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The role of endoplasmic reticulum chaperone glucose-regulated 78-kilodalton (GRP78) in lung cancer
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The role of endoplasmic reticulum chaperone glucose-regulated 78-kilodalton (GRP78) in lung cancer
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Content
THE ROLE OF ENDOPLASMIC RETICULUM CHAPERONE GLUCOSE-
REGULATED 78-KILODALTON (GRP78) IN LUNG CANCER
by
Daisy Flores Rangel
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 2019
Copyright 2019 Daisy Flores Rangel
ii
DEDICATION
To my husband Sal, my mom Luz, and my dad Manuel
iii
ACKNOWLEDGEMENTS
I thank God for helping me accomplish this feat. I am very grateful for this
opportunity and for all of the blessings in my life.
I thank my mentor Dr. Amy Lee, for her guidance and support through these years.
I am grateful for her commitment to helping me grow not only as a scientist and leader
but also as a person with confidence and resilience.
I am also very thankful to my committee members Drs. Louis Dubeau and Ite
Offringa for their guidance, suggestions, and support. Dr. Dubeau taught me the
pathology of the lung and he also determined lung cancer grade for all lung tissues
analyzed in this study. Dr. Offringa provided me with the BEAS-2B and H522 cell lines
as well as the protocol and tools to perform the intratracheal intubations that were
performed on mice. I would also like to thank all current and past members of the Lee
Lab for helpful discussions as well as academic and moral support. I especially thank Dat
Ha for helping me run some of the Western blots, Charles Tseng for insightful
discussions, Willy Tsai for assisting me with the co-immunoprecipitations, Anthony
Carlos for helping me with immunofluorescent experiments, Richard Van Krieken for his
contribution working with the BEAS-2B cell line, and Han Wang who worked with
various lung cancer cell lines to confirm my findings. I also want to thank Mario Pulido
iv
for teaching me how to perform intratracheal intubations on mice and Daniel Mullen for
helping with the TCGA data analysis of patients with lung adenocarcinoma.
This dissertation would not have been possible without the financial support of
the Supplemental grant CURE scholar program. I am also grateful for technical support
and advice from USC staff and members of the various Lab Cores that helped me
complete my experiments including Lillian Young with her assistance in
immunohistochemistry staining, Michaela Mac Veigh for training me on how to use the
confocal microscope, and Ryan Park, from the USC Molecular Imaging Center, for
performing PET/CT imaging on mice. I also want to thank Dr. Robert Maxson for letting
me use his compound and light microscope and Drs. Zea Borok and Beiyun Zhu for
providing the SPC-Cre mice.
To the most important people in my life, I would like to thank my husband who
has supported me and has been my rock throughout this journey. To my mom and dad
who have encouraged me and gave me their love throughout my life. To my sisters,
Haydee, Lesly, and Jennifer, my goddaughters Elena, Emma, and Lucia, my brothers-in-
law, Juan Carlos, Michael, and Arturo, and the rest of my family for their unconditional
love and support.
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 Endoplasmic reticulum 1
1.1.1 Endoplasmic reticulum homeostasis 1
1.1.2 GRP78 in the ER 3
1.1.3 Other functions of GRP78 and cancer 6
1.2 The lungs and GRP78 8
1.2.1 Pulmonary function 8
1.2.2 ER and the lungs 10
1.2.3 GRP78 and the lungs 11
1.3 Lung cancer, GRP78, and KRAS 13
1.3.1 Lung cancer and GRP78 13
1.3.2 KRAS and lung cancer 18
1.4 Models of lung cancer 19
1.4.1 Mouse models for lung studies 19
1.4.2 Conditional KRAS mouse models 21
Chapter 2: GRP78 plays an essential role in KRAS-induced lung cancer
24
2.1 Introduction 24
2.2 Materials and methods 27
2.3 Results 32
2.3.1 Creation of the mouse model with GRP78 deletion
and activation of oncogenic KRAS (Kras
G12D/+
;Grp78
f/f
)
32
2.3.2 GRP78 insufficiency suppresses tumor burden in Kras-
driven lung tumorigenesis
34
2.3.3 Histological grade and weight change differences across
all mice analyzed
36
2.3.4 Depletion of Grp78 results in KRAS reduction
40
2.4 Discussion 42
vi
Chapter 3: GRP78 happloinsufficiency in AT2 cells prolongs survival in
mice with KRAS-induced lung cancer
46
3.1 Introduction 46
3.2 Materials and methods 48
3.3 Results 52
3.3.1 Creation of the SPC-Cre mouse model with biallelic
deletion of GRP78
52
3.3.2 Early deaths of SPC-Cre mice with wild-type levels of
GRP78
54
3.3.3 GRP78 happloinsufficiency in AT2 cells reduces
histological grade
55
3.4 Discussion
59
Chapter 4: GRP78 co-localizes with KRAS and is required for plasma
membrane localization and signaling
63
4.1 Introduction 63
4.2 Materials and methods
4.3 Results
66
4.3.1 GRP78 is required for the integrity of KRAS and co-
localizes at the ER cytosolic interphase
69
4.3.2 GRP78 is required for plasma membrane localization of
KRAS and signaling
72
4.3.3 KRAS signaling affected by GRP78 knockdown can be
rescued
75
4.3.4 Effects of GRP78 on EGFR 80
4.3.5 Human expression data and GRP78 83
4.4 Discussion 85
Chapter 5: Conclusions and Perspectives
90
BIBLIOGRAPHY 104
vii
LIST OF TABLES
Table 1.1: Primer sequences for PCR using DNA extracted from lungs and tails
to detect the Kras
G12D
and Grp78 floxed alleles
28
Table 2.1: Primer sequences for PCR using DNA extracted from tails to detect
the SPC-Cre allele
52
Table 3.1: Primer sequences for real-time PCR used to detect GRP78, KRAS,
ICMT, and β-actin
69
viii
LIST OF FIGURES
Figure 1.1: GRPs in the unfolded protein response (A. Lee 2014)
5
Figure 2.1: Mating strategy for the generation of Kras
G12D/+
;Grp78
+/+
,
Kras
G12D/+
;Grp78
f/+
, and Kras
G12D/+
;Grp78
f/f
mice
33
Figure 2.2: Confirmation of mouse models and timeline of analysis
34
Figure 2.3: Comparative analysis of lungs of K78
+/+
, K78
f/+
, and K78
f/f
mice
35
Figure 2.4: Tumor burden analysis of the lungs of K78
+/+
, K78
f/+
, and K78
f/f
mice
37
Figure 2.5: PET/CT scan of K78
+/+
, K78
f/+
, and K78
f/f
mice
38
Figure 2.6: Tumor grade and mouse weight change of K78
+/+
, K78
f/+
, and K78
f/f
mice
39
Figure 2.7: Reduced KRAS in lungs of K78
f/f
mice 41
Figure 3.1: Breeding scheme for generating mice for the SPC-Cre model
53
Figure 3.2: Representative genotypes of the SPC-Cre mice and timeline of
analysis
54
Figure 3.3: Gross appearance of internal organs and mouse weight change in
SPC-Cre mice
55
Figure 3.4: Analysis of survival of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice
56
Figure 3.5: GRP78 and pathology of the lungs of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice
57
Figure 3.6: Histological grade of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice
58
Figure 4.1: Complex formation of GRP78 and KRAS
71
Figure 4.2: GFP-truncated KRAS (GFP-tK) after GRP78 knockdown in A427
cells
72
Figure 4.3: ICMT and GRP78 in A427 cells
74
Figure 4.4: Analysis of mRNA in A427 human lung adenocarcinoma cells
76
Figure 4.5: A427 human lung adenocarcinoma cells rescue of KRAS 78
ix
Figure 4.6: KRAS downstream signaling after GRP78 siRNA knockdown
treatment
79
Figure 4.7: Summary of effects of GRP78 knockdown
80
Figure 4.8: EGFR and KRAS in A427 cells after GRP78 knockdown
82
Figure 4.9: EGFR and KRAS in A549 cells after GRP78 knockdown
84
Figure 4.10: GRP78 mRNA expression in human lung adenocarcinoma
85
Figure 5.1: ER stress markers in A427 cells after GRP78 siRNA treatment for
48 hrs
96
Figure 5.2: A549 analysis and signaling after GRP78 knockdown 99
Figure 5.3: KRAS analysis in H460 and H522 lung cell lines 100
x
Abstract
The endoplasmic reticulum (ER) is an essential organelle important for the
manufacturing of lipids and proteins and it specializes in proper protein folding and
secretion. Various studies show that the function of ER chaperones is not limited to
simply helping nascent peptides fold but that these proteins have key roles in human
diseases such as cancer, diabetes, obesity, tumor immunity, and mammalian development.
To investigate the link between the ER chaperone, glucose-regulated protein 78 kDa
(GRP78), also known as binding immunoglobulin protein (BiP), and lung tumorigenesis,
several transgenic mouse models where fragments of the gene encoding for GRP78,
called, heat shock protein family A, member 5 (HSPA5), were conditionally deleted, have
been constructed for investigation in vivo. In addition, we used human lung cell lines for
in vitro studies.
Lung cancer is the leading cause of cancer related deaths worldwide, claiming
more than 1.6 million deaths annually and approximately 153,718 deaths in the United
States alone. Mutations in the proto-oncogene KRAS occur in 10 to 30% of lung
adenocarcinomas, classified as a non-small cell lung cancer (NSCLC). Here we report the
requirement of GRP78 for the development and progression of lung adenocarcinoma as
identified through the generation of two separate mouse models containing floxed Grp78
or K-ras Lox-Stop-Lox G12D (K-ras
LSL-G12D
) alleles. The first mouse model was intubated
intratracheally to introduce Cre directly into the lungs for the creation of the following
xi
mice: (1) Kras
G12D/+
Grp78
+/+
(referred to as KGrp78
+/+
or K78
+/+
), (2) Kras
G12D/+
Grp78
f/+
(referred to as KGrp78
f/+
or K78
f/+
), and (3) Kras
G12D/+
Grp78
f/f
(referred
to as KGrp78
f/f
or K78
f/f
). Tamoxifen injection activated Cre in mice of the second model,
since the Cre gene was driven by a 3.7 kb human surfactant protein C and estrogen
receptor (SPC-CreER
T2
) promoter was expressed in alveolar type 2 (AT2) cells of the
lung. Mice used in model 2 were of the following genotypes: (1) SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
Grp78
+/+
(referred to as CKGrp78
+/+
or CK78
+/+
), (2) SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
Grp78
f/+
(referred to as CKGrp78
f/+
or CK78
f/+
), and (3) SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
Grp78
f/f
(referred to as CKGrp78
f/f
or CK78
f/f
). Consistent with published reports
that GRP78 is upregulated in human lung adenocarcinoma, strong immunostaining for
GRP78 was detected in the K78
+/+
mice, and in comparison, the staining intensity for
GRP78 decreased in the lungs of K78
f/+
and a further decrease in K78
f/f
mice. Supporting
the notion that decreased GRP78 expression leads to decreased proliferation in
pulmonary alveolar cells harboring a mutant KRAS, we observed decreased
immunostaining of the proliferation marker Ki67 in the lungs of K78
f/+
and further
decrease in K78
f/f
mice compared to K78
+/+
mice. Moreover, pathological review
revealed increased progression in histological grade such that adenocarcinoma was
detected as early as 16 weeks post Cre activation in K78
+/+
mice, compared to 22 weeks
in K78
f/+
mice, yet it was not detected in K78
f/f
mice analyzed. This difference in tumor
burden and histological grade was also confirmed by 18F-FDG PET/CT analysis. This
suppressive effect of heterozygous knockout of GRP78 is even more pronounced in our
xii
second mouse model where both the heterozygous CK78
f/+
and the homozygous CK78
f/f
knockouts showed prolonged survival compared to CK78
+/+
. Towards understanding the
mechanism where GRP78 suppresses mutant KRAS driven lung tumorigenesis, we
discovered that depletion of GRP78 leads to lowering of KRAS protein levels both in
vivo and in three human lung cancer cell lines harboring KRAS mutations, namely, A427
(KRAS
G12D
), A549 (KRAS
G12S
), and H460 (KRAS
Q61H
). Our biochemical studies showed
that KRAS can form a complex with GRP78 and confocal imaging further revealed co-
localization of the two proteins in the ER. Strikingly, we also detected a decrease in
isoprenylcysteine carboxyl methyltransferase (ICMT), the ER resident protein
responsible for posttranslational methylation of the isoprenylated C-terminal cysteine of
KRAS. The reduction of KRAS and ICMT proteins in GRP78 knockdown lung cancer
cell lines is not at the mRNA level and could be partially rescued by the proteasome and
lysosomal inhibitors, MG132 and 3-MA, respectively. We also used the GFP-tK
construct, which contains GFP fused to the last 17 amino acids of the KRAS C-terminus,
as these sequences are necessary and sufficient for KRAS modification in the ER and
translocation to the plasma membrane. Interestingly, in cells with GRP78 knockdown
treatment, GFP-tK levels were decreased as observed by Western blot and translocation
to the plasma membrane was not observed. We also detected decreased signaling of two
major proliferative signaling pathways activated by KRAS at the plasma membrane,
PI3K/AKT and RAF/MEK/ERK, indicating that GRP78 is required for the activation of
these signaling pathways. EGFR is also commonly mutated in lung adenocarcinoma and
xiii
can be abundantly overexpressed in non-small cell lung cancer patients. Moreover, EGFR
activation has been shown to be important for activation of KRAS whether KRAS is
wild-type or mutated. Our results also demonstrate that depletion of GRP78 affects the
translocation of EGFR to the plasma membrane. In addition, we also independently
confirmed upregulation of GRP78 in human lung cancer through analysis of the TCGA
database. Collectively, these studies reveal GRP78 as a novel regulator for KRAS-
induced lung tumorigenesis and expand our understanding of the biological function of
GRP78, as well as highlight the potential of GRP78 as a therapeutic target to battle lung
cancer.
1
Chapter 1
Overview and Introduction
1.1 Endoplasmic reticulum
1.1.1 Endoplasmic reticulum homeostasis
The endoplasmic reticulum (ER) is responsible for the synthesis, modification and
delivery of proteins, as well as sterols and lipids, in the secretory pathway and the
extracellular space. Proteins destined for the secretory pathway enter the pathway through
the ER (Schröder and Kaufman 2005). A predominantly hydrophobic signal peptide
sequence directs the nascent polypeptide chain into the ER. In the ER, proteins fold into
their native conformation and undergo post-translational modifications. Chaperones
within the lumen of the ER assist in folding of the newly synthesized polypeptides and
prevent aggregation of unfolded or misfolded proteins (Ni and Lee 2007). Incompletely
folded proteins are retained in the ER to be properly folded or are targeted for
degradation (Ellgaard et al. 1999).
There is quality control in the ER to avoid accumulation of unfolded proteins or
the folding of incompetent proteins, both of which can lead to stress or perturbations of
the normal state of the ER. If the ER experiences stress, it responds by activating the
unfolded protein response (UPR). This ER stress can be triggered by the accumulation of
folded-incompetent proteins, accumulation of unfolded or aggregated proteins. This
2
disturbance in ER homeostasis can be the origin of many diseases and developmental
abnormalities (Schröder and Kaufman 2005). Various physiological conditions have been
discovered to impact the ER such that the demand of protein folding exceeds the capacity
of the ER leading to ER stress. For example, differentiation of B-cells into highly
secretory plasma cells (Iwakoshi et al. 2003), infection caused by viruses or microbes
(Watowich et al. 1991; Walther-Larsen et al. 1993). In both yeast and mammalian
systems, ER stress can be induced by pharmacologic agents that cause accumulation of
misfolded or underglycosylated proteins. In higher eukaryotes there are other unique
stress inducers, including the microenvironment of solid tumors (glucose starvation, low
pH and hypoxia), embryonic development and mood-altering drugs (alcohol, valproate)
(A S Lee 2001). To overcome these stressors, upon activation of the UPR, various
signaling cascades are activated in efforts to restore the ER back to its normal
physiological state and homeostasis. Two mechanisms are activated during the UPR. The
first is the upregulation of molecular chaperones and foldases to help increase the folding
capacity as well as the increase of the size of the ER. The second is the downregulation of
protein synthesis at both the transcription and translational level. ER-associated
degradation is also activated to target unfolded proteins for degradation in order to
decrease the protein folding load. In higher eukaryotes, if these responses do not alleviate
ER stress, the UPR can also induce apoptotic pathways.
3
1.1.2 GRP78 in the ER
Glucose regulated proteins (GRPs) are stress inducible chaperones important for
assisting with protein folding during stress conditions in the ER. GRPs are mostly
localized to the ER and mitochondria but can translocate to the cell membrane during
conditions of ER stress. Aside from assisting with protein folding, GRPs also have
functions in controlling signaling, proliferation, invasion, apoptosis, inflammation, and
immunity (A. Lee 2014). The most abundant ER chaperone and UPR master regulator is
glucose-regulated protein 78-kDa (GRP78) also known as binding immunoglobulin
protein (BiP). Encoded by the HSPA5 gene, GRP78 recognizes newly synthesized
proteins starting from their translocation across the ER membrane. GRP78 can bind to a
wide array of proteins and binds with low affinity to the hydrophobic residues on the
surface of proteins. GRP78 is also responsible for maintaining the permeability barrier of
the ER during protein translocation (Ma and Hendershot, 2004). To prevent malfolded
proteins from being expelled from the ER, GRP78 assists with the controlled process of
guiding malfolded proteins for degradation in the ER-associated degradation system
(ERAD), whereby malfolded proteins translocate to the cytosol through the ERAD
machinery to be degraded by cytosolic proteasomes (Ushioda et al. 2008).
GRP78 is the main regulator of the UPR and the apoptotic machinery that is
associated with the ER because of its interactions with three ER-resident stress sensor
proteins, namely, inositol-requiring kinase 1 (IRE1), activating transcription factor 6
4
(ATF6) and PKR-like eukaryotic initiation factor 2αkinase (PERK). During non-stress
conditions, GRP78 is bound to these three and in doing so prevents their activation. As
shown in Figure 1.1 (A. Lee, 2014), during ER stress conditions, GRP78 is titrated away
from its associations with these three proteins, freeing them for activation of the UPR
(Rutkowski and Kaufman, 2004). GRP78 is upregulated to increase its role as a
chaperone binding unfolded and misfolded proteins during ER stress (Figure 1.1). As the
UPR ensues, both PERK and IRE1 dimerize and activate downstream signaling that lead
to attenuation of translation to minimize protein load and increase ERAD function to
eliminate misfolded or unfolded proteins. ATF6 is an ER-transmembrane-activating
transcription factor and after the UPR is activated, it is cleaved in the golgi compartment
yielding the cytosolic nuclear form that translocates to the nucleus and upregulates
transcription of UPR genes. Genes that encode molecular chaperones are upregulated to
increase the folding capacity of the ER (Liu et al. 2018). If the protein folding defect is
not corrected, the UPR can also activate CHOP, a pro-apoptotic transcription factor.
Additionally, prolonged UPR activation leads to apoptotic cell death via activation of
caspases 7 and 12. GRP78 releases these caspases upon ER stress, and during prolonged
UPR, activation of these caspases leads to apoptotic cell death (A. Lee, 2014). Also
shown in Figure 1.1, GRP78 can translocate to other compartments during ER stress,
such as, the mitochondria and cell surface, and can be cleaved to enter the cytosol. Not
only can GRP78 escape to the cell surface upon ER stress, it can regulate cell signaling,
proliferation, apoptosis and immunity (Tsai et al., 2017).
5
Figure 1.1 GRPs in the unfolded protein response (A. Lee 2014). ER luminal GRP78 functions as a UPR
signaling regulator by binding to and maintaining the ER stress sensors PERK, ATF6 and IRE1 in inactive
Figure 1.1 GRPs in the unfolded protein response (A. Lee 2014). GRP78 in the ER functions as a UPR
regulator by binding to PERK, ATF6, IRE1 to prevent their activation. Upon ER stress, the UPR is
triggered and GRP78 is titrated away to bind to misfolded proteins, resulting in PERK and IRE1 self-
dimerization that triggers the activation of their downstream signaling pathways, leading to arrest of protein
translation and activation of the protein degradation machinery, ERAD. The UPR also generates the active
nuclear form of ATF6 (ATF6(N)) and can also induce transcription of the pro-apoptotic transcription factor
CHOP. Following release of caspase 7 and 12 from GRP78, the UPR can trigger apoptosis.
GRP78 does not contain a classical transmembrane domain and as such it interacts with
cell surface proteins such as GPI-anchored proteins. Cell surface GRP78 has been shown
to act like a multifunctional receptor and regulate signaling pathways, such as activating
6
the PI3K/AKT signaling for cancer survival and proliferation (Gonzalez-Gronow et al.
2009; Ni, Zhang, and Lee 2011). The mechanism whereby ER stress induces cell surface
translocation of GRP78 has been elucidated in recent years. It was identified that GRP78
requires its substrate binding domain for translocation and it exists mainly as a peripheral
protein (Tsai et al., 2015). In addition, GRP78 translocates to the cell surface via IRE1
binding and activating SRC, the nonreceptor protein-tyrosine kinase belonging to the
SRC family kinases (SFK) (Tsai et al., 2017; Yeatman, 2004). In summary, GRP78 is
not only critical for protein folding, quality control in the ER, aiding in the maturation of
proteins destined to the plasma membrane and regulating the UPR, but is also an
important regulator of various signaling pathways via surface translocation during ER
stress.
1.1.3 Other functions of GRP78 and cancer
GRP78 is required for many other essential cellular processes, such as protecting
the inner cell mass from apoptosis during early mouse embryonic development (Luo et al.
2006). Grp78
-/-
mice die before embryonic E3.5, demonstrating its essential role for cell
growth and survival (Luo et al., 2006; Ni et al., 2011). GRP78 is also important in cell
proliferation and maintenance of hematopoietic homeostasis (Wey, Luo, and Lee 2012).
GRP78 is a critical component of the ER and plays a key role in maintaining its integrity,
as it also binds to ER calcium to help store it and suppresses stress-induced autophagy (Li
et al. 2008). Basal levels of GRP78 are maintained under normal physiological conditions,
7
however, in tumorigenic cells, various stressful conditions exist that can activate the UPR
and increase GRP78, such as hypoxia, nutrient deprivation, pH changes, or poor
vascularization. GRP78 has been observed in proliferating and dormant cancer cells as
well as tumor initiating cells (A. Lee 2014), and protective effects in these cells was
observed. To identify the role of GRP78 in different organs, various transgenic mouse
models have further revealed context-dependent roles of GRP78. For example, biallelic
conditional knockout of Grp78 impeded PTEN null-induced leukemogenesis in the
hematopoietic system. This demonstrates that in the hematopoietic system, GRP78 is a
critical effector of leukemia progression in part through regulation of the PI3K/AKT
pathway (Wey at al., 2012). Yet knockout of Grp78 leads to differentiation of progenitor
cells in the intestine and esophagus (Heijmans et al., 2013; Rosekrans et al., 2014). In
another mouse model, GRP78 was shown to be a regulator for PTEN-loss mediated liver
injury and cancer progression (Chen et al. 2013). Furthermore, GRP78 was found to be
important in acinar-to-ductal metaplasia in the pancreas, which is regarded as a precursor
to pancreatic ductal adenocarcinoma, a highly lethal disease (Shen et al. 2017). In
pancreatic cancers, up to 90% of cancers contain KRAS mutations. As a result, KRAS
oncogenic function was activated in a mouse study from our lab, along with heterozygous
depletion of p53 and GRP78. Reduced proliferation and tumor burden was observed
after depletion of GRP78 in these mice and pancreata weight was normal, along with
decreased signaling of AKT, S6, ERK and STAT3. Collectively, these mice studies
support the pro-proliferative function of GRP78. Although GRP78 knockout embryos
8
are lethal in mice, heterozygous Grp78 mice expressing approximately 50% levels are
viable and phenotypically normal (Luo et al. 2006). Interestingly, aged mice with
prolonged GRP78 haploinsufficiency that were followed up to 2 years of age revealed
that body weight, organ development and integrity were not impaired (Lee et al. 2017).
Additionally, no significant effect on cancer incidence and inflammation was observed
between male and female mice that were analyzed. This indicates that depleting GRP78
that can be accomplished by therapeutic drugs to target certain cancers can be promising
for patients.
1.2 The lungs and GRP78
1.2.1 Pulmonary function
The respiratory system composed of the nasal passage, oral cavity, pharynx,
larynx, trachea, bronchi and bronchioles and the alveoli are responsible for taking in
oxygen and expelling carbon dioxide. According to the National Institute of Health, brain
cells start dying less than 5 minutes after oxygen supply stops. The lungs are
continuously exposed to the outside environment and the alveolar membrane, the major
site of gas exchange to and from the bloodstream, is the largest surface of body in contact
with the outside environment (Martin and Frevert, 2005). The alveolar epithelium is
comprised of two main cell types, alveolar type 1 (AT1) and type 2 (AT2) cells (Ward
and Nicholas, 1984). AT1 cells are flattened squamous cells that provide the major
9
surface area for gas exchange and AT2 cells are cuboidal, surfactant-producing cells that
serve as progenitors of AT1 cells during development and after injury (Barkauskas et al.
2013). In the adult lung, the surface area is approximately 90 m
2
, larger than the gut (10
m
2
) and skin (2m
2
) (Kopf et al. 2015). As such, the lungs are important in innate
immunity, functioning to detect and expel harmful microbes and agents, as well as
organic and inorganic materials. To expel threats, large particles are deposited in the
nasopharynx and tonsillar regions, which can be cleared by coughing or sneezing for
example. Further down the respiratory tract unwanted agents are captured on the
mucociliary surface and through movement of cilia are expelled to the upper airways.
Lung resident macrophages and dendritic cells take up and remove solid and liquid
particles, allergens and airborne microbes. To maintain lung compliance or the proper
ability of the lungs to stretch and expand, the lungs rely on pulmonary surfactant, a
surface-active lipoprotein complex that is both important in immunity and to prevent the
collapse of airways. Moreover, surfactant decreases alveolar surface tension. Collectins,
surfactant proteins, lipopolysaccharide binding proteins (LBP), and other proteins in the
innate immune system bind to bacterial cell walls and work to enhance the
responsiveness of alveolar macrophages (Brass et al. 2008). During infection or exposure
to unwanted environmental factors, inflammatory responses help promote clearance.
However, inappropriate acute or long-term inflammation can lead to disease such as
edema, asthma, fibrosis and emphysema (Kopf et al. 2015). Anti-inflammatory
macrophages, lung dendritic cells and airway epithelial cells are responsible for proper
10
homeostasis, all in efforts to maintain proper gas exchange. Although it is known that
inhaled pollutants and cigarette smoke are major causes of lung disease, additional effects
due to problems with protein folding in the ER can also drive the development of lung
disease.
1.2.2 ER and the lungs
Protein misfolding in the endoplasmic reticulum can lead to various lung diseases
from acute lung injury to pulmonary fibrosis to cancer (S. J. Marciniak, 2017). If the
protein load exceeds the capacity of the rate of protein folding in the ER, stress can
trigger inflammatory signaling which can lead to the development of lung disease. For
example, obesity is a significant risk factor for acute respiratory distress syndrome. Once
adipose tissue becomes sufficiently expanded in an obese individual, the surplus of
dietary lipids are stored in alternative tissues, leading to tissue inflammation. Specifically,
the effects of lipids on membrane fluidity, which helps modulate protein trafficking and
function in the ER, lead to ER stress. In addition, elevated levels of saturated fatty acids
in the serum of mice cause pulmonary endothelial cell dysfunction by inducing ER stress
and promote acute lung injury (Shah et al. 2017). ER stress is also associated with the
development and progression of idiopathic pulmonary fibrosis (IPF) (Burman et al. 2018).
IPF is a progressive age-related disease that is characterized by difficult or labored
breathing, exercise intolerance, low concentration of oxygen in the blood, and respiratory
failure (Mora et al. 2017). The pathological manifestation is the presence of fibrotic
11
remodeling in the distal lung parenchyma and can include collagen deposition from
activated fibroblasts (Burman et al. 2018). ER stress in alveolar epithelial cells is
prominent in IPF (Lawson et al. 2008). Lung carcinoma cells expressing mutant
surfactant protein C (SFTPC, which is produced by AT2 cells) show an accumulation of
mutant protein in the ER and subsequent ER stress and proinflammatory signaling. In
addition, UPR markers are also prominently expressed in alveolar epithelial cells in lung
samples from patients with SFTPC mutation-associated fibrosis (Lawson et al. 2008;
Maguire et al. 2014).
1.2.3 GRP78 and the lungs
The role of GRP78 in lung homeostasis has been investigated. Neonate mice with
a knock-in expressing a mutant form of GRP78 lacking the ER retrieval amino acid
sequence Lys-Asp-Glu-Leu (KDEL) suffered respiratory failure due to the inability of
AT2 cells to secrete pulmonary surfactant (Mimura et al. 2007). Levels of surfactant
protein C (SPC) were reduced and the lamellar body was malformed indicating that
GRP78 is important in surfactant production. Interestingly, this mutant form of GRP78
was sufficiently functional to allow lung development before birth. In another study,
knockout mice with conditional deletion of GRP78 in AT2 cells was made possible using
the SFTPC-cre mice to create cGrp78
f/f
mice (Flodby et al. 2016). cGrp78
f/f
mice died
shortly after birth. Lungs at embryonic day 12 (E12) displayed distal airspaces and at E18,
airspaces were enlarged and irregularly shaped. Moreover, lungs harvested from embryos
12
at E14 and E18 had increase of GRP94 as well as the UPR marker, p-eIF2α, indicative of
activation of IRE1. Expression of CHOP, the pro-apoptotic marker, was also increased at
E18. Grp78 KO also had altered ER structures, as the ER was abnormally expanded, had
fewer lamellar bodies and had apoptotic features. TUNEL staining revealed that E18
lungs had an increased number of apoptotic cells within saccules. This was confirmed by
immunohistochemical staining of caspase 3 positive cells in airspaces but not in
bronchiolar epithelial cells. In addition, Grp78 KO resulted in reduced expression of AT1
and AT2 cell markers. Collectively, these data reveal the role of GRP78 in maintaining
ER homeostasis and alveolar cell survival during lung development. Furthermore, since
AT2 cells are secretory cells, these are more sensitive to GRP78 deficiency, resulting in
the increase of ER stress and apoptosis of alveolar epithelial cells.
The role of GRP78 has also been investigated in the context of acute
inflammatory injury of lung epithelium. The vascular endothelium lining the pulmonary
blood vessels is the major barrier protecting fluid entry into airspaces of the lung
(Leonard et al. 2019). Upon infection into the pulmonary circulation such as exposure to
lipopolysaccharides (LPS) from Gram-negative bacteria, lung vascular endothelial cells
lining the blood capillaries secrete inflammatory and chemotactic substances and suffer
from loss of barrier integrity (Gross et al. 2018). The disruption of the pulmonary
endothelial barrier and inflammatory phenotype are features of acute lung injury, which
is a common cause of respiratory failure in critically ill patients. In a study involving
mice exposed to aerosolized LPS to induce acute lung inflammation and injury, mice
13
were also treated with Subtilase cytotoxin (SubAB), the holoenzyme that cleaves and
inactivates GRP78, or the mutant form SubA
A272
B (Leonard et al. 2019). Lung
inflammation and injury were significantly inhibited in SubAB treated mice but not in
those treated with SubA
A272
B. Analysis of lung homogenates demonstrated that
inflammatory mediators VCAM-1 and IL-1β were induced after LPS treatment but were
strongly inhibited in mice treated with SubAB. Furthermore, infiltration of the
inflammatory cells, neutrophils, was also inhibited in SubAB treated mice. Analysis of
bronchoalveolar lavage demonstrated an increase in albumin levels after LPS treatment,
indicating microvascular leakage. However, in SubAB treated mice, the response was
inhibited, unlike in SubA
A272
B treated mice. Additionally, lung compliance was protected
in mice pretreated with SubAB after LPS treatment. These findings demonstrate that
GRP78 has a critical role in mediating endothelial cell permeability and inflammation
and as such, is a critical determinant of lung vascular inflammation and acute injury
(Leonard et al. 2019).
1.3 Lung cancer, GRP78, and KRAS
1.3.1 Lung cancer and GRP78
Lung cancer is the leading cause of cancer-related deaths worldwide, claiming
more than 1.6 million deaths annually (Torre et al, 2015). According to the Centers for
Disease Control and Prevention (CDC) and the National Cancer Institute (NCI), in 2015,
14
the latest year for which incidence data are available, 153,718 deaths in the United States
were caused by lung and bronchus cancers and lung cancer was the top cancer by death
rates. Lung and bronchus cancers rate third in new cancer cases, behind breast cancer in
females, and prostate cancer in males. Lung cancer incidence and mortality patterns
follow temporal patterns of cigarette smoking (Pass et al. 2010). The incidence of lung
cancer is associated with the amount and duration of cigarette smoked, and this
association is well established. It is predicted that by 2030, tobacco will be responsible
for 10% of all deaths globally, and an estimated 8.3 million people will die globally from
tobacco-induced deaths (Mathers et al. 2006). 50% of people who smoke will die of a
tobacco-related disease such as heart disease, stroke, lung cancer, lower respiratory
infections and chronic obstructive pulmonary disease (COPD). Additionally, smokers
will lose on average 10 years of life (Minn et al. 2007). Lung cancer is also estimated to
cause approximately 17 – 26,000 deaths in nonsmokers annually in the United States
(Thun et al. 2006). Secondhand smoke can partially explain these deaths, however, other
occupational and environmental exposures as well as genetic factors are associated with
lung cancer. For example, exposure to naturally occurring elements such as asbestos,
arsenic, beryllium, chloromethyl ether, chromium, diesel exhaust, nickel, radon, silica,
and vinyl chloride are all associated with increased lung cancer risk (Pass et al. 2010).
The two major disease subtypes of lung cancer are non-small cell lung cancer
(NSCLC) as the predominant subtype accounting for 80 to 85% of cases and small cell
15
lung cancer (SCLC) accounting for 15 to 20% of human cases. The disease categories of
lung cancer are classified based on histological, clinical and neuroendocrine
characteristics. Molecular studies further classify lung cancer subtypes. The two most
common subtypes of NSCLC are adenocarcinoma (ADC) and squamous cell carcinoma
(SCC) (Pass et al. 2010). Like SCC, ADC is linked to smoking, however, ADC is also
prevalent in nonsmokers and in women (Ramalingam et al. 1998). ADC is defined by
mucin production or glandular differentiation. The World Health Organization further
classifies ADC into papillary, acinar, bronchioloalveolar, solid with mucin production,
and mixed patterns as the most commonly observed. Less common variants include fetal,
mucinous cyst-adenocarcinoma, mucinous colloid, signet ring, and clear cell ADC
(Travis et al. 2015).
Many reports throughout the years have demonstrated that GRP78 is
overexpressed in cancerous tissues over normal tissues or benign neoplasms (Uramoto et
al. 2007; Wang et al. 2005; Chae et al. 2012). GRP78 has been reported to be highly
expressed in various lung cancer lesions, such as in non-small cell lung cancer patients
with adenocarcinoma or squamous cell carcinoma (Imai et al. 2017; Ma et al. 2015; Chae
et al. 2012) as well as adenosquamous cell carcinoma and small cell carcinoma (Uramoto
et al. 2007). However, the clinicopathological significance of GRP78 expression in lung
cancer patients is controversial. One of the earliest reports describing overexpression of
GRP78 in 132 lung cancer patients with various lung cancer lesions including
16
adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, carcinoid,
large cell carcinoma and small cell carcinoma, reported that patients with a positive
GRP78 expression tended to show a better prognosis than those with negative expression
of GRP78 (Uramoto et al. 2007). However, multiple reports have claimed the opposite.
Lung cancer patients with adenocarcinoma expressing higher GRP78 had considerably
shorter overall survival compared to those with low to undetected levels (Chae et al.
2012). Additionally, overall survival in patients with higher GRP78 expression was
significantly poorer in a study involving 163 patients (Ma et al. 2015). Another report
involving 220 patients claimed GRP78 expression was significantly higher in patients
with pulmonary adenocarcinoma and had shorter disease-free survival and poor prognosis
(Kwon et al. 2018). Another study analyzed 369 patients with pulmonary
adenocarcinoma and 246 patients with squamous cell carcinoma reported that elevated
levels of GRP78 was a poor prognostic factor (Imai et al. 2017). Elevated levels of
GRP78 have also been associated with lung metastasis (Lizardo et al. 2016). More than
90% of cancer deaths are the result of metastasis occurring in the patient. The lung is a
common site of metastasis for solid tumors such as breast, prostate, melanoma, and
pediatric osteosarcoma (Khanna et al. 2014; Lizardo et al. 2016). Although most tumor
cells that metastasize to the lung undergo apoptosis, highly metastatic cells can resist
apoptosis (Hong et al. 2012). This suggests that highly metastatic cells are better adapted
to meet the hostile microenvironment of the lung. In efforts to identify the role that
GRP78 has in metastatic growth in the lung microenvironment, four highly metastatic
17
cell lines were investigated in mice. GRP78 was upregulated in these cell lines along with
ER-stress responsive genes (Lizardo et al. 2016). Doxycycline-inducible shRNA to
decrease GRP78 was activated and the metastatic ability of human osteosarcoma MG63.3,
a normally highly metastatic cell line, was inhibited. IT-139, an anti-tumor ruthenium
small molecule inhibitor that targets GRP78 (Bakewell et al. 2018) was also introduced in
these mice. Treatment with IT-139 also diminished the metastatic potential of another
highly metastatic osteosarcoma cell line 143B. This indicates that GRP78 upregulation is
required for malignant osteosarcoma cells to metastasize to the lung. Moreover,
inhibition of GRP78 in the mouse models also led to decreased tumor burden and
increased life span (Lizardo et al. 2016).
GRP78 has also been linked to drug resistance in lung cancer cell lines
(D’Abrosca et al. 2019; Dadey et al. 2017; Lin et al. 2011; Wang et al et al. 2008).
Traditional treatments for patients with NSCLC were commonly cisplatinum-based
combination therapies. Although cisplatin is a potent anti-cancer drug, its benefits were
limited by the resistance that eventually developed. Patients had a limited outcome, with
a 5-year survival limited to less than 1% (D’Abrosca et al. 2019). Chemotherapy efficacy
limited by resistance has also been reported in patients with small cell lung cancer.
Another common therapeutic drug used in the treatment of lung cancers is VP-16,
otherwise known as etoposide. SCLC cell line NCI-H446 was treated with VP-16 and
subjected to downregulation of GRP78, and inhibition of GRP78 resulted in increased
18
sensitivity to VP-16 treatment (Wang et al. 2008). In another study, antibodies to target
GRP78 were investigated for their effects of ionizing radiation on NSCLC cell lines both
in vivo and in vitro (Dadey et al. 2017). NSCLC cell lines were treated with anti-GRP78
antibodies and were evaluated for proliferation rates, colony formation, cell death and
PI3K/AKT/mTOR signaling. Cell proliferation and colony formation were attenuated
while apoptosis was enhanced, and PI3K/AKT/mTOR signaling was suppressed. A549
lung carcinoma cells were injected in the right hind limb of nude mice and tumors were
irradiated with 3 fractions of 3 Gy over the span of 24 hrs. Post irradiation, anti-GRP78
antibodies were injected and tumors were harvested post 48 hrs. Treatment with antibody
against GRP78 enhanced efficacy of radiation. In addition, tumor growth was delayed in
mouse xenograft models. These data demonstrate that targeting GRP78 offers a new
strategy to enhance therapy for lung cancer patients undergoing radiation (Dadley et al.
2017).
1.3.2 KRAS and lung cancer
The oncogene, KRAS, was originally identified in sarcoma-inducing retroviruses in rats.
Orthologs of these viral oncogenes were then identified in DNA fragments of human
cancers. The Kirsten sarcoma virus gene, KRAS, is located on chromosome 12p12.1 and
encodes a 21 kDa protein with guanine nucleotide-binding activity (Kirsten and Mayer,
1967; Tsuchida and Uesugi 1981). KRAS belongs to the RAS family of GTPases that
function as binary switches that cycle between active, when bound to guanosine
19
triphosphate (GTP) and inactive, when bound to guanosine diphosphate (GDP) states
(Tsuchida et al. 2016). Mutations in the proto-oncogene KRAS occur in 10 to 30% of
lung adenocarcinomas (Shigematsu et al. 2005). KRAS is most commonly mutated in
lung adenocarcinoma and its activation likely occurs in the early stages of tumorigenesis
(Guerra et al. 2003, Jackson et al. 2001). Indeed, lung tumors have high rates of
mutations in KRAS (Gibbons et al. 2014) and in mice, expression of mutant KRAS in the
alveolar epithelium leads to lung adenocarcinoma (Fisher et al. 2001; Johnson et al. 2001;
Jackson et al. 2001).
1.4 Models of lung cancer
1.4.1 Mouse models for lung studies
To better understand lung cancer development and progression and to develop
better treatments to combat this deadly disease, various mouse models have been
developed. Primary lung tumors in mice share histogenic, molecular, and morphological
resemblance to human lung adenocarcinoma (Malkinson, 1992). The common models to
induce lung cancer are carcinogen or chemically-induced, environmentally-induced such
as with radiation or viruses, genetically-induced such as through transgenic expression,
and orthotopically-induced such as by injection of human tumor cells into the lungs (Pass
et al. 2010). Mouse strains are categorized into sensitive, intermediate, and resistant. The
use of environmental agents such as viruses have the advantage over chemical methods
20
of inducing lung cancer since they are not strain-dependent. Mice have been successfully
used to induce lung cancer after exposure to viruses and produce reproducible lung
tumors that develop NSCLC. Moreover, lung tumor development in mice allows for
studies of late stage progression and decreased metastatic potential. In addition,
noninvasive imaging such as PET/CT scan can be used to study disease progression in
mice. The development of genetic mouse models that can mimic the human situation has
allowed the identification of various genes that drive lung cancer development and
progression. For example, three important tumor suppressor genes have been discovered
to be inactivated in adenocarcinoma, namely, p53 at 50-70%, retinoblastoma at 23-57%,
and p16 at 55%. Increases in protein expression of CCND1 (35-55%), CRK (8-30%),
EGFR (40-65%), and HER2 (16-38%) have also been described for adenocarcinoma.
Oncogenic alterations in adenocarcinoma also include mutations in KRAS (10-30%),
EGFR (10-40%), as well as amplification of MET (20%). KRAS mutations are most
commonly found in codons 12, 13 and 61, with G12, G13 and Q61 being the most
commonly affected (Cox et al., 2014). The top mutations include G12C, G12D, and
G12V. Mice harboring the latent G12D oncogenic mutation of the KRAS gene were
developed (K-rasLA) (Johnson et al., 2001) and mice developed lung tumors as early as 1
week after birth. The problems that arose from these mice were several, including the
short lifespan of the tumor-bearing mice as well as the inability of the lung cancer to
metastasize. The ability to conditionally activate oncogenic activity arose from the
development of mice by Jackson et al. (2001) with the introduction of a Lox-Stop-Lox
21
(LSL) codon containing a removable transcriptional termination stop element. Upon
removal of the stop codon, with the introduction of adenovirus-Cre recombinase through
intranasal administration, mice developed lung cancers (Jackson et al. 2001). These
KRAS transgenic mice were further crossed with various other transgenic mice in order
to investigate the tumorigenicity and metastatic potential, such as a p53 mutation leading
to the development of metastatic lung cancer.
1.4.2 Conditional KRAS mouse models
Conditional genetic models of human NSCLC in mice using the K-ras Lox-Stop-
Lox G12D (K-ras
LSL-G12D
) oncogenic activation have made it possible to study lung
cancer development and progression. The ability to introduce engineered adenovirus-Cre
by intratracheal intubation directly into the mouse lungs (DuPage et al. 2009) allows for
the stop codon to be removed to activate oncogenic activity of K-ras
LSL-G12D
.
Adenoviruses can infect a wide range of tissues and do not incorporate their genome into
the host genome. The LSL cassette was introduced into the first intron of the Kras gene.
The LSL cassette creates a null version of the gene, since it contains both transcriptional
and translational stop elements flanked by the LoxP sites that are recognized by Cre
recombinase. Therefore, until Cre is present, the oncogenic Kras cannot be activated. Of
note, mice can only be heterozygous for the K-ras
LSL-G12D
allele, since Kras null mice are
not viable (Johnson et al. 1997). As early as 4 weeks post intranasal instillation with 5 x
10 PFU
8
of Adenovirus-Cre, K-ras
LSL-G12D
mice had numerous lesions and cobblestone
22
appearance on the lung surface (Jackson et al. 2001). Lungs analyzed 6 weeks post
instillation resulted in three types of lesions, namely, atypical adenomatous hyperplasia
(AAH), epithelial hyperplasia (EH) of the bronchioles, and adenomas. The AAH present
in the mice closely resembled AAH in humans, which is proposed to be the precursor of
adenocarcinoma (Kerr et al. 2001). On the contrary, EH lesions have not been identified
in humans. At 12 weeks postinfection, AAH continued to be present, however, adenomas
became the major subtype. Adenomas of the lung are defined as neoplasms with papillary,
solid, or mixed architecture that distort alveolar septae (Jackson et al. 2001) and represent
the intermediate step between AAH and adenocarcinoma. Moreover, the adenomas
primarily identified in the K-ras
LSL-G12D
mice were predominantly papillary subtype.
Immunohistochemical analysis revealed that AAH lesions and adenomas were positive
for surfactant protein C (SPC), a marker of alveolar type 2 cells, as well as positive for
the clara cell antigen, CC10, a marker for clara or club cells, suggesting that hyperplasias
are related to clara cells and alveolart type II cells (Jackson et al. 2001). At 16 weeks
adenocarcinomas were present and AAH lesions were rare supporting that these lesions
are precursors to more advanced tumors. Adenocarcinomas showed numerous mitoses,
nuclear pleomorphisms with enlarged nuclei and prominent nucleoli.
The ability to cross other mouse models with K-ras
LSL-G12D
mice has made it
possible to identify the role and function of various players involved in the development
and progression of lung cancer. In this study, the role of GRP78 in K-ras
LSL-G12D
mice
23
will be investigated to determine the role of GRP78 in lung cancer development and
progression. Studying the role of GRP78 in mice as well as in human lung cancer cell
lines is particularly valuable to delve further into the many aspects of lung cancer
development and progression, which can lead to novel treatments for lung cancer.
24
Chapter 2
GRP78 plays an essential role in KRAS-induced
lung cancer
2.1 Introduction
Non-small cell lung cancer accounts for most lung cancer cases around the world
(Román et al. 2018). However, the distribution of various histological subtypes of lung
cancer varies widely between countries. For example, lung adenocarcinoma is more
common in western (26%) than in Asian (11%) populations as well as in smokers (30%)
than nonsmokers (10%) (Garrido et al. 2017). The incidence of ADC is increasing,
particularly among females (Nagy-Mignotte et al. 2011). A higher incidence of
adenocarcinoma compared to squamous cell carcinoma, with more than a 5-fold,
difference is reported in females from China, Japan, and Saudi Arabia (Cheng et al. 2016).
However, in countries like Belarus, India, the Netherlands, and the Russian Federation,
squamous cell carcinoma has a higher incidence (Cheng et al. 2016). Lung cancer rates
are affected by levels of socioeconomic development since almost half of all lung cancer
cases occur in countries ranked as medium to low on the Human Development Index
(HDI) as measured by population health, knowledge, and living standards of the country
(Ferlay et al. 2012). Lung cancer will continue to be a health problem and preventative
strategies as well as improved therapeutic options need to be established to combat this
deadly disease.
25
As KRAS mutations are detected in over one third of lung adenocarcinomas and
limited treatments are available for KRAS-mutated lung cancer patients, this present
study will focus on the context of KRAS-induced ADC in efforts to better understand the
role of ADC development and progression. Furthermore, understanding the molecular
moieties required for ADC will be important for the development of effective therapeutic
strategies to combat this disease.
Secretory and membrane proteins are synthesized in the ER, a specialized perinuclear
organelle. Protein chaperones located within the lumen of the ER assist in folding of
newly synthesized polypeptides and prevent aggregation of unfolded or misfolded
proteins (Ni and Lee 2007). GRP78, an essential chaperone, is also required for the
integrity of the ER structure (Miao Wang et al. 2010). Furthermore, GRP78 senses ER
stress and serves as a master regulator of the UPR when the protein load exceeds the
protein folding capacity of the ER (Wang et al. 2009). GRP78 assists in transporting
misfolded proteins in the ER lumen to the cytosol for proteasome-mediated degradation
(Ushioda et al. 2008). Professional secretory cells expand their ER capacities to adapt to
the increased demand in protein folding (Federovitch, Ron, and Hampton 2005). Proper
ER function is essential for lung function and homeostasis. Distinct secretory cells of the
airway lining mucosa help keep inhaled air moist and free of harmful dust particles,
organisms, and gases (Jeffery et al. 1992). In addition, secretory cells such as alveolar
type 2 cells prevent the collapse of the lung and allow for proper alveolar function.
Moreover, mucus-secreting cells act as pluripotent stem cells during development and in
26
the adult lung and function following mucosal injury (Jeffery et al. 1992). Human airway
secretory cells also include non-ciliated clara cells, also known as club cells, and dense-
core granulated neuroendocrine cells. Club cells protect the bronchiolar epithelium by
secreting proteins as well as solutions like pulmonary surfactant. Club cells detoxify
harmful substances inhaled into the lungs and act as stem cells multiplying into ciliated
cells to replenish the bronchiolar epithelium (Fernández-Valdivia et al. 2006).
Biosynthesis and secretion of large proteins requires molecular chaperones and folding
enzymes in the ER. Thus, GRP78 may be a key determinant for lung function as well as
in lung cancer development and progression.
To directly investigate the function of GRP78 in vivo in Kras-induced lung cancer,
we created mouse models with knockout floxed alleles of Grp78. Previous studies
suggest that in adult mice, the requirement of GRP78 for cell survival appears to be cell
type specific and content dependent (Luo et al. 2006; Wang et al. 2010; Fu et al. 2008).
In the present study towards understanding the role of GRP78 in lung cancer, we
conditionally depleted GRP78 expression in the lungs by intratracheally intubating mice
and introducing Adenovirus-Cre recombinase directly into the lungs. Simultaneously,
Adenovirus-Cre also removed the stop codon in order to activate oncogenic activity of K-
ras
LSL-G12D
. The conclusion that GRP78 function is essential for KRAS-induced lung
cancer is reported here. The implications of these results and the novel finding of the
requirement of GRP78 function in the lungs will be discussed.
27
2.2 Materials and methods
Generation of lung conditional knockout mice
The generation of Grp78
f/f
mice, on a mixed C57BL6;129Sv background, was described
previously (Luo et al. 2006; Fu et al. 2008). Briefly, mouse Grp78 contains 8 exons,
where the critical ATPase domain lies between exons 3-5 and the peptide binding domain
between exons 6-8. In transgenic mice, exons 5 through 7 are flanked by loxP sites. Thus,
introduction of Cre will remove these exons and create a non-functional GRP78. Mice
containing the K-ras Lox-Stop-Lox G12D (K-ras
LSL-G12D
) allele have been described
previously (Jackson et al., 2001). The LSL cassette was introduced into the first intron of
the Kras gene. The LSL cassette creates a null version of the gene, since it contains both
transcriptional and translational stop elements flanked by the LoxP sites that are
recognized by Cre recombinase. Thus, upon Cre activation, mutated KRAS will be
expressed and oncogenic activation will occur. Kras
G12D/+
Grp78
f/+
mice (referred to as
KGrp78
f/+
or K78
f/+
) were generated by breeding Kras
G12D/+
mice with the Grp78
f/f
mice.
The KGrp78
f/f
(K78
f/f
) and KGrp78
+/+
(KG78
+/+
) were generated by breeding Kras
G12D/+
Grp78
f/+
mice with Grp78
f/+
mice. Polymerase chain reaction (PCR) was performed
on DNA extracted from mouse tails to determine the genotype of mice. Primers PF3 and
PR3, previously described in Luo et al. 2006, were used to identify the floxed Grp78
allele. PF3 and PTR primers were used to identify the Grp78 knockout allele. KRAS
primer sequences, Kras1 and Kras2, were obtained from the Jacks Lab (https://jacks-
28
lab.mit.edu/protocols/genptyping/kras_cond) and were used to identify the wild-type or
recombined Kras alleles. PCR was performed on DNA extracted from the lung to
determine if oncogenic KRAS was activated and GRP78 was knocked out. Primer
sequences used to detect genotype, floxed or knockout alleles are listed in Table 1.1. In
addition, mice weight was recorded weekly post Cre-activation.
PF3 5’-CCCAGGTCAAACACAAGGATGTTCTTC-3’
PR3 5’-GATTTGAACTCAGGACCTTCGGAAGAGCAG-3’
PTR 5’-TCGTATAGCATACATTATACGAAGTTATACA-3’
Kras1 5’-GTCTTTCCCCAGCACAGTGC-3’
Kras2 5’-CTCTTGCCTACGCCACCAGCTC-3’
Table 1.1 Primer sequences for PCR using DNA extracted from lungs and tails to detect the K-ras
LSL-G12D
and Grp78 floxed alleles
Adenovirus-Cre Activation and Tissue Processing
Male and female mice were both analyzed in this study. Adenovirus-Cre recombinase
was obtained from the Gene Transfer Vector Core at the University of Iowa. The
Adenovirus-Cre is Ad5CMVCre with E1a deletion and partial deletion of E1b and E3.
2x10^7 pfu were administered per mouse. The intratracheal intubation protocol was
developed in the lab of Dr. Anton Berns at the Netherlands Cancer Institute in
Amsterdam and administration of Adenovirus-Cre was performed on 10-week-old mice
under anesthesia using a combination of ketamine and xylazine. Mice were euthanized at
12, 16, or 22-week time points post Cre intubation. After mice were euthanized, lungs
were removed and flushed with PBS by injecting PBS solution into the right ventricle
29
three times. Lungs were then fixed and flushed with 10% formalin three times and placed
in 10% formalin overnight. The next day, lungs were transferred to 25% ethanol for 2 hrs,
followed by 50% ethanol for 2 hrs, and then 75% ethanol overnight. The next day, the
gradient was continued by placing lungs in 90% ethanol for 2 hrs followed by 100%
ethanol for 2 hrs, before placing them in xylene solution twice for 1 hr each incubation
time. Next, lungs were placed in paraffin inside a vacuum chamber for 1 hr and this last
step was repeated. Infiltrated lung tissues were then embedded into wax blocks and
sectioned at 7 µm. The entire lung was cut for all mice analyzed.
Quantification of tumor burden
All lung tissue slides stained with hemotoxylin and eosin were evaluated for
tumor burden using the histogram function of the Adobe Photoshop CS5 Imaging
software (San Jose, CA). This software function can mine staining intensities. Tumor
burden was calculated as the histogram value of tumorigenic areas over the histogram
value of the entire lung. Each mouse had six cross-section lung slides analyzed that were
each separated by ~ 700 µm. All the sampled images were in the same position relative to
the dorsal-ventral axis and were approximately similar in size across all mouse lungs
analyzed.
Grading of pulmonary tumors
Grading criteria were based on those used by DuPage et al. 2009 and were performed by
30
Dr. Louise Dubeau, certified pathologist, in a blind review. Lesions not visible with the
naked eye that showed focal thickening and hypercellularity of the alveolar walls and
uniformly staining nuclei showing no variation in size and not completely obliterating the
alveolar space were scored as atypical adenomatous hyperplasia. Lesions forming
macroscopically visible solid nodules obliterating the alveolar spaces, and showing more
prominent nucleoli and slightly larger but uniform nuclei lacking atypia, were scored as
adenomas. Lesions showing prominent nuclear atypia characterized by variation in
nuclear sizes, shapes, and staining intensities typically associated with larger nucleoli
were scored as adenocarcinomas. Invasive carcinomas, showing invasive edges and
destruction of normal structures were not seen in this study.
Tissue immunostaining and imaging
Paraffin-embedded tissue sections were first deparaffinized and rehydrated.
Antigens were retrieved by incubating the slides with Retrievagen A Solution (BD
PharMingen) at ~95°C for 30 min. Slides were then cooled to room temperature for 1 hr.
Slides were then washed with distilled water and PBS. To eliminate endogenous
peroxidase activity, slides were washed with 3% hydrogen peroxide solution in PBS for 5
min. Slides were then blocked to prevent non-specific binding of antibodies with goat
serum (ABC Elite Kit, Vector Laboratories). After blocking, lung tissue sections were
incubated at 4°C overnight with primary antibodies. For immunohistochemistry (IHC),
the antibodies used were: GRP78 (1:500, Abcam #ab108613, Cambridge, MA), Ki67
31
(1:200, Thermo Fisher Scientific #RM-9106, Waltham, MA). The next day, slides were
washed three times for 5 min in PBS. Sections were then developed according to the
ABC Elite Kit manufacturer’s protocol. Next, slides were counterstained with
hematoxylin, dehydrated, and cover slipped. Negative controls were from adjacent tissue
sections and were processed similarly but without primary antibody. For
immunofluorescent staining of mouse lungs, tissues were first treated with GRP78 (1:200,
Santa Cruz Biotechnology, H-129) and secondary antibody with Alexa Fluor 594 (1:500,
ThermoFisher Scientific). Slides were then blocked with Mouse on Mouse blocking kit
(M.O.M Blocking Kit, VectorLabs, Burlingame, CA) followed by the KRAS antibody
(ThermoFisher #415700, Waltham, MA) and the secondary antibody Alexa Fluor 488
(1:500, ThermoFisher Scientific). Immunofluorescent imaging was performed using a
Zeiss LSM 510 confocal microscope with LSM 510 Version 4.2 SP1 acquisition software,
and the images were analyzed with ZEN lite imaging software (ZEISS, Thornwood, NY)
and Adobe Photoshop CS5 (San Jose, CA).
18F-FDG PET/CT imaging of lungs
2-deoxy-2-[fluorine-18]fluoro-D-glucose integrated computed tomography (18F-
FDG PET/CT) scanning of lungs was performed with Rigaku CT Lab GX scanner. Mice
were placed in an induction chamber with 2% isoflurane in oxygen for inhalant
anesthesia and during intravenous CT contrast administration via i.v. Mice were imaged
pre and post-contrast (5 min post i.v. with ExiTron 12000 contrast agent). Contrast agent
32
was administered to aid with vascular contrast from tumor tissue. Scans were acquired at
12, 16, and 22 weeks post Adenovirus-Cre administration using the following parameters:
90kVp, 88uA, 50um resolution and scanned and reconstructed using the Rigaku software.
CT data was segmented for lung tissue and tumor tissue using Amira software (Zuse
Institute Berlin, Thermo Fisher Scientific).
Statistical analysis
A two-tailed Student’s t test was applied for all pairwise comparisons. Data are
expressed as mean ± S.E.M.
2.3 Results
2.3.1 Creation of the mouse model with GRP78 deletion and activation of oncogenic
KRAS (Kras
G12D/+
;Grp78
f/f
)
Activating mutations in KRAS codon 12 are common in human lung
adenocarcinoma (Cancer Genome Atlas Research Network, 2014). In mice, the Kras
G12D
mutation induces spontaneous ADC (Jackson et al. 2001). In order to investigate the role
of GRP78 in ADC development, we used the breeding scheme shown in Fig.2.1 to
generate the mouse cohorts for conditionally depleting either one or both alleles of Grp78
in the lungs of mice heterozygous for the Kras
G12D
allele. The genotypes of mice were
confirmed by PCR and amplified product was run on an agarose gel to resolve the
fragments (Figure 2.2A). Mice with the correct genotype were then intubated by
33
intratracheal intubation and had Adenovirus-Cre directly inserted into the lungs. Lungs
were then harvested at 12, 16, and 22 weeks post Cre intubation (Figure 2.2B).
Figure 2.1 Mating strategy for the generation of Kras
G12D/+
;Grp78
+/+
, Kras
G12D/+
;Grp78
f/+
, and
Kras
G12D/+
;Grp78
f/f
mice.
To examine the levels of GRP78 in the mouse lung tissues,
immunohistochemistry was performed. Consistent with published reports that GRP78 is
upregulated in human lung adenocarcinoma, GRP78 was strongly detected in K78
+/+
lungs, and in comparison, the staining intensity for GRP78 decreased in the lungs of
K78
f/+
mice and further decreased in K78
f/f
mice (Figure 2.3A). Staining for Ki67, a
marker of cell cycle activity, in 5 random high-power fields of lung tissues 16 weeks after
Cre intubation showed an average of 46 positive cells in K78
+/+
mice, 27 positive cells in
K78
f/+
mice, and 12 positive cells in K78
f/f
mice (Figure 2.3B, C). Representative
examples of Ki67 immunostains are shown in (Figure 2.3B).
34
Figure 2.2 Confirmation of mouse models and timeline of analysis. (A) Representative genotypes of the
Adenovirus-Cre mice using DNA extracted from tail to confirm Grp78 floxed or WT alleles and from the
lung after Cre activation to confirm Kras G12D or WT alleles and knockout of Grp78. (B) Timeline of
mice analysis begins at 10 wk when adult mice are intubated with Adenovirus-Cre and the lungs are
harvested and processed for analysis at 12, 16, 22 weeks post Cre-activation.
2.3.2 GRP78 insufficiency suppresses tumor burden in Kras-driven tumorigenesis
Histological sections of lung were stained with hematoxylin and eosin (H&E) and
the entire lung surface area was imaged for each slide. Lung tissues were cut in a dorsal-
ventral plane at 7 µm thickness. Each mouse lung had six cross-section lung slides
analyzed that were each separated by ~ 700 µm. Tumor burden was analyzed by taking
the H&E slides and using the histogram function of the Adobe Photoshop CS5 Imaging
software (San Jose, CA). This software function can mine for staining intensities. Tumor
burden was calculated as histogram value of tumorigenic areas over the histogram value
of the entire lung. All the sampled images were in the same position relative to the
dorsal-ventral axis and were approximately similar in size across all mouse lungs
analyzed.
35
Figure 2.3 Comparative analysis of the lungs of K78
+/+
, K78
f/+
, and K78
f/f
mice. (A)
Immunohistochemistry (IHC) of GRP78 in lungs of KGrp78
+/+
, KGrp78
f/+
, and KGrp78
f/f
mice 12 weeks
post Adenovirus-Cre activation by intratracheal intubation. (Scale bar, 100 µm). (B) Representative
immunostains for Ki67 in lungs of mice of each genotype 22 weeks post Cre activation. (C) Quantitation of
Ki67 positive cells using 5 random high-power fields of lung tissues after 22 weeks post Cre activation.
Data are presented as mean ± s.e.m, *p<0.05 and **p<0.01.
Tumor burden as a percent of tumor area over total lung area was graphed and
presented according to genotype at the respective time points of analysis (Figure 2.4A).
Tumor burden was reduced in K78
f/+
and even more so in K78
f/f
, compared to K78
+/+
mice at all time points of analysis. Tumor burden was statistically significantly different
between K78
+/+
and both K78
f/+
and K78
f/f
mice. Representative images of tumor burden
are shown in Figure 2.4B, showing the darker regions of the lungs as abnormal and
tumorigenic. The histological grade of the tumors and abnormal pathology of the lung
were also analyzed. Mice were also imaged by 18F-FDG PET/CT scan and followed over
time as they were imaged at 12, 16, and 22-weeks post Cre activation. Two K78
f/f
mice
36
were imaged along with one of each K78
+/+
and K78
f/+
mice, only one of the K78
f/f
mice
is shown since it is closer to the average tumor burden presented in Figure 2.4A, the
other K78
f/f
mice imaged had close to zero percent tumor burden (data not shown).
Tumor volume is represented in golden color while blue is normal lung (Figure 2.5A).
The percentage of tumor burden was quantified using the PET/CT analysis software.
Lung volume to tumor volume ratio in the K78
+/+
mouse increased from 25.84% in 12
weeks post Cre activation to 51.95% at 16 weeks, to 59.54% at 22 weeks. In contrast, the
K78
f/+
mouse began at 12.95% at 12 weeks, 15.84% at 16 weeks, and 31.70% by 22
weeks post Cre activation. The K78
f/f
mouse began at 9.3% at 12 weeks, 12.98% at 16
weeks, and 9.60% was recorded for 22 weeks post Cre activation for the first mouse.
Lungs of mice that were imaged by PET/CT were then sectioned and stained by H&E
stain to determine pathology grade of each genotype (Figure 2.5B).
2.3.3 Histological grade and weight change differences across all mice analyzed
All mice were analyzed and categorized according to grading criteria described in
DuPage et al. 2009. Monitoring tumor development and progression using histological
technique is important though difficult. This NSCLC model generates a multi-focal
disease and as such tumors are heterogenous and tumors don’t always progress in the
37
Figure 2.4 Tumor burden analysis of the lungs of K78
+/+
, K78
f/+
, and K78
f/f
mice. (A) Quantitation of
tumor burden for all three genotypes in K78
+/+
, K78
f/+
, and K78
f/f
mice at all time points analyzed. The
numbers for the K78
+/+
, K78
f/+
, K78
f/f
mice: at 12 weeks (n=9, 8 and 9, respectively), 16 weeks (n=8, 9 and
11, respectively), and 22 weeks (n=9, 8 and 8, respectively). Data are presented as mean ± s.e.m, *p<0.05
and **p<0.01. (B) Cross section of whole lungs at 2X magnification of H&E stain. Mice are under their
respective genotype at 12, 16, or 22 weeks post Cre intubation.
same way or at the same time. Therefore, to follow the changes observed histologically,
we followed a four-stage grading system like that used by DuPage et al. 2009. We
labeled our first stage as normal (N), followed by the earliest lesion as atypical
adenomatous hyperplasia (AAH). AAH have uniform nuclei with focal thickening and
hypercellularity of the alveolar walls that showed no variation in size and do not
completely obliterate the alveolar space. The next grade was classified as adenomas
(Adm) containing slightly enlarged nuclei with prominent nucleoli but uniform nuclei
lacking atypia and lesions forming macroscopically visible solid nodules.
38
Figure 2.5 18F-FDG PET/CT scan of K78
+/+
, K78
f/+
, and K78
f/f
mice. (A) Mice were imaged by 18F-FDG
PET/CT scan and followed over time as they were imaged at 12, 16, and 22-weeks post Cre activation.
Only one mouse per genotype was included and analyzed. Tumor volume is represented in golden color
while blue is normal lung. (B) Hematoxylin and Eosin stain of the lung sectioned is indicated by the red
line in part (A) for the 22-week time point and imaged at 20X magnification. ADC: adenocarcinoma; Adm:
adenoma. Contributions: Scanned images for part (A) were performed by Ryan Park at the USC Molecular
Imaging Center.
39
Figure 2.6 Tumor grade and mouse weight change of K78
+/+
, K78
f/+
, and K78
f/f
mice. (A) Hematoxylin and
eosin staining of representative images of normal lung pathology (N), atypical adenomatous hyperplasia
(AAH), adenoma, adenocarcinoma (ADC). (Scale bar, 100 µm). (B) Histological examination revealed
differences in histological grade of lungs analyzed in all mice. (C) Mouse weight change post Cre
activation in all mice analyzed.
The most advanced grade we observed was classified as adenocarcinomas (ADC)
containing a high degree of cellular pleomorphism and nuclear atypia characterized by
variation in nuclear sizes, shapes, and staining intensities typically associated with larger
nucleoli were scored as adenocarcinomas. Adenocarcinomas invading across the
mesothelium into the pleural cavity were not observed in this study. Representative
images of the various grades we observed are represented in Figure 2.6A. All mice lungs
were analyzed and categorized according to the highest grade observed and graphed in
40
Figure 2.6B. KGrp78
+/+
had the highest number of mice lungs categorized as
adenocarcinomas. Depletion of GRP78 reduced the grade. For example, in K78
f/+
mice,
only at 22 weeks post Cre activation did mice begin to show adenocarcinoma while in all
of the K78
f/f
mice, no mice had adenocarcinoma. Mice weight was recorded weekly after
Cre activation and weight change is reported as a percentage difference from the start of
treatment to the endpoint at analysis time. Consistent with tumor burden and grade of
lung tumorigenesis, K78
+/+
experienced weight loss compared to K78
f/+
and K78
f/f
mice
(Figure 2.6C). At initial time point past Cre activation, mice showed an upward trend of
positive weight change, however, K78
+/+
mice demonstrated weight loss as time
progressed.
2.3.4 Depletion of Grp78 results in KRAS reduction
K78
+/+
showed robust expression of GRP78 and KRAS, and co-localization of the
two proteins was detected in the peri-nuclear region, suggesting interaction between these
proteins at the ER cytosolic interphase where KRAS undergoes post-translational
modification (Figure 2.7). Interestingly, levels of KRAS decreased in parallel to those of
GRP78 in K78
f/+
and K78
f/f
mice lung tissues. Figure 2.7A shows representative images
of K78
+/+
, K78
f/+
, and K78
f/f
mice at 22 weeks post Cre activation. Figure 2.7B
represents magnified images of lung tissue of K78
+/+
mice at 22 weeks post Cre
activation. Arrows indicate co-localization observed between GRP78 and KRAS.
41
Figure 2.7 Reduced KRAS in lungs of K78
f/f
mice. (A) Immunofluorescent co-staining of GRP78 and
KRAS in lung tissues of K78
+/+
, K78
f/+
, K78
f/f
mice at 22 wks post Cre activation by intratracheal
administration of Adenovirus-Cre. (Scale bar, 25 µm). (B) Magnified view of immunofluorescence in (A)
top panel demonstrating GRP78 and KRAS in lung tissues of K78
+/+
mice. (Scale bar, 25 µm).
42
2.4 Discussion
GRP78 is a major chaperone in the ER and is also a regulator of ER stress
signaling (Ni and Lee, 2007; Rutkowski and Kaufman, 2004; Pfaffenbach and Lee, 2011;
Luo and Lee, 2012). However, its role in KRAS-induced lung tumorigenesis is not
known. In the present study, we used a conditional knockout mouse model to directly
investigate the requirement of GRP78 in lung tumorigenesis initiated by the activation of
oncogenic KRAS. Tumor burden was decreased in mice depleted of GRP78, suggesting
that lung cancer cells require GRP78 to survive and grow during KRAS-induced lung
tumorigenesis. Suppression of KRAS-induced cancer has been previously reported during
conditions of GRP78 depletion. Suppression of Kras-driven pancreatic tumorigenesis by
Grp78 heterozygosity was reported by the Lee lab (Shen et al. 2017). In the reported
mouse model of pancreatic ductal adenocarcinoma, the pancreatic and duodenal
homeobox 1 promoter-driven Cre-recombinase was activated to delete one allele of p53
and Grp78 while simultaneously activating the Kras
LSL-G12D
allele, in mice labeled as
PKC78
f/+
. Mice containing wild-type levels of GRP78 labeled as PKC, had detectable
pancreatic ductal adenocarcinoma by 3 months and rapid tumor growth post Cre
activation, yet PKC78
f/+
mice maintained normal pancreata during the early months. In
addition, PKC78
f/+
pancreata also had suppressed proliferation as measured by Ki67
staining. These data support a role for GRP78 in the initiation process of pancreatic
adenocarcinoma. In our study, Ki67 was reduced in the lungs of K78
f/+
mice and even
43
more so in K78
f/f
compared to the lungs of K78
+/+
mice. Tumor burden was significantly
suppressed in the lungs of K78
f/f
mice compared to K78
f/+
and K78
+/+
mice. The results of
the PET/CT scan also support a reduced tumor burden. For example, the K78
+/+
mouse
tumor burden average for 12 weeks was around 22%, and increased to almost 40% at 16
weeks, and around 65% at 22 weeks post Cre activation (Figure 2.4A). Tumor burden
average for the K78
f/+
mouse was also similar since tumor burden at 22 weeks post Cre
activation was approximately 30%. Although a limited number of mice were imaged by
PET/CT, the percentage of tumor burden supported the data in Figure 2.4. These data
support the role of GRP78 in the initiation of lung adenocarcinoma.
Tumor progression and histological phenotype were also evaluated in this study.
Consistent with reduced tumor burden of K78
f/+
and K78
f/f
mice compared to the K78
+/+
mice, pulmonary lesions appeared earlier in K78
+/+
than in mice with mutant GRP78
alleles. Lungs of K78
f/f
were either histologically normal or showed atypical
adenomatous hyperplasia 12 weeks post intubation. Half of the K78
+/+
mice and 20% of
the K78
f/+
showed adenomas at this time point. By 16 weeks post Cre activation,
adenocarcinoma was detectable in 30% of the K78
+/+
mice, yet at this same time point, no
adenocarcinoma was observed for either K78
f/+
or K78
f/f
mice. Adenocarcinoma was first
observed in K78
f/+
mice by week 22 post intubation; however, it was never detected in
K78
f/f
mice. Mice that were imaged by PET/CT were also analyzed by H&E to determine
histological grade. In support of the histological data reported in Figure 2.6B, tumor
44
burden was increased for K78
+/+
mice at all time points analyzed compared to K78
f/+
or
K78
f/f
mice. In addition, adenocarcinoma was predominant in K78
+/+
mice and only
adenomas were observed in K78
f/+
or K78
f/f
mice. Importantly, partial reduction of
GRP78 expression in K78
f/+
mice with loss of only one Grp78 allele was sufficient to
impede tumorigenesis. GRP78 haploinsufficiency has no harmful effect on cell
homeostasis and organ function in both young and old mice (Lee et al. 2017). This
implies that therapeutic agents able to partially suppress GRP78 expression or activity
can potentially block lung ADC development without harming normal organs. GRP78
inhibitors are in development (Lee, 2014, Cerezo et al. 2016). A small molecule inhibitor
IT-139, which has completed Phase I clinical trial with efficacy and low toxicity,
preferentially suppressed GRP78 expression in tumors but not in adjacent normal cells in
xenograft models (Bakewell, 2018).
In efforts to identify the mechanism by which GRP78 affects KRAS-induced lung
tumorigenesis, immunofluorescent staining was performed to determine if levels of
KRAS were affected. Surprisingly, KRAS levels decreased in parallel with GRP78. In
addition, co-localization was apparent in the perinuclear region. This data provided the
first hint that GRP78 is required for the integrity of KRAS in lung cancer. For KRAS to
activate and function as a GTPase signaling protein, it needs to reach the plasma
membrane and pass through the endoplasmic reticulum, the site where it gets modified.
The KRAS membrane targeting signals are located within the last 17 C-terminal
45
sequences containing the –CAAX motif (Hancock et al. 1990). For KRAS to localize to
the plasma membrane, the newly synthesized KRAS protein is first farnesylated in the
cytosol by a prenyl transferase, followed by the removal of the –AAX motif by the RAS-
converting CAAX-endopeptidase I (RCE-1) and methylesterfication of the farnesylated
cysteine by isoprenylcysteine carboxylmethyltransferase (ICMT), with both processes
taking place at the cytosolic surface of the endoplasmic reticulum (ER) where RCE-1 and
ICMT are located. Upon completion of CAAX processing, KRAS traffics to the plasma
membrane independent of the conventional exocytic pathway, bypassing the Golgi
(Apolloni et al. 2000). Thus, the ER is a major site for the modification and maturation of
KRAS that is critical for its trafficking, localization, stability and ultimately, function.
This data suggests the chaperone function of GRP78 could aid in the modification
processes of KRAS as it goes through the ER. In the subsequent chapters, further
mechanistic insights will shed light on the role of GRP78 on KRAS function and stability.
In summary, depletion of GRP78 blocks lung tumorigenesis induced by the
activation of oncogenic KRAS. The identification of GRP78 as a critical player for
KRAS-induced lung cancer suggests that it could be targeted to reduce lung cancer
mortality in patients suffering from lung adenocarcinoma containing a KRAS mutation.
46
Chapter 3
GRP78 happloinsufficiency in AT2 cells prolongs survival in
mice with KRAS-induced lung cancer
3.1 Introduction
The Adenovirus-Cre recombinase-controlled tumor model made it possible to
conditionally deplete the Grp78 allele and simultaneously remove the Kras stop codon to
allow for the expression of oncogenic mutated Kras
LSL-G12D
. However, mice inhaling the
virus cannot limit the effects of Cre to only the population of cancer-initiating cells. In
addition, intubation may not reach all lung surface areas equally in all mice even though
the same amount of virus is injected per mouse. Thus, to address the limitations of the
first mouse model, a second mouse model was created. For the second model, referred to
as the SPC-Cre model, mice contain the Cre gene driven by a 3.7 kb human surfactant
protein C promoter and fused to the estrogen receptor (SPC-CreER
T2
). In this model, only
SPC promoter activation of Cre will take place in the population of cells expressing
surfactant protein C (SFTPC). Studies to determine cells of origin of NSCLC in the
mouse lung with Kras mutations have described alveolar type 2 (AT2) cells and
bronchioalveolar stem cells (BASCs) as SFTPC-positive cells that can give rise to lung
adenocarcinoma (Kim et al. 2005; Hanna and Onaitis 2013). Further studies described
BASCS as double-positive cells (DPCs) shown to express the clara cell marker, Clara
47
Antigen 10 (CC10), and the SFTPC marker, and these cells, located at the
bronchioalveolar junction (BADJ), when plated in Matrigel could give rise to clara, AT2,
and alveolar type 1 (AT1) cells (Kim et al. 2005). Moreover, CC10-positive cells
considered putative cells capable of initiating Kras mutant adenocarcinoma in mice, also
include the cell population of clara cells throughout the airway, putative BASCs at the
BADJ, and about 10% of AT2 cells (Hanna and Onaitis 2013). Lineage-tracing
experiments performed to track cells identified as AT2 cells, showed these giving rise to
adenocarcinoma in Kras
G12D
mice, and although SFTPC-positive and SFTPC-negative
clara cells initiated hyperplasia, they did not advance to adenocarcinoma. Thus, AT2 cells
were concluded to be the cells of origin of Kras-induced adenocarcinomas (Xu et al.
2012). In this study, SPC-CreER
T2
mice crossed with the Kras
G12D/+
mice from the
Adenovirus-Cre model described in the previous chapter, generated SPC-CreER
T2/+
;
Kras
LSL-G12D/+
; Grp78
+/+
(CK78
+/+
), SPC-CreER
T2/+
; Kras
LSL-G12D/+
;Grp78
f/+
(CK78
f/+
),
and SPC-CreER
T2/+
;Kras
LSL-G12D/+
;Grp78
f/f
(CK78
f/f
).
In the present study, aimed at understanding the role of GRP78 in lung cancer
even further, we conditionally depleted GRP78 expression in the lungs by injecting
tamoxifen via intraperitoneal (IP) injection. Simultaneously, Cre also removed the stop
codon in order to activate oncogenic activity of K-ras
LSL-G12D
allele. The conclusion that
GRP78 function is essential for KRAS-induced lung cancer is further confirmed here, as
well as increased survival in mice with lower levels of GRP78. The implications of these
48
results and requirement of GRP78 function in the AT2 cells of the lung will be discussed.
3.2 Materials and methods
Mice
Mice generated for the first mouse model (Adenovirus-Cre model 1) were used to
generate the second mouse model (SPC-Cre model 2). The Kras
G12D/+
;Grp78
f/+
mice of
mixed C57BL/6;129/Sv background were crossed with SPC-Cre-ER
T2;Cre/Cre
;Grp78
f/f
mice that were generated by the lab of Dr. Zea Borok. The first offspring of mice were
SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
f/+
and SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
f/f
. All
mice for this second mouse model were heterozygous for the Cre gene. To generate SPC-
Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
+/+
, the mice from model 1, Kras
G12D/+
;Grp78
+/+
mice
were crossed with SPC-Cre-ER
T2;Cre/Cre
;Grp78
f/+
mice. Genotyping was performed by
PCR using genomic DNA extracted from mouse tails as described in Fu et al. 2008 and
the primers PF3 and PR3 previously described in Luo et al. 2006, were used to determine
the floxed Grp78 allele. Genomic DNA was also extracted from lungs to identify the
Grp78 KO band and the PF3 and PTR primers were used to identify the Grp78 knockout
allele (Luo et al. 2006). Genomic DNA, extracted from mouse tails, was used to
determine genotype status of KRAS. Sequences of primers Kras1 and Kras2 were
obtained from the Jacks Lab (https://jacks-lab.mit.edu/protocols/genptyping/kras_cond)
and were used to identify the wild-type or recombinant Kras alleles. Genomic DNA
49
extracted from the lungs was used to detect if oncogenic Kras was activated after the
removal of the stop codon. Primer sequences used to detect the genotype and floxed or
knockout alleles are listed in Table 1.1. Genomic DNA was also used to determine if the
Cre allele was present in mice of model 2. Primers for the detection of the SPC-Cre allele
or wild-type are listed in Table 2.1. Mouse weight was also recorded weekly post Cre-
activation. All protocols for animal use were reviewed and approved by the USC
Institutional Animal Care and Use Committee.
Tissue Processing
Male and female mice were both analyzed in this study. Mice were euthanized and their
lungs were removed and flushed with PBS by injecting 1X PBS solution into the right
ventricle three times. Lungs were then fixed and flushed with 10% formalin three times
and placed in 10% formalin overnight. The next day, lungs were transferred to 25%
ethanol for 2 hrs, followed by 50% ethanol for 2 hrs, and then 75% ethanol overnight.
The third day, the gradient continued by placing lungs in 90% ethanol for 2 hrs followed
by 100% ethanol for 2 hrs, before placing it in xylene solution twice for 1 hr each
incubation time. Next, lungs were placed in paraffin inside a vacuum chamber for 1 hr
and this last step was repeated. Infiltrated lung tissues were then embedded into wax
blocks and sectioned at 7 µm. The entire lung was cut and approximately 50 slides per
mouse were kept for further staining.
50
Quantification of tumor burden
All slides were evaluated for tumor burden from the hematoxylin and eosin
staining of lung tissues using the histogram function of the Adobe Photoshop CS5
Imaging software (San Jose, CA). This software function can mine for staining intensities.
Tumor burden was calculated as histogram value of tumorigenic areas over the histogram
value of the entire lung. Each mouse had six cross-section lung slides analyzed that were
each separated by ~ 700 µm. All the sampled images were in the same position relative to
the dorsal-ventral axis and were approximately similar in size across all mouse lungs
analyzed.
Grading of pulmonary tumors
Grading criteria were based on those used by DuPage et al. 2009 and were performed by
Dr. Louise Dubeau, certified pathologist, in a blind review. Lesions not visible with the
naked eye that showed focal thickening and hypercellularity of the alveolar walls and
uniformly staining nuclei showing no variation in size and not completely obliterating the
alveolar space were scored as atypical adenomatous hyperplasia. Lesions forming
macroscopically visible solid nodules obliterating the alveolar spaces showing more
prominent nucleoli and slightly larger, but uniform nuclei lacking atypia were scored as
adenomas. Lesions showing prominent nuclear atypia characterized by variation in
nuclear sizes, shapes, and staining intensities typically associated with larger nucleoli
51
were scored as adenocarcinomas. Invasive carcinomas, showing invasive edges and
destruction of normal structures were not seen in this study.
Tissue immunostaining and imaging
For immunostaining, paraffin-embedded tissue sections were first deparaffinized
and rehydrated. Antigens were retrieved by incubating the slides with Retrievagen A
Solution (BD PharMingen) at ~95°C for 30 min. Slides were then cooled to room
temperature for 1hr. Slides were then washed with distilled water and PBS. To eliminate
endogenous peroxidase activity, slides were then washed with 3% hydrogen peroxide
solution in PBS for 5 min. Next, slides were blocked to prevent non-specific binding of
antibodies with goat serum (ABC Elite Kit, Vector Laboratories). After blocking, lung
tissue sections were incubated at 4°C overnight with primary antibodies. For
immunohistochemistry (IHC), the antibodies used were: GRP78 (1:500, Abcam
#ab108613, Cambridge, MA). The next day, slides were washed three times for 5 min in
PBS. Sections were then developed according to the ABC Elite Kit manufacturer’s
protocol. Slides were then counterstained with hematoxylin, dehydrated, and cover
slipped. Negative control sections were from adjacent tissue sections and were processed
similarly but without primary antibody. Slides were imaged using a Leica S8 APO stereo
microscope and the Leica compound microscopes at the Tissue Imaging Core Facility of
the University of Southern California, Research Center for Liver Diseases (P30
DKO048522).
52
Statistical analysis
A two-tailed Student’s t test was applied for all pairwise comparisons. Data are
expressed as mean ± S.E.M. Log-rank (Mantel-Cox) test was used for survival data.
3.3 Results
3.3.1 Creation of the SPC-Cre mouse model with biallelic deletion of GRP78
The breeding scheme for the generation of the following four groups of mice
cohorts is shown in Figure 3.1. They include: 1) heterozygous allele of SPC-Cre-
ER
T2;Cre/+
and Kras
G12D/+
but wild-type GRP78 (SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
+/+
)
2) with heterozygous floxed allele of Grp78
f/+
(SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
f/+
)
and 3) with homozygous floxed alleles of Grp78
f/f
; 4) and mice without the Kras
G12D/+
alleles as negative controls (SPC-Cre-ER
T2;Cre/+
;Grp78
+/+
). Primers to confirm the Kras
and Grp78 alleles were the same as used for the Adenovirus-Cre mouse model 1 and are
listed in Table 1.1. Primers to confirm presence of SPC-Cre-ER
T2;Cre/+
are listed in Table
2.1. Presence of a band using the SPC-KO Forward primer and the SPC-KO Reverse
primer indicated that the indicated allele was present. Presence of a band using the SPC-
WT Forward with Reverse indicated that the wild-type allele was present.
SPC-KO Forward 5’-TGAGGTTCGCAAGAACCTGATGGA-3’
SPC-KO Reverse 5’-ACCAGCTTGCATGATCTCCGGTAT -3’
SPC-WT Forward 5’- TGGTTCCGAGTCCGATTCTTC -3’
SPC-WT Reverse 5’- CCTTTTGCTCTGTTCCCCATTA -3’
Table 2.1 Primer sequences for PCR using DNA extracted from tails to detect the SPC-Cre allele.
53
Figure 3.1 Breeding scheme for generating mice for the SPC-Cre model. Mice in bolded black box (SPC-
Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
f/+
, or (CK78
f/+
)), green box (SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
f/f
(CK78
f/f
)), and red box (SPC-Cre-ER
T2;Cre/+
;Kras
G12D/+
;Grp78
+/+
(CK78
+/+
)), were used for mouse model 2 and
injected with tamoxifen. Mice lacking the Kras
G12D/+
gene were used as negative controls.
Adult mice at 10 weeks of age were injected with tamoxifen at 1mg per day for two
consecutive days. Due to the breeding scheme, CK78
f/+
and CK78
f/f
mice were generated
first and were analyzed at 12 weeks since that was the time we observed presence of lung
abnormalities from mouse model 1. Mouse genotypes were confirmed for all mice
analyzed (Figure 3.2A). Mouse tails were used to extract genomic DNA to confirm
floxed alleles of Grp78 and presence of the SPC-Cre-ER
T2;Cre/+
allele. DNA isolated from
lung extracts was used to determine knockout of Grp78 and recombined Kras
G12D/+
allele.
Once CK78
+/+
mice were generated, we noted that mortality started at around 3 weeks
post tamoxifen injection and none of these mice survived past 8 weeks post tamoxifen
injection. Thus, the timeline of model 2 mice analysis is highlighted in Figure 3.2B.
54
Figure 3.2 Representative genotypes of the SPC-Cre mice and timeline of analysis. (A) DNA extracted
from tail was used for PCR to confirm Grp78 floxed or WT alleles. Genomic DNA extracted from the lung
to perform PCR to determine Cre activation and confirm Kras G12D or WT alleles and knockout of Grp78.
(B) Timepoint of analysis of SPC-Cre mice. Adult mice of approximately 10 weeks of age were injected
with tamoxifen and lungs were harvested at the indicated week time points post injection.
3.3.2 Early deaths of SPC-Cre mice with wild-type levels of GRP78
We performed autopsies to determine the cause of CK78
+/+
mice early death.
Autopsy examination of the CK78
+/+
mice revealed that the abnormalities were confined
to the lungs, which showed multiple tumor nodules, while the rest of the organs appeared
normal (Figure 3.3A). For comparison, a sibling mouse (C78
+/+
) was euthanized the
same day the CK78
+/+
mouse died and the lungs of both were harvested immediately after
death. Lungs of the sibling mouse lacking the activation of Kras
G12D/+
oncogenic activity
were normal Figure 3.3A. Consistent with increase tumor burden and early death,
CK78
+/+
mice experienced weight loss Figure 3.3B post tamoxifen injection. Survival of
55
mice in model 2 was graphed using the Kaplan-Meier curve and survival length times
were significantly different between CK78
+/+
mice and both CK78
f/+
and CK78
f/f
mice
(Figure 3.4).
Figure 3.3 Gross appearance of internal organs and mouse weight change in SPC-Cre mice. (A) Gross
appearance of internal organs of C78
+/+
and CK78
+/+
mice 8 weeks post Cre activation by tamoxifen
injection. The mice were siblings and C78
+/+
was euthanized the same day the CK78
+/+
mouse died. Lungs
of the C78
+/+
show a smooth and glistening surface of uniform color while those of the CK78
+/+
mouse
show multiple tumor nodules (represented by arrows). Representative photographs of eviscerated lungs,
hearts, spleens, and livers. (B) Total body weight changes of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice during
treatment.
3.3.3 GRP78 happloinsuffiency in AT2 cells reduces histological grade
In accordance with reports of human survival data, patients with increased GRP78
staining of lung adenocarcinoma tissues, by immunohistochemistry, had shorter survival.
Similarly, CK78
+/+
mice with shorter survival observed in this study, also demonstrated
56
elevated levels of GRP78 compared to CK78
f/+
and CK78
f/f
mice (Figure 3.5).
Figure 3.4 Analysis of survival of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice. (A) Kaplan-Meier survival curve of
CK78
+/+
(n=11), CK78
f/+
(n=11) and CK78
f/f
(n=13) mice. Male and female mice were combined. CK78
f/+
and CK78
f/f
are statistically significantly different from CK78
+/+
(**: P = 0.003) and (**: P < 0.0001)
respectively, as analyzed using log-rank Mantel-Cox test.
Representative GRP78 staining and histology of these lung sections were shown in
Figure 3.5. For example, CK78
+/+
mice exhibited robust GRP78 staining at 8 weeks
following tamoxifen injection (Figure 3.5A), corresponding with large tumor formation
consisting of adenoma and adenocarcinoma in the lungs. CK78
f/+
mice showed reduction
of GRP78 expression, which was further decreased in CK78
f/f
mice (Figure 3.5B).
Tumor formation and grade were much reduced in CK78
f/+
and CK78
f/f
genotypes, with
CK78
f/+
mice showing predominantly adenomas while CK78
f/f
lesions were primarily
atypical alveolar hyperplasia even at 12 weeks after tamoxifen injection. Histological
grade was investigated, and lung slides of all mice were given a grade according to the
57
same rubric applied to mice in model 1 (Figure 3.6). Although CK78
+/+
mice did not live
Figure 3.5 GRP78 and pathology of the lungs of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice. (A)
Immunohistochemistry (IHC) of GRP78 in lungs of CK78
+/+
mice at 8 wks post tamoxifen I.P. Magnified
box area to the right (Scale bar, 100 µm). On bottom panel, cross section of whole lungs with magnified
boxed area representing Hematoxylin and Eosin (H&E) stain. (B) IHC staining comparing lungs of CK78
f/+
and CK78
f/f
mice, both at 12 wks post tamoxifen I.P. injection. Magnified box area to the right (Scale bar,
200 µm). On bottom panel, cross section of whole lungs with magnified boxed area representing H&E stain.
ADC: adenocarcinoma; Adm: adenoma; AAH: atypical adenomatous hyperplasia.
past 8 weeks post Cre activation, 15% of the mice had adenocarcinoma and the rest of the
58
85% were adenomas. CK78
f/+
and CK78
f/f
mice that were analyzed were euthanized at 12
weeks post Cre activation and examined for lung pathology revealed that none had
adenocarcinoma. 25% of CK78
f/+
mice were categorized as atypical alveolar hyperplasia
and 75% as adenomas. For CK78
f/f
mice, 60% were categorized as atypical alveolar
hyperplasia and 40% as adenomas (Figure 3.6). Collectively, these data reveal that
GRP78 happloinsufficiency in AT2 cells is sufficient to suppress mutant KRAS-mediated
lung tumorigenesis.
Figure 3.6 Histological grade of CK78
+/+
, CK78
f/+
, and CK78
f/f
mice. Histological examination revealed
differences in histological grade of lungs analyzed in all mice.
3.4 Discussion
In this present study, we used a different conditional knockout mouse model to
59
directly investigate the requirement of GRP78 for lung tumorigenesis activated by
oncogenic KRAS. Similarly, to our results in the first mouse model, depletion of GRP78
reduced tumor burden in these mice and decreased the histology grade of the lung tumors
that developed. This mouse model targeted depletion of GRP78 in alveolar type 2 cells,
the cells that have been identified as cancer-initiating cells leading to the development of
lung adenocarcinoma (Lin et al. 2012; Kim et al. 2005). Consistent with published reports
that GRP78 is upregulated in human lung adenocarcinoma, GRP78 was strongly detected
in the CK78
+/+
lungs, and in comparison, the staining intensity for GRP78 decreased in
the lungs of CK78
f/+
mice and even further decreased in CK78
f/f
mice. The proliferation
marker, Ki67, also was decreased in CK78
f/f
mice compared to CK78
f/+
and CK78
+/+
(data not shown). This suggests that GRP78 is important in AT2 cells for growth and
survival and most importantly in the initiation of lung adenocarcinoma. CK78
f/f
and
CK78
f/+
mice had a longer survival time compared to CK78
+/+
mice, which did not live
past 8 weeks post tamoxifen injection. This data supports the reports that in human
patients with increased levels of GRP78 in lung tumors had shorter survival (Kwon et al.
2018; Chae et al. 2012). Mice survival has been reported to be affected by the amount of
virus administered as well as the mouse model chosen. For example, survival was
reported to differ between the Kras
LSL-G12D/+
mouse versus the Kras
LSL-G12D/+
;P53
f/f
mouse
which contained floxed tumor suppressor p53 allele. The median survival with
Adenovirus-Cre was 185 days for the Kras
LSL-G12D/+
mouse and 76 days for Kras
LSL-
G12D/+
;P53
f/f
(DuPage et al. 2009). In this study, the CK78
f/f
and CK78
f/+
mice lived well
60
past 30 weeks, or 210 days. However, CK78
+/+
mice did not live past 18 weeks, or 126
days, less than that for previously reported Kras
LSL-G12D/+
mice. Another factor that can
influence survival is virus titer, which can result in differences in tumor burden as
quantified by the number of tumors observed in the lungs per mouse. For example,
administration of 2.5x10^7 infectious particles of Adenovirus-Cre (University of Iowa,
Gene Transfer Vector Core) was reported to generate greater than 200 tumors per mouse,
and accordingly, the number of tumors decreased with lower doses of virus administered.
In this current study, the same amount of tamoxifen was administered per mouse to keep
treatments consistent. Interestingly, CK78
+/+
mice were suspected of dying in utero
because although Mendelian genetics would expect the generation of these mice to be
close to 1/4, in reality, mice were generated closer to a 1/6 ratio. This gave the first hint
that the Sftpc-Cre-ERT2 promoter might be leaky, meaning it does not require tamoxifen
to become activated. Indeed, this was the case, as many Sftpc
+/creERT2
mice that were not
injected with tamoxifen but had the oncogenic KRAS allele (CK78
+/+
), developed tumors
without tamoxifen injection (data not shown). The Sftpc
+/creERT2
mice we used were
developed by breeding Sftpc
+/creERT2
mice that were originally created for studying
integrin in adult distal lung epithelial population with regenerative potential in mice, thus,
the leaky promoter may have gone unnoticed (Chapman et al. 2011). Sftpc
+/creERT2
mice in
this study were crossed with Grp78
f/f
mice and all of the mice in this study, namely,
CK78
+/+
, CK78
f/+
, and CK78
f/f
were heterozygous for the Cre allele. Although we did not
inject tamoxifen in mice containing two Cre alleles, it might have been necessary to
61
determine if the effects of having either heterozygous or homozygous levels of Cre could
have changed the results we observed. The effects of the Cre allele are important, as it
has been reported by the Jackson Laboratory that Cre alone can produce a phenotype and
differences in Cre expression can result if Cre is transmitted from the female or male
parent. For example, the Jackson Laboratory reports that Cre is more efficient when
transmitted maternally. We did ensure that females used in the parental crosses contained
the Cre allele. Although we did not investigate the effects of Cre alone, we can assume
that the effects of Cre contributed equally to all genotypes studied. It is also important to
note the issues created using tamoxifen. For example, it has been reported that tamoxifen-
dependent inducible Cre systems are at risk of creating genome instability and additional
endocrine effects (Koitabashi et al. 2009; Loonstra et al. 2001). We used the dose
recommended by the developers of the SFTPC mouse model, namely 1 mg per mouse,
thus, in a 20 g mouse, final concentration would be 50 mg/kg, less than the commonly
used concentrations ranging between 75-100 mg/kg or higher for mice containing
CreERT2 (Madisen et al. 2010; Jahn et al. 2018). Although only one dose of tamoxifen
was tested in the mice of our study, mortality associated with the use of tamoxifen was
not observed in either of the CK78
f/+
, and CK78
f/f
mice. Furthermore, autopsies in
CK78
+/+
mice that died as early as three weeks post tamoxifen injection revealed
abnormal lungs with tumors while the rest of the organs remained normal, when
compared to sibling mice lacking the Kras
G12D
allele (C78
+/+
) (Figure 3.3A).
Furthermore, weight loss was only observed in CK78
+/+
mice but not in CK78
f/+
or
62
CK78
f/f
mice (Figure 3.3B). This data indicate that GRP78 haploinsufficiency in lung
alveolar AT2 cells is sufficient to halt lung tumor progression and prolong survival. As
the major chaperone with potent anti-apoptotic properties and a signaling regulator,
GRP78 is thought to protect tumor cells from ER stress and the host cell defense, thereby
promoting tumor growth. This is consistent with our observation that GRP78 was
upregulated in tumors as was observed in the first mouse model as well.
In summary, our studies reveal a function of GRP78 in lung tumorigenesis.
Future studies are required to address how depleting GRP78 affects KRAS and initiates
lung adenocarcinoma. In the next chapter, the use of lung adenocarcinoma human cell
line, A427, harboring the same KRAS
G12D
mutation, as in our mouse models, was used to
offer an experimental system for further imaging and biochemical analysis.
63
Chapter 4
GRP78 co-localizes with KRAS and is required for plasma
membrane localization and signaling
4.1 Introduction
The finding of robust expression of GRP78 and KRAS and co-localization of the two
proteins in the peri-nuclear region in K78
+/+
mice lung tissues, suggested that interaction
between these two proteins is taking place at the ER cytosolic interphase where KRAS
undergoes post-translational modification. Second, we discovered that the levels of
KRAS decreased in parallel to those of GRP78 in K78
f/+
and K78
f/f
mice, providing the
first hint that GRP78 is required for the integrity of KRAS in lung cancer. To test
whether these in vivo observations in mice apply to human lung cancer, we utilized the
human lung ADC cell line A427. It harbors the same KRAS
G12D
mutation as our mouse
model so we can use it as an experimental system in efforts to identify the mechanism
whereby GRP78 affects KRAS-induced lung cancer.
The KRAS oncogene is mutated more frequently than any other oncogene in human
cancers (Tsai et al. 2015). The KRAS transcript is alternatively spliced in the fourth exon
to yield two products, namely, KRAS4A and KRAS4B. In humans, KRAS4A is reported
to be expressed in the gastrointestinal tract and to a lesser extent in the lung and other
tissues of endodermal origin but not in skeletal muscle or the nervous system (Plowman
64
et al. 2006). KRAS4A mRNA has been reported to be expressed at lower levels
compared to KRAS4B (Pells et al. 1997). Both splice variants are oncogenic when KRAS
is mutated. In the lung, the predominant protein splice variant of KRAS expressed is
KRAS4B, as such we focused on this variant and hereafter will be referred to as KRAS.
KRAS is a membrane-associated GTPase signaling protein that promotes proliferation
and cell survival. The KRAS membrane-targeting signals are located within the last 17
amino acids of the C-terminal sequences containing the –CAAX motif (Hancock et al.,
1990). For KRAS to localize to the plasma membrane, the newly synthesized KRAS
protein is first farnesylated in the cytosol, followed by the removal of the –AAX motif
and methylesterfication, by isoprenylcysteine methyltransferase (ICMT), of the
farnesylated cysteine, with both processes taking place at the cytosolic surface of the
endoplasmic reticulum (ER) by enzymes localized there. Upon completion of CAAX
processing, KRAS traffics to the plasma membrane independent of the conventional
exocytic pathway, bypassing the Golgi (Apollloni et al. 2000). Properly modified, KRAS
contains hydrophobic molecules that require a cytosolic chaperone to protect the lipid
residues. One of those chaperones is the δ-subunit of cGMP phosphodiesterase type 6
(PDE6δ); however, KRAS4A does to not bind to PDE6δ (Tsai et al. 2015).
Another abundant mutation in lung adenocarcinoma is that of epidermal growth
factor receptor (EGFR). EGFR is a member of the ErbB family of proteins containing the
four receptor tyrosine kinases, EGFR (ErbB1), Her2 (Neu, ErbB2), Her3 (ErbB3), and
Her4 (ErbB4) (Yarden and Sliwkowski 2001). KRAS is a downstream factor of EGFR
65
(Shigematsu et al. 2005; Schmid et al. 2009). The importance of the EGFR-KRAS
network in lung ADC tumorigenesis was described in Desai et al. (2014). AT2 self-
renewal was found to be selectively induced by EGFR ligands in vitro and oncogenic
Kras
G12D
in vivo. A signal transduced by the EGFR-KRAS network controls self-renewal,
and is hijacked during oncogenesis and AT2 stem-cell activity. Recent work has
challenged the notion that KRAS upon acquiring activating mutations would lead to
constitutive oncogenic activation. Rather, mutated KRAS is not just locked in its active
form but can be activated by upstream EGFR (Moll et al. 2018; Kruspig et al. 2018).
Results, by both Moll et al. (2018) and Kruspig et al. (2018), demonstrate the
involvement of the ErbB family such that when a pan inhibitor targeting ERbB members
was used, a decrease in lung tumorigenesis was observed. As a result of the importance
of EGFR, and its family members, in lung cancer, we also investigated the effects of
GRP78 knockdown on EGFR and we identified that EGFR localization is affected by
depletion of GRP78.
The ER is a major site for the modification and maturation of KRAS. Protein folding
quality control exists in the ER to avoid accumulation of misfolded and unfolded proteins.
An important contributor to this quality control system is GRP78 (Ni and Lee 2007; Lee
2014). GRP78 has been demonstrated to be essential for many cellular processes and this
raises the question whether GRP78 is required for KRAS maturation and function. To test
directly whether GRP78 is a pivotal player in the maturation, translocation, and function
of KRAS, we treated A427 cells with siRNA targeting GRP78 for proper knockdown.
66
Additionally, we used a truncated KRAS protein, referred to below as (GFP-tK). GFP-tK
was constructed fusing GFP to the last 17 amino acids of the KRAS C-terminus, which
contains the sequence motifs required for KRAS modification in the ER and translocation
to the plasma membrane (Apolloni et al. 2000). Interestingly, just as it was observed in
the mouse lung tissues, depletion of GRP78 in the lung adenocarcinoma A427 cells led to
decreased levels of KRAS as well as GFP-tK (using anti-GFP antibody). In addition, we
also observed that the ER membrane-bound ICMT also decreased after GRP78
knockdown. Our studies provide new evidence that GRP78 is required for KRAS
translocation to the plasma membrane as well as for proper KRAS function. The
implications of these results and the requirement of GRP78 function for KRAS will be
discussed.
4.2 Materials and methods
Cell lines and cell culture
The cells A549 and H460 were acquired from ATCC. H522 and BEAS-2B cells were a
kind gift from Dr. Ite Offringa. A427 was a kind gift from Dr. David Shackelford at
UCLA. All cells were cultured according to recommendations from ATCC and validated
by Sanger Sequencing. All cells tested negative for mycoplasma contamination.
Transfection of siRNA
Lipofectamine RNAiMAX (Invitrogen) was used according to manufacturer’s
67
recommendations to introduce siRNA to all cell lines. Custom siRNAs were purchased
from GE Healthcare Dharmacon, Inc. (Chicago, IL). The control (siCtrl) sequence is as
follows: 5’- GAGAUCGUAUAGCAACGGU -3’and the siRNA targeting GRP78 (si78)
sequence: 5’-GGAGCGCAUUGAUACUAGA-3’.
Protein extraction and immunoblot analysis
The cell lysates were prepared for Western blot analysis as described (Li et al. 2008).
Lysates were then run on gradient gels (4-15% Mini-Protean TGX, BioRad, #4561086).
The following primary antibodies were used: GRP78 (1:1000, mAb159, Parkash Gill),
GAPDH (1:2000,), p-ERK (Thr202/Tyr204, 1:1000, Cell Signaling #9106), ERK (1:1000,
Cell Signaling #9102), p-AKT (Ser473, 1:1000, Cell Signaling #9271) and AKT (1:1000,
Cell Signaling #9272) KRAS (KRAS-2B, 1:1000, Proteintech #16155-1-AP). The
secondary antibodies used in this study are as follows: horseradish peroxidase conjugate
goat anti-mouse, anti-rabbit (1:1000, Santa Cruz Biotechnology #sc-2005, and #sc-2004).
Protein levels were visualized and quantitated by ChemiDoc™ XRS+ Imager (Bio-Rad
Laboratories, Irvine, CA).
HA-KRAS and GFP-tK plasmids
The plasmids containing HA-tagged full-length wild-type (HA-KRAS
WT
) or G12D
mutated (HA-KRAS
G12D
) KRAS-4B sequence were acquired from Dr. Dominic Esposito
at the NCI Ras Initiative at the Frederick National Laboratory for Cancer Research
68
(Bethesda, Maryland). The previously described GFP-tK plasmid (Apolloni et al. 2000)
was acquired from Dr. John F. Hancock.
Gene expression data
Data measured in fragments per kilobase million upper quartile normalized (FPKM-UQ),
was downloaded for lung adenocarcinoma samples from the The Cancer Genome Atlas
(TCGA) using the TCGAbiolinks package and log2 transformed. After removing
duplicate tumor samples taken from the same subject, a t-test was performed to compare
mean GRP78 expression from normal (n=59) and tumor (n=513) samples.
Immunofluorescent staining
The immunofluorescent (IF) staining was performed as described previously (Fu
et al. 2008). For IF staining in A427 cells, the antibodies used were: GRP78 (1:200, anti-
GRP78 monoclonal antibody Mab159, described in Liu et al. 2013), KRAS (1:200, anti-
rabbit polyclonal antibody, Proteintech, 16155-1-AP), and ICMT (1:200, polyclonal
antibody, Bioss Antibodies, bs-8019R). IF imaging was performed using a Zeiss LSM
510 confocal microscope with LSM 510 Version 4.2 SP1 acquisition software, and the
images were analyzed with ZEN lite imaging software (ZEISS, Thornwood, NY) and
Adobe Photoshop CS5 (San Jose, CA). To perform IF staining on A427 and A549 cells
to detect EGFR, the following antibody was used: EGFR (1:200, anti-EGFR D38B1,
anti-rabbit, Cell Signaling, 4267).
69
Statistical analysis
Statistical analysis was performed with either a 2-tailed Student’s t-test or a One-
Way Anova. Data represented as mean ± s.e.m. *p < 0.05, **p < 0.01, and ***p < 0.001.
Real-time quantitative PCR
RNA was extracted from A427 cells and reverse-transcription and real-time PCR
were performed as previously described (Ni et al. 2009). Primers used for ICMT and
KRAS have been previously described by Etichetti et al. (2019) and Guarnaccia et al.
(2018), respectively. All primers used for the real time-PCR assay are listed in Table 3.1
GRP78 Forward 5’ - GGTGAAAGACCCCTGACAAA - 3’
GRP78 Reverse 5’ - GTCAGGCGATTCTGGTCATT - 3’
KRAS Forward 5’- GACTGAATATAAACTTGTGGTAGTTGGA -3’
KRAS Reverse 5’- CATATTCGTCCACAAAATGATTCTGA -3’
ICMT Forward 5’- CAGTGGAGTGTACGCTTGGT -3’
ICMT Reverse 5’- AGAATCGCCACACTGTCAGG -3’
β-actin Forward 5’- TCCCTGGAGAAGAGCTACGA -3’
β-actin Reverse 5’- AGCACTGTGTTGGCGTACAG -3’
Table 3.1 Primer sequences for real-time PCR used to detect GRP78, KRAS, ICMT and β-actin.
4.3 Results
4.3.1 GRP78 is required for the integrity of KRAS and co-localizes at the ER
cytosolic interphase
To test whether the observation that GRP78 and KRAS co-localize in the perinuclear
70
region in mice also applies to human lung cancer, a lung adenocarcinoma cell line
containing the KRAS
G12D
mutation, A427, was used as an experimental system for further
imaging and biochemical analysis. Similar to the in vivo model, we observed co-
localization of KRAS and GRP78 in A427 by immunofluorescent staining. Co-
localization between KRAS and GRP78 was readily detected in the ER region (Figure
4.1A). Furthermore, after knockdown of GRP78 via treatment of siRNA targeting GRP78
for 48 hrs, endogenous KRAS levels decreased, confirming the result we observed in vivo
whereby depletion of Grp78 resulted in decreased levels of Kras in mouse lung tissues.
To test biochemically whether GRP78 forms complex with KRAS, we co-transfected
FLAG-GRP78 (F-78) expression vector with either HA-tagged wild-type or G12D
mutated Kras cDNA expression vectors, namely, HA-KRAS
WT
(HA-K
WT
) and HA-
KRAS
G12D
(HA-K
G12D
), into 293T cells which exhibited high transfection efficiency. The
cell lysates were subjected to immounoprecipitation with the anti-Flag antibody, followed
by Western blots to detect the presence of the HA-KRAS that was pulled down with F-78.
We observed that both HA-KRAS
WT
and HA-KRAS
G12D
were able to form complex with
F-78. Only the co-IP using HA-KRAS
G12D
is shown (Figure 4.1B).
To confirm if
endogenous levels of KRAS were decreased after GRP78 knockdown as was seen in
Figure 4.1A, a Western blot was performed (Figure 4.1C). Quantitation of KRAS
relative levels based on the Western blot were also graphed (Figure 4.1C, Right).
71
Figure 4.1 Complex formation of GRP78 and KRAS. (A) Immunofluorescent stain of GRP78 (red) and
endogenous KRAS (green) in A427 cells under conditions of knockdown via siRNA targeting GRP78 (si78)
or control scramble sequence siRNA (siCtrl) for 48 hrs. Scale bar is 7 µm and is applicable to all sections.
(B) Whole-cell lysate from 293T cells expressing full length GRP78 tagged with FLAG, FLAG-GRP78 (F-
78) with full length G12D mutant KRAS tagged with HA, HA-KRAS
G12D
(HA-K
G12D
) were subjected to
immunoprecipitation with IgG and anti-FLAG. The immunoprecipitate and the whole cell-lysate were
probed with FLAG-GRP78 and HA antibodies by Western blot analysis. (C) Western blot of endogenous
KRAS levels, GRP78, and GAPDH after A427 cells were subjected to siRNA treatment targeting Grp78
(si78) or control scramble sequence (siCtrl) for 48 hrs. Quantitation of relative KRAS levels from the
western blot in (C) are shown on the right based on five independent experiments. Data are presented as
mean ± s.e.m, ***p<0.001. Additional contributions: (A) D.F.R. performed experiment and imaged cells
three times. The fourth time, D.F.R. performed experiment and Anthony Carlos imaged cells. (B) D.F.R.
72
performed Co-IP, twice and the third and fourth times were performed by Willy Tsai. (C) D.F.R. performed
experiment 4 times, the fifth time, D.F.R. performed experiment and Dat Ha ran the Western blot.
4.3.2 GRP78 is required for plasma membrane localization of KRAS and signaling
GFP-tK was constructed by fusing the green fluorescent protein (GFP) to the last
17 amino acids of KRAS at its C-terminus, which contains the sequence motifs required
for KRAS modification in the ER and translocation to the plasma membrane (Hancock et
al. 1991; Apolloni et al. 2000). The last 17 amino acids encode the lysine residues at the
C-terminus as well as the CAAX motif that gets modified so that KRAS can reach the
plasma membrane. The polybasic domain created by the six-lysine residues creates a
positive charge that allows these KRAS sequences to localize to the plasma membrane.
The CAAX motif, where C is a cysteine, A an aliphatic amino acid such as alanine,
isoleucine, leucine, proline, and valine, and X is any amino acid, is the site of post-
translational modifications required for efficient binding of KRAS to the plasma
membrane. Both the polybasic domain and the CAAX with the posttranslational
modifications are necessary and sufficient to target KRAS to the plasma membrane
(Hancock et al. 1991). The construct of the truncated KRAS containing only the last 17
amino acids fused to GFP is shown Figure 4.2A. To determine if indeed GFP-tK signal
could reach the plasma membrane in A427 cells, transfection of the GFP-tK construct
was performed. A427 cells were first treated with siRNA treatment targeting GRP78 for
32 hrs followed by 16 hrs of transfection with the GFP-tK construct for a total
73
Figure 4.2 GFP-truncated KRAS (GFP-tK) after GRP78 knockdown in A427 cells. (A) The GFP-tK
construct has the last 17 amino acids of KRAS-4B attached to the C-terminus region of GFP. The last 14
amino acids are shown to point out the six lysine residues required for KRAS to attach to the plasma
membrane as well as the CAAX motif where C = cysteine, A = aliphatic, X = any amino acid. (B) Western
blot of A427 cells after knockdown for 32 hrs preceded transfection of GFP-tK for 16 hrs for a total length
of knockdown treatment 48 hrs. Antibodies used were GFP-tK (anti-GFP), GRP78 and GAPDH. Data are
presented as mean ± s.e.m, *p<0.05. (C) Immunofluorescent staining after A427 cells were subjected to the
same treatment as in (B) to stain for GRP78 and GFP-tK (anti-GFP). Scale bar is 7 µm and is applicable to
all sections. Additional contributions: (B) D.F.R. performed experiments twice and third time D.F.R
performed experiment and Dat Ha ran Western blot. (C) D.F.R performed experiment and Anthony Carlos
imaged cells.
knockdown time of 48 hrs. GFP-tK translocation to the plasma membrane was observed
in control A427 cells Figure 4.2C. However, GFP-tK was not able to reach the plasma
membrane in knockdown treated cells (si78) Figure 4.2C, indicating that GRP78 is
required for the translocation of GFP-tK to the plasma membrane. Furthermore, Western
blot revealed that GFP-tK levels were decreased after probing with anti-GFP antibody
(Figure 4.2B Left). Quantitation of relative GFP-tK protein levels was graphed based on
three independent experiments (Figure 4.2B Right). The modifications of the CAAX
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motif are to first link a prenoid derivative as a thioether to the cysteine residue (Casey et
al. 1989). At the ER interphase, KRAS undergoes the removal of the -AAX motif via
activity by RCE1 (Gutierrez et al. 1989).
Figure 4.3 ICMT and GRP78 in A427 cells. (A) Immunofluorescent stain of GRP78 (green) and
endogenous ICMT (red) in A427 cells. Scale bar is 7 µm and is applicable to all sections. (B) Western blot
levels of ICMT, GRP78 and GAPDH under conditions of knockdown via siRNA targeting GRP78 (si78) or
control scramble sequence siRNA (siCtrl) for 48 hrs. Quantitation of ICMT levels based on two
independent experiments is graphed on the right. Data are presented as mean ± s.e.m, *p<0.05. Additional
contributions: (A) D.F.R performed experiment and Anthony Carlos imaged the cells. (B) D.F.R.
performed experiment and Dat Ha ran Western blot.
At the ER interphase, ICMT allows for the methyl-esterification of the carboxyl group of
the now C-terminal cysteine residue (Clarke et al. 1988). As a result, we then questioned
whether GRP78 could localize with ICMT as well. A427 cells were also used to co-stain
75
GRP78 and ICMT and co-localization was observed in the perinuclear region (Figure
4.3A). ICMT was also probed via Western blot to determine if ICMT levels were affected
after GRP78 knockdown treatment in A427 cells, and indeed protein levels were
decreased (Figure 4.3B Left). Quantitation was performed based on two independent
experiments performed on A427 cells (Figure 4.3B Right).
To test whether the knockdown of GRP78 affected the levels of KRAS and ICMT mRNA,
a real time-PCR analysis was performed. We observed that mRNA levels of KRAS and
ICMT were not affected after GRP78 knockdown and relative mRNA levels were
quantified based on three independent experiments run as triplicates per run and levels
were normalized to β-actin (Figure 4.4).
4.3.3 KRAS signaling affected by GRP78 knockdown can be rescued
The potent, reversible, and cell-permeable proteasome inhibitor MG132, as well
as the lysosomal inhibitor, 3-methyladenine (3-MA), have been reported to protect
against KRAS protein degradation (Shin et al. 2018; Shukla Neoplasia 2014). In A427
cells, we observed that both MG132 and 3-MA could partially rescue KRAS levels in the
GRP78 knockdown cells, consistent with the notion that GRP78 knockdown destabilizes
KRAS through protein degradation (Figure 4.5).
Two major proliferative signaling pathways activated by KRAS at the plasma
membrane are the PI3K/AKT and RAF/MEK/ERK pathways (Pylayeva-Gupta et al.
76
2011). We observed in A427 cells that knockdown of GRP78 resulted in decreased levels
of p-AKT and p-ERK, suggesting that both signaling pathways are negatively affected by
loss of GRP78 (Figure 4.6A). To test if downstream signaling is indeed occurring due to
KRAS activation, HA-full length mutated KRAS (HA-K
G12D
) was introduced in the
normal bronchial epithelium human cell line, BEAS-2B.
Figure 4.4 Analysis of mRNA in A427 human lung adenocarcinoma cells. Relative mRNA level of GRP78,
KRAS, and ICMT, after 48 hrs of knockdown using siRNA targeting GRP78. The data are presented as
mean ± s.e., *p<0.05.
Transfection with HA-KRAS
G12D
resulted in increased activation of both p-AKT and p-
ERK. However, upon depletion of GRP78 by knockdown, levels of both p-AKT and p-
ERK were decreased (Figure 4.6B). Quantitation was based on three independent
experiments whereby p-ERK was normalized to total ERK and p-AKT was normalized to
total AKT.
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Depletion of GRP78 leads to the loss of KRAS and ICMT and to the inhibition of
KRAS localization to the plasma membrane as well as downstream signaling of the two
of the many effectors activated by KRAS, mainly, p-ERK and p-AKT. Figure 4.7
summarizes the effects of GRP78 on KRAS activity (Left) and the effects of GRP78
depletion (Right).
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Figure 4.5 A427 human lung adenocarcinoma cells rescue of KRAS. Western blot analysis using
antibodies against GRP78, KRAS, ICMT, and GAPDH. Partial rescue of KRAS after treatment with
MG132 or 3-MA for 4 hrs, preceded by 44 hrs of knockdown, for a total of 48 hrs of knockdown treatment.
Quantitation of GRP78, KRAS, and ICMT are shown on the bottom. Additional contributions: D.F.R.
performed experiment and Dat Ha ran Western blot.
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Figure 4.6 KRAS downstream signaling after GRP78 siRNA knockdown treatment. (A) Representative
Western blots of p-AKT (Ser473), total AKT (t-AKT), p-ERK (Thr202/204), and total ERK (t-ERK) in
A427 cells. Quantitation shown on the right, normalizing p-AKT over total AKT and p-ERK over total
ERK, based on two independent experiments. Protein levels of GRP78 after 72 hrs of siRNA treatment
targeting GRP78. (B) Representative Western blot of GRP78, p-AKT (Ser473), total AKT (t-AKT), p-ERK
(Thr202/204), and total ERK (t-ERK) after transfection with full length HA-tagged to mutated KRAS (HA-
KRAS
G12D
) in BEAS-2B cells. Cells were treated with siRNA targeting GRP78 (si78) and control
scrambled sequence (siCtrl) for 32 hrs then HA-KRAS
G12D
was added for 16 hrs, for a total transfection
time of 48 hrs. Quantitation shown on the right, normalizing p-AKT over total AKT and p-ERK over total
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ERK, was based on three independent experiments. Data are presented as mean ± s.e., *p<0.05. Additional
contributions: (B) Richard Van Krieken performed experiment and ran Western blot.
Figure 4.7 Summary of effects of GRP78 knockdown. (A) Conditions of non-ER stress leading to the
proper response when GRP78 is present in the cell. GRP78 affects 1) levels of KRAS, 2) localization of
KRAS to the plasma membrane, 3) levels of ICMT, 4) levels of phosphorylated AKT, and 5) levels of
phosphorylated ERK. (B) Depletion of GRP78 results in 1) decrease in KRAS levels, 2) decreased
localization of KRAS to the plasma membrane (PM), 3) reduction in ICMT, 4) phosphorylated-AKT, and 5)
phosphorylated-ERK.
4.3.4 Effects of GRP78 on EGFR
The epidermal growth factor receptor, EGFR, is a transmembrane protein with
cytoplasmic kinase activity and is important in activating growth factor signaling. EGFR
is commonly mutated in lung cancer and more than 60% of NSCLCs express EGFR (da
Cunha Santos et al. 2011). Tyrosine kinase inhibitors (TKIs) have been developed for
lung cancer patients and TKIs have been shown to be effective in patients with tumors
containing activating mutations in the tyrosine kinase domain of EGFR. As a result,
identification of EGFR mutations is utilized in clinical practice (Roengvoraphoj et al.
2013). However, despite these advances, prognosis remains unfavorable because of the
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occurrence of either intrinsic or acquired resistance (Morgillo et al. 2016). Thus, we also
investigated whether GRP78 depletion affects EGFR. First, we took A427 cells and
treated the cells with siRNA targeting GRP78 or a scrambled control sequence for 48 hrs
and EGFR was found to localize at the plasma membrane. However, after GRP78
knockdown, EGFR localization was disturbed and was not able to reach the plasma
membrane (Figure 4.8A). In addition, just as observed previously, KRAS signal was
decreased after GRP78 knockdown treatment (Figure 4.8A). The results were also
confirmed after attempting to transfect full length HA-tagged KRAS for 16 hrs following
knockdown treatment for 32 hrs for a total of 48 hrs of knockdown treatment in A427
cells. Although the HA-KRAS was found to localize in the perinuclear region, EGFR was
localized to the plasma membrane under siCtrl conditions, yet it became mislocalized
after si78 treatment (Figure 4.8B). Another lung cancer cell line containing a KRAS
mutation (G12S) (KRAS
G12S
), A549, was also investigated and treated with si78 and
siCtrl RNAs. EGFR was also internalized after GRP78 knockdown compared to the
control (Figure 4.9A) in A549 cells. Interestingly, KRAS was also depleted in A549 after
treatment with si78 (Figure 4.9B), supporting the results we observed in A427 cells.
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Figure 4.8 EGFR and KRAS in A427 cells after GRP78 knockdown. (A) A427 cells were treated with
siRNA targeting GRP78 (si78) and control scramble sequence (siCtrl) for 48 hrs and stained by
immunofluorescence to determine localization of EGFR (B) A427 cells were transfected with HA-tagged
full length wild-type KRAS (HA-KRAS) for 16 hrs, preceded by 32 hrs of knockdown, for a total of 48 hrs
of knockdown treatment. EGFR and HA antibody were used to stain A427 cells.
83
EGFR undergoes N-glycosylation modification at the ER before localizing to the plasma
membrane. The N-glycosylation determines membrane interactions and arrangement of
the ligand-binding EGFR ectodomain (Kaszuba et al. 2015). Western blot analysis
revealed that although the levels of EGFR remained unchanged after GRP78 knockdown,
the amount of underglycosylated EGFR did increase as noticed by the smaller size band
detected (Figure 4.9C). This data suggests that EGFR is not being properly modified, or
glycosylated, as a result of GFP78 absence and as such cannot reach the plasma
membrane. GRP78 knockdown treatment was extended to 48 and 72 hrs and
underglycosylation of EGFR was detected for both time points (Figure 4.9C).
4.3.5 Human expression data and GRP78
Consistent with published reports that GRP78 is upregulated in human lung
adenocarcinoma, as well as in our mouse models, we independently confirmed this
through analysis of the The Cancer Genome Atlas (TCGA) data (Figure 4.10).
Expression data from 513 tumor tissues were compared to 59 normal adjacent tissues.
After removing duplicate tumor samples taken from the same subject, a t-test was
performed to compare mean GRP78 expression.
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Figure 4.9 EGFR and KRAS in A549 cells after GRP78 knockdown. (A) A549 cells were treated with
siRNA targeting GRP78 (si78) and control scramble sequence (siCtrl) for 48 hrs and stained by
immunofluorescence to determine localization of EGFR and GRP78 (A) and KRAS and EGFR (B). (C)
Western blots of EGFR, GRP78, and GAPDH after A549 cells were subjected to siRNA treatment
targeting Grp78 (si78) or control scramble sequence (siCtrl) for 48 hrs and 72 hrs.
85
Figure 4.10 GRP78 mRNA expression in human lung adenocarcinoma. (A) GRP78 mRNA expression in
normal (n=59) and human lung adenocarcinoma tumor tissue (n=513) p = 5.648^-49. Data generated at
UNC; platform: Illumina HiSeq and obtained from the Cancer Genome Atlas (TCGA). Additional
contributions: Daniel Mullen downloaded data set and performed analysis.
4.4 Discussion
KRAS is most commonly mutated in lung ADC and its activation likely occurs in the
early stages of tumorigenesis (Cancer Genome Atlas 2014; Guerra et al. 2003; Jackson et
al. 2001). While progress has been made towards targeting KRAS and its oncogenic
signaling cascade, curbing mutant KRAS-driven tumorigenesis is elusive since most
inhibitors are rendered ineffective due to compensatory mechanisms. Thus, it is important
to identify alternative approaches to target KRAS. Another important target is EGFR.
86
EGFR is commonly mutated in lung adenocarcinoma, but it is also abundantly
overexpressed in NSCLC patients (Suzuki et al. 2005). It has been reported that EGFR
mutations located in the tyrosine kinase domain in exons 18-21 are associated with
improved response to EGFR inhibitors (Lynch et al. 2004). However, KRAS mutations
are associated with poor response to EGFR-directed tyrosine kinase inhibitor therapies
(Massarelli et al. 2007; Eberhard et al. 2005). Resistance to EGFR inhibitors is also
problematic, as secondary mutations have been proven to be involved after EGFR-
directed therapies. Therefore, treatments that can affect both EGFR and KRAS
simultaneously would be effective for patients battling NSCLC.
GRP78 is a stress-inducible multi-functional protein, which is emerging as an
exciting new target for anti-cancer therapy (Lee, 2014). In addition to being a critical ER
chaperone and pivotal regulator of the UPR and apoptotic machinery that is associated
with the ER, it can be actively translocated to other cellular locations and secreted, thus
assuming a wide range of functions that control cellular signaling, proliferation, invasion,
apoptosis, inflammation and immunity, impacting cancer progression and therapeutic
resistance (Ni et al. 2011; Tsai et al. 2018). Tissue-specific ablation of GRP78 using
genetically engineered mouse models established the requirement of GRP78 in Pten-null
driven cancers and PI3K/AKT signaling (Fu et al. 2008; Lee, 2014). Moreover, GRP78
haploinsufficiency in the pancreatic ductal adenocarcinoma mouse model, Pdx1-Cre;
KrasG
12D/+
;p53
f/+
reduced proliferation and suppression of AKT, S6, ERK, and STAT3
as well as EGFR, indicating that Grp78 heterozygosity can suppress tumorigenesis driven
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by Kras mutation (Shen et al. 2017). However, the role of GRP78 in lung cancer was not
known. The proliferation of lung adenocarcinoma requiring GRP78 (as shown in the
mouse models with decreased proliferation as measured by Ki67) coupled with the
observation that GRP78 is upregulated in human lung cancer, which we independently
confirmed through analysis of the TCGA data (Figure 4.10) and most importantly,
depletion of KRAS after knockout of GRP78 in mouse lung tissues, raise the interesting
possibility that the maturation of KRAS in the ER may require GRP78. Our studies
revealed several exciting new findings that are summarized below.
In our effort to understand the mechanism by which GRP78 suppress mutant KRAS-
driven lung tumorigenesis, we discovered that depletion of GRP78 leads to lowering of
KRAS protein levels both in vivo and in the A427 cell line, which contains the same
KRAS mutation as in the mouse models, Kras
G12D/+
. Our biochemical studies showed
that KRAS can form a complex with GRP78 and confocal imaging further revealed co-
localization of the two proteins in the ER. The reduction of KRAS protein in GRP78
knockdown the lung cancer cell line is not at the mRNA level, and it could be partially
rescued by the proteasome inhibitor, MG132 and lysosome inhibitor 3-MA. Furthermore,
KRAS cannot traffic to the plasma membrane in GRP78 knockdown lung cancer cells.
Thus, one scenario could be that GRP78 is an obligatory partner of the KRAS complex
on the ER, without which KRAS cannot be processed normally for its routing to the
plasma membrane and its stability. Correspondingly, GRP78 knockdown human lung
cancer cells showed reduced ERK and AKT signaling downstream of KRAS activation in
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A427 cells. To determine if KRAS signaling was being affected by the knockdown of
GRP78, we used BEAS-2B cells, a human cell line derived from normal bronchial
epithelium. Transfection with the full-length HA-KRAS
G12D
lead to expression and
activation of two downstream effectors of KRAS, namely, p-ERK and p-AKT. Depletion
of GRP78 decreased the levels of KRAS and downstream signaling, p-ERK and p-AKT.
Thus, agents targeting GRP78 not only can eliminate the protective properties of GRP78
but can also disrupt KRAS stability, trafficking, and signaling. Further support for this
was our investigation of the ER resident protein ICMT, which is key for the proper
modification of KRAS before migrating to the plasma membrane. Just like KRAS, ICMT
decreased after depletion of GRP78. In addition, both GRP78 and ICMT were found to
co-localize in the ER region. These data support the role of GRP78 in the modification
process of KRAS as it goes through the ER. A key finding would have been to detect
differences in modification of KRAS after the depletion of GRP78, such as detecting the
absence of the carboxymethylation at the cysteine residue located at the C-terminus of
KRAS. Although a small shift in KRAS was detected by running A427 cell lysate after
GRP78 knockdown by Western blot analysis (data not shown), antibodies to detect this
KRAS modification are not available. Moreover, mass spec was also out of the limits of
this study due to the lysine residue providing a strong positive charge of KRAS at the C-
terminus.
Depletion of GRP78 also affected the localization of EGFR in the lung cancer cell
lines investigated, A427 and A549. Although the levels of EGFR did not decrease after
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depletion of GRP78, the increase of underglycosylated EGFR and mislocalization of
EGFR after GRP78 knockdown signify that GRP78 is also important for the proper
maturation of EGFR in the ER. The cells demonstrating EGFR localization in part (A)
and (B) of Figure 4.9 were subjected to 48 hrs of knockdown siGRP78 and showed clear
mislocalization EGFR as it was found to be around the perinclear region suggesting that
underglycosylation of EGFR prevents it from reaching the plasma membrane. Although
the Western blots demonstrating underglycosylated EGFR for the si78 conditions for 48
hrs were not as prominent as for the 72 hrs, imaging the cells after 72 hrs knockdown
treatment would most likely result in a more pronounced phenotype. GRP78 has been
found to form a direct complex with EGFR in N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG) treated cells (G. Niu, Shang, and Yu, 2006). GRP78 was also found to form a
complex with EGFR under 2-deoxyglucose induced stress conditions (Cai et al. 1998).
Although not directly investigated, GRP78 could also potentially facilitate the
stabilization of EGFR at the cell surface, since GRP78 is also translocated to the cell
surface (Tsai et al. 2018). These new findings warrant vigorous future investigations.
90
Chapter 5
Conclusions and Perspectives
The glucose-regulated proteins are stress-inducible molecular chaperones that
mainly reside in the ER but can also translocate to the cell surface, the nucleus,
mitochondria, and even be secreted (Lee, 2014). Since the ER plays an important role in
many essential cellular processes, the disorder of ER homeostasis and stress are
intensively involved in various tissues and disorders, such as cancer, embryogenesis,
maintenance of the hematopoietic system, neurodegenerative disorders, inflammation,
and diabetes mellitus (Shen et al. 2017; Ni and Lee, 2007; Yoshida et al. 2007; Luo et al.
2006; Wey, Luo, and Lee, 2012; Luo et al. 2011) with roles in regulating oncogenic
signaling (Tsai et al. 2018; Luo and Lee, 2013). In this dissertation, we set forth to
investigate the roles of GRP78 in lung cancer mediated by oncogenic activity of KRAS
through the creation of conditional knockout mouse models as well as in vitro models
using human-derived lung cell lines.
In Chapters 2 and 3, we found that GRP78 is required for Kras-driven pulmonary
tumorigenesis. We used two different approaches to induce Cre-mediated recombination
in mice in order to remove the stop codon that prevents expression of the mutated Kras
while simultaneously removing the Grp78 floxed allele. In this first mouse model,
consistent with published reports that GRP78 is overexpressed in lung adenocarcinoma
tissues, we detected strong immunostaining of GRP78 in K78
+/+
mice, and in comparison,
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the staining intensity for GRP78 decreased in the lungs of K78
f/+
and further decreased in
K78
f/f
mice. The proliferation marker Ki67 demonstrated decreased cell cycle activity in
lung tissues of K78
f/f
mice when compared to K78
+/+
and K78
f/+
mice. Histological
examination revealed that consistent with increased tumor burden, pulmonary lesions
appeared earlier in K78
+/+
mice. Moreover, histology grade revealed increased
progression in grade such that adenocarcinoma was detected as early as 16 weeks post
Cre activation in K78
+/+
mice, compared to 22 weeks in K78
f/+
mice, while it was not
detected in K78
f/f
mice. This difference in tumor burden and histology grade was also
confirmed by 18F-FDG PET/CT analysis. Surprisingly, we also showed that depleting
GRP78 decreased the levels of KRAS, providing the first evidence that GRP78 is
required for KRAS stability. Collectively, we provided evidence that depleting GRP78
can delay the progression of lung adenocarcinoma driven by oncogenic KRAS.
Chapter 3 of this dissertation focuses on the second approach we used to induce
Cre-mediated recombination to activate expression of mutated Kras while simultaneously
knocking out Grp78. This second mouse model was created because of the limitations of
the first mouse model such that mice inhaling the virus cannot limit the effects of Cre to
only the population of cancer initiating cells. In addition, intubation may not reach all
lung surface areas equally in all mice even though the same amount of virus is injected
per mouse. As a result, we tested the effect of an inducible conditional knockout of
GRP78 targeted to lung alveolar type 2 cells (AT2) cells carrying a Kras
G12D
mutation on
92
lung tumorigenesis. Despite the differences between the two mouse models, the second
mouse model supported the results of the first mouse model, in that heterozygous
depletion of GRP78 resulted in decreased tumor burden and halted lung tumor
progression. Interestingly, CK78
f/+
and CK78
f/f
mice had prolonged survival. Autopsy
examination of the CK78
+/+
mice revealed that the abnormalities were confined to the
lungs, which showed multiple tumor nodules. In addition, tumor grade was more severe
compared to CK78
f/+
and CK78
f/f
mice despite having a shorter on average life span.
To understand the mechanisms whereby GRP78 insufficiency blocks
tumorigenesis in lungs carrying an activating Kras mutation, results from our in vitro
model are described in Chapter 4. Just as observed in mouse lung tissues, depletion of
GRP78 in A427 resulted in decreased levels of KRAS. Moreover, co-localization of
KRAS and GRP78 was also observed at the perinuclear region. Interestingly, ICMT was
also found to co-localize with GRP78 at the perinuclear region and levels of ICMT
decreased after GRP78 knockdown in A427 cells. Consistent with the notion that GRP78
knockdown destabilizes KRAS and ICMT through protein degradation, we observed that
both MG132 and 3-MA could partially rescue both KRAS and ICMT levels. KRAS
modifications take place at the outer leaflet of the ER and modifications are necessary for
KRAS to translocate to the plasma membrane. Interestingly, the truncated KRAS
fragment bound to GFP containing the last 17 amino acids, which are necessary and
sufficient to allow for translocation to the plasma membrane, was decreased in A427 cells
93
after depletion of GRP78. In further support that KRAS could not activate downstream
signaling in the absence of GRP78, we detected decreased p-AKT and p-ERK after
knockdown treatment. The results were further confirmed in BEAS-2B cells that also had
decreased signaling of p-AKT and p-ERK even after introduction of the full-length
mutated HA-KRAS
G12D
construct. Thus, these results indicate that GRP78 is required for
the two major KRAS-mediated proliferative signaling pathways. GRP78 has been shown
to co-stain with p-AKT in both endometrial cancer and pancreatic adenocarcinoma (Lin
et al. 2015; Hill et al. 2012). Furthermore, decreased activation of p-AKT and p-ERK was
observed in a pancreatic adenocarcinoma in vivo mouse model with the Kras activating
mutation (Shen et al. 2017). Although our study provides evidence that the chaperone
function of GRP78 at the ER affects KRAS maturation and thus its activity, resulting in
the decrease of both p-ERK and p-AKT, evidence that GRP78 can re-localize to the cell
surface where it can interact with different ligands and cell surface proteins, such as PI3K,
to promote phosphatidylinositol (3,4,5)-trisphosphate formation, have been previously
reported (Zhang et al. 2013). Although cell surface GRP78 was not investigated in this
current study, it would be interesting to investigate if GRP78 on the cell surface can be in
complex with KRAS or other cell surface receptors to mediate AKT activation, and
possibly that of ERK as well, to promote lung cancer growth.
GRP78, as a functional chaperone, participates in the folding of early protein
intermediates. It has abundant client proteins, including growth factors, which must be
94
secreted, and/or growth factor receptors, which must be matured. The observation that
GRP78 also affects the translocation of EGFR to the plasma membrane helps give insight
into how GRP78 helps decrease lung tumorigenesis and decrease tumor burden in mice.
Although attempts to stain EGFR in the lung tumor tissues were unsuccessful, the results
of EGFR mislocalization in two different lung cancer cell lines harboring KRAS
mutations, mainly (G12D) in A427 and (G12S) in A549, provide evidence that GRP78 is
important in the maturation of EGFR. EGFR, being a part of the ErbB family of tyrosine
receptors, has been shown to be a key receptor in the activation of KRAS and its
downstream signaling pathways (Desai et al. 2014). However, in the clinic, EGFR
inhibitors have been unsuccessful in patients carrying KRAS mutations and patients with
EGFR mutations eventually acquire resistance (Massarelli et al. 2007; Eberhard et al.
2005). Two independent studies confirmed the role of the other non-EGFR members of
the ErbB family in the failure of the first generation of tyrosine kinase inhibitors.
However, the importance of targeting the ErbB family has been recently studied. Through
mouse models, patient-derived and cell line-derived xenografts, as well as in vitro
experiments it was demonstrated that the U.S. Food and Drug Administration approved
pan-ErbB inhibitor, afatinib, impaired KRAS-driven lung tumorigenesis (Moll et al. 2018;
Kruspig et al. 2018). As a result of these latest findings, it would be interesting to
investigate if GRP78 also has a role in the localization and modification of the rest of the
ErbB family members.
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GRP78 is the master regulator of the unfolded protein response and upon ER
stress, GRP78 is titrated away from the three ER stress sensors activating downstream
pathways (Luo and Lee, 2012). Complete loss of GRP78 has been reported to induce ER
stress in various organs and cell lines including the lung, the bone marrow cells, the
intestine, the uterus, HEK293 cells, Purkinje cells (Wang et al. 2010; Flodby et al. 2016;
Heijmans et al. 2013; Li et al. 2008; Lin et al. 2015; Wey, Luo, and Lee 2012). We
investigated whether ER stress might play a role in A427 cells, and indeed upregulation
of various ER stress markers was observed by Western blot. Upregulation of p-eIF2α was
observed, which is a downstream effector of PERK, and increased production of CHOP,
a downstream target of eIF2α phosphorylation. Induction of pro-apoptotic markers,
cleaved PARP and cleaved caspase-7, was also detected in GRP78 knockdown A427
treated cells (Figure 5.1). Induction of eIF2α and CHOP under ER stress contributes to
suppression of metabolic genes during endoplasmic stress. Therefore, the diminished
lung tumorigenesis in GRP78-deficient KRAS
G12D
mice could also be due to increased
CHOP protein levels as well as cleaved PARP and caspase activity.
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Figure 5.1 ER stress markers in A427 cells after GRP78 siRNA treatment for 48 hrs. (A) Expression of the
ER stress markers by Western blot. (B) Splicing of XBP-1 by mRNA analysis performed by reverse
transcriptase (RT-PCR). Additional contributions: (A) and (B) D.F.R. performed experiment and Dat Ha
ran Western blot with the help of Priscilla Chan.
Our results from our study point to a possible early treatment method for combating
lung cancer. Lung cancer has a poor prognosis since over half of the patients die within a
year of diagnosis and the 5-year survival is less than 18% (Zappa and Mousa, 2016).
Moreover, approximately 40% of newly diagnosed lung cancer patients are categorized
as stage IV, where the treatment option for these patients is merely to prolong survival
and improve the quality of life. Depending on the stage at which the patient is diagnosed,
several regimens of platinum-based treatments are preferred. For example, the American
97
Society of Clinical Oncology states that treatment for patients with a performance status
of 0 or 1, should be cisplatin or carboplatin plus paclitaxel, gemcitabine, docetaxel,
vinorelbine, irinotecan, or permetrexed (Masters et al. 2015). Patients with
adenocarcinoma, may benefit from permetrexed. Recent studies have shown that patients
with RAS mutations may benefit from inhibitors against KRAS downstream effectors
such as c-RAF inhibitors (Sanclemente et al.2018). Although several drugs that target
other KRAS downstream effectors such as those involved in the MAPK and PI3K
pathways have been tested, their high toxicity has prevented them from being
implemented in the clinic (Lujambio, 2018). Inhibitors that target the farnesyl
transferases were initially praised, however, after failure in the clinic, it was found that
KRAS could also be activated by another modification, namely by geranylgeranylation
(Appels et al. 2005). Interestingly, inhibitors targeting the KRAS G12C mutation have
been in development and validated in vitro and in vivo (Misale et al. 2019). Recent
announcements by pharmaceutical companies Amgen (Thousand Oaks, CA) and Mirati
Therapeutics, Inc. (San Diego, CA) revealed KRAS inhibitors in clinical trials targeting
KRAS
G12C
. Effectiveness of the KRAS inhibitors as well as the side effects will remain to
be seen. Success of this inhibitor might propel the manufacturing of other inhibitors that
can target other mutations such as KRAS
G12D
. Currently in Phase II clinical trials, the
small molecule, IT-139 a first-in-class ruthenium-based drug, developed for the treatment
of solid cancers was successfully studied in a Phase I clinical trial (Trondl et al. 2014).
IT-139 preferentially suppresses GRP78 expression in tumors but not in adjacent cells of
98
xenograft models (Bakewell et al. 2018). Intriguing questions remain for GRP78 in lung
cancer, such as whether cell surface GRP78 can be detected in mouse or human lung
cancer cell lines and whether the application of the GRP78 antibody treatments would
affect their signaling and survival. Monoclonal antibodies have been developed to target
cell surface GRP78, and they have been shown to compromise growth of various cancers
both in vitro and in vivo (de Ridder et al. 2011; Lin et al. 2015; Liu et al. 2013).
Additionally, the monoclonal IgG antibody, MAb159, was reported to have high affinity
towards GRP78 and specifically recognized cell surface GRP78. MAb159 triggered
GRP78 endocytosis and inhibited PI3K signaling without compensatory MAPK pathway
activation. Furthermore, the humanized form of MAb159 had desirable pharmacokinetic
properties and was not toxic in mice (Liu et al. 2013). Interestingly, we detected cell
surface GRP78 in A549 cells by Western blot after treatment with the ER stress inducer
thapsigargin, the non-competitive inhibitor of the sarco/endoplasmic reticulum Ca
2+
ATPase, as well as treatment with tunicamycin, which inhibits N-linked glycosylation
and thereby blocks protein folding and transit through the ER (data not shown). A more
in-depth analysis is warranted to determine if cell surface GRP78 is detected in other lung
adenocarcinoma cell lines containing the KRAS
mutations, such as A427, and whether
inhibitors that target cell surface GRP78 can have the same effects as those observed in
this study after knockdown of GRP78.
Our most significant finding was the role of GRP78 in KRAS stability and its ability
to traffic to the plasma membrane. However, questions remain about whether the results
99
observed in A427 can also be observed in other lung cell lines. Immunofluorescence in
A549 lung carcinoma cell lines harboring the KRAS
G12S
mutation also showed KRAS
levels to be depleted after GRP78 knockdown (Figure 4.9). Western blots also revealed
depletion of KRAS and ICMT in A549 cells after knockdown for 48 hrs (data not shown).
We also transfected the GFP-tK construct into A549 cells and observed levels decreased
by Western blot. Moreover, both p-ERK and p-AKT were decreased in A549 after
GRP78 knockdown treatment for 48 hrs (Figure 5.2).
Figure 5.2 A549 analysis and signaling after GRP78 knockdown. (A) A549 cells were treated with siRNAs
targeting GRP78 and analyzed for GRP78, phosphorylated-AKT (p-AKT), phosphorylated -ERK (p-ERK)
and their respective total levels, t-AKT and t-ERK as well as GAPDH, by Western blot. (B) A549 cells
were treated with siRNA targeting GRP78 for 32 hrs followed by 16 hrs of transfection with full GFP-tK
for a total knockdown time of 48 hrs. Western blot is shown using antibodies against GRP78, GFP and
GAPDH.
Moreover, we also detected decreased KRAS levels in the lung cancer cell line H460,
which contains KRAS
Q61H
but not in H522, which is wild-type for KRAS. We cannot
100
conclude that GRP78 only affects mutated KRAS however, since the antibodies we used
for Western blot detect both wild-type and mutant KRAS. In addition, A427 cells are
heterozygotes for the KRAS allele, and depleting GRP78 by more than 80% decreased
KRAS by more than 50%, indeed closer to 80%. Although the cell lines investigated here
have numerous mutations that limit any conclusions we can make, we can speculate that
cell lines with KRAS mutations are sensitive to GRP78 depletion. However, further
studies involving other NSCLC cell lines harboring KRAS mutations such as H2122
(KRAS
G12C
) and H358 (KRAS
G12C
) are warranted. Another approach worth investigating
can be to isolate normal lung cells and transform them to express only the KRAS
mutation by the introduction of full-length mutated HA-KRAS
G12D
and HA-KRAS
WT
in
order to determine the effects of GRP78 depletion.
Figure 5.2 KRAS analysis in H460 and H522 lung cell lines. Western blot analysis of GRP78, KRAS, and
GAPDH after GRP78 knockdown (si78) and control (siCtrl) treatment for 48 and 72 hrs.
Previous studies have demonstrated that GRP78 is required for ER integrity and
stress-induced autophagy, since ER is a putative membrane source for generating the
autophagosomal double membrane (Li et al. 2008). It has been discovered that cancer
101
cells with RAS activation require autophagy for maintenance of functional mitochondria,
for tolerance of metabolic stress and for tumorigenesis. Indeed, in a KRAS-driven
NSCLC mouse model with deletion of the autophagy-related-7 (atg7), autophagy
deficiency altered KRAS
G12D
-induced carcinomas to rare predominantly benign
oncocytomas. Moreover, these KRAS
G12D
autophagy-deficient mice had accumulation of
defective mitochondria and reduced tumor growth (Guo et al. 2013). Autophagy is a
protective process in cancer development and therapy that is activated in response to
stress in order to recycle cellular components and to maintain homeostasis (Eng et al.
2016). Autophagy requires the fusions of atuophagosomes with early and late endosomes
for proper function (Razi et al. 2009). MAP kinase and autophagy pathways have been
shown to cooperate and maintain RAS mutant cancer cell survival (Lee et al. 2019).
Studies in mouse models indicate autophagy can restrict tumor initiation by regulating
DNA damage and oxidative stress. In addition, some tumors rely on autophagy for tumor
promotion and maintenance. The clathrin-dependent pathway leads to KRAS signaling
on late endosomes en route to lysosomes (Lu et al. 2009). EGF treatment led to the
translocation of KRAS to endosomal membranes from early to late endosomes and then
to lysosomes. However, KRAS is less retained in the endosomes compared to HRAS or
NRAS, probably as a result of a faster recycling to the plasma membrane (Jiang and
Sorkin, 2002). Furthermore, KRAS has been shown to be refractory to ubiquitylation,
indicating that its interaction with endosomal membranes might not be as stable.
However, KRAS-mediated MAPK signaling was observed in late endosomes (Lu et al.
102
2009). In one study for example, after 30 min of EGF stimulation, p-ERK1/2 was co-
localized with GFP-KRAS at the endosome, and signal was still detected even after 60
min of stimulation. Interestingly, immunoelectron microscopy and gold-labelled
antibodies have demonstrated that GRP78 is present in the lumen of endosomal vesicles
(Tamayo et al. 2011). Thus, it will be of great interest to determine the role of GRP78 in
autophagy and KRAS function.
Another intriguing question is if GRP78 plays a role in the prenyl-binding protein
PDEδ-KRAS complex. PDEδ facilitates the diffusion of KRAS in the cytoplasm and is a
guanine nucleotide dissociation inhibitor (GDI)-like solubilizing factor that aids
translocation of RAS proteins to the plasma membrane (Chandra et al. 2012; Zhang et al
2004; Nancy et al. 2002). Indeed, small molecule inhibition of the KRAS-PDEδ
interaction impairs oncogenic signaling (Zimmerman et al. 2013). As we have
demonstrated in this current study, GRP78 is required for the maturation and
translocation of KRAS to the plasma membrane. Thus, it will be interesting to investigate
whether GRP78 has a role in aiding PDEδ-KRAS function.
UPR components and ER chaperones are crucial for ER homeostasis and evidence
demonstrating GRPs are also functional on the cell surface suggest they are central for
many cellular processes and signaling pathways. Studies in this dissertation expand our
understanding of GRP78 in regulating lung tumorigenesis induced by KRAS oncogenic
activation. Furthermore, this study provides evidence that GRP78 is key for proper
103
KRAS function and as such illuminate the potential for GRP78 as a therapeutic target to
battle lung cancer.
104
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Abstract (if available)
Abstract
The endoplasmic reticulum (ER) is an essential organelle important for the manufacturing of lipids and proteins and it specializes in proper protein folding and secretion. Various studies show that the function of ER chaperones is not limited to simply helping nascent peptides fold but that these proteins have key roles in human diseases such as cancer, diabetes, obesity, tumor immunity, and mammalian development. To investigate the link between the ER chaperone, glucose-regulated protein 78 kDa (GRP78), also known as binding immunoglobulin protein (BiP), and lung tumorigenesis, several transgenic mouse models where fragments of the gene encoding for GRP78, called, heat shock protein family A, member 5 (HSPA5), were conditionally deleted, have been constructed for investigation in vivo. In addition, we used human lung cell lines for in vitro studies. ❧ Lung cancer is the leading cause of cancer related deaths worldwide, claiming more than 1.6 million deaths annually and approximately 153,718 deaths in the United States alone. Mutations in the proto-oncogene KRAS occur in 10 to 30% of lung adenocarcinomas, classified as a non-small cell lung cancer (NSCLC). Here we report the requirement of GRP78 for the development and progression of lung adenocarcinoma as identified through the generation of two separate mouse models containing floxed Grp78 or K-ras Lox-Stop-Lox G12D (K-rasLSL-G12D) alleles. The first mouse model was intubated intratracheally to introduce Cre directly into the lungs for the creation of the following mice: (1) KrasG12D⁄+Grp78+/+ (referred to as KGrp78+/+ or K78+/+), (2) Kras G12D/+Grp78f/+ (referred to as KGrp78f/+ or K78f/+), and (3) Kras G12D/+Grp78f/f (referred to as KGrp78f/f or K78f/f). Tamoxifen injection activated Cre in mice of the second model, since the Cre gene was driven by a 3.7 kb human surfactant protein C and estrogen receptor (SPC-CreERT2) promoter was expressed in alveolar type 2 (AT2) cells of the lung. Mice used in model 2 were of the following genotypes: (1) SPC-Cre-ERT2
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Asset Metadata
Creator
Rangel, Daisy Flores
(author)
Core Title
The role of endoplasmic reticulum chaperone glucose-regulated 78-kilodalton (GRP78) in lung cancer
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
07/15/2019
Defense Date
05/20/2019
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
endoplasmic reticulum,GRP78,KRAS,lung cancer,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Offringa, Ite (
committee chair
), Dubeau, Louis (
committee member
), Lee, Amy (
committee member
)
Creator Email
daisyfloresrangel@gmail.com,djflores@usc.edu
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https://doi.org/10.25549/usctheses-c89-183547
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UC11660539
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etd-RangelDais-7551.pdf (filename),usctheses-c89-183547 (legacy record id)
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etd-RangelDais-7551.pdf
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183547
Document Type
Dissertation
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Rangel, Daisy Flores
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(contributing entity),
University of Southern California Dissertations and Theses
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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...
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Tags
endoplasmic reticulum
GRP78
KRAS
lung cancer