yi Zhang, Liu, Ni, Gill, & Lee, 2010). For example, in PTEN loss driven
48
prostate cancer and leukemia, a partial reduction of GRP78 suppresses AKT activation (Fu
et al., 2008; Wey, Luo, Tseng, et al., 2012). But, how GRP78 affects AKT activation could
be multifactorial. Although GRP78 is generally regarded as an ER resident protein, a
subfraction of GRP78 localizes at the cell surface in selected cell types, notably cancer
cells (Shin et al., 2003; Yi Zhang et al., 2010). Cell surface GRP78, through its interaction
with different ligands and cell surface proteins, can mediate important signal transduction
pathways including PI3K/AKT and this could play a major role since MAb159 targeting
cell surface GRP78 is highly effective in suppressing AKT activation in a variety of tumors,
including EAC. However, it is also possible that, as a major molecular chaperone in the
ER, GRP78 may be required for processing important growth factors and the cell surface
expression of their corresponding receptors, which might regulate AKT activation.
One proposed mechanism in which cell surface GRP78 activates AKT is by
complexing with PI3K to promote PIP3 formation (Yi et al., 2013). Consistent with this
notion, co-localization of GRP78 with pAKT on the cell surface in Pten-null EAC was
detected, similar to the phenomena seen in pancreatic adenocarcinoma (Hill et al., 2012).
Collectively, we report here for the first time that, in this murine Pten-null EAC,
endometrium-specific GRP78 deletion is able to suppress AKT and S6 activation. Our
targeted genetic deletion of GRP78 using floxed murine models provides compelling
evidence to explore and identify pharmacologic means to block GRP78 and further
represents a therapeutic opportunity to exploit this target (A. S. Lee, 2014; Sato, Yao, Arap,
& Pasqualini, 2010).
49
Chapter 3
Glucose-regulated protein 78 Haploinsufficiency Suppresses
Pancreatic Tumorigenesis Driven by Kr a s Mutation and p53
Heterozygosity
3.1 Introduction
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest disease
with overall 5-year survival rate <10% (Siegel et al., 2016). In patients whose tumors are
resectable and receive adjuvant chemotherapy, the 5-year survival rate is up to 20%
(Neoptolemos et al., 2004). Therefore, new approaches in early diagnostic and preventive
strategies are urgently needed, and identifying key players in tumor initiation as well as
tumor growth will shed light on the new approaches.
PDAC is believed to arise from three preneoplastic lesions, namely pancreatic
intraepithelial neoplasm (PanIN), the most common precursor lesions observed in humans,
as well as mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm
(IPMN) (Ying et al., 2016). During the progression from precursor lesions to PDAC, the
lesions acquire genetic mutations, which include activation of Kras (90% of tumors),
inactivation of Cdkn2a (95% of tumors), mutation of Tp53 (50-75% of tumors) and
inactivation of Dpc4 (50% of tumors) (Hidalgo, 2010). However, the cell of origin in
PDAC has been elusive. While the histological appearance of PDAC suggests a ductal cell
of origin, mounting evidence are suggesting that PDAC precursor cells primarily arise from
duct-like state of acinar cells and such process of acinar cells transdifferentiating into ductal
50
cells is called, acinar-to-ductal metaplasia (ADM) (Kopp et al., 2012).
To study PDAC in vivo, genetically engineered mouse models have been developed.
A pancreatic cancer mouse model mimicking human PDAC has been established using
pancreatic and duodenal homeobox 1 promoter driven Cre-recombinase (Pdx1-Cre) to
conditionally activate Kras and delete one allele of p53, resulting in mPanIN at 2 months
of age and PDAC at 4-5 months of age (Bardeesy et al., 2006).
The 78 kDa glucose-regulated protein (GRP78), also referred to as BiP/HSPA5, has
been regarded as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic
properties (Reddy et al., 2003; Zhou et al., 2011) and a master regulator of ER stress
signaling (A. S. Lee, 2014; Miao Wang, Wey, Zhang, Ye, & Lee, 2009). Tumor cells are
subjected to ER stress. As an adaptive measure, cancer cells, as well as stroma cells in the
tumor microenvironment, turn on the unfolded protein response (UPR) (B Luo & Lee, 2012;
Ron & Walter, 2007). ER stress induction of GRP78 in cancer cells represents a major pro-
survival response (A. S. Lee, 2014). While GRP78 is generally retained in the ER by the
KDEL retrieval machinery (Munro & Pelham, 1986), evidence has emerged that, under
pathophysiologic conditions, a subfraction of GRP78 re-localizes to the surface of specific
cell types, in particular cancer cells (Arap et al., 2004; Gonzalez-Gronow, Selim, Papalas,
& Pizzo, 2009; C. Liu, Bhattacharjee, Boisvert, Dilley, & Edgington, 2003; Ying Liu et al.,
2007; Miao Wang et al., 2009). We recently determined that ER stress can actively promote
cell surface localization of GRP78 (Yi Zhang et al., 2010). Cell surface proteome profiling
of tumor cells revealed a relative abundance of heat shock chaperones and glucose-
regulated proteins, including GRP78 (Shin et al., 2003). Importantly, preferential
expression of GRP78 on the surface of tumor cells but not in normal organs enables specific
51
tumor targeting by circulating ligands without a harmful effect on normal tissues (Arap et
al., 2004; Kim et al., 2006; Ying Liu et al., 2007). Cell surface GRP78 has been shown to
form complexes with specific cell surface proteins and regulate signal transduction
(Gonzalez-Gronow et al., 2009; Ni et al., 2011a; Miao Wang et al., 2009), such as
interacting with PI3K, impacting downstream AKT activation (Yi et al., 2013). Thus, the
discovery of GRP78 beyond the ER and its signaling function at the cell surface represents
a paradigm shift on its biological properties (Ni et al., 2011a) and opens up a unique
opportunity for a novel anti-cancer strategy. While mice with complete knockout of GRP78
are embryonic lethal, mice with one half GRP78 are viable (S. Luo et al., 2006). Thus,
models using GRP78 heterozygous mice could determine its role in specific cancers with
minimal effect on the normal organ physiology.
For human pancreatic cancer, GRP78 has been identified as a novel biomarker by
several independent studies. High level of GRP78 was detected by MALDI-Ion Mobility
Separation-Mass Spec Imaging in human PDAC validated by immunohistochemical (IHC)
staining (Djidja et al., 2009). Proteomic and tissue array profiling of microdissected
pancreatic cancer nests showed significantly higher level of GRP78 compared to normal
pancreatic ducts, and IHC staining revealed 69.3% of human PDAC stained positive for
GRP78 in the cytosol (Cui et al., 2009). GRP78 was reported to be highly upregulated in
the ductal structures of both human and murine ADM and PDAC lesions and co-
localization with activated AKT is evident on the cell surface (Hill et al., 2012). Another
study stained tissue microarray by IHC and showed an elevated GRP78 level in PDAC
compared to ducts in non-tumor adjacent ducts (Z. Niu et al., 2015). The upregulated level
of GRP78 correlated with shorter overall survival of the patients. In cell culture studies,
52
downregulation of GRP78 in human PDAC cells resulted in decreased proliferation and
invasion (Z. Niu et al., 2015). Blockage of GRP78 induction in glucose-deprived PANC-1
human pancreatic cells led to cytotoxicity (Cha, Yoon, Son, & Park, 2009). Furthermore,
GRP78 is upregulated in gemcitabine-resistant PDAC cells and knockdown of GRP78
compromised their chemoresistance (Gifford et al., 2016). Collectively, these studies
strongly suggest that GRP78 is commonly expressed in PDAC and ablation of GRP78 may
impede PDAC.
In this study, we report that homozygous knockout of GRP78 showed reduced
weight and fat infiltration in the pancreas. However, pancreas with one allele of Grp78
deleted displayed normal morphology. Strong GRP78 was detected not only in mPanIN,
but also in the acinar cells of PKC mice. To test the role of GRP78 in pancreatic
tumorigenesis, we generated Pdx1-Cre;Kras
G12D/+
;p53
f/+
(referred to as PKC mice) and
Pdx1-Cre;Kras
G12D/+
;p53
f/+
;Grp78
f/+
(referred to as PKC78
f/+
mice). The PKC78
f/+
pancreas showed decreased tumor area, associating with significantly prolonged survival.
In understanding the mechanism, we determined that GRP78 heterozygous pancreas
exhibited reduced proliferation, AKT/S6 and ERK activation. Furthermore, we discovered
that GRP78 haploinsufficiency, while having no effect on ductal formation under normal
physiologic conditions, suppressed ADM in response to stimulation by TGFα. In PKC78
f/+
mice, reduced ADM was also observed, supporting a novel role of GRP78 in tumor
initiation process of PDAC.
53
3.2 Materials and methods
Mice
Pdx1-Cre mice on C57BL6 background were crossed with Grp78
f/f
mice (S. Luo et
al., 2006) to generate Pdx1-Cre;Grp78
f/+
and Pdx1-Cre;Grp78
f/f
mice (Figure 3.1A). The
breeding between Kras
G12D/+
;p53
f/f
mice on C57BL/6;129/SV background with Pdx1-
Cre;Grp78
f/+
mice to generate Pdx1-Cre;Kras
G12D/+
;p53
f/+
(referred to as PKC) and Pdx1-
Cre;Kras
G12D/+
;p53
f/+
;Grp78
f/+
mice (referred to as PKC78
f/+
) were demonstrated in
Figure 3.1B. Genotyping was performed by PCR using mouse tail genomic DNA and
pancreas as previously described (Chen, Tseng, et al., 2014; Ying Liu et al., 2015). The
Kras
G12D
was detected using 5′-AGCCACCATGGCTTGAGTAAGTC-3′ as forward PCR
primer and 5′-CCTTTACAAGCGCACGCAGACTG-3′ as reverse primer. All protocols
for animal use and euthanasia were reviewed and approved by the University of Southern
California Institutional Animal Care and Use Committee.
54
Figure 3.1 Mating scheme. A. Pdx1-Cre mice were mated with Grp78
f/f
(78
f/f
) mice to generate Pdx1-
Cre;Grp78
f/+
and Pdx1-Cre;Grp78
f/f
mice. B. Kras
G12D/+
mice were mated with p53
f/f
mice to generate
Kras
G12D/+
p53
f/f
mice. Pdx1-Cre;Grp78
f/+
were mated with Kras
G12D/+
p53
f/f
mice to generated Pdx1-
Cre;Kras
G12D/+
;p53
f/+
and Pdx1-Cre;Kras
G12D/+
;p53
f/+
;Grp78
f/+
mice.
Tissue processing and histology
Both male and female mice were euthanized and pancreas were isolated. Collected
samples were either frozen in liquid nitrogen for biochemical analysis or fixed in 10% zinc
formalin (Sigma-Aldrich, St. Louis, MO) for tissue analysis. Paraffin-embedded tissues
were sectioned at 4 μm.
Western blot analysis
Tissue lysates were subjected to SDS-PAGE and Western blot analysis as described
previously (Fu et al., 2008). Primary antibodies used were GRP78 (1:1000), STAT3
(1:1000), BD Biosciences, San Jose, CA, p-ERK (1:1000), ERK (1:1000), p-STAT3
(1:1000), p-AKT (Ser 473, 1:1000), AKT (1:1000) from Cell Signaling, Danver, MA, and
β-actin (1:5000, Sigma-Aldrich, St. Louis, MO).
Tissue section staining
The paraffin sections were first deparaffinized and rehydrated. After washing with
double distilled water and PBS, the slides were incubated in 3% H2O2 in PBS for 15
minutes to eliminate endogenous peroxidase activity and washed twice in PBS. The
antigens were retrieved by means of incubation of slides in Retrievagen A Solution (BD
Biosciences) at 95–100 °C for 30 minutes. Slides were then allowed to cool at room
temperature for 1 hour in Retrievagen A Solution. The slides were subsequently washed
55
twice in PBS and blocked with the 1.5% goat serum of ABC Elite kit diluted in PBS. After
blocking, sections were incubated overnight with monoclonal rabbit anti-p-eIF2α (D9G8,
Cell Signaling Technology) diluted at 1:50 in blocking solution (ABC Elite Kit, Vector
Laboratories) at 4 °C. After three 5-minute washings in PBS, sections were then incubated
with biotinylated anti-rabbit secondary antibody (1:200 dilution, ABC Elite Kit, Vector
Laboratories) for 2 hours at room temperature. After three 5-minute PBS washings,
sections were incubated in ABC reagents from the ABC Elite Kit for 30 minutes, according
to manufacturer’s protocol. After 5minute PBS+0.1% Tween-20 washing and two 5-minute
PBS washings, the slides were developed with the DAB reagent from Peroxidase Substrate
Kit Vector Laboratories. Slides were then counter-stained with hematoxylin, washed with
distilled water, dehydrated, and cover slipped. Negative control slides were processed
without primary antibody.
Primary antibodies used were against GRP78 (1:100, MAb159, Dr. Parkash Gill)
for immunofluorescent staining, GRP78 (1:500) from Abcam (Cambridge, MA) for
immunohistochemical staining, pan cytokeratin (1:50) from Abcam, p-eIF2α (1:50),
phospho-S6 (1:200), p-ERK (1:200), from Cell Signaling Technology, GRP94 (1:250)
from Enzo Life Sciences (Farmingdale, NY), Amylase (1:50), CHOP (1:50) from Santa
Cruz Biotechnology (Dallas, TX), Ki67 (1:200) from Thermo Scientific (Fremont, CA),
and α-Smooth Muscle Actin (1:2000, Sigma-Aldrich). Immunofluorescence was analyzed
using a Zeiss LSM 510 confocal microscope with LSM 510 Version 4.2 SP1 acquisition
software. Confocal images were acquired with 10X, 20X or 40X oil lens. Images were then
processed with LSM Image Browser R4.2 and Adobe Photoshop CS5.
56
Primary acinar cell explant cultures and immunofluorescent staining
Primary acinar cell explant cultures were prepared by modifying published
protocols (Gout et al., 2013; Sawey, Johnson, & Crawford, 2007). Briefly, pancreas from
8-week-old mice were isolated and minced into small pieces of 1-3 mm
3
. After
centrifugation, pancreas fragments were subject to enzymatic dissociation by collagenase
IA and mechanical dissociation by pipetting back-and-forth in sterile pipettes of decreasing
size. Enzymatic dissociation was stopped by buffered washing solution (HBSS medium,
Corning, NY), supplemented with 5% FBS and 1% HEPES) and filtered through 100µm
nylon cell strainer (Corning). The filtrate was resuspended in Waymouth MB752/1 medium
(Gibco, Thermo Fisher) supplemented with 1% FBS, 1% penicillin and streptomycin,
0.1mg/mL soybean trypsin inhibitor (Santa Cruz Biotechnology) and dexamethasone
1µg/mL (Sigma-Aldrich). An equal volume of Type I collagen (2.5mg/mL rat tail, Corning)
was added to the cellular suspension, seeded into 48 well plates, and incubated at 37°C in
a humidified atmosphere of 5% CO2 for 30 minutes. After the suspension solution formed
gel, 250 µL of supplemented Waymouth medium were added to each 48 well. Recombinant
human TGFα (50ng/mL, Novoprotein, Summit, NJ) were added to induce acinar cell
transdifferentiation. Cultures were maintained at 37°C in a humidified atmosphere of 5%
CO2 up to 5 days with medium replaced every other day.
Immunostaining of collagen discs were described previously (Sawey et al., 2007).
For storage collagen discs were removed from the 48-well plate and fixed in
methanol/DMSO (4:1) overnight at 4ºC. Disks were stored in 100% methanol at -20ºC. For
staining, discs were rehydrated and washed with PBST (PBS + 0.5% Triton X-100). Next,
discs were blocked with PBSBT (PBS + 0.5% Triton X-100 + 2% BSA) for 2 hours at
57
room temperature and then incubated sequentially with the primary and secondary
antibodies diluted in PBSBT overnight at 4ºC on shaker. Between antibody incubations,
discs were washed in PBST 6 x 10 minutes. Lastly, discs were mounted using Vectashield
containing DAPI (Vector Laboratories).
Glucose tolerance test
Glucose tolerance test was described previously (Ye et al., 2011). After overnight
fasting, mouse tail blood was measure for glucose by OneTouch Ultra System (Lifescan,
Inc., Milpitas, CA, USA). Then, mice were subjected to intraperitoneal injection of glucose
(1 mg/g body weight), followed by blood glucose measurement at 30, 60, 90 and 120
minutes.
Statistical analysis
Statistical analysis was performed with log-rank (Mantel-Cox) test for survival data
or 2-tailed Student’s t-test as indicated.
3.3 Results
3.3.1 Grp 78 homozygous deletion induced fat infiltration in the pancreas
First, we generated Pdx1-Cre;Grp78
f/f
mice (Figure 3.1A), referred to below as
c78
f/f
mice. Pdx1 promoter is activated at embryonic day 8.5, leading to gene ablation in
both endocrine and exocrine compartment of the pancreas (Hingorani et al., 2003). At both
2 and 3 months, the c78
f/f
pancreata were smaller (Figure 3.2A) and lighter (Figure 3.2B)
58
than WT pancreata. Histological examination of pancreas sections stained with H&E
revealed two differences. First, c78
f/f
pancreata had smaller area compared to WT pancreata.
Second, part of c78
f/f
pancreata acinar cells were replaced with adipocytes (arrow head),
whereas ducts (black arrows) and islets of Langerhans (red arrows) were intact (Figure
3.2C). Furthermore, we determined GRP78 level in those sections by
immunohistochemical (IHC) staining and observed comparable GRP78 expression in WT
and c78
f/f
pancreata (Figure 3.2D), suggesting that the acinar cells in c78
f/f
pancreata might
have escaped the Cre recombination. The ducts and islets within adipocytes also expressed
GRP78 (Figure 3.2D, black and red arrow respectively). Some c78
f/f
mice exhibit
hunchback and ill after 3 months.
59
Figure 3.2 Generation of P d x 1 - Cre-mediated Grp 78 homozygous knockout mouse model. A.
Representative gross anatomy of pancreata of the indicated genotypes at 2 and 3 months. Scale bar, 1 cm. B.
The ratio of pancreas weight over body weight in WT and c78
f/f
mice at 2 months (n=14 and 9, respectively)
and 3 months (n=7 and 6, respectively). The data are presented as mean ± s.e., *p<0.05 and ** p<0.01. C.
H&E staining of WT and c78
f/f
pancreata at 2 and 3 months. Scale bar, 200 µm. D. IHC analysis of GRP78
in WT and c78
f/f
pancreata at 3 months.
60
3.3.2 Grp 78 heterozygous deletion in the pancreas
Since the c78
f/f
pancreata had fat infiltration, we explored whether one half GRP78
would affect normal pancreas development using the Pdx1-Cre;Grp78
f/+
mice, referred to
below as c78
f/+
pancreata. The genotypes of the mice were determined by PCR of tail
genomic DNA (Figure 3.3A). The size and weight of WT and c78
f/+
pancreata were similar
at 2, 5 and 9 months (Figure 3.3B and C). Histological analysis of pancreatic sections with
H&E staining similar morphology between WT and c78
f/+
pancreata at both 2 and 5 months
(Figure 3.3D). We also confirmed reduced GRP78 level in c78
f/+
pancreata compared to
WT pancreata and examined ductal marker pan-cytokeratin (panCK), which indicated
normal duct morphology (Figure 3.3E). Furthermore, we quantitated the number of ducts
in WT and c78
f/+
pancreata and observed similar number of ducts in both genotypes at 2
and 5 months (Figure 3.3F). Lastly, glucose tolerance was tested and results showed that
while the c78
f/+
mice metabolized glucose similar to WT, the c78
f/f
mice had lower tolerance
for glucose compared to WT mice (Figure 3.4).
61
Figure 3.3 Generation of P d x 1 - Cre-mediated Grp 78 heterozygous knockout mouse model. A.
Representative mouse tail PCR genotyping of the indicated alleles. B. Representative gross anatomy of
pancreata of the indicated genotypes at 2, 5 and 9 months. Scale bar, 1 cm. C. The ratio of pancreas weight
62
over body weight in WT and c78
f/+
mice at 2 months (n=14 and 7, respectively), 5 months (n=4 and 3,
respectively) and 9 months (n=2 and 4, respectively). The data are presented as mean ± s.e. D. H&E staining
of WT and c78
f/+
pancreata at 2 and 5 months. Scale bar, 200 µm. E. IHC analysis of GRP78 and pan-
cytokeratin (panCK) in WT and c78
f/+
pancreata at 5 months. F. Quantitation of the number of ducts per 10x
images at 2 and 5 months. The data are presented as mean ± s.e.
Figure 3.4 Analysis of glucose tolerance. Glucose tolerance test on 8-week-old WT (n=11), c78
f/+
(n=3),
c78
f/f
(n=9) mice.
3.3.3 PKC pancreata exhibit ER stress
First, we examined the expression of GRP78 and UPR markers in the PKC mice.
Immunofluorescent (IF) and IHC staining revealed that GRP78 is elevated in mPanIN
compared to normal ducts (Figure 3.5A and F). Elevated phospho-eIF2α was detected in
PKC pancreas compared to WT pancreas (Figure 3.5A). CHOP was detected in neither of
WT and PKC pancreas (Figure 3.5H). Intriguingly, upregulation of GRP78 is detected in
the acinar cells of PKC pancreata (Figure 3.5B and F), prior to the formation of mPanIN
and PDAC. To study the role of Grp78 heterozygosity in the pancreas, Pdx1-Cre;Kras
G12D/+
;p53
f/+
;Grp78
f/+
mice (referred to as PKC78
f/+
) were generated to deplete one half of
GRP78 (Figure 3.1B). Littermates lacking Pdx1-Cre transgene were used as WT controls.
63
Genotypes of the mice was validated by performing PCR with genomic DNA extracted
from the pancreas of the mouse cohorts (Figure 3.5C). At 2-5 months, PKC pancreata was
considerably larger and heavier than WT (Figure 3.5D and E). However, PKC78
f/+
pancreata were smaller than that of PKC between 2-5 months (Figure 3.5D and E).
Reduced GRP78 level was confirmed in both acinar and ductal cells in PKC 78
f/+
pancreata
compared to PKC pancreata at least up to 5 months (Figure 3.5F). In addition, we also
analyzed the level of another ER chaperone GRP94. At 5 months, GRP94 is elevated in
mPanIN and reduction of GRP78 did not induce compensation of GRP94 in PKC78
f/+
pancreas (Figure 3.5G).
64
Figure 3.5 Analysis of GRP78 and UPR markers in PKC and PKC78
f/+
pancreata. A. H&E,
immunofluorescent (IF) staining of GRP78, and IHC of p-eIF2α in pancreata of indicated genotypes. Scale
bar, 100 µm. B. IHC of GRP78 of indicated genotypes at 2, 3 and 4 months. Scale bar, 100 µm. C.
Representative PCR genotyping of indicated alleles from genomic DNA extracted from the pancreas. D.
Representative gross anatomy of pancreata of the indicated genotypes at 2, 3, 4 and 5 months. Scale bar, 1
cm. E. The ratio of pancreas weight over body weight in WT, PKC and PKC78
f/+
mice at 2 months (n=10, 10
and 9, respectively), 3 months (n=18, 15 and 11, respectively), 4 months (n=15, 8 and 8, respectively), 5
months (n=12, 8 and 8, respectively). The data are presented as mean ± s.e., *p<0.05 and ** p<0.01. IHC of
F. GRP78 and G. GRP94 of indicated genotypes at 5 months. Scale bar, 100 µm. H. IHC staining of CHOP
65
in pancreata of indicated genotypes with positive control from PTEN-null uterus. Scale bar, 100 µm.
3.3.4 Grp 78 heterozygosity in the pancreas delayed PKC-driven tumorigenesis and
prolonged survival
We examined the histology of pancreatic sections stained with H&E. At 2 months,
both PKC and PKC78
f/+
exhibited small early lesions (Figure 3.6A). At 3 months, while 3
out of 6 PKC mice had tumor area more than 20% of total area, 5 out of 5 PKC78
f/+
pancreata had tumor area less than 10% of total area (Figure 3.6A and B). At 4 months,
whereas 4 out of 5 PKC had tumor area more than 20%, 5 out of 5 PKC78
f/+
had tumor
area less than 20% of total area (Figure 3.6A and B). At 5 months, 3 out of 6 PKC78
f/+
pancreata had similar tumor area compared to PKC, whereas the other 3 PKC78
f/+
pancreata had tumor area less than 20% of total area (Figure 3.6A and B). In agreement
with the reduced tumor area, PKC78
f/+
mice showed significantly prolonged life span than
PKC mice (p = 0.029, log-rank test) (Figure 3.6C).
66
Figure 3.6 Reduced pancreatic tumorigenesis in PKC78
f/+
pancreata. A. H&E staining of indicated
genotypes at 2, 3 and 4 months. Scale bar, 500 µm. B. The ratio of tumor area to total pancreas area in PKC
and PKC78
f/+
pancreata at indicated ages. C. Kaplan-Meier survival curve of WT (n=20), PKC (n=19) and
PKC78
f/+
(n=15). p = 0.029, log-rank (Mantel-Cox) method.
3.3.5 Suppression of proliferation and oncogenic signaling in PKC78
f/+
pancreata
To confirm the tumor size, we further analyzed the morphology of the tumor by
IHC of epithelial cell marker and stroma cell marker. Consistent with H&E, IHC staining
of epithelial cell marker - panCK and activated myofibroblast marker - α-smooth muscle
actin showed decreased tumor in PKC78
f/+
pancreata compared to that of PKC (Figure
3.7A). To understand the mechanism(s) whereby GRP78 haploinsufficiency suppressed
PKC tumor progression, we measured proliferation by Ki67 staining and observed a
67
reduction in PKC78
f/+
compared to PKC pancreata in both acinar (68%) and ductal cells
(52%) (Figure 3.7B and C). IHC staining of cleaved caspase-3 showed minimal apoptosis
in PKC and PKC78
f/+
pancreata (Figure 3.7E), indicating tumor suppression is not due to
apoptosis of the pancreatic tissue.
Next, we characterized the signaling pathways in PKC78
f/+
pancreata. PI3K/S6,
ERK, STAT3 and β-catenin pathways have been reported to be activated in PDAC
(Corcoran et al., 2011; Eser et al., 2013; Morris, Wang, & Hebrok, 2010; Yaqing Zhang et
al., 2013). PKC78
f/+
pancreata displayed reduced S6 activation in both acinar and ductal
cells (Figure 3.7D). ERK activation, STAT3 activation and β-catenin level were less in
PKC78
f/+
pancreata (Figure 3.7D).
68
Figure 3.7 Characterization of proliferation, apoptosis and signaling pathways in PKC78
f/+
pancreata.
A. IHC staining of pan-cytokeratin (panCK) and α-smooth muscle actin (α-SMA) of the indicated genotypes
at 3 months. Scale bar, 400 µm. B. IHC staining of Ki67 of the indicated genotypes at 3 months. Scale bar,
50 µm. C. Quantitation of Ki67 positive cells in the pancreas sections PKC (n=3) and PKC78
f/+
(n=3) at 3
months. D. IHC staining of phospho-S6 (Ser235/236), phospho-ERK (Thr202/Tyr204), phospho-STAT3
(Tyr705) and β-catenin. Scale bar, 100 µm. E. IHC staining of cleaved caspase-3 of the indicated genotypes
at 3 months. Scale bar, 100 µm.
69
3.3.6 Reduction of ADM in PKC78
f/+
pancreata and in c 78
f / +
acinar cells induced by
TGF α
Acinar-to-ductal metaplasia (ADM) have been discovered to be the primary
mechanism for pancreatic cancer initiation (Kopp et al., 2012). Strong GRP78 expression
was observed in both human and mouse ADM (Hill et al., 2012). Given the elevated GRP78
level in PKC acinar cells, and the reduced tumor area when GRP78 is reduced to one half,
we hypothesized that GRP78 upregulation may be needed for ADM in the pancreas. This
implies that Grp78 heterozygosity may attenuate this tumor initiation step of pancreas
tumorigenesis. To test this, we first compared the amount of ADM, which is characterized
by acinar clusters with both markers, by double IF staining of acinar marker (amylase) and
ductal marker (panCK). PKC78
f/+
pancreata showed decreased panCK
+
only cell and ADM
than that of PKC (Figure 3.8A and B).
70
Figure 3.8 Analysis of acinar-to-ductal metaplasia in WT, PKC and PKC78
f/+
pancreata. A. Double
staining of amylase (green) and panCK (red) of the indicated genotypes at 3 months (n=3 per genotype).
Nuclei stained with DAPI (blue). Scale bar, 50 µm. B. Quantitation of ADM and panCK positive only area
over total area. The data are presented as mean ± s.e., *p<0.05 and ** p<0.01.
To confirm the results observed in PKC78
f/+
pancreata, we investigated the impact
of GRP78 haploinsufficiency on the ductal formation transdifferentiating from the acinar
cells stimulated by growth factor. We isolated pancreatic acinar cells from WT and c78
f/+
mice for 3D explant culture in collagen with transforming growth factor α (TGFα)
induction (Figure 3.9A). Reduced number of ducts was transdifferentiated from acinar
cells in c78
f/+
compared to WT pancreata at day 3, shown in light microscope and IF
staining of panCK (red) (Figure 3.9B). The acinar clusters undergoing ADM, characterized
by expressing both acinar (amylase in green) and ductal (panCK in red) markers, was also
26% less in c78
f/+
compared to WT pancreata at day 3 (Figure 3.9C). In addition,
quantitation of ducts formed from acinar cells demonstrated similar ducts at day 1, however,
at day 3, c78
f/+
acinar clusters gave rise to 46% less ducts (Figure 3.9D). The reduction of
GRP78 in the isolated c78
f/+
acinar cells was confirmed by western blot (Figure 3.9E) and
the downstream pathways of TGFα induction, namely, ERK, AKT and STAT3 were
examined. TGFα stimulated an upregulation of p-ERK and p-AKT, but not p-STAT3 at day
2. Interestingly, we observed 35% drop at day 0 and 68% drop at day 2 of p-ERK in c78
f/+
acinar clusters compared to that of WT (Figure 3.9E). While p-AKT level was similar
between the two genotypes at day 0, c78
f/+
acinar clusters had a 43% reduction in p-AKT
at day 2 compared to that of WT (Figure 3.9E). STAT3 activation was similar in both
genotypes. Thus, corresponding to GRP78 haploinsufficiency, decreased AKT activation
71
and ERK activation induced by TGFα in the c78
f/+
acinar cells was observed.
Figure 3.9 Acinar transdifferentiation of WT and c7 8
f/+
pancreata. A. Scheme of in vitro acinar-to-ductal
metaplasia experiment. B. Light microscope picture and IF of panCK (red) with nuclei stained with DAPI
(blue) of the indicated genotypes at day 3. Black arrows denote ducts and white arrows denote acinar clusters
with panCK expression. Scale bar, 50 µm. C. Double IF staining of amylase (green) and panCK (red) of the
indicated genotypes at day 3. Nuclei stained with DAPI (blue). Scale bar, 100 µm. D. Quantitation of the
percentage of ducts over total acinar structures of the indicated genotypes at day 1 and 3. The data are
presented as mean ± s.e., ** p<0.01 (student’s t-test). E. Representative western blots of GRP78, p-AKT (Ser
473), AKT, p-ERK (Thr202/204), ERK, p-STAT3 (Tyr705) and STAT3 of the indicated genotypes at day 0,
1, 2, and 3. Corresponding quantification were displayed in bar graph. The data are presented as mean ± s.e.,
* p<0.05 (student’s t-test).
72
3.4 Discussion
GRP78 is a key mediator for ER homeostasis and has been discovered to serve
important roles in organ homeostasis as well. Whole body knockout of GRP78 resulted in
embryonic lethality with reduced proliferation and increased apoptosis (S. Luo et al., 2006).
Depletion of GRP78 in the Purkinje cells led to ER dilation, retarded growth and cerebellar
atrophy (M Wang et al., 2010). Inducible ablation of GRP78 in the adult hematopoietic
system reduced the hematopoietic stem cells pool and activated unfolded protein response
(Wey, Luo, & Lee, 2012). Adipose-specific knockout of GRP78 led to lipoatrophy (G Zhu
et al., 2013). Liver-specific loss of GRP78 induced fat accumulation and moderate liver
injury (Chen, Zhu, et al., 2014; Ji et al., 2011). Loss of GRP78 in the intestinal epithelium
activated ER stress and lost intestinal epithelial stemness (Heijmans et al., 2013). Depletion
of GRP78 in the mouse uterus resulted in reduced size and glands (Lin et al., 2015; Shen
et al., 2016). However, not all organs are affected by loss of GRP78, the prostate epithelium
appeared normal without GRP78 (Fu et al., 2008).
In this study, we report that the depletion of GRP78 in the pancreas starting
embryonic day 8.5 led to decreased pancreas weight, induced fat infiltration. Interestingly,
examination of GRP78 expression revealed similar level of GRP78 between WT and c78
f/f
pancreata, in which the acinar cells likely escaped the Cre recombinase. The observation
of fat infiltration could be due to two possible mechanisms. One is the depletion of GRP78
induced extensive acinar cell death and the adipocytes replaced the acinar cell positions.
Another possible mechanism is the loss of GRP78 induced transdifferentiation of acinar
cells into adipocytes. The two possibilities are not unprecedented. Pancreas-specific
deletion of Prox1 (homeodomain transcription factor 1), a critical regulator of vertebrate
73
organogenesis, led to X-gal negative fat infiltration in the pancreas, indicating the
adipocytes did not originate from acinar cells (Westmoreland et al., 2012). On the other
hand, conditional inactivation of c-Myc in the pancreas transdifferentiated acinar cells into
adipocytes, confirmed by YFP reporter (Bonal et al., 2009). Thus, lineage tracing will help
determine the cell of origin in the adipocytes induced by GRP78 ablation.
In accordance with other studies (Djidja et al., 2009; Gifford et al., 2016; Z. Niu et
al., 2015), IHC staining revealed upregulated level of GRP78 in pancreatic malignancy
compared to normal ducts. To characterize the expression of GRP78 in pancreatic cancer
compared to normal pancreas, tissue-microarray based IHC may be a better approach than
analyzing mRNA database of PDAC and normal pancreas. The former method allows
comparison of PDAC to normal ducts under the microscope, whereas the latter is a
comparison of a mixture of different cell types in normal and PDAC samples. Specifically,
normal pancreas samples is likely to be composed of high GRP78 expressing acinar cells
and low GRP78 expressing ductal cells, and PDAC samples is likely composed of low
GRP78 expressing stromal cells, which could account for more than 90% of tumor volume
(Xie & Xie, 2015) and PDAC cells.
Strikingly, GRP78 was highly elevated not only in the PDAC cells, but also in the
PKC acinar cells. The acinar cells of the exocrine pancreas have abundant ER to produce
huge amount of digestive enzymes. Its protein synthesis rate is higher than all other adult
tissues, therefore they are highly dependent on ER functions and particularly susceptible
to ER stress (Kubisch & Logsdon, 2008). One method for acinar cells to deal with stress,
such as pancreatitis, is to transiently transdifferentiate into ductal cells (Morris, Cano,
Sekine, Wang, & Hebrok, 2010; Murtaugh & Keefe, 2014). Hence, upregulated GRP78
74
may assist the acinar cells to cope with ER stress and maintain normal ER activity.
Elevation of p-eIF2α, one marker of unfolded protein response, was also observed.
Together, these data indicate that the PKC pancreata are under ER stress.
Our work provides the first evidence that Grp78 heterozygosity can suppress
tumorigenesis driven by Kras mutation. While decreasing GRP78 triggered compensation
of another ER chaperone GRP94 in the mouse embryo and Purkinje cells (S. Luo et al.,
2006; M Wang et al., 2010), interestingly reducing GRP78 to one half in the pancreas did
not induce such compensation. GRP78 haploinsufficiency exhibited reduced tumor size,
proliferation and prolonged survival, which opened up the opportunity to target PDAC with
small molecule inhibitors that could suppress GRP78 to basal level. Indeed, IT-139, a
ruthenium-based drug proven to inhibit GRP78 expression, sensitized gemcitabine-
resistant PDAC cells (Gifford et al., 2016). Another small molecule inhibitor targeting
GRP78, HA15, triggered ER stress and induced cell death in gemcitabine-resistant PDAC
cell (Cerezo et al., 2016). The upregulation of GRP78 in PKC acinar cells and decreased
tumor area in PKC78
f/+
pancreata, raised the possibility that GRP78 may contribute to the
tumor initiation process - acinar-to-ductal metaplasia. Reduced amount of ADM was
detected in PKC78
f/+
pancreata compared to that of PKC. Consistent with previous study
(Ye, Mareninova, et al., 2010), Grp78 heterozygous pancreas is phenotypically normal,
which opens a unique window to reducing GRP78 to one half without harming normal
pancreas functions. However, GRP78 haploinsufficiency compromised the potential of
acinar cell transdifferentiating into ductal cells stimulated by TGFα, which strongly
suggest a role of GRP78 in pancreatic tumor initiation. In understanding the mechanism,
ERK and AKT activations were inhibited in c78
f/+
acinar cells, which likely contribute to
75
suppression of ADM.
In summary, homozygous deletion of Grp78 in the pancreas led to fat infiltration.
Pancreas specific heterozygous deletion of Grp78 suppressed PDAC progression, and
pancreatic tumor initiation. Therefore, targeting GRP78 in PDAC may not only suppress
tumor growth, but also inhibit new tumor forming with minimal damage to normal
pancreas. Agents inhibiting GRP78 expression to basal level are of interest.
76
Chapter 4
Conclusions and Perspectives
The glucose-regulated proteins (GRPs) are stress-inducible molecular chaperones
that belong to the heat shock protein (HSP) family. They mainly reside in the ER, however,
they are also secreted outside the cell and detected in other compartment of the cell, such
as the cell surface, nucleus, mitochondria (A. S. Lee, 2014). As their role in cancer is
emerging, the GRPs have been implicated to participate in the cancer cell proliferation,
apoptosis, angiogenesis, invasion and metastasis (A. S. Lee, 2014). However, the roles of
GRP94 and GRP78 in the endometrial cancer and pancreatic cancer are still unclear. In this
dissertation, we set forth to investigate the role of GRP94 in endometrial cancer and the
role of GRP78 haploinsufficiency in pancreatic cancer. These studies expands our
understanding in the function of GRPs in tumors driven by either PTEN-null or Kras
mutation.
In the first part of this dissertation (Chapter 2), we investigated the role of GRP94
in endometrial epithelial tumor. The model system we employed was progesterone-receptor
promoter driven Cre recombinase to specifically delete the target genes in the mouse uteri
and we generated two uterus-specific knockout mouse model with deletion of Grp94 alone
(c94
f/f
), or in combination of the tumor suppressor Pten (cP
f/f
94
f/f
). First, we characterized
the expression of GRP94 in human endometrioid adenocarcinoma (EAC). We showed that
Grp94 mRNA is elevated in human EAC compared to that of normal samples, and strong
GRP94 is detected in human EAC tissue. Knocking down GRP94 in the PTEN-null human
EAC cell lines reduced their proliferation. Next, we demonstrated that ablation of GRP94
77
in the uterus induced spontaneous squamous cell metaplasia (SCM) at 8 weeks of age,
which was associated with decreased active nuclear β-catenin. Furthermore, depletion of
both PTEN and GRP94 suppressed PTEN-null EAC and accelerated induction of SCM,
which coincided with attenuated β-catenin, AKT signaling and decreased proliferation. In
contrast to myometrial invasion detected in seven out of ten cP
f/f
uteri, the cP
f/f
94
f/f
uteri
showed no myometrial invasion, which correlated with wild-type level of E-cadherin
expression in SCM. Notably, the size of cP
f/f
94
f/f
uteri was considerably larger than cP
f/f
uteri past 8 weeks, and histological examination revealed massive expanded but mature
glands with abundant secretion in cP
f/f
94
f/f
uteri. Collectively, in Chapter 2, we provided
the first evidence that GRP94 deficiency can delay the development of cancer driven by
loss of PTEN.
Metaplasia is defined as the replacement of one differentiated cell type with another.
Squamous cell metaplasia, replacing the previous columnar epithelial cells, can be
observed occasionally in EAC, chronic irritation and progestin effects (Anderson et al.,
2002; Y . Lee et al., 1983; Miranda & Mazur, 1995). Interference with the Wnt/β-catenin
pathway, namely deletion of Wnt4, depletion of β-catenin, activation of β-catenin by
knocking out Apc, and depletion of PTEN have been reported to result in SCM (Franco et
al., 2011; Jeong et al., 2009; van der Zee et al., 2013; Villacorte et al., 2012). Although
reports have unveiled important pathways inducing SCM, little is known about the cell of
origin in SCM. One possible mechanism is the squamous cells may come from adjacent
cervix, which is lined by squamous cells. Another possibility is the squamous cells may
arise from existing cell types in the mouse uterus. One study grafted uterus of the
embryonic day 18.5 mice into renal capsule of ovariectomized nude mice, and upon
78
depletion of β-catenin by Keratin 5-Cre (active at embryonic day 16.5), observed SCM
(Villacorte et al., 2012). Grafting the uterus to renal capsule decreased the possibility of the
migration of squamous cells from the cervix, yet SCM was still evident in those samples
lending support to the latter possibility. Since GRP94 deficiency attenuated active nuclear
β-catenin, it is likely that ablation of GRP94 utilize similar mechanism inducing SCM to
the depletion of β-catenin. Thus, we argue that the SCM likely arise from existing cells in
the uterus.
In case of SCM originating from the uterus, the squamous cells could either derive
from transdifferentiation of the columnar epithelial cells, or emerge from the
stem/precursor cells of the uterus that alternatively initiate a different differentiation
pathway. To test the possibility of transdifferentiation, double staining of columnar cells
marker and squamous cell marker were performed. In the 8-week cP
f/f
94
f/f
uterus, majority
of the cells expressed squamous marker and cells expressing columnar marker were located
in the luminal layer. Although at a low frequency, cells expressing both markers were
detected. These data suggest that transdifferentiation from columnar cells to squamous cells
contribute to SCM, however, its low frequency indicate that this might not be the only
mechanism giving rise to such big population of squamous cells. Furthermore, the
distribution of squamous cells near the basal layer and the columnar cells near the luminal
layer, suggests that squamous cells arising from stem/precursor cells were replacing the
columnar cells. This hypothesis is not unprecedented. Depletion of GRP94 in the liver led
to hyperproliferation of liver progenitor cells with impaired cell adhesion molecules (Chen,
Tseng, et al., 2014). GRP94 deficiency resulted in increased hematopoietic stem and
progenitor cell pool attributed to impaired interaction of hematopoietic stem cells with the
79
niche and loss of cell surface integrin α4 (Biquan Luo et al., 2011). Hence, both
mechanisms could contribute to SCM in cP
f/f
94
f/f
uterus. The lineage tracing technology,
such as YFP or Rosa26, and transplantation of the naïve c94
f/f
uterus that has not developed
SCM to the kidney capsule, could provide evidence on whether the SCM derive from the
uterus or the cervix.
How could loss of GRP94 change the differentiating fate of stem/progenitor cells?
One possible explanation is that depletion of GRP94 may modulate the cell surface proteins,
and interrupt its communication with surrounding environment, further leading to the
change of its cell polarity and differentiation fate. GRP94 loss in a prostate cancer cell line
has been reported to impede the trafficking of integrin α2, αL, changing the cell polarity
from bipolar to multi-polar morphology (Ghosh et al., 2016). GRP94 has been reported to
selectively regulate many integrin expression in hematopoiesis (Staron et al., 2010). For
cancer cells to migrate and invade, they switch their usage of integrins (Guo & Giancotti,
2004). Hence, ablating GRP94 may provide the advantage of disrupting physical adhesion
of multiple integrins at once, as well as shutting down integrin-mediated signaling. Indeed,
silencing GRP94 reduced breast cancer cell migration (Dejeans et al., 2012). A more recent
study indicated that knocking down GRP94 in prostate cancer cells hampered their
migratory potential and interfered with the cancer cell polarity (Ghosh et al., 2016). In
Chapter 2, while Pten-null driven EAC invaded into the myometrium after 8 weeks of age,
GRP94 depleted uteri induced squamous cells leading to well-differentiated glandular
structures and were devoid of myometrial invasion. Due to the induction of another cell
type and less malignant lesions, we were unable to conclude the function of GRP94 on
invasion. Therefore, the functions of GRP94 in cancer cell migration and invasion should
80
be further validated. First, GRP94 expression pattern in human invasive versus non-
invasive cancers could be examined by comparing protein level in invasive/metastatic
cancer cells with non-invasive cancer cells, and by IHC staining of human tissue samples.
Second, the effect of GRP94 loss in cancer cell invasion, could be tested using invasion
transwells. Third, GRP94 inhibition in cancer cell metastasis could be tested in mouse
xenograft models injected with metastatic cells lines. Fourth, the role of GRP94 in cancer
metastasis could be further explored using mouse models which would develop metastatic
cancers. Lastly, potent GRP94 inhibitors could be tested to impede cancer cell migration,
invasion and metastasis in cell culture and mouse xenografts. The role of GRP94 loss in
cancer invasion and metastasis warrants further investigation.
The cP
f/f
94
f/f
uteri present several distinct features. First, ablation of GPR94 in
PTEN-null driven EAC delayed the tumor, whereas GRP94 deficiency in PTEN-null liver
accelerated the tumor (Chen, Tseng, et al., 2014). Knockout of GRP94 in the PTEN-null
uteri attenuated AKT activation and β-catenin activation, while such decrease was absent
in the liver model. Knockout of GRP94 in the hematopoietic system increased AKT
activation (Biquan Luo, Tseng, Adams, & Lee, 2013). Ablation of GRP94 in the gut and
the multiple myeloma disease models led to reduction of β-catenin pathway (Hua et al.,
2013; B. Liu et al., 2013). Together, the fact that disruption of GRP94 in different organs
led to distinct outcomes, suggest the function of GRP94 is context-dependent. Second, the
reduced AKT activation and β-catenin activation were observed in the squamous cells, but
absent in the glandular cells. This observation raised the possibility that the squamous cell
may have escaped Cre recombinase. Therefore, we first confirmed GRP94 loss in the SCM
and also further demonstrated PTEN loss via IHC staining at both 4 and 8 weeks (Figure
81
4.1) and western blot analysis. Although bearing the same genetic mutations, squamous
cells and glandular cells exhibit different response in signaling pathways to loss of PTEN
and GPR94. The squamous cells may be more resistant to PTEN loss, that is no increase
in p-AKT after deleting Pten, or more responsive to GRP94 loss, that is decreased p-AKT
level after deleting Grp94. To test this hypothesis, induction of SCM by mechanisms other
than downstream of GRP94, PTEN and β-catenin, combining with Pten deletion, may
determine whether it is the squamous cells that are resistant to PTEN loss. Third, while
both GRP78 and GRP94 deficient uteri were smaller in size and showed a decrease in the
number of glands, GRP94 uteri were lined with squamous cells instead of a single layer of
columnar cells. Furthermore, cP
f/f
94
f/f
and cP
f/f
78
f/f
uteri also showed distinct phenotypes.
While the cP
f/f
78
f/f
uteri were devoid of EAC up to 8 months (Lin et al., 2015), the cP
f/f
94
f/f
uteri did develop less aggressive and small EAC at 4 weeks and showed low-grade less
aggressive lesions at 8 weeks. Both uteri showed decreased AKT activation, however, the
cP
f/f
94
f/f
uteri also reduced β-catenin activation. Together, these data suggest that GRP94
and GRP78 have distinct and non-compensatory functions in endometrial tumorigenesis,
albeit they are coordinately upregulated in ER stress.
82
Figure 4.1 IHC staining of PTEN. IHC staining of PTEN of the indicated genotypes at 4 and 8 weeks. Scale
bar, 200µm.
As GRP94 emerging to be a novel target of cancer suppression, small molecule
inhibitors suppressing GRP94 have been developed (Duerfeldt et al., 2012; Muth et al.,
2014; Patel et al., 2013). GRP94 inhibitor WS-13 have been reported to regulate plasma
membrane HER2 expression of breast cancer cells in a tumor-specific manner (Patel et al.,
2013), and induce apoptosis and block multiple myeloma cell growth (Hua et al., 2013).
GRP94 has also been detected on the cancer cell surface, facilitating HER2 dimerization
in breast cancer cells and enhancing invasion and metastatic potential in liver cancer cells
(Altmeyer et al., 1996; Hou et al., 2015; X. Li et al., 2015; Patel et al., 2013). Targeting
83
cell surface GRP94 with a specific monoclonal antibody led to decreased cell growth in
vitro, as well as suppression of breast and liver tumor growth in vivo (Hou et al., 2015; X.
Li et al., 2015). Another GRP94-specific antibody increased the sensitivity of melanoma
cells bearing BRAF mutations to BRAF inhibitors, and restored the sensitivity of those
resistant to BRAF inhibitors (Sabbatino et al., 2015). Taken together, targeting GRP94
holds great potential in combatting cancer.
The previous studies investigating the role of GRP78 in various cancer mouse were
mostly driven by loss of PTEN. It is critical to explore the role of GRP78 in cancers driven
by other genetic alterations beyond PTEN loss. The second part of this thesis (Chapter 3)
focuses on determining the role of GRP78 in pancreas homeostasis and pancreatic
adenocarcinoma (PDAC). We employed the pancreatic and duodenal homeobox 1
promoter driven Cre recombinase, to specifically delete the target genes in the mouse
pancreas. We created two pancreas-specific knockout mouse model with deletion of one or
both alleles of Grp78 (c78
f/+
and c78
f/f
). Unexpectedly, depletion of GRP78 in the pancreas
showed smaller size, less tolerance for glucose and fat infiltration. Pancreas with
heterozygous Grp78 presented normal morphology and similar number of ducts to WT
controls. To study PDAC, we generated a mouse model combining activation of Kras
G12D/+
,
deletion of one allele of p53 and Grp78 (PKC78
f/+
). GRP78 haploinsufficiency resulted in
reduced pancreas size, weight and prolonged the survival of the PKC78
f/+
mice compared
to that of PKC mice. Further examination of the PKC78
f/+
pancreas revealed decreased
percent tumor area, compromised proliferation and less S6, ERK, STAT3, and β-catenin
activation. Based on these observations, we hypothesized that Grp78 heterozygosity may
mediate the tumor initiation step of pancreatic tumorigenesis: acinar-to-ductal metaplasia
84
(ADM). We discovered that reduced amount of ADM in PKC78
f/+
pancreata and one half
GRP78 suppressed the potential of isolated acinar cells transdifferentiating into ductal cells
induced by TGFα. Collectively, in Chapter 3, we presented the first evidence that Grp78
heterozygosity can delay the development of pancreatic cancer driven by activation of
oncogene Kras. Exploring the functions of GRP78 in more Kras-driven tumor, such as
lung cancer and colorectal cancer, and cancers driven by other oncogenes would further
broaden our understanding of GRP78 in cancer.
The pancreas is composed of endocrine and exocrine compartments, where the
acinar cells produce and secrete digestive enzymes that are drained into the intestine
through pancreatic ducts. The acinar cells of the exocrine pancreas have abundant ER to
support its prominent role in protein synthesis. Its protein synthesis rate is higher than all
other adult tissues, therefore they are particularly susceptible to ER stress (Kubisch &
Logsdon, 2008). ER stress-response mechanisms have been linked to acute pancreatitis, a
disease arising from pancreatic acinar cells (Kubisch & Logsdon, 2008). As the most
abundant chaperone in the ER and the master regulator of unfolded protein response,
depletion of GRP78 in the pancreas resulted in reduced exocrine pancreas area and fat
infiltration. Notably, ducts and islets of the Langerhans were intact inside the adipose tissue,
indicating that it is the acinar cells that were absent and replaced by adipocytes.
Interestingly, GRP78 level in c78
f/f
was comparable to that in WT, which indicated that the
acinar cells present in c78
f/f
pancreas have escaped the Cre recombinase. The fact that the
existing acinar cells in c78
f/f
pancreas expressed GRP78, suggests a possibility that the
acinar cell may require GRP78 to for their survival. This hypothesis can be tested by
silencing GRP78 in cultured acinar cells and examine their survival, and investigating
85
earlier time points of the c78
f/f
pancreas. In addition, repopulation of GRP78 was also
observed in homozygous knockout of GRP78 in the liver with Albumin-Cre (Chen, Zhu, et
al., 2014), in the mammary glands with MMTV-Cre (Genyuan Zhu et al., 2014), in the
adipose with aP2-Cre (G Zhu et al., 2013), and in the intestine with Ah1-Cre (Heijmans et
al., 2013).
Another intriguing observation is the fat infiltration in GRP78 deficient pancreas.
Fat infiltration in the pancreas is known to be associated with aging, type 2 diabetes, and
metabolic syndrome in humans (Hori et al., 2014; Prachayakul & Aswakul, 2015; Smits &
van Geenen, 2011). It has been reported in genetic knockout mouse models as well.
Altering genes in the TGFβ family (Böttinger et al., 1997; Hashimoto et al., 2006) and β-
catenin signaling (Bonal et al., 2009; Morris, Cano, et al., 2010) rendered reduced acinar
mass with adipose replacement. Interference with intrinsic factors involved in the acinar
cell differentiation, such as E2F1/E2F2 and Prox1, also resulted in fat infiltration (Iglesias
et al., 2004; Westmoreland et al., 2012). Deletion of the Shwachman-Bodian Diamond
syndrome gene (Sbds) in mice mimicked the features of Shwachman-Bodian Diamond
syndrome, including fat infiltration (Tourlakis et al., 2012). To determine the cell of origin
for adipocytes, studies have performed lineage tracing and double staining of acinar cell
marker and adipocyte marker. On one hand, in the pancreas with c-Myc inactivation,
lineage tracing suggested that the adipocytes were derived from acinar cells (Bonal et al.,
2009). On the other hand, in Prox1 deleted and Sbds deleted pancreata, lineage tracing
indicated otherwise (Tourlakis et al., 2012; Westmoreland et al., 2012). Two mechanisms
exist leading to fat infiltration in the pancreas and are context-dependent. In case of GRP78
depletion, if the adipocytes stemmed from the c78
f/f
acinar cells, they are more likely to
86
exhibit low GRP78 expression. As shown in Chapter 3, the adipose tissue expressed
abundant GRP78. Given the critical role of GRP78 in acinar cells, these data favor the
replacement mechanism. However, we could not rule out the possibility of re-expression
of GRP78 after transdifferentiation from acinar cells to ductal cells. Double staining of
acinar and adipose markers and lineage tracing will provide more solid evidence. In
addition, the c78
f/f
displayed slower metabolism for glucose. While this could be attributed
to the reduced pancreas size, the intact islets in the fat infiltration part, suggest the decrease
in total islet numbers may be minor. The role of GRP78 in the pancreatic endocrine
compartment warrants further study.
Grp78 heterozygosity have been reported to suppress PTEN-null driven prostate
tumorigenesis, leukemogenesis, but not endometrioid and liver tumorigenesis (Chen, Zhu,
et al., 2014; Fu et al., 2008; Lin et al., 2015; Wey, Luo, Tseng, et al., 2012). In Chapter 3,
we further explored the role of GRP78 heterozygosity in pancreas tumorigenesis driven by
activation of Kras oncogene and heterozygosity of p53. Deleting one allele of Grp78 in the
pancreas compromised the ADM process induced by TGFα. When acinar cells reprogram
into ductal cells, they switch to a different set of proteins, for instance, the acinar cells
expressing amylase will lose this expression and start to express ductal markers such as
cytokeratin 19 (L. Zhu, Shi, Schmidt, Hruban, & Konieczny, 2007). Therefore, under
pathological conditions, partial reduction of GRP78 may delay this protein switching by
slowing down protein folding in the acinar cells.
Furthermore, the heterozygosity of Grp78 in acinar cells hampered the upregulation
of ERK and AKT signaling, but not STAT3, which might also contribute to the decrease in
ADM. Grp78 heterozygosity have been reported to inhibit AKT activation (Fu et al., 2008;
87
Wey, Luo, Tseng, et al., 2012). Interestingly, GRP78 has been shown to co-stain with p-
AKT in PDAC and endometrial cancer (Hill et al., 2012; Lin et al., 2015). Multiple
mechanisms may contribute to GRP78 affecting AKT activation. Evidence are
accumulating that under pathophysiological conditions, a subfraction of GRP78 can re-
localize to the cell surface of specific cells types, in particular cancer cells (A. S. Lee, 2014;
R. Liu et al., 2013; Misra et al., 2006; Shin et al., 2003; Yi Zhang et al., 2010). Cell surface
GRP78 could mediate important signaling pathways including PI3K/AKT by interacting
with different ligands and cell surface proteins. One proposed mechanism is that the cell
surface GPR78 complexes with PI3K to promote phosphatidylinositol (3,4,5)-
trisphosphate formation (Yi et al., 2013). On the other hand, it is also possible that GRP78
may be in charge of processing important growth factors or the cell surface expression of
their corresponding receptors, which might mediate AKT activation.
How could GRP78 regulate ERK activation? Silencing of GRP78 in hepatocellular
carcinoma cells and glioblastoma cells reduced ERK activation (H. Li et al., 2012; L. H.
Zhang, Yang, Zhang, Cheng, & Zhang, 2011). Association of GPR78 and activated α2-
macroglobin facilitated the interaction between EGFR and c-Src (Zhao et al., 2015).
Additionally, GRP78 has been found to form direct complex with EGFR in N-methyl-N’-
nitro-N-nitrosoguanidine (MNNG) treated cells (G. Niu, Shang, & Yu, 2006) and with
underglycosylated form of EGFR under 2-deoxyglucose induced stress condition (Cai,
Tomida, Mikami, Nagata, & Tsuruo, 1998). Taking these studies into account, GRP78 may
help the proper folding of EGFR in the ER, and/or facilitate the stabilization of cell surface
EGFR, therefore, partial reduction of GRP78 may reduce EGFR expression and diminish
its downstream signaling such as p-ERK.
88
TGFα binds to its receptor EGFR and activates downstream signals including RAS
and AKT (Chong & Jänne, 2013). To mimic the pancreatic cancer driven by oncogenes, in
vitro ADM experiment could be performed using Pdx1-Cre;Kras mutant mice compared
with cKras78
f/+
mice, in which Kras mutation would replace TGFα to induce ductal
differentiation. To further mimic PDAC mouse model in our study, PKC and PKC78
f/+
mice could be utilized to conduct in vitro ADM experiment. However, coupling Kras
mutation with p53 heterozygosity may accelerate the process and younger mice, earlier
time points should be considered. In addition, ADM can also be induced in vivo by ligating
pancreatic ducts and such procedure has been utilized to compare the ADM potential
between mice with different genotype (Hamamoto et al., 2002; Wei et al., 2016). Thus, the
impact of Grp78 heterozygosity hampering ADM could be further substantiated by
replacing TGFα with Kras mutation in the in vitro acinar differentiation experiment, and
by triggering in vivo ADM through pancreatic ductal ligation. Intriguingly, to study the
transdifferentiation of acinar cells to ductal cells, RNA sequencing and phospho-kinase
protein microarray of the samples before, in the middle and after the in vitro ADM, could
provide a comprehensive view of the players and critical signaling pathways participating
the ADM process. Including samples of the c78
f/+
mice could provide clues on proteins and
signaling pathways affected by GRP78 heterozygosity during the process.
Pancreatitis is linked with an increased risk of pancreatic cancer, but most people
with pancreatitis never develop pancreatic cancer (Lowenfels et al., 1993). In response to
cerulein-induced pancreatitis, wild-type mouse acinar cells may assume a transient duct-
like morphology (Morris, Cano, et al., 2010; Strobel et al., 2007). However, when
combined with mutations of Kras, the Kras mutations could hijack the regeneration process
89
after pancreatic injury, stabilizing the duct-like morphology and promoting further PanIN
and PDAC formation (Guerra et al., 2007; Morris, Cano, et al., 2010). Grp78 heterozygous
whole body knockout mice have been reported to exacerbate pancreatitis in a
C57BL6/129sv genetic background, however not in a C57BL6 genetic background (Ye, et
al. 2010). Further investigation revealed that it is the general ER chaperone balance, rather
than GRP78 alone, contributes to the protection of experimental pancreatitis (Ye, et al.
2010). Downregulation of GRP78 in a rat pancreatic acinar cell line treated with cerulein
alone or in combination with LPS (lipopolysaccharide) significantly promoted apoptosis
(Yong Liu et al., 2014). Hence, in case of pancreatitis, reducing GRP78 to one half may
induce apoptosis of the acinar cells, depriving ADM of the source from which to develop.
In addition, pancreatic injury created by extrinsic insults, such as cerulein, may utilize a
different mechanism promoting ADM from the pancreatic malignancy created by intrinsic
genetic mutation. It will be of interest to determine the role of Grp78 heterozygosity in
pancreatic injury induced PDAC.
Manipulating GRP78 level revealed the roles of GRP78 in PDAC. Downregulation
of GRP78 in human PDAC cells resulted in decreased proliferation and invasion (Z. Niu
et al., 2015). Blockage of GRP78 induction in glucose-deprived PANC-1 human pancreatic
cells led to cytotoxicity (Cha et al., 2009). Furthermore, GRP78 is upregulated in
gemcitabine-resistant PDAC cells and knockdown of GRP78 compromised their
chemoresistance (Gifford et al., 2016). In this study, GRP78 heterozygosity not only
impeded the tumor initiation process - ADM and reduced proliferation in acinar cells, but
also decreased proliferation in ductal cells and hampered signaling pathways. Collectively,
these studies strongly suggest that partial reduction of GRP78 may have dual effects on
90
pancreatic tumorigenesis: hampering growth of already formed PDAC, as well as
suppressing the initiation of new tumors from acinar cells.
Our results from Chapter 3 shed light on possible early prevention method for
pancreatic tumorigenesis and possible approach treating PDAC. Combination of
gemcitabine with EGFR inhibitor (Erlotinib) gave disappointing clinical results with only
modest improvement in the survival of PDAC patients in a Phase III clinical trial (Moore
et al., 2007). Two independent studies have reported that subgroups of PDAC patients and
defined molecular subtypes, responded to the EGFR inhibitor (Collisson et al., 2011;
Jimeno et al., 2008). GRP78 haploinsufficiency may influence unexplored downstream
targets other than EGFR. Agents inhibiting GRP78 expression have great potential treating
PDAC. Other than the inhibitors discussed in Chapter 1, a first-in-class ruthenium-based
drug, IT-139, has been developed to treat solid cancer and successfully studied in Phase I
clinical trial (Trondl et al., 2014). It could induce reactive oxygen species and inhibit
GRP78 expression in tumor cells (Trondl et al., 2014). Treating drug-resistant human
PDAC cell with IT-139 restored its sensitivity to gemcitabine, associating with reduced
AKT activation (Gifford et al., 2016). A more recent study showed single agent HA15 was
able to inhibit GRP78 ATPase activity, exerting ER stress and eventually leading to
apoptosis and autophagy in seven different cancer cells, including one gemcitabine-
resistant PDAC cell line (Cerezo et al., 2016). With the emerging role of cell surface
GRP78, GRP78 was reported to co-localize with p-AKT on PDAC cell surface (Hill et al.,
2012). Cell surface GRP78 is selectively present on malignant cells, but absent on normal
cells, mediating signal transduction critical for cancer cells survival and proliferation (A.
S. Lee, 2014; Ni, Zhang, & Lee, 2011b). As discussed in Chapter 1, monoclonal antibodies
91
targeting cell surface GRP78 have been reported to compromise growth of various cancers
both in vitro and in vivo (de Ridder et al., 2012; Hensel et al., 2013; Lin et al., 2015; R. Liu
et al., 2013; Rasche et al., 2013). GRP78 was found to co-localize with p-AKT on the cell
surface of mouse PDAC tissue (Hill et al., 2012). Intriguing questions remain, such as
whether the cell surface form of GRP78 can be detected by others on mouse or human
PDAC cells; whether applying GRP78-specific monoclonal antibodies to human PDAC
cells or mouse PDAC model would diminish PDAC. Agents targeting cell surface or
endoplasmic reticulum form of GRP78 hold great promise as potential therapeutic target
and warrant rigorous testing.
In Chapter 2 and Chapter 3, both studies involved metaplasia. First, in the normal
development of the mouse uterus, GRP94 depletion triggered squamous cell metaplasia
replacing the previous columnar and glandular epithelial cells. In contrast, GRP78 ablation
in the uterus, did not lead to squamous cell metaplasia. During the normal development of
the pancreas, ablation of GRP78 rendered fat infiltration replacing the previous acinar cells.
GRP78 heterozygous pancreas displayed normal morphology. Thus, in the normal
development of organs, GRP78 and GRP94 may mediate the homeostasis of the organs
and disturbance of such balance may trigger metaplasia. Second, in the course of
tumorigenesis, GRP94 loss accelerated squamous cell metaplasia in PTEN-null uterus, and
such replacement of cells resulted in reduced proliferation, well-differentiated glands and
no myometrial invasion. GRP78 loss, inhibited the PTEN-null driven endometrial cancer
without induction of squamous cells (Lin et al., 2015). Partial reduction of GRP78 in the
pancreas delayed the acinar-to-ductal metaplasia induced by either TGFα or Kras mutation,
leading to reduced tumor. These results indicate that while GRP94 could induce metaplasia,
92
GRP78 could not induce but could interfere with the metaplasia process, and they may
assume distinct roles during the metaplasia process in a context-dependent manner. IHC
staining of GRP94 revealed an upregulation of its expression in mPanIN compared to
normal ducts (Chapter 3). GRP94 expression was observed in 52% of the human PDAC
samples and downregulation of GRP94 in PDAC cells increased apoptosis (Pan, Erkan,
Streit, Friess, & Kleeff, 2009). Hence, investigating the function of GRP94 in the normal
development and tumorigenesis of the pancreas will expand our knowledge about the role
of GRP94 in ADM and PDAC.
GRP78 acts as the master regulator of unfolded protein response, and upon ER
stress GRP78 dissociates from the three ER stress sensors activating their downstream
pathways (B Luo & Lee, 2012). Indeed, complete loss of GRP78 resulted in ER stress in
the HEK293 cells, the Purkinje cells, the bone marrow cells, the intestine, the uterus and
the lung (Flodby et al., 2016; Heijmans et al., 2013; J. Li et al., 2008; Lin et al., 2015; M
Wang et al., 2010; Wey, Luo, & Lee, 2012). However, heterozygous deletion of Grp78
induced minimal, if any, ER stress in the mouse embryonic fibroblasts, the Purkinje cells,
and the pancreas (S. Luo et al., 2006; M Wang et al., 2010; Ye, Mareninova, et al., 2010).
In addition, depletion of GRP94 has no effect on ER stress in the mouse embryonic
fibroblasts (Mao et al., 2010). Therefore, the squamous cell metaplasia induced by GRP94
loss and the impairment of acinar-to-ductal metaplasia by GRP78 haploinsufficiency is
unlikely to be related to ER stress.
Collectively, studies in this dissertation revealed the complex roles of GRP94 and
GRP78 in regulating metaplasia, organ homeostasis and tumorigenesis. Moreover, these
studies expand our understanding on biological functions of glucose-regulated proteins, as
93
well as illuminate the potentials of GRP94 and GRP78 as therapeutic targets for combatting
endometrial and pancreatic cancers.
94
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Abstract (if available)
Abstract
The glucose-regulated proteins (GRPs), including GRP78 and GRP94, are stress-inducible molecular chaperones belonging to the heat shock protein family. The GRPs primarily reside in the ER, however, they are also detected in other compartments of the cell and even secreted outside the cell. Apart from their traditional function as ER chaperones, evidence are emerging that GRPs are elevated in various cancers associating with aggressive properties. ❧ Endometrial carcinoma is the most prevalent gynecologic cancer in the United States. The tumor suppressor gene Pten (phosphatase and tensin homolog) is commonly mutated in the more common type 1 (endometrioid) subtype. Here we report that expression profiles from the Cancer Genome Atlas (TCGA) showed significantly increased Grp94 mRNA levels in endometrial tumor versus normal tissues, correlating with highly elevated GRP94 protein expression in patient samples and the requirement of GRP94 for maintaining viability of human endometrioid adenocarcinoma (EAC) cell lines. Through generation of uterus-specific knockout mouse models with deletion of Grp94 alone (c94f/f) or in combination with Pten (cPf/f94f/f), we discovered that c94f/f uteri induced squamous cell metaplasia (SCM) and reduced active nuclear β-catenin. The cPf/f94f/f uteri showed accelerated SCM and suppression of PTEN-null driven EAC, with reduced cellular proliferation, attenuated β-catenin signaling and decreased AKT/S6 activation in the SCM. In contrast to single PTEN knockout uteri (cPf/f), cPf/f94f/f uteri showed no decrease in E-cadherin level and no invasive lesion. Collectively, our study implies that GRP94 downregulation induces SCM in EAC and suppresses AKT/S6 signaling, providing a novel mechanism for suppressing EAC progression. ❧ Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest disease with <10% overall 5-year survival rate. High GRP78 expression in PDAC was reported in several studies. Here, we investigated the role of GRP78 in pancreatic cancer using the established PKC model (Pdx1-Cre
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Shen, Jieli
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Core Title
The role of glucose-regulated proteins in endometrial and pancreatic cancers
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Keck School of Medicine
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Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
07/22/2017
Defense Date
05/31/2016
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endometrial cancer,glucose-regulated proteins,GRP78,GRP94,OAI-PMH Harvest,pancreatic cancer
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Dubeau, Louis (
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), Lee, Amy S. (
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), Stiles, Bangyan L. (
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friendjjshen@gmail.com,jielishe@usc.edu
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etd-ShenJieli-4606.pdf (filename),usctheses-c40-278447 (legacy record id)
Legacy Identifier
etd-ShenJieli-4606.pdf
Dmrecord
278447
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Shen, Jieli
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
endometrial cancer
glucose-regulated proteins
GRP78
GRP94
pancreatic cancer