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Tight junction protein CLDN18.1 attenuates malignant properties and related signaling pathways of human lung adenocarcinoma in vivo and in vitro
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Tight junction protein CLDN18.1 attenuates malignant properties and related signaling pathways of human lung adenocarcinoma in vivo and in vitro
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Content
Tight junction protein CLDN18.1 attenuates malignant properties and
related signaling pathways of human lung adenocarcinoma in vivo
and in vitro
By
Jiao (Jane) Luo
A Dissertation Presented to the
Faculty of The Graduate School of
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)
August 2018
2
Table Contents
Chapter 1 Introduction ............................................................................... 9
1.1 Claudins and their role in barrier function ..................................................................... 9
1.2 Claudins in carcinogenesis ........................................................................................... 11
1.3 CLDN18.1 in lung alveolar epithelium .......................................................................... 12
1.4 Cross-talk between CLDN18.1 and YAP/TAZ in lung cancer ..................................... 13
1.5 Purpose of the study ...................................................................................................... 15
Chapter 2 Materials and Methods ........................................................... 16
2.1 Animal procedures ......................................................................................................... 16
2.2 Micro-computed tomography (CT) imaging ................................................................. 16
2.3 Preparation of plasmids and lentiviral particles .......................................................... 17
2.4 Cell culture procedures and generation of stable cell lines ....................................... 18
2.5 Methylation, transient transfections and luciferase assays ....................................... 19
2.6 RNA isolation and quantitative real-time PCR ............................................................. 20
2.7 Immunofluorescence and confocal microscopy ......................................................... 21
2.8 Cell counting and proliferation assay .......................................................................... 21
2.9 Soft agar anchorage-independent growth assay ........................................................ 22
2.10 Migration and invasion assays ................................................................................... 22
2.11 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay ... 23
2.12 Microarray analysis ...................................................................................................... 23
2.13 Reverse phase protein array (RPPA) analysis ........................................................... 23
2.14 Statistical analyses ...................................................................................................... 24
3
Chapter 3 CLDN18 suppresses lung carcinogenesis in vivo ............... 25
3.1 Aged Cldn18
-/-
mice develop multiple lung tumors histologically consistent with
adenocarcinoma ................................................................................................................... 25
3.2 Loss of CLDN18 expression in tumors of LuAd patients ........................................... 28
3.3 Clinical relevance of CLDN18 expression in LuAd patients ....................................... 30
3.4 Repletion of CLDN18.1 in human LuAd cells inhibits tumor growth ......................... 33
3.4.1 Generation of stable human LuAd cell line with inducible CLDN18.1 expression 33
3.4.2 Expression of CLDN18.1 in human LuAd cells suppresses xenograft tumor
growth .................................................................................................................................... 35
Chapter 4 CLDN18.1 inhibits malignant properties of LuAd cells in
vitro ............................................................................................................ 37
4.1 CLDN18.1 inhibits cell growth and proliferation of LuAd cells in vitro ..................... 37
4.2 Induced expression of CLDN18.1 decreases migration and invasion of LuAd cells 42
4.3 CLDN18.1 reduces anchorage-independent growth of LuAd cells ........................... 43
Chapter 5 Molecular mechanisms mediating the tumor suppressor
role of CLDN18.1....................................................................................... 46
5.1 CLDN18.1 suppresses IGF-1R/AKT signaling .............................................................. 46
5.1.1 Loss of Cldn18 activates IGF/AKT signaling in whole lung and AT2 cells ............ 46
5.1.2 Expression of CLDN18.1 in LuAd cells inhibits IGF-1R/AKT activity ..................... 49
5.2 Inhibition of YAP/TAZ activity contributes to the tumor suppresser role of
CLDN18.1 ............................................................................................................................... 52
5.2.1 Tumors in both Cldn18
-/-
mice and human LuAd show increased YAP activity .... 52
4
5.2.2 CLDN18.1 inhibits YAP/TAZ signaling in LuAd cell lines ........................................ 55
5.3 Contribution of YAP/TAZ to CLDN18.1-mediated regulation of AKT ......................... 56
Chapter 6 Discussion ............................................................................... 59
6.1 Model for the role of CLDN18.1 in regulation of lung tumorigenesis ........................ 59
6.2 Molecular mechanisms underlying CLDN18 regulation of IGF-1R/AKT .................... 61
6.3 Molecular mechanisms underlying CLDN18.1 regulation of YAP/TAZ ..................... 65
6.4 Cross-talk between YAP/TAZ and IGF-1R/AKT signaling ........................................... 68
6.5 Regulatory mechanism of CLDN18.1 expression in the lung .................................... 69
6.6 Conclusions and Perspectives ..................................................................................... 69
Appendix ................................................................................................... 71
Appendix A: RT-PCR primer sequences ........................................................................................... 71
Appendix B: siRNA sequences ........................................................................................................ 71
Appendix C: List of genes differentially expressed in AT2 cells from Cldn18
-/-
versus wild-type mice
(FC>2, p<0.05) ................................................................................................................................ 72
Appendix D: List of 77 proteins altered with Dox treatment in H23/C18 cells ................................. 76
Appendix E: List of 80 proteins altered in AT2 cells isolated from Cldn18
-/-
mice ............................. 80
References ................................................................................................ 84
5
List of Figures and Tables
Figure 1 Molecular composition of the tight junction .................................................................. 10
Figure 2 H&E staining of Cldn18
-/-
mouse lung tumor ................................................................ 25
Figure 3 Ex-vivo micro-CT lung images ..................................................................................... 26
Figure 4 Reduced CLDN18 expression in human LuAd ............................................................ 29
Figure 5 CLDN18 expression level is associated with lung tumor stage and patient survival .... 30
Figure 6 CLDN18.1 expression in LuAd is inversely correlated with patient mortality and
promoter methylation. ......................................................................................................... 32
Figure 7 Generation of Dox-inducible CLDN18.1 expressing LuAd cell line .............................. 34
Figure 8 Induction of CLDN18.1 suppresses xenograft tumor growth ....................................... 36
Figure 9 CLDN18.1 inhibits growth of H23/C18 cells ................................................................. 37
Figure 10 CLDN18.1 inhibits growth of H358/C18 cells ............................................................. 38
Figure 11 CLDN18.1 does not affect apoptosis ......................................................................... 39
Figure 12 CLDN18.1 inhibits proliferation of LuAd cells ............................................................. 40
Figure 13 Dox has no effect on proliferation by parental H23 cells ............................................ 41
Figure 14 CLDN18.1 decreases migration of LuAd cells ........................................................... 42
Figure 15 CLDN18.1 decreases invasion of LuAd cells ............................................................. 43
Figure 16 CLDN18.1 suppresses colony forming ability of LuAd cells ....................................... 44
Figure 17 Dox does not affect anchorage-independent growth in parental H23 cells ................ 45
Figure 18 IPA analysis of differentially expressed genes in AT2 cells from Cldn18
-/-
and WT
mice .................................................................................................................................... 47
Figure 19 Loss of Cldn18 activates IGF/AKT signaling .............................................................. 48
Figure 20 IPA analysis of differentially expressed proteins in H23/C18 in the presence or
absence of Dox ................................................................................................................... 49
Figure 21 CLDN18.1 suppresses IGF-1R and AKT phosphorylation of LuAd cells ................... 50
6
Figure 22 CLDN18.1 does not change ERK1/2 phosphorylation in H23/C18 cells .................... 50
Figure 23 Dox treatment of the H23/C18 cells in the presence of IGF-1 ................................... 51
Figure 24 Loss of Cldn18 increases nuclear YAP in mouse lung .............................................. 53
Figure 25 Nuclear YAP is increased in human LuAd ................................................................. 54
Figure 26 CLDN18.1 downregulates the expression of TAZ and YAP ....................................... 55
Figure 27 CLDN18.1 suppresses expression of YAP/TAZ target genes .................................... 56
Figure 28 Role for YAP/TAZ in CLDN18.1-mediated AKT inactivation in H23/C18 cells ........... 58
Figure 29 Working model for the tumor suppressor activity of CLDN18.1 in LuAd .................... 61
Figure 30 Interactions of epithelial junctional modules with the core Hippo pathway ................ 66
Table 1 Tumor number and volume in aged Cldn18
-/-
mice ....................................................... 27
7
Abstract
Claudins are a family of transmembrane proteins integral to the structure and function of
tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important
features of invasive and metastatic cancer cells. We recently observed that aged
Cldn18
-/-
mice have increased propensity to develop lung adenocarcinoma (LuAd),
suggesting a tumor suppressor role for CLDN18. CLDN18.1 is the lung-specific isoform
of CLDN18 while the other isoform, CLDN18.2, is normally expressed in the stomach.
The goal of this study was to further explore the tumor suppressor role of CLDN18.1 in
LuAd as well as to investigate the underlying mechanisms. We found that CLDN18.1
expression is markedly decreased in LuAd patients in a stage-dependent manner and its
expression also correlates inversely with promoter methylation, suggesting that
CLDN18.1 expression is regulated by DNA methylation in LuAd. Analyses of LuAd patient
cohorts further shows that CLDN18.1 expression correlates with patient survival,
suggesting a protective role of CLDN18.1 in lung cancer. Furthermore, when restored in
LuAd cells that have lost expression, CLDN18.1 significantly attenuates malignant
properties including xenograft tumor growth in vivo as well as cell proliferation, migration,
invasion and anchorage-independent colony formation in vitro. Based on high throughput
analyses of Cldn18
-/-
murine lung alveolar epithelial type II cells, as well as CLDN18.1-
repleted human LuAd cells, we hypothesized, and confirmed by Western analysis,
inhibition of insulin-like growth factor 1 receptor (IGF-1R) and AKT phosphorylation by
CLDN18.1. Additionally, consistent with recent data in the Cldn18
-/-
murine model, re-
expression of CLDN18.1 suppressed YAP/TAZ target genes in human LuAd cells in vitro,
potentially contributing to its tumor suppressor activity. Analysis of LuAd cell line, in which
8
YAP and/or TAZ are silenced with siRNA, demonstrates that inhibition of TAZ, and
possibly YAP, may be involved in CLN18.1-mediated AKT inactivation. Taken together,
these data indicate a tumor suppressor role for CLDN18.1 in LuAd, mediated by a
regulatory network that encompasses YAP/TAZ, IGF-1R and AKT signaling.
9
Chapter 1 Introduction
1.1 Claudins and their role in barrier function
Tight junctions (TJ) constitute the most apical intercellular junctional complex in epithelial
and endothelial cells (1). As shown in Figure 1, TJs are comprised of multiple proteins
that include integral membrane and cytoplasmic/scaffolding proteins (2). Membrane
proteins such as occludin, claudins and junctional adhesion molecules (JAMs) mediate
cell-cell contact, paracellular permeability and barrier function. Scaffolding proteins such
as zonula occludens 1 (ZO-1), ZO-2 and ZO-3 link the transmembrane proteins to the
actin cytoskeleton (1–4).
Claudins are of particular interest because of their critical role in the assembly and
function of TJs (2). There are 27 known mammalian claudin family members, of which 26
have been identified in humans (2). The exact combination of claudin proteins within a
given tissue is thought to determine the selectivity and integrity of the tight junctions. Apart
from their barrier function in paracellular solute trafficking, claudins also define the
boundary between the basolateral and apical membranes, thus enabling the normal
transcellular transport characteristic of polarized epithelia (5,6). The general structure of
all claudins consists of four transmembrane helices with two extracellular loops and one
intracellular loop (2). The extracellular loops in neighboring cells interact with each other
to form a barrier that restricts paracellular ion, solute and liquid trafficking (2). Of interest,
expression of specific family members determines distinctive paracellular permeability
properties of various tissues. For example, CLDN2, known as one of the ‘pore-forming’
10
claudins, is highly expressed in leaky epithelia, including the proximal tubule of the kidney
and the intestinal crypts. It forms loose barriers thus facilitating paracellular trafficking (2).
CLDN5, in contrast, is predominantly known as one of the ‘barrier-forming’ claudins. It is
highly expressed in endothelium and appears to be particularly important in vascular
endothelium of the blood-brain barrier to prevent the permeability of macromolecules.
(1,2,7) This feature of CLDN5 is not universal since increased levels of CLDN5 have been
shown to decrease barrier function in airway and alveolar epithelia making them leaky (8).
In summary, claudins play crucial roles in the control of paracellular transport and the
maintenance of cell polarity and the specific pattern of claudins expressed determines the
barrier properties of a given tissue.
Figure 1 Molecular composition of the tight junction
Tight junctions consist of two main groups of proteins: transmembrane proteins (e.g. claudins
and occludin) and adaptor proteins (e.g. ZO-1, ZO-2, ZO-3, cingulin). From Yu (2008) Biochim
Biophys Act. (4).
11
1.2 Claudins in carcinogenesis
Aberrant expression of claudins is observed in various cancers and their role in
carcinogenesis is thought to be context-dependent. For example, CLDN3 and CLDN4 are
frequently upregulated in ovarian, breast, prostate and pancreatic tumors (6-8), while
CLDN7 is downregulated in breast cancer but elevated in stomach cancer (9,10). In vitro
modulation of claudins in cancer cells has provided strong evidence for an association
between claudin proteins and tumorigenesis. For example, downregulation of CLDN3 or
CLDN4 in ovarian cancer promotes tumor growth and metastatic behavior in vivo (9). In
breast cancer, low expression of CLDN3, CLDN4, and CLDN7 corresponds to high grade
malignancy (10,11). In contrast, overexpression of CLDN1 in colon cancer cells with low
CLDN1 expression (SW480 cells) is sufficient to enhance the ability of the cells to form
tumors and/or metastasize in vivo (12). Interestingly, CLDN6 has been reported to play
opposite roles in different cancers. Decreased expression of CLDN6 enhances
anchorage-independent growth and promotes invasiveness in breast cancer cells while
its overexpression increases cell invasion, migration, and proliferation potential in gastric
cancer cell lines (13). Similarly, CLDN7 has a dual role of both tumor suppressor and
tumor promoter, enhancing cell growth and metastatic behavior of esophageal squamous
cell carcinoma, while inhibiting colon cancer progression and invasiveness (14,15). In
summary, changes in claudin expression are commonly observed in a variety of tumors
and these changes are both positively and negatively associated with tumor formation.
These findings suggest the possibility that claudins may serve as prognostic biomarkers
in a variety of cancers. However, a causal role for claudin proteins and a unifying
hypothesis regarding their role in regulating carcinogenesis remains to be determined.
12
Knockout and overexpression studies have demonstrated that claudin proteins regulate
a number of cellular signaling cascades implicated in cancer development and
progression. For example, CLDN1 overexpression modulates Notch signaling in a matrix
metallopeptidase 9 (MMP-9)-dependent manner to promote colon cancer development
(16). In human liver cells, CLDN1 promotes invasive behavior by activating the c-Abl-PKC
signaling pathway (17). Moreover, association between CLDN1 and Src proteins in a
complex with ZO-1 drives colon cancer cells to undergo metastasis presumably in a
claudin-1/Src/PI3k-Akt/Bcl-2-dependent manner (18). Thus deregulation of claudin
expression and/or cellular distribution has inherent potential for altering cellular functions
including cell proliferation, migration, and invasion by modulating key signaling cascades
(1,19). Nonetheless, a question remains as to how claudin dysregulation leads to
disruption of signaling pathways in different tumor types and how these changes
potentially contribute to tumor formation.
1.3 CLDN18.1 in lung alveolar epithelium
Human lung alveolar epithelium primarily expresses three claudins: CLDN3, CLDN4 and
CLDN18 (8,20,21). Of these, only CLDN18.1 is unique to the lung, suggesting a lung-
specific function (21–23). Alternative usage of the two promoters of CLDN18 gives rise to
two splice variants, CLDN18.1 and CLDN18.2, each with a unique exon 1 spliced to
common exons 2 through 5 (22). The two variants show strict lineage specificity in normal
human tissues. CLDN18.1 is primarily expressed in lung epithelium while CLDN18.2 is
normally confined to differentiated epithelial cells of the gastric mucosa (22,24).
13
Recently generated Cldn18
-/-
mice, in which both Cldn18.1 and Cldn18.2 isoforms are
globally deleted show increased solute and ion permeability, consistent with the known
barrier function of claudins (23). Interestingly, Cldn18
-/-
mice also show enlarged lungs,
likely due to increased proliferation of lung alveolar epithelial type II (AT2) cells, believed
to be the progenitor cells of distal lung epithelium (24). Underlying mechanistic studies
revealed that loss of Cldn18 leads to the activation of Yes-associated protein (YAP)
signaling, which has a recognized role in regulating stem/progenitor cell proliferation and
organ size (24,25). Since CLDN18.2 is not normally expressed in the lung, we attributed
the phenotypes observed in Cldn18
-/-
mice lung to the loss of Cldn18.1.
Numerous cancer therapeutic studies have focused on CLDN18.2 due to its frequent
ectopic expression in pancreatic, esophageal, ovarian and lung cancers (26,27), whereas
investigation of CLDN18.1 in cancer has been limited. In the Cldn18
-/-
mouse model, we
observed that aged mice have increased propensity to develop lung adenocarcinoma
(LuAd) (24), suggesting that this TJ protein may play a tumor suppressor role in human
lung alveolar epithelial cells.
1.4 Cross-talk between CLDN18.1 and YAP/TAZ in lung cancer
As shown in our recent study, loss of Cldn18.1 leads to activation of YAP, a transcriptional
co-activator that plays a critical role in stem/progenitor cell proliferation and organ size
regulation. Moreover, activation of YAP and its paralog, transcriptional co-activator with
PDZ-binding motif (TAZ), is widespread in many human tumors including lung cancer
14
(28–32). Immunohistochemical (IHC) studies of lung cancer have shown that elevated
expression/nuclear localization of YAP or TAZ correlates with malignant features such as
high histological grade, late stage, lymph-node metastasis and poor patient outcome
(33,34). Knockdown of either is sufficient to inhibit cell proliferation, invasion and
clonogenicity of LuAd cell lines (33,35). YAP/TAZ are transcriptional coactivators that
shuttle between the cytoplasm and the nucleus, where they interact with other
transcription factors, and in particular TEA domain (TEAD) family members (25).
Sustained activation of YAP/TAZ is involved in regulation of a transcriptional program that
promotes aberrant cell proliferation, suppresses apoptosis and expands cancer stem cells
(36–39). Therefore, a better understanding of the cross-talk between CLDN18.1 and
YAP/TAZ may suggest innovative strategies to treat cancer.
YAP has previously been shown to upregulate insulin-like growth factor (IGF) signaling
by transcriptionally activating the expression of IGF-1 and its receptor, insulin-like growth
factor 1 receptor (IGF-1R). Moreover, IGF signaling has been implicated in induction and
maintenance of a range of different malignancies including lung cancer (23,24). Clinically,
increased plasma levels of IGF-1 have been associated with an elevated risk of lung
cancer. One of the major downstream effectors of IGF signaling is the phosphoinositide
3-kinase (PI3K)/AKT pathway, aberrant activation of which has been reported in more
than 40% of LuAd cases from The Cancer Genome Atlas (TCGA) network (cBioportal.org)
(27). Moreover, an abnormally activated PI3K/AKT pathway is one of the mechanisms of
acquired resistance to tyrosine kinase inhibitors (TKi) in LuAd patients (40). The known
interactions between YAP and IGF-1 signaling and involvement of both pathways in lung
15
carcinogenesis suggest the importance of investigating cross-talk between YAP and IGF-
1R/AKT signaling activity in the Cldn18
-/-
mouse model in which YAP is activated.
1.5 Purpose of the study
The goal of this study is to investigate a tumor suppressor role for CLDN18.1 in LuAd and
elucidate underlying mechanisms. We will ascertain its clinical relevance to human LuAd
by comparing expression levels of CLDN18.1 between normal and LuAd patient samples
as well as examining the relationship between CLDN18.1 expression and patient survival.
Results of these studies will allow us to explore its potential application as a prognostic
biomarker. We will evaluate effects of CLDN18.1 on the malignant phenotype in vitro
using overexpression approaches with inducible human LuAd cancer cell lines and in vivo
using a xenograft mouse model. We will also investigate the signaling cascades regulated
by CLDN18.1 using high-throughput analysis approaches to assess changes in pathway-
based expression profiles in order to identify mechanisms underlying the tumor
suppressor function of CLDN18.1 in LuAd.
16
Chapter 2 Materials and Methods
2.1 Animal procedures
Lung AT2 cells and whole lung extracts were harvested from mice with global deletion of
both Cldn18 isoforms. Generation of these mice, extraction of the lungs, and preparation
of primary AT2 cells were as previously described (23). In vivo tumorigenicity assays were
carried out in female athymic nude mice (Jackson Laboratories, Bar Harbor, ME). Briefly,
LuAd cells were grafted subcutaneously in the flanks of eight-week-old mice (1x10
6
cells
per flank). For doxycycline (Dox)-induced gene expression in the xenografts, mice were
fed a Dox-containing diet (625 mg/kg) (Teklad Diets, Madison, WI; #TD.01306). Tumor
length (l) and width (w) were measured weekly, and tumor volume (V) was calculated as
V = lw
2
/2 (41). Mice were euthanized after 6 weeks and tumors were excised and weighed.
All animal studies were performed in compliance with the University of Southern California
Institutional Animal Care and Use Committee guidelines.
2.2 Micro-computed tomography (CT) imaging
Lungs were fixed and inflated with 4% paraformaldehyde (PFA) at 20 cm H
2
O pressure.
After overnight fixation, lungs were rinsed with phosphate-buffered saline (PBS) then 50%
ethanol before incubation through a serial ethanol gradient (70%, 80%, 90% and 100%)
for 2 hr each at 4°C. Following this, lung samples were incubated with 100%
hexamethyldisilazane overnight before air drying (42). Lung specimens were scanned at
17
an isotropic resolution of 10 microns at 45kVp, 200mAs (43). CT image raw data were
analyzed using AMIRA software (FEI, Hillsboro, OR) to create volume renderings (44).
For radiographic measurement of lung tumors, regions of high density on acquired CT
images were automatically detected, counted and volumetrically quantified. A CT
threshold (320 Hounsfield unit (HU)) was used to segment lung tumor from normal lung
areas.
2.3 Preparation of plasmids and lentiviral particles
For promoter methylation studies, the CLDN18.1 sequence between positions -300 and
-1 relative to the transcription start site was PCR-amplified using human genomic DNA
as template and the primer pair 5’-AGTCTGGTTTAAGACAGAGCAC-3’ and 5’-
GCCGAAGGTGTGAAGCTAA-3’. The amplicon was ligated into the TA cloning vector
(Invitrogen, Carlsbad, CA; #K4500-01), the insert was excised using Acc65I and BamHI,
and directionally cloned into Acc65I/BamHI-digested CpG-free pCpGL-basic luciferase
vector (gift from Dr. Peter Jones, Van Andel Research Institute, Grand Rapids, MI) to
yield pCpGL-300. For Dox-inducible CLDN18.1 expression, the Myc-Flag-tagged coding
sequence of CLDN18.1 was cloned into the SpeI/XbaI-digested lentiviral entry vector
pEN_TmiRc3 (ATCC, Manassas, VA; MBA-248), and the resulting plasmid was
recombined using the Gateway system (Invitrogen; #11791-020) with the pSLIK-Hygro
destination vector (ATCC; MBA-237) to yield pSLIK-Tet-rtTA-CLDN18.1-Hygro. All
constructs were verified by sequencing. For preparation of lentiviral particles, HEK293T
cells were co-transfected with pSLIK-Tet-rtTA-CLDN18.1-Hygro, the pCMV8.91
packaging plasmid and the pMDG envelope plasmid, using the calcium chloride method
18
(45). Culture medium containing virus particles was harvested after 48 hours and virus
titer (typically 10
8
lentiviral particles/ml) was determined using a p24 Elisa Assay Kit (Cell
Biolabs, San Diego, CA; #VPK-108-H).
2.4 Cell culture procedures and generation of stable cell lines
Primary human AT2 cells were isolated from remnant transplant lungs as previously
described (47). The isolation procedure follows Institutional Review Board-approved
protocols for the use of human source material in research (HS-07-00660). Human H23
(#CRL-5800) and H358 (#CRL-5807) LuAd cell lines from the American Type Culture
Collection (ATCC, Manassas, VA) were cultured in RPMI-1640 medium (Invitrogen;
#11875-119) supplemented with 4 mM L-glutamine, 10% fetal bovine serum (FBS)
(Hyclone, Logan, UT; #SH30071.02), 100 U/ml penicillin and 100 μg/ml streptomycin.
Both cell lines were authenticated at the Genetic Core of the University of Arizona based
on polymorphic short tandem repeat (STR) loci. To generate stable H23 and H358
subclones with Dox-inducible CLDN18.1, cells were transduced with the pSLIK-Tet-rtTA-
CLDN18.1-Hygro lentivirus in the presence of 10 µg/ml polybrene at MOI=2. Transduced
cells were selected with 100-150 μg/ml of hygromycin (Life Technologies, Carlsbad, CA;
#10687-010) for 10 days, resulting in H23/C18 and H358/C18 Dox-inducible CLDN18.1-
expressing sublines. All studies were performed with cells pretreated with Dox for 2 days
to induce CLDN18.1 expression. Mouse lung epithelial (MLE-15) cells (gift from Dr.
Jeffrey Whitsett, University of Cincinnati) were cultured in HITES medium (RPMI 1640,
10 nM hydrocortisone, 5 μg/ml insulin, 5 μg/ml human transferrin, 10 nM β-estradiol, 5
μg/ml selenium, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml
19
streptomycin) supplemented with 2% FBS (46). Human embryonic kidney HEK293T cells
(ATCC; #CRL-1573) were grown in Dulbecco's modified Eagle's medium (DMEM)
(Sigma-Aldrich, St. Louis, MO; #5796) supplemented with 4 mM L-glutamine, 10% FBS,
100 U/ml penicillin and 100 μg/ml streptomycin.
2.5 Methylation, transient transfections and luciferase assays
Prior to transfection, the plasmids pCpGL and pCpGL-300 (20 µg) were each incubated
for 48 hours at 37°C with 50 units of methyltransferase SssI (New England Biolabs,
Ipswich, MA; #M0226L) and 160 μM S-adenosylmethionine (SAM) (New England
Biolabs; #B9003S). Control, unmethylated DNA was prepared by incubating the plasmids
as above, except the methyltransferase was omitted. Completeness of methylation was
verified using the methylation-sensitive restriction enzyme HhaI. MLE-15 cells were
seeded in 24-well plates (6x10
4
cells/well) and transfected after 24 hours with 0.75 μg/well
of either methylated or unmethylated pCpGL or pCpGL-300 firefly luciferase plasmids,
along with 50 ng Renilla luciferase control vector using Superfect reagent (Qiagen,
Valencia, CA; #301307). Reporter activity was determined 48 hr later with the Dual-
Luciferase Reporter System (Promega, Madison, WI; #E1960). siRNA transfections of
H23/C18 cells were performed with Lipofectamine RNAi-MAX (Thermo Fisher, Waltham,
MA; #13778030) according to the manufacturer’s instructions. Non-targeting (control) and
YAP and TAZ siRNAs were purchased from Dharmacon (Chicago, IL). Sequences of
siRNAs used are provided in Supplemental Table 1.
20
2.6 RNA isolation and quantitative real-time PCR
Total RNA was extracted from cultured H23/C18 cells and from human AT2 cells using
RNeasy Plus Mini Kit (Qiagen, Germantown, MD; #74134). cDNA synthesis was carried
out with Superscript (Invitrogen; #18080-051). qRT-PCR was performed with a thermal
cycler (Applied Biosystems, Foster City, CA; 7900HT) followed by data analysis using
SDS2.0 software (Applied Biosystems). Primer sequences are provided in Supplemental
Table 2.
Western analysis
Protein extracts (10-60 μg) in 2% sodium dodecyl sulfate (SDS) buffer containing
protease and phosphatase inhibitors (Calbiochem, Billerica, MA; #539134) were resolved
by SDS-PAGE and transferred to PVDF membranes (Bio-Rad; #162-0177) as previously
described. Proteins were visualized using an enhanced chemiluminescence kit (Pierce,
Rockford, IL; #32106) and an Azure Imager c300, followed by quantitation with Azurespot
software (Azure Systems, Dublin, CA). Sources of antibodies and their dilution factors
were as follows: anti-CLDN18 (Life Technologies, Grand Island, NY; #700178; 1:500),
anti-lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA; #sc20681; 1:2000), anti-α-
tubulin (Sigma-Aldrich, #6-11B-1; 1:5000), anti-b-actin (Abcam, Cambridge, MA;
#ab8227; 1:5000) and anti-GAPDH (Ambion, Foster City, CA; #AM4300; 1:2000). Anti-
phospho-IGF-1R (#3027; 1:200), anti-IGF-1R (#3018; 1:200), anti-phospho-S127-AKT
(#4911; 1:200), anti-AKT (#3477; 1:500), anti-Erk1/2 (#4695; 1:200), anti-phospho-Erk1/2
(#4370; 1:200), anti-YAP (#4912; 1:200), anti-p-YAP (#4911; 1:200) and anti-TAZ
((#4883; 1:200) were from Cell Signaling Technology (Beverly, MA).
21
2.7 Immunofluorescence and confocal microscopy
For immunostaining, 6x10
4
H23/C18 or H23 cells were seeded on 12 mm Transwell filters
(Corning, Corning, NY; #3401) and cultivated for two days before fixation with 4% PFA.
After antigen retrieval (Vector Laboratories, Burlingame, CA; #H-3300), filters were
incubated with primary Abs overnight at 4°C. After washing with Tris-buffered saline with
0.05% Tween 20, filters were incubated for 1 hr with biotinylated anti-rabbit or -mouse
IgG (1:300, Vector Laboratories; #BA-1000 or #BA-2000) followed by 10 min incubation
with Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA; #016-
160-084; 1:300). Cells were viewed using a Nikon Eclipse 80i microscope with
epifluorescence optics and images were captured with a cooled CCD camera (QImaging,
Surrey, BC, Canada). Confocal images were captured using a ZEISS LSM 510 confocal
system (Carl Zeiss, Jena, Germany). Primary Abs were the same as those used for
Western analysis.
2.8 Cell counting and proliferation assay
For cell growth assays, H23/C18 and H23 cells pre-treated with Dox or vehicle were
seeded in 24 well plates at a density of 6x10
4
cells/well. Cells were then counted daily
using a Z1 Coulter particle counter (Beckman Coulter, Brea, CA) up to day 5. To evaluate
proliferation of H23 and H23/C18 cells, 5-ethynyl-2’-deoxyuridine (EdU; 10 µM) was
added to the culture medium 1 hr before fixation with 4% PFA. EdU incorporation was
detected with the Click-iT Plus EdU Imaging Kit (Life Technologies, #10337) according to
manufacturer’s instructions. To determine the percentage of EdU
+
cells, five random
22
pictures of each repeat were taken using the 40X lens of a Nikon Eclipse 80i microscope.
Numbers of EdU
+
cells were counted in each of five fields. A total of ~2,000 cells from
each repeat were counted. Percentage of EdU
+
is determined by the number of EdU
+
cells over the total number of cells.
2.9 Soft agar anchorage-independent growth assay
H23 and H23/C18 cells were suspended in growth medium containing 0.3% agar (Fisher
Scientific, Hampton, NH; #BP1423-500) and 1.5 ml (4x10
4
cells) was added per well (22
mm) in 12-well plates. Prior to seeding, each well was prepared with a 1.5 ml basal layer
of 0.6% agar in growth medium. Cells were incubated at 37°C with a change of media
supplemented with Dox or vehicle every other day. Colonies were fixed in 4% PFA after
14 days and stained with crystal violet. Colonies were imaged for number and size
analyses. Five random pictures of each repeat were taken using the 10X lens of a Nikon
Eclipse 80i microscope (Nikon Instruments, Melville, NY). Colony number and size were
quantified using ImageJ software (National Institutes of Health).
2.10 Migration and invasion assays
For migration assays, 6x10
4
H23/C18 cells were seeded in the upper chamber of a non-
coated FluoroBlok 12 mm insert (Corning; #62406-504). Inserts coated with Matrigel
(Corning; #354234) were used for invasion assays. After incubation at 37°C for 48 hrs in
the presence or absence of Dox, cells were stained with 4 µg/ml Calcein-AM (VWR,
Radnor, PA; #89044-502) for 1 h and fluorescence was measured from the bottom of the
23
plates using the CytoFluor4000 fluorescence plate-reader (Applied Biosystems) at
wavelengths of 490/520 (Ex/Em).
2.11 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Detection of apoptosis based on labeling of DNA strand breaks was performed using the
In-Situ Cell Death Detection Kit (#11684795910, Roche Diagnostics, Indianapolis, IN)
according to the manufacturer's instructions.
2.12 Microarray analysis
Total RNA was extracted from AT2 cells from wild type and Cldn18
-/-
mice using an
RNeasy kit (Qiagen) and global expression profiling was performed in quadruplicate using
Mouse Ref8 v2.0 BeadChips (IIlumina, San Diego, CA) by the Southern California
Genotyping Consortium at the University of California, Los Angeles (UCLA). Raw data
processing was done using GenomeStudio (Illumina) and extended analyses were
carried out using R (version 2.11.1) as previously described (47). The complete
microarray dataset has been deposited to Gene Expression Omnibus (GEO) with the
accession number GSE106233.
2.13 Reverse phase protein array (RPPA) analysis
H23/C18 cells with and without Dox and wild type and Cldn18
-/-
knockout mouse AT2 cells
were subjected to RPPA analyses at the Functional Proteomics RPPA Core Facility, MD
Anderson Cancer Center. Raw data were normalized for protein loading and antibody
24
variation. Normalized log-transformed values were used for determination of differences
between samples.
2.14 Statistical analyses
Unless otherwise specified, data are presented as mean ± standard error of the mean
(SEM). Significance (p < 0.05) was determined by two-sided t-test for comparison of two
group means and Z-test for comparisons between normalized data. Significance (p < 0.05)
for ≥ 3 group means with one and two factors was determined by one-way and two-way
analysis of variance (ANOVA), respectively. Post-hoc analyses were performed with
Fisher’s least significant difference (LSD) test. Significance (p < 0.05) for Kaplan-Meier
curves was determined using the log-rank test. The Ingenuity Pathway Analysis (IPA)
package (http://www.ingenuity.com) was used for analysis of differentially expressed
RNAs (microarray) and proteins (RPPA). Fisher’s exact test as implemented in IPA
software was used to calculate p-values of significance level 0.05.
25
Chapter 3 CLDN18 suppresses lung carcinogenesis in vivo
3.1 Aged Cldn18
-/-
mice develop multiple lung tumors histologically consistent with
adenocarcinoma
We recently observed development of LuAd in aging mice deficient in Cldn18 and
confirmed using hematoxylin and eosin (H&E) staining as shown in Figure 2. We found
that tumors were generally in a sub-pleural location and typically adenocarcinomas with
papillary features. To better quantify the tumors, we utilized micro-CT to visualize the
tumors in 3D. Representative micro-CT images show that Cldn18
-/-
mice have multiple
lung tumors at 16 months of age while no visible tumors are observed in WT mice (Figure
3). Table 1 summarizes the tumor volume and size in lungs of 3 wild-type (WT) and
Cldn18
-/-
mice lung.
Figure 2 H&E staining of Cldn18
-/-
mouse lung tumor
H&E staining of a representative LuAd in a Cldn18
-/-
mouse at three different magnifications,
shows peripheral/sub-pleural location in lung (left panel) and papillary features (middle and right
panels). Scale bars from left to right = 1 mm, 200 μm and 100 μm.
26
Figure 3 Ex-vivo micro-CT lung images
Lung specimens from age-paired (15-16 months) WT and Cldn18
-/-
mice (n = 3) were scanned.
Upper panel (i and ii): 3D reconstruction of representative WT and Cldn18
-/-
mouse lungs. Scale
bar = 5 mm. Lower panel (iii and iv): Representative coronal sections of micro-CT images
corresponding to lungs in upper panel are shown. Tumors in Cldn18
-/-
lungs are indicated by
arrows.
i ii
iv
iii
27
Table 1 Tumor number and volume in aged Cldn18
-/-
mice
28
3.2 Loss of CLDN18 expression in tumors of LuAd patients
To further investigate the association between levels of CLDN18 expression and
tumorigenesis, we analyzed CLDN18 expression using two human LuAd patient cohorts:
microarray data from 58 matched human LuAd and adjacent non-tumor lung (NTL)
samples from the Canary Foundation/Early Detection Research Network (CF/EDRN),
and RNA-Seq data from 287 LuAd and 19 NTL deposited in The Cancer Genome Atlas
(TCGA). Analysis of gene expression data from these datasets shows significant
reduction of CLDN18 mRNA in human LuAd compared to non-tumor lung tissue (Figure
4 A-B). This observation is verified by double immunofluorescence staining for CLDN18
(red) and NKX2-1 (green), which is an AT2 cell marker in distal lung. Staining shows
decreased CLDN18 protein expression in human LuAd (Figure 4 C) compared to adjacent
non-tumor lung, consistent with a tumor suppressor role for CLDN18 in LuAd.
A B
29
Figure 4 Reduced CLDN18 expression in human LuAd
A-C. Loss of CLDN18 expression in human LuAd. Cldn18 mRNA expression is reduced based
microarray data from 58 matched micro-dissected LuAd and adjacent lung (A) and RNA-seq data
from 287 LuAd and 19 NTL based upon data generated by The Cancer Genome Atlas (TCGA)
Research Network (B). Paired t-test for microarray data and unpaired t-test for RNA-seq data. C.
Representative immunofluorescence staining for CLDN18 (red) and NKX2-1 (green) shows
decreased CLDN18 protein expression in human LuAd (lower panel) compared to adjacent non-
tumor lung (top panel), n = 3. Scale bar = 50 μm. DAPI is used as the nuclear counterstain.
C
C
LuAd
LuAd tumor
adjacent lung
30
3.3 Clinical relevance of CLDN18 expression in LuAd patients
To study the clinical relevance of changes in CLDN18 expression in LuAd patients, we
utilized the microarray patient cohort dataset (same dataset used for Figure 4 A) to
investigate the relationship between CLDN18 expression and lung tumor stages. CLDN18
expression was found to be decreased in LuAd patients in a stage-dependent manner
(Figure 5 A). In further support of a tumor suppressor role for CLDN18 in LuAd,
comparison of the survival rates of patients expressing CLDN18.1 mRNA at high versus
low levels in the TCGA LuAd patient cohort indicates that higher CLDN18.1 expression
is associated with significantly better survival (Figure 5 B). In contrast to CLDN18.1,
expression of the stomach-specific CLDN18.2 isoform, which is often ectopically
expressed in LuAd and other cancers, was not associated with LuAd patient survival (data
not shown). These observations suggest a protective role of CLDN18.1 in lung
carcinogenesis and a potential application of CLDN18 as a prognostic marker for LuAd.
A B
Normal Stage I Stage II Stage III Stage IV
CLDN18 mRNA
expression
14
12
10
8
31
Figure 5 CLDN18 expression level is associated with lung tumor stage and
patient survival
A. Stage-dependent decreases in CLDN18 mRNA expression using microarray data from 58
matched human LuAd and adjacent NTL samples fit with an exponential regression model (p <
1.75e-32). B. Kaplan-Meier curves for 502 patients in the TCGA LuAd cohort (p < 0.003, log-
rank test).
Interestingly, methylation of a CpG island near the CLDN18.1 transcription start site was
found to be increased in tumor versus normal tissue in the TCGA LuAd patient cohort
(Figure 6 A-B), while in vitro methylation of the CLDN18.1 promoter resulted in strong
transcriptional inhibition of a linked luciferase reporter (Figure 6 C). Furthermore,
methylation of the CpG island near the CLDN18.1 transcription start site was inversely
correlated with CLDN18 mRNA expression in the TCGA LuAd patient cohort (Figure 6 D).
A
B
32
Figure 6 CLDN18.1 expression in LuAd is inversely correlated with patient
mortality and promoter methylation.
A. Methylation of the CLDN18.1 promoter in the TCGA LuAd patient cohort. Data are from 286
LuAd samples and 19 adjacent non-tumor tissues, with boxes representing the 25% to 75%
quartiles and lines within the boxes depicting median values. Data are for probe cg10602180
(depicted in the schematic) (*, p < 0.0001, unpaired two-tailed t-test). B. Schematic of CLDN18
gene located in chromosome 3 indicating its two isoforms (lung-specific CLDN18.1 and stomach-
specific CLDN18.2). A CpG island is located in the CLDN18.1 promoter region (modified from
NCBI Reference Sequence). C. In vitro methylation inhibits activity of the CLDN18.1 promoter.
CpGL-300 contains the CpG-rich 300-bp CLDN18.1 promoter fragment in the CpG-less-luciferase
vector CpGL. The indicated plasmids were methylated or mock-methylated and transfected into
MLE-15 cells. * indicates p < 0.05 compared to unmethylated CpGL-300, n = 3, two-way ANOVA.
D. Analysis of the correlation between CLDN18.1 mRNA expression and methylation of its
promoter CpG island (r = 0.5476, p < 0.0001).
C
C
D
33
3.4 Repletion of CLDN18.1 in human LuAd cells inhibits tumor growth
3.4.1 Generation of stable human LuAd cell line with inducible CLDN18.1
expression
To address the role of CLDN18 in suppressing human lung carcinogenesis in vivo, we
conditionally expressed CLDN18.1 in the H23 human LuAd cell line and tested
tumorigenicity in a mouse xenograft model. Cells were transduced with a doxycycline
(Dox)-inducible CLDN18.1 lentiviral vector, yielding cells that were termed H23/C18.
Dox-induced CLDN18.1 expression was confirmed by qRT-PCR, Western analysis and
immunofluorescence (Figure 7 A-C). CLDN18.1 was induced two days prior to
implantation by treating cells with 0.5 µg/ml Dox, a concentration that increased
CLDN18.1 expression to levels comparable to that seen in primary human AT2 cells
(Figure 7 A).
34
Figure 7 Generation of Dox-inducible CLDN18.1 expressing LuAd cell line
A-C. H23/C18 cells were treated with Dox at the indicated concentrations and CLDN18.1
expression was assessed by qRT-PCR (A), Western analysis (B) and immunofluorescence (C).
Human lung alveolar type II (hAT2) cells were used for reference in (A). In C, bar = 10 μm.
A
B
A
C
B
35
3.4.2 Expression of CLDN18.1 in human LuAd cells suppresses xenograft tumor
growth
Dox-treated and control cells were inoculated subcutaneously into the flanks of nude mice
and CLDN18.1 induction continued in vivo by administration of Dox in the diet. Tumors
were detectable in the control group after 2 weeks and their mean volume reached 314 ±
19.90 mm
3
by 6 weeks after inoculation. In contrast, H23/C18
cells in which CLDN18.1
was induced by Dox did not form tumors until 3 weeks, and their mean volume was only
98 ± 19.36 mm
3
at 6 weeks after inoculation. Tumor weight at 6 weeks was 136 ± 20.35
mg in the control group versus 49 ± 9.22 mg in the CLDN18.1-expressing group (Figure
8 C). As expected, Dox affected neither the size nor the weight of xenograft tumors formed
by the parental (non-transduced) H23 cells, attributing the inhibition of xenograft tumor
growth to CLDN18.1 induction (Figure 8 A-C). The ~70% decrease in tumor volume and
weight (p<0.05) in the Dox-treated group (Figure 8 A-C) confirms a tumor suppressor role
for CLDN18.1 in human LuAd cells in vivo.
A
36
Figure 8 Induction of CLDN18.1 suppresses xenograft tumor growth
A-C. Nude mice were injected with H23/C18 or parental H23 cells and CLDN18.1 expression
was induced by Dox. Tumor volume (D), measured every week, was decreased in Dox-treated
H23/C18 cells compared to other conditions at the same time point (*, p < 0.05, n ≥ 3, two-way
ANOVA). Mice were euthanized 6 weeks after injection and nodules were photographed (E),
excised and weighed (F) (* indicates p < 0.05 compared to H23 cells and untreated H23/C18
cells, n ≥ 3, two-way ANOVA).
B
A
C
C
37
Chapter 4 CLDN18.1 inhibits malignant properties of LuAd
cells in vitro
4.1 CLDN18.1 inhibits cell growth and proliferation of LuAd cells in vitro
In pursuit of cellular mechanisms underlying the tumor suppressor role of CLDN18 in
human LuAd cells, we investigated effects of Dox-induced CLDN18.1 expression on
malignant properties of H23/C18 cells. As shown in Figure 9, Dox treatment reduced the
number of H23/C18
cells on day 3 by ~50%. Dox-induced CLDN18.1 expression resulted
in similar growth inhibition of another human LuAd cell line, H358/C18 (Figure 10 A-B). A
TUNEL assay revealed no difference in apoptosis between Dox-treated and untreated
H23/C18
cells (Figure 11). In contrast, assessment of cell proliferation based on EdU
incorporation demonstrated decreased numbers of EdU
+
cells in Dox-treated compared to
non-treated H23/C18
cells (Figure 12 A-B). As control, Dox treatment did not affect the
number of Edu
+
cells in cultures of parental H23 cells (Figure 13 A-B).
Figure 9 CLDN18.1 inhibits growth of H23/C18 cells
Cell numbers were counted on days 1-5. * indicates p < 0.05 compared to untreated cells at the
same time point, n = 3, two-way ANOVA.
Day
38
Figure 10 CLDN18.1 inhibits growth of H358/C18 cells
A. Immunofluorescence staining of CLDN18 in H358/C18 cell cultures treated with Dox (1
µg/ml) or vehicle. Bar =10 μm. B. Cell number was counted on days 1-5. * indicates p < 0.05
compared to no Dox treatment at the same time point, n = 3, two-way ANOVA.
A
B
A
Day
39
Figure 11 CLDN18.1 does not affect apoptosis
Representative images (A) and quantitative analysis (B) of TUNEL staining of H23/C18 cells in
the presence and absence of Dox.
B
A
A
40
Figure 12 CLDN18.1 inhibits proliferation of LuAd cells
A-B. Cell proliferation was assessed based on Edu incorporation. Shown are representative
fluorescence images with EdU staining in green and CLDN18.1 staining of the same cells in red
(A; bar = 20 μm) and quantitative analysis of the percentage of EdU
+
cells (B). * indicates p <
0.05, n = 3, unpaired two-tailed t-test.
A B
*
41
Figure 13 Dox has no effect on proliferation by parental H23 cells
A. Representative fluorescence images of EdU
+
(green) labeling of H23 cells treated with Dox or
vehicle. Bar = 20 μm. B. Quantitative analysis of the percentage of EdU
+
cells.
A B
42
4.2 Induced expression of CLDN18.1 decreases migration and invasion of LuAd
cells
We next investigated effects of CLDN18.1 on cell migration and invasion using Boyden
chamber assays. Dox-induced CLDN18.1 expression decreased the number of cells that
crossed the membrane in the migration assay by ~2-fold (Figure 14 A-B). Similarly, Dox
decreased the number of cells that crossed the Matrigel-coated membrane in the invasion
assay by ~16-fold (Figure 15 A-B).
Figure 14 CLDN18.1 decreases migration of LuAd cells
A-B. The effects of Dox on migration was assessed in Transwell assays by staining of cells that
crossed the membrane with Calcein AM dye. Shown are representative images of cells that
crossed the membrane (A; bar = 100 μm) and quantitative analyses of the relative fluorescent
units (RFU) (B). * indicates p < 0.05, n = 3, unpaired two-tailed t-test.
A
B
*
43
Figure 15 CLDN18.1 decreases invasion of LuAd cells
A-B. The effects of Dox on invasion was assessed in Transwell assays by staining of cells that
crossed the membrane with Calcein AM dye. Shown are representative images of cells that
crossed the membrane (A; bar = 100 μm) and quantitative analyses of the data (B). * indicates
p < 0.05, n = 3, unpaired two-tailed t-test.
4.3 CLDN18.1 reduces anchorage-independent growth of LuAd cells
We next tested the effect of CLDN18.1 on anchorage-independent growth in H23/C18
cells. As shown in Figure 16 A-C, CLDN18.1 markedly inhibited both the number and size
of colonies formed by H23/C18 cells in soft agar. As control, Dox treatment did not affect
anchorage-independent growth of parental H23 cells (Figure 17 A-C).
A B
+
44
Figure 16 CLDN18.1 suppresses colony forming ability of LuAd cells
A-C. Dox decreases anchorage-independent growth. Colonies formed in soft agar in the absence
and presence of Dox are shown at low (top, whole well), medium (middle, bar = 200 μm) and
high magnification (bottom, bar = 50 μm), and the data are quantitated for colony number (B)
and size (C). In B and C, * indicates p < 0.05, n=3, unpaired two-tailed t-test.
A
B
C
45
Figure 17 Dox does not affect anchorage-independent growth in parental H23
cells
A-C. Colonies formed in soft agar by H23 cells in the presence or absence of Dox are shown at
low (top, whole well), medium (middle, bar = 200 μm) and high magnification (bottom, bar = 50
μm), and the data are quantitated for colony number (B) and size (C), n = 3, unpaired two-tailed
t-test.
C
A B
46
Chapter 5 Molecular mechanisms mediating the tumor
suppressor role of CLDN18.1
5.1 CLDN18.1 suppresses IGF-1R/AKT signaling
We employed two experimental systems, primary AT2 cells from Cldn18
-/-
mice and
H23/C18 cells with Dox-inducible CLDN18.1, to pursue pathways involved in the tumor
suppressor function of CLDN18 in lung alveolar epithelial cells in an unbiased fashion.
5.1.1 Loss of Cldn18 activates IGF/AKT signaling in whole lung and AT2 cells
First, we investigated global gene expression in AT2 cells freshly isolated from Cldn18
-/-
versus wild-type control mice. Ingenuity Pathway Analysis (IPA) of 135 differentially
expressed genes suggested that insulin-like growth factor 1 (IGF-1) signaling is the most
altered pathway in AT2 cells isolated from Cldn18
-/-
mice (Figure 18). Indeed, Western
blot analysis with anti-phospho-IGF-1R and pan-IGF-1R Abs indicates specific
upregulation of the phosphorylated active receptor in E18 lung from Cldn18
-/-
as
compared to wild type mice (Figure 19 A). Quantitative analysis indicates ~1.7-fold
increase in IGF-1R phosphorylation/activation upon loss of Cldn18 (Figure 19 B). We also
used the same Western blot to assess the effect of Cldn18
loss on phosphorylation
(activation) of AKT, a major downstream effector of IGF-1R. As expected, activation of
IGF-1R was associated with a 2-fold activation of AKT (Figure 19 C).
47
Figure 18 IPA analysis of differentially expressed genes in AT2 cells from
Cldn18
-/-
and WT mice
IPA analysis of 135 genes differentially expressed in AT2 cells freshly isolated from Cldn18
-/-
and
WT mice (FC > 2, p < 0.05). Line graph represents the ratio between the number of genes in a
given pathway that are present in the list of differentially regulated genes divided by the total
number of genes that make up that pathway in the reference gene set. The significance values
for the canonical pathways represented by bars was calculated using right-tailed Fisher's exact
test.
48
Figure 19 Loss of Cldn18 activates IGF/AKT signaling
A. Western blot analysis using anti-phospho-IGF-1R and pan-IGF-1R Abs in wild type (n=6) and
Cldn18
-/-
(n=7) E18 mice lung. B-C. Quantitation of IGF-1R and AKT phosphorylation. In B and
C, * indicates p < 0.05, n = 3, Z-test.
A
B
C
49
5.1.2 Expression of CLDN18.1 in LuAd cells inhibits IGF-1R/AKT activity
In an independent approach to identify pathways potentially mediating the tumor
suppressor function of CLDN18.1 in LuAd, we subjected Dox-treated and control
H23/C18 human LuAd cells to functional proteomics analysis using RPPA of over 300
antibodies. Supplemental Table 4 lists 77 proteins that were altered by Dox treatment
(fold change ≥ 1.2), and IPA analysis of these proteins again suggested that IGF-1 and
AKT signaling were among the pathways most strongly affected by CLDN18.1 (Figure
20). Indeed, Western analysis demonstrated that Dox-mediated CLDN18.1 induction in
H23/C18 cells resulted in a ~2 fold decrease in the phosphorylation of both IGF-1R and
AKT (Figure 21). In contrast, CLDN18.1 induction in the H23/C18 cells did not alter
ERK1/2 phosphorylation (Figure 22).
Figure 20 IPA analysis of differentially expressed proteins in H23/C18 in the
presence or absence of Dox
The top 77 proteins affected by Dox in H23/C18 cells were interrogated using IPA analysis (FC >
1.2, p < 0.05). The significance values for the canonical pathways represented by bars was
calculated using right-tailed Fisher's exact test.
50
Figure 21 CLDN18.1 suppresses IGF-1R and AKT phosphorylation of LuAd cells
A-C. Representative western blots (A) and quantitative analysis (B-C) of IGF-1R and AKT
phosphorylation in Dox-treated versus control H23/C18 cells. In B and C, * indicates p < 0.05, n
= 3, Z-test.
Figure 22 CLDN18.1 does not change ERK1/2 phosphorylation in H23/C18 cells
A-B. Western (A) and quantitative analysis (B) demonstrate that Dox-induced CLDN18.1 in
H23/C18 cells does not affect ERK1/2 phosphorylation.
A B
C
A
B
51
To further understand how CLDN18.1 regulates the IGF-1R/AKT axis in human LuAd cells,
we tested whether IGF-1 could overcome CLDN18.1-mediated inhibition of IGF-1R/AKT
signaling. Treatment of H23/C18 cells with recombinant IGF-1 (5 ng/ml) resulted in the
expected increase in both IGF-1R and AKT phosphorylation (Figure 23, lane 3 versus 1).
Consistent with Figure 21 A, Dox-mediated CLDN18.1 expression in the absence of IGF-
1 resulted in decreased phosphorylation of both IGF-1R and AKT (Figure 23, lane 2 versus
1). Interestingly, however, Dox-mediated CLDN18.1 expression in the presence of IGF-1
resulted in downregulation of AKT phosphorylation without any significant change in IGF-
1R phosphorylation (Figure 23, lane 4 versus 3), suggesting that CLDN18.1-mediated
inactivation of AKT may employ an alternative pathway in addition to the well-established
IGF-1R/AKT axis.
Figure 23 Dox treatment of the H23/C18 cells in the presence of IGF-1
H23/C18 cells were treated with Dox to induce CLDN18.1 and/or IGF-1, followed by Western
analysis with anti-phospho-IGF-1R Abs, anti-phospho-AKT Abs and the respective pan-Abs.
52
Note persistent CLDN18.1-mediated AKT inactivation, but not IGF-1R inactivation, in the
presence of IGF-1 ligand.
5.2 Inhibition of YAP/TAZ activity contributes to the tumor suppresser role of
CLDN18.1
5.2.1 Tumors in both Cldn18
-/-
mice and human LuAd show increased YAP activity
We have shown that enhanced YAP activity contributes to lung enlargement and
increased AT2 cell proliferation in Cldn18
-/-
mice (24). Sustained or elevated YAP
expression levels have also been noted in a wide range of cancers including lung cancer.
Gene amplifications and epigenetic modulation of the YAP locus in cancer are similarly
prevalent, which implies that YAP-mediated transcriptional activity is important for the
development and sustainability of neoplasia. Therefore, we wondered whether YAP
signaling was also involved in tumorigenesis in the lungs of Cldn18
-/-
mice. We harvested
lungs from aged WT and Cldn18
-/-
mice and conducted YAP staining. Tumors and
surrounding tissues in Cldn18
-/-
mice both show strong nuclear YAP while control mice
lung show more cytoplasmic YAP (Figure 24), suggesting that YAP-mediated increases
in proliferation following CLDN18 downregulation may contribute to tumorigenesis.
Consistent with the observations in Cldn18
-/-
mice, staining of YAP in LuAd samples
revealed increased nuclear distribution compared to non-tumor lung samples (Figure 25).
53
Figure 24 Loss of Cldn18 increases nuclear YAP in mouse lung
Immunofluoresence shows nuclear YAP (red) in Cldn18
-/-
lungs in areas both with and without
tumor. Lower panel shows higher magnification views of rectangle in upper panel. 4',6-diamidino-
2-phenylindole (DAPI) is the nuclear counterstain. n = 2. Scale bar = 20 μm.
WT
Cldn18
-/-
non-tumor
Cldn18
-/-
tumor
54
Figure 25 Nuclear YAP is increased in human LuAd
IF shows nuclear YAP is increased in human LuAd compared to normal lung. Lower panel shows
higher magnification views of rectangle in upper panel. DAPI is used as the nuclear counterstain.
n = 2. Scale bar: 20 μm.
55
5.2.2 CLDN18.1 inhibits YAP/TAZ signaling in LuAd cell lines
CLDN18 downregulates YAP in murine AT2 cells (24) and YAP and TAZ in human
H23/C18 cells. Given the known cross-talk between AKT and YAP/TAZ (48–54), we
wondered whether YAP and/or TAZ could also play a role in CLDN18.1-mediated AKT
regulation in human LuAd cells. We first tested the effect of CLDN18.1 expression in LuAd
cells on the levels of YAP and TAZ, as well as their downstream targets. Western analysis
demonstrates strong downregulation of TAZ, with weaker downregulation of YAP (Figure
26), and qRT-PCR analysis indicates 3-5 fold inhibition of YAP/TAZ target gene
expression by CLDN18.1 (Figure 26), indicating negative regulation of YAP/TAZ by
CLDN18.
Figure 26 CLDN18.1 downregulates the expression of TAZ and YAP
Representative western blot and quantitative analysis of TAZ and YAP in Dox-treated versus
control H23/C18 cells. * indicates p < 0.05, n = 3, Z-test.
56
Figure 27 CLDN18.1 suppresses expression of YAP/TAZ target genes
qRT-PCR analysis of YAP/TAZ target gene expression in Dox-treated versus untreated H23/C18
cells. * indicates p < 0.05, n = 3, Z-test.
5.3 Contribution of YAP/TAZ to CLDN18.1-mediated regulation of AKT
We then tested by siRNA silencing the potential roles of YAP and TAZ in regulating AKT.
As shown in Figure 28 A and B, TAZ depletion resulted in AKT inactivation (lane 3) to
levels comparable to those observed after Dox-induced CLDN18.1 expression (lane 5),
indicating that TAZ stimulates AKT and that its inhibition by CLDN18.1 (Figure 28 B) may
thus play a role in CLDN18.1-mediated AKT inactivation. Compared to TAZ silencing,
the effect of YAP silencing on AKT phosphorylation was difficult to assess because,
consistent with previous studies, YAP silencing strongly induced TAZ (Figure 28 A and B,
lane 2 and 6), potentially resulting in two opposing effects: direct AKT inactivation as a
57
result of decreased YAP and indirect (TAZ-dependent) AKT activation. Nevertheless,
stimulation of AKT by YAP is suggested by lack of AKT activation in siYAP-transfected
cells (Figure 28 A and B, lane 2 versus 1) despite a robust (likely compensatory) increase
in TAZ expression upon YAP silencing. In addition to establishment of a
CLDN18.1/YAP/TAZ/AKT axis, the YAP/TAZ silencing study also demonstrates that
CLDN18.1 further inhibits AKT activation in cells depleted of both YAP and TAZ (Figure
28 A and B, lane 8 versus 4), indicating that inhibition of YAP/TAZ is insufficient for
execution of the full negative regulation of AKT by CLDN18.1 and consistent with residual
activation via a CLDN18.1/IGF1-R/AKT axis. These results suggest that
CLDN18.1/YAP/TAZ/AKT and CLDN18.1/IGF1-R/AKT axes regulate AKT activity via two
parallel pathways (Figure 29), although we cannot rule out additional interactions between
the two pathways.
58
Figure 28 Role for YAP/TAZ in CLDN18.1-mediated AKT inactivation in H23/C18
cells
A. H23/C18 cells were treated with Dox to induce CLDN18.1 while YAP and/or TAZ were silenced
with siRNA, followed by Western analysis with the indicated antibodies. B. Quantitative analysis
of AKT activation based on optical density of the pAKT and total AKT bands. *, p < 0.05 compared
to control with no Dox treatment; ┼, p < 0.05 compared to siTAZ/YAP with no Dox treatment, n =
4, two-way ANOVA
B
A
59
Chapter 6 Discussion
6.1 Model for the role of CLDN18.1 in regulation of lung tumorigenesis
In a recent study in Cldn18
-/-
mice, we showed a critical non-redundant role for CLDN18
in regulating alveolar epithelial TJ composition and permeability properties (28). In
addition, loss of Cldn18 led to lung enlargement due to increased abundance and
proliferation of AT2 cells, suggesting a novel role for CLDN18 in regulating lung epithelial
progenitor cell homeostasis. Further study also revealed involvement of the Hippo-YAP
pathway in CLDN18-mediated organ size regulation and stem cell homeostasis.
Interestingly, Cldn18
-/-
mice showed increased propensity to develop LuAd with age.
Analyses of LuAd patient cohorts showed LuAd stage-dependent reduction of CLDN18.
Furthermore, low levels of CLDN18.1 expression were associated with poor LuAd patient
survival, suggesting a clinically significant role of CLDN18. In this study, we demonstrate
a tumor suppressor role of CLDN18 in human LuAd. By restoration of CLDN18.1
expression in human LuAd cell lines to levels observed in normal AT2 cells, we
discovered a strong inhibition of tumor growth in a mouse xenograft model. Contributing
to this tumor suppressor activity, repletion of CLDN18.1 in LuAd cells resulted in
decreased proliferation, migration, invasion and anchorage-independent colony formation
in vitro.
A role for AKT downstream of CLDN18.1 was indicated by functional proteomics analysis
of >300 proteins in H23/C18 human LuAd cells, in which CLDN18.1 was induced by Dox.
PI3K/AKT was the pathway most enriched in the set of 77 proteins altered in response to
60
CLDN18.1 induction. A 2-fold decrease in AKT phosphorylation, with no change in ERK
phosphorylation, was confirmed by Western analysis. Interestingly, studies in Cldn18
-/-
mice showed that AKT phosphorylation is also regulated by CLDN18 in non-malignant
cells from both embryonic lungs and adult AT2 cells and may therefore be involved in
tumor initiation associated with loss of CLDN18. These results are consistent with
inhibition of AKT recently observed in A549 LuAd cells upon CLDN18.1 expression
(55,56).
Several mechanisms could account for attenuation of AKT activity by CLDN18. By
utilizing high-throughput data including microarray and protein array analyses of AT2
cells from Cldn18
-/-
mice and a human LuAd cell line, respectively, we found that the IGF-
1R/AKT pathway is consistently implicated in the tumor suppressor activity of CLDN18.1.
In addition, immunofluorescence of both tumors from Cldn18
-/-
mice and human LuAd
patient samples showed increased nuclear YAP, suggesting enhanced YAP activity
following the loss of CLDN18. Restoration of CLDN18.1 in human LuAd cell lines
decreased the expression of TAZ and to a lesser extent YAP and their downstream target
genes, confirming a role of CLDN18.1 in suppressing YAP/TAZ signaling in malignant
cells. By using siRNA silencing of YAP and TAZ we found a potential role of YAP and
TAZ in regulating AKT activity, suggesting a CLDN18.1—YAP/TAZ—AKT axis. These
data indicate a tumor suppressor role for CLDN18.1 in LuAd mediated by a regulatory
network that encompasses YAP/TAZ, IGF-1R and AKT signaling (Figure 29). Additional
potential interactions between the CLDN18.1/YAP/TAZ/AKT and the CLDN18.1/IGF1-
R/AKT axes remain to be elucidated. Molecular mechanisms by which CLDN18.1
integrates regulation of YAP/TAZ on the one hand, and IGF-1R on the other hand, to
61
inactivate AKT remain largely unknown.
Figure 29 Working model for the tumor suppressor activity of CLDN18.1 in LuAd
CLDN18.1 attenuates a number of tumorigenic properties in lung alveolar epithelial cells.
Claudin-18 Inhibits AKT via effects on both IGF-1R and YAP/TAZ. Additional potential
interactions between the CLDN18.1/YAP/TAZ/AKT and the CLDN18.1/IGF1-R/AKT axes remain
to be elucidated.
6.2 Molecular mechanisms underlying CLDN18 regulation of IGF-1R/AKT
The IGF-1 pathway has been implicated in induction and maintenance of a range of
different malignancies including lung cancer (57,58). Survival signals emanating from the
62
IGF-1R are primarily through two downstream signaling pathways: the
phosphatidylinositol-3 kinase-AKT (PI3K/AKT) pathway and the Ras-Raf-MEK-ERK
pathway (also known as the mitogen-activated protein kinase (MAPK) pathway), resulting
in increased cell proliferation, apoptosis inhibition and cancer cell motility (59). Due to the
central role of the IGF-1R in cell cycle progression and transformation, targeting IGF-1R
may constitute an important paradigm in preventing oncogenesis (60). The PI3K/AKT
pathway is a signal transduction pathway involved in the regulation of multiple cellular
functions including cell proliferation, survival, differentiation, adhesion, motility and
invasion. The PI3K/AKT pathway is frequently activated in NSCLC (40,61). Aberrant
activation of the PI3K/AKT pathway has been reported in more than 40% of LuAd cases
from TCGA network (62,63). Moreover, an abnormally activated PI3K/AKT pathway is
one of the mechanisms of acquired resistance to tyrosine kinase inhibitors (TKi) in LuAd
patients(40). A novel finding in the present work is the inhibitory effect of CLDN18.1 on
IGF-1R and AKT. A decrease in IGF-1R and AKT phosphorylation was observed in
H23/C18 LuAd cells in vitro in response to Dox-induced CLDN18.1 expression, and IGF-
1R and AKT are more highly phosphorylated in lungs isolated from Cldn18
-/-
compared to
control mice. In contrast to AKT, ERK phosphorylation was not altered upon CLDN18.1-
mediated IGF-1R inactivation in H23 cells, suggesting that PI3K/AKT rather than the
MAPK pathway is involved in CLDN18.1-mediated IGF-1R inhibition. This is also
supported by recent work in A549 LuAd cells, which indicated no significant effects on
phosphorylation of either EGFR or downstream kinases such as Raf, MEK1, ERK1/2,
JNK and p38 (55).
Inactivation of IGF-1R by junctional proteins is not without precedent. For example, E-
63
cadherin, an essential adhesion junctional protein as well as a tumor suppressor that is
silenced in many cancers, can negatively regulate IGF-1R in MDCK cells (64). In that
study, IGF-1 ligand could barely activate the IGF-1R in MDCK cells unless the cells were
treated with E-cadherin neutralizing antibody or calcium depletion. To test whether E-
cadherin can disrupt the binding of IGF-1 to its receptor, researchers incubated cells with
labeled IGF-1 and covalently fixed the ligand bound to IGF-1R. They found ligand-bound
IGF-1R was readily detected in cells incubated with the E-cadherin neutralizing antibody
or calcium depletion, while ligand-bound receptor was barely detectable in cells grown
under normal conditions. Moreover, they found that the number of IGF-1R on the cell
surface was independent of E-cadherin activity, suggesting E-cadherin-dependent
inhibition of IGF-1R activation was due to impaired binding affinity of IGF-1 ligand to its
receptor. Strikingly, the cellular response to 500 ng/ml of IGF-1 was similar to that
produced by 5 ng/ml of IGF-1 under conditions of calcium depletion. This observation
excluded the possibility that decreased IGF-1R activity is due to insufficient IGF-1 ligand.
However, this explanation may not be applicable to the CLDN18.1-mediated IGF-1R
activity loss. As shown in Figure 24, adding IGF-1 ligand to the culture medium could
reverse the inhibitory effect of CLDN18.1 on IGF-1R activity. IGF-1R phosphorylation
level is altered but total IGF-1R protein remains unchanged (Figure 22 A and Figure 24
A). IGF-1R mRNA (data not shown) also remains the same in the Cldn18
-/-
mouse model
and human LuAd cell line model, suggesting that the inhibitory effect of CLDN18.1 on
IGF-1R activity is not through accelerating IGF-1R turnover or internalization or through
inhibiting IGF-1R transcription.
One possible mechanism by which CLDN18.1 inhibits IGF-1R activity could be through
64
dephosphorylating the receptor. As shown in Figure 4, claudin proteins have long
intracellular COOH-terminal tails where a PDZ-binding motif is located. This motif binds
to PDZ domains on several TJ scaffolding proteins, such as ZO-1, ZO-2, and ZO-3 (ZOs)
and MUPP1 (65). Interestingly, there is evidence that ZO-1 co-precipitates IGF-1R (66),
suggesting that CLDN18.1 could be in the same complex with IGF-1R through ZO-1 via
the PDZ domain. Although there is no direct evidence showing that ZO-1
dephosphorylates IGF-1R, studies in colorectal and pancreatic cancer models have
suggested that ZO-1 interacts with the EGFR and overexpression of ZO-1 in multiple
myeloma cells inhibits EGFR phosphorylation (48). PTEN, a protein tyrosine phosphatase
(PTP), harbors a functional PDZ domain-binding motif at its C-terminal tail (67); therefore,
although a direct interaction between ZO-1 and PTEN has not been reported, it might
occur in one of the remaining PDZ domains in ZO-1. Thus, CLDN18.1 inhibiting IGF-1R
activity could be through recruiting PTPs such as PTEN via ZO-1 to the proximity of IGF-
1R to dephosphorylate the receptor. Moreover, in addition to providing a basis for
assembly of multiprotein complexes at the cell cytoplasmic surface, ZOs are known to be
engaged in the transmission of signals from the plasma membrane to the nucleus to
regulate gene expression. ZO-1 is associated with the transcription factor ZONAB, while
ZO-2 is associated with the transcription factors Jun, Fos and C/EBP (3,68), providing a
mechanism of how CLDN18.1 protein transmits extracellular signals to regulate
homeostasis at the transcriptional level. The scaffolding proteins could provide platforms
that bring CLDN18.1 to the proximity of diverse signaling molecules including IGF-1R and
thus regulate a variety of cellular and biological events. However, delineation of precise
mechanisms whereby CLDN18.1 regulates IGF1-R activity will require further
65
investigation.
6.3 Molecular mechanisms underlying CLDN18.1 regulation of YAP/TAZ
Several studies have demonstrated that components of the Hippo pathway , including the
downstream components Lats and YAP, interact with membrane-associated proteins,
including proteins found in tight junctions and adherens junctions (Figure 30) (69–71). In
addition, there is accumulating evidence for Hippo kinase-independent modulation of YAP
activity through direct interactions with cell membrane- and cytoskeletal- associated
proteins that regulate apical-basolateral polarity and cell-cell contact resulting in
cytoplasmic and/or membrane sequestration (72). In this regard, AJ proteins E-cadherin
and α-catenin inhibit YAP signaling by both Hippo-independent and -dependent
mechanisms (73,74). For example, α-catenin and YAP interact independent of
phosphorylation by Hippo kinases, whereas p-LATS is required for E-cadherin-mediated
contact inhibition of YAP (75).
66
Figure 30 Interactions of epithelial junctional modules with the core Hippo
pathway
Epithelial tight junction (TJ) and adherens junction (AJ) complexes may function together to sense
the integrity of the epithelial layer and regulate the activity of YAP/TAZ through the core Hippo
pathway. Adapted from Barry M. Gumbiner. Journal of Cell Science (2014) (69).
Our previous work also demonstrates that CLDN18 inhibits YAP activity in murine AT2
cells (24). Loss of Cldn18 leads to increased nuclear YAP and expression of its
downstream target genes, while overexpression of CLDN18 reverses these effects. Co-
immunoprecipitation (co-IP) demonstrated interaction among p-YAP, CLDN18, ZO-1 and
p-LATS1/2 in WT AT2 cells. Furthermore, when overexpressed, CLDN18 and YAP co-
localized at sites of cell-cell contact, suggesting sequestration of p-YAP at TJ. Decreased
67
p-YAP in Cldn18
-/-
lungs and AT2 cells and decreased p-LATS/p-YAP interaction in
membranes of Cldn18
-/-
AT2 cells, suggest that CLDN18 is required for Hippo signaling
and further that CLDN18 serves a scaffolding function that promotes interaction between
the Hippo kinases and p-YAP at TJ. However, since CLDN18 does not contain putative
motifs that can interact with YAP, it is likely that the interaction between CLDN18 and p-
YAP is mediated through other TJ scaffolding proteins such as angiomotin (AMOT) or
ZO-1, which have been shown to interact with YAP. Understanding specific protein-
protein interactions that mediate CLDN18 and p-YAP/YAP interaction requires further
investigation.
Many lines of evidence point to the importance of deregulated Hippo signaling in lung
cancer (28,32,76,77). YAP/TAZ are both highly expressed in LuAd in humans (78,79). In
addition, knocking down either YAP or TAZ in LuAd cells is sufficient to inhibit cell
proliferation, invasion and anchorage-independent growth in vitro and tumor growth in
mice in vivo (76,77,80). In this study, we found that YAP/TAZ changed in both cellular
distribution and transcriptional activity in cancer cells when CLDN18 is overexpressed,
which is consistent with our previous study showing that YAP is activated in Cldn18
-/-
mice. While inhibition of YAP by CLDN18.1 in human H23/C18 LuAd cells was modest,
we observed strong inhibition of its paralog TAZ as well as YAP/TAZ target genes in these
cells (Figure 29). We suggest that CLDN18.1-mediated downregulation of TAZ plays a
role in AKT inactivation because silencing of TAZ mimicked the CLDN18.1-mediated AKT
inactivation (Figure 29). Specific effects on YAP versus TAZ may be context-dependent
and vary among species and experimental systems.
68
6.4 Cross-talk between YAP/TAZ and IGF-1R/AKT signaling
Molecular mechanisms by which CLDN18.1 integrates regulation of YAP/TAZ on the one
hand, and IGF-1R on the other hand, to inactivate AKT (Figure 30) remain largely
unknown. There is evidence that CLDN18 physically interacts with YAP (24) and with
phosphoinositide-dependent kinase-1 (PDK1) which acts upstream of AKT (55),
suggesting direct regulatory mechanisms. Additionally, cross-talk between YAP/TAZ and
IGF-1R/AKT signaling in LuAd may result in regulation of one pathway by the other.
Similar crosstalk has been observed in other contexts. For example, it was shown that
activation of RTKs such as EGFR and IGF-1R results in dissociation of complexes
containing PDK1 and Hippo pathway components, leading to YAP/TAZ activation
(52,59,69). Reciprocally, there is evidence that YAP/TAZ target genes indirectly regulate
the IGF-1R/AKT pathway, for example, through increased expression of IGF-1 and its
binding proteins (48,53) as well as the RTK/PI3K adaptor protein GAB2 (81) and the
PTEN-targeting miR29 (48,82). Finally, it is also plausible that PI3K/AKT stabilizes TAZ
through inhibitory phosphorylation of glycogen synthase kinase-3 beta (GSK3ß) and thus
attenuation of the phosphorylation of the N-terminal phosphodegron in TAZ (49) . In
addition to delineating the relationships among the effects of CLDN18.1 on IGF-1R,
YAP/TAZ and AKT, future studies will need to address their relative contributions to the
various tumor suppressor properties of CLDN18.1 in LuAd.
69
6.5 Regulatory mechanism of CLDN18.1 expression in the lung
Regulation of CLDN18.1 expression in the lung is not well understood. Promoter activity
of claudin-18 has been reported to be increased by NKX2-1 in lung NCI-H441 cells (83).
However, staining of human LuAd samples showed strong expression of NKX2-1 and
lung cancer cells including A549 and PC-3 express significant amounts of NKX2-1 mRNA
(24,84). Furthermore, overexpression of NKX2-1 failed to increase the amount of
endogenous claudin-18 in A549 cells (55). A promoter CpG island was identified in the
human CLDN18.1 gene and methylation of that CpG island is significantly higher in LuAd
tumors compared to adjacent non-tumor tissues, suggesting the possibility that
expression of CLDN18.1 is down-regulated by DNA methylation in lung cancer. However,
the cause of increased CLDN18.1 DNA methylation in human LuAd needs to be further
investigated.
6.6 Conclusions and Perspectives
In this study, we provide several lines of evidence to support an important role of
CLDN18.1 in suppressing lung cancer. The tumor suppressor activities of CLDN18.1
observed include inhibition of xenograft tumor growth in vivo, attenuation of various
malignant phenotypes in vitro and regulation of multiple oncogenic pathways. We
demonstrate that CLDN18.1 inhibits the IGF-1R/AKT and the YAP/TAZ/AKT axes and
that high CLDN18.1 expression is associated with better LuAd patient survival. Both in
vitro and in vivo data strongly support a tumor suppressor role for CLDN18.1 in LuAd and
expression of CLDN18.1 may be of prognostic value in LuAd management. Importantly,
70
the fact that CLDN18.1 can affect multiple cancer-survival pathways makes it a potential
therapeutic target for refractory or drug-resistant cancers.
71
Appendix
Appendix A: RT-PCR primer sequences
Appendix B: siRNA sequences
72
Appendix C: List of genes differentially expressed in AT2 cells from Cldn18
-/-
versus
wild-type mice (FC>2, p<0.05)
Total RNA was extracted from AT2 cells from wild type and Cldn18
-/-
mice using an RNeasy
kit and global expression profiling was performed in quadruplicate using Mouse Ref8 v2.0
BeadChips (IIlumina, San Diego, CA) by the Southern California Genotyping Consortium at
the University of California Los Angeles (UCLA). Raw data processing was done using
GenomeStudio (Illumina) and extended analyses were carried out using R. The complete
microarray dataset has been deposited to Gene Expression Omnibus (GEO) with the
accession number GSE106233.
Upregulated genes
Gene
symbol
Entrez Gene Name
Fold
Change
H2-Ab1 major histocompatibility complex, class II, DQ beta 1 7.61
H2-Aa major histocompatibility complex, class II, DQ alpha 1 3.58
Psca prostate stem cell antigen 3.53
Cxcl14 C-X-C motif chemokine ligand 14 3.38
Slc26a4 solute carrier family 26 member 4 3.34
H2-Ea major histocompatibility complex, class II, DQ E 3.29
Chia chitinase, acidic 3.27
Nnat neuronatin 3.06
Pla2g1b phospholipase A2 group IB 3.05
Clu clusterin 2.94
Napsa napsin A aspartic peptidase 2.90
Lcn2 lipocalin 2 2.73
Psmb1 proteasome subunit beta 1 2.67
Cxcl14 C-X-C motif chemokine ligand 14 2.67
Ear4 eosinophil-associated, ribonuclease A family, member 4 2.53
H2-Ab1 major histocompatibility complex, class II, DQ beta 1 2.48
Kcnk1 potassium two pore domain channel subfamily K member 1 2.45
Rasl11a RAS like family 11 member A 2.45
Ear2 eosinophil-associated, ribonuclease A family, member 2 2.37
Kcnj15 potassium voltage-gated channel subfamily J member 15 2.36
Lgi3 leucine rich repeat LGI family member 3 2.33
Tspan8 tetraspanin 8 2.31
Itgae integrin subunit alpha E 2.27
Ccl17 C-C motif chemokine ligand 17 2.27
73
Napsa napsin A aspartic peptidase 2.26
Per2 period circadian regulator 2 2.25
Adora2b adenosine A2b receptor 2.25
Spp1 secreted phosphoprotein 1 2.24
Aqp5 aquaporin 5 2.22
Tlr2 toll like receptor 2 2.19
Stbd1 starch binding domain 1 2.17
Apoc1 apolipoprotein C1 2.15
Rasl11b RAS like family 11 member B 2.15
Napsa napsin A aspartic peptidase 2.14
Irx2 iroquois homeobox 2 2.10
Rbpjl
recombination signal binding protein for immunoglobulin kappa J region
like 2.10
Krt79 keratin 79 2.09
Calca calcitonin related polypeptide beta 2.08
Cdh16 cadherin 16 2.07
Per2 period circadian regulator 2 2.06
Ctsk cathepsin K 2.06
Hsph1 heat shock protein family H (Hsp110) member 1 2.05
Lamb3 laminin subunit beta 3 2.04
Arfgef3 ARFGEF family member 3 2.04
Kng2 kininogen 2 2.04
Arhgap8 Rho GTPase activating protein 8 2.03
Il33 interleukin 33 2.03
Pon3 paraoxonase 3 2.01
Lamp3 lysosomal associated membrane protein 3 2.01
Downregulated genes
Gene
symbol
Entrez Gene Name
Fold
Change
Cldn18 claudin 18 -11.93
Egr1 early growth response 1 -10.35
Cyr61 cysteine rich angiogenic inducer 61 -6.86
Fos Fos proto-oncogene, AP-1 transcription factor subunit -6.56
Cyr61 cysteine rich angiogenic inducer 61 -4.93
Slc4a1 solute carrier family 4 member 1 (Diego blood group) -4.84
Cyr61 cysteine rich angiogenic inducer 61 -4.38
Acta1 actin, alpha 1, skeletal muscle -4.35
Alas2 5'-aminolevulinate synthase 2 -4.14
Igfbp2 insulin like growth factor binding protein 2 -3.99
Snca synuclein alpha -3.84
Myl1 myosin light chain 1 -3.66
Egr2 early growth response 2 -3.29
Spon2 spondin 2 -3.24
Lor loricrin -3.08
74
Bex2 brain expressed X-linked 1 -3.05
Rsad2 radical S-adenosyl methionine domain containing 2 -3.05
Igfbp2 insulin like growth factor binding protein 2 -2.94
Snca synuclein alpha -2.90
Krt13 keratin 13 -2.89
St8sia4 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 -2.68
Arrdc3 arrestin domain containing 3 -2.67
Pcdh17 protocadherin 17 -2.64
Tmem106b transmembrane protein 106B -2.56
Hbb-b1 hemoglobin subunit beta -2.54
Tnnc2 troponin C2, fast skeletal type -2.49
Krtdap keratinocyte differentiation associated protein -2.45
Ppbp pro-platelet basic protein -2.44
Klk1b22 kallikrein related peptidase 3 -2.41
Lce3f late cornified envelope 3A -2.39
Gucy1a3 guanylate cyclase 1 soluble subunit alpha -2.36
Asprv1 aspartic peptidase, retroviral-like 1 -2.35
Pcolce2 procollagen C-endopeptidase enhancer 2 -2.34
Faim3 Fc fragment of IgM receptor -2.32
Crct1 cysteine rich C-terminal 1 -2.32
Ltf lactotransferrin -2.30
Ppbp pro-platelet basic protein -2.30
Pcdh1 protocadherin 1 -2.30
Igfbp3 insulin like growth factor binding protein 3 -2.29
Ehd4 EH domain containing 4 -2.28
Flt1 FMS-related tyrosine kinase 1 -2.28
Slc25a37 solute carrier family 25 member 37 -2.28
Bex4 brain expressed X-linked 4 -2.26
Klf2 Kruppel like factor 2 -2.25
Ets1 ETS proto-oncogene 1, transcription factor -2.24
Hlx H2.0 like homeobox -2.24
Fam198b family with sequence similarity 198, member B -2.24
Rptn repetin -2.24
Mylpf myosin light chain, phosphorylatable, fast skeletal muscle -2.21
Sema5a semaphorin 5A -2.19
Ptprb protein tyrosine phosphatase, receptor type B -2.19
Junb JunB proto-oncogene, AP-1 transcription factor subunit -2.17
Synm synemin -2.17
Pdgfb platelet derived growth factor subunit B -2.17
Zeb2 zinc finger E-box binding homeobox 2 -2.15
Atp2a1 ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 -2.14
Tnni2 troponin I2, fast skeletal type -2.13
Ankrd1 ankyrin repeat domain 1 -2.12
Mzb1 marginal zone B and B1 cell-specific protein 1 -2.11
Ece1 endothelin converting enzyme 1 -2.11
Uap1 UDP-N-acetylglucosamine pyrophosphorylase 1 -2.10
Myh8 myosin heavy chain 8 -2.08
75
Angpt2 angiopoietin 2 -2.08
Satb1 SATB homeobox 1 -2.08
Clec1a C-type lectin domain family 1 member A -2.07
Camp cathelicidin antimicrobial peptide -2.07
Prickle1 prickle planar cell polarity protein 1 -2.07
Igfbp5 insulin like growth factor binding protein 5 -2.06
Tcf4 transcription factor 4 -2.06
Plvap plasmalemma vesicle associated protein -2.05
Bmper BMP binding endothelial regulator -2.04
Arhgef15 Rho guanine nucleotide exchange factor 15 -2.04
Hoxa5 homeobox A5 -2.04
Mapt microtubule associated protein tau -2.04
Hspb6 heat shock protein family B (small) member 6 -2.04
Scgb3a1 secretoglobin family 3A member 1 -2.03
Cldn5 claudin 5 -2.03
Dgkg diacylglycerol kinase gamma -2.03
Krt4 keratin 4 -2.03
Serpinb12 serpin family B member 12 -2.01
Lce3b late cornified envelope 3C -2.01
Hbb-b1 hemoglobin subunit beta -2.01
Hoxb5 homeobox B5 -2.00
76
Appendix D: List of 77 proteins altered with Dox treatment in H23/C18 cells
H23/C18 cells were treated with Dox or vehicle and frozen cell pellets were submitted for
protein array analysis by the Functional Proteomics RPPA Core Facility, MD Anderson
Cancer Center. Cell extracts were printed on slides in serial dilutions and probed with 306
antibodies. Raw data processing was performed by the core facility and Level 3 protein
measurements were used to evaluate Dox-induced changes.
Gene
Name
FC (Dox vs.
control)
Validation
Status
Catalog # Company
Antibody
Origin
DUSP4/M
KP2
DUSP4 -2 V 5149 CST Rabbit
NDRG1
(phospho
T346)
NDRG1 -1.6 V 3217 CST Rabbit
AKT
(phospho
S473)
AKT1 -1.6 V 9271 CST Rabbit
TAZ TAZ -1.4 V 4883 CST Rabbit
HES1 HES1 -1.4 V 11988 CST Rabbit
Caveolin-
1
CAV1 -1.4 V 3238 CST Rabbit
IGFBP2 IGFBP2 -1.3 V 3922 CST Rabbit
AKT
(phospho
T308)
AKT1 -1.3 V 2965 CST Rabbit
eEF2K EEF2K -1.2 V 3692 CST Rabbit
beta
Catenin
CTNNB1 -1.2 V 9562 CST Rabbit
Transferri
n
Receptor
TFRC -1.2 V 22500002
Novus
Biologicals
Rabbit
Rb
(phospho
S807/S81
1)
RB1 -1.2 V 9308 CST Rabbit
PTEN PTEN -1.2 V 9552 CST Rabbit
PKA RI
alpha
PRKAR1A -1.2 V 5675 CST Rabbit
Notch1 NOTCH1 -1.2 V 3268 CST Rabbit
Mcl 1 MCL1 -1.2 V 5453 CST Rabbit
77
MDM2
(phospho
S166)
MDM2 -1.2 V 3521 CST Rabbit
HSP27
(phospho
S82)
HSBP1 -1.2 V 2401 CST Rabbit
Glycogen
Synthase
GYS1 -1.2 V 3886 CST Rabbit
Cyclin B1 CCNB1 -1.2 V 1495-1 Epitomics Rabbit
Chk1
(phospho
S296)
CHEK1 -1.2 V ab79758 Abcam Rabbit
Bcl2A1 BCL2A1 -1.2 V PAB8528 Abnova Rabbit
Axl AXL -1.2 V 8661 CST Rabbit
Annexin I ANXA1 -1.2 V 610066
BD
Biosciences
Mouse
53BP1 TP53BP1 -1.2 V 4937 CST Rabbit
p70 S6
Kinase
(phospho
T389)
RPS6KB1 1.2 V 9205 CST Rabbit
Smad4 SMAD4 1.2 V sc-7966 Santa Cruz Mouse
SOD1 SOD1 1.2 V 4266 CST Mouse
SF2/ASF SRSF1 1.2 V 32-4500 Invitrogen Mouse
SCD SCD 1.2 V sc-58420 Santa Cruz Mouse
Rad50 RAD50 1.2 V 05-525 Millipore Mouse
PARP-1 PARP1 1.2 V sc-7150 Santa Cruz Rabbit
N-Ras NRAS 1.2 V sc-31 Santa Cruz Mouse
ErbB2/HE
R2
ERBB2 1.2 V
MS-325-
P1
Lab Vision Mouse
ERCC1 ERCC1 1.2 V sc-17809 Santa Cruz Mouse
CD49b ITGA2 1.2 V 611016
BD
Biosciences
Mouse
CD31 PECAM1 1.2 V M0823 Dako Mouse
Bcl2 BCL2 1.2 V M0887 Dako Mouse
Annexin
VII
ANXA7 1.2 V 610668
BD
Biosciences
Mouse
ADAR1 ADAR 1.2 V ab88574 Abcam Mouse
Src SRC 1.3 V 05-184 Millipore Mouse
ERRFI1/M
IG6
ERRFI1 1.3 V
WH00542
06M1
Sigma-Aldrich Mouse
Gab2 GAB2 1.3 V 3239 CST Rabbit
GSK-
3alpha/bet
a
GSK3A/G
SK3B
1.3 V sc-7291 Santa Cruz Mouse
E2F-1 E2F1 1.3 V sc-251 Santa Cruz Mouse
78
Cyclophili
n F
PPIF 1.3 V ab110324 Abcam Mouse
BAP1 BAP1 1.3 V sc-28383 Santa Cruz Mouse
Estrogen
Receptor
ESR1 1.4 V RM-9101 Lab Vision Rabbit
VDAC1/P
orin
VDAC1 1.5 V ab14734 Abcam Mouse
p70/S6K1 RPS6KB1 1.8 V ab32529 Abcam Rabbit
Rb RB1 1.2 Q 9309 CST Mouse
Caspase-
8
CASP8 1.2 Q 9746 CST Mouse
eEF2 EEF2 -1.3 C 2332 CST Rabbit
PAR PAR -1.3 C
4336-
BPC-100
Trevigen Rabbit
PAICS PAICS -1.3 C
HPA0358
95
Sigma-Aldrich Rabbit
LDHA LDHA -1.3 C 3582 CST Rabbit
mTOR
(phospho
S2448)
MTOR -1.2 C 2971 CST Rabbit
beta Actin ACTB -1.2 C 4970 CST Rabbit
TSC1/Ha
martin
TSC1 -1.2 C 4906 CST Rabbit
TRIM25 TRIM25 -1.2 C ab167154 Abcam Rabbit
Merlin/NF
2
NF2 -1.2 C 22710002
Novus
Biologicals
Rabbit
IGFRb IGF1R -1.2 C 3027 CST Rabbit
Connexin
43
CNST43 -1.2 C 3512 CST Rabbit
CDK1 CDK1 -1.2 C ab32384 Abcam Rabbit
ARID1A ARID1A -1.2 C
HPA0054
56
Sigma-Aldrich Rabbit
Stat3 STAT3 1.2 C 4904 CST Rabbit
RPA32
(Phospho
S4/S8)
RPA2 1.2 C
A300-
245A
Bethyl Rabbit
IRF-1 IRF1 1.2 C sc-497 Santa Cruz Rabbit
Hif-1
alpha
HIF1A 1.2 C 610958
BD
Biosciences
Mouse
H2AX
(phospho
S140)
H2AFX 1.2 C MA1-2022
Pierce
Biotechnology
Mouse
Epithelial
Membran
e Antigen
EMA 1.2 C
M061329-
2
Dako Mouse
Beclin BECN1 1.2 C sc-10086 Santa Cruz Goat
Ubiquityl
Histone
H2B
H2BFM 1.3 C 05-1312 Millipore Mouse
79
PRAS40 AKT1S1 1.3 C AHO1031 Invitrogen Mouse
HSP27 HSBP1 1.3 C 2402 CST Mouse
YAP
(phospho
S127)
YAP1 -1.2 E 4911 CST Rabbit
VHL/EPP
K1
EPPK1 1.3 E 556347
BD
Biosciences
Mouse
80
Appendix E: List of 80 proteins altered in AT2 cells isolated from Cldn18
-/-
mice
AT2 cells isolated from WT or Cldn18
-/-
mice and frozen cell pellets were submitted for
protein array analysis by the Functional Proteomics RPPA Core Facility, MD Anderson
Cancer Center. Cell extracts were printed on slides in serial dilutions and probed with 306
antibodies. Raw data processing was performed by the core facility and Level 3 protein
measurements were used to evaluate Dox-induced changes.
Official Ab Name
Gene
Name
FC (Cldn18
-/-
vs. WT)
Validation
Status
Catalog
#
Company
Antibody
Origin
Histone H3
H3F3A
,
H3F3B
-5.2 V ab1791 Abcam Rabbit
Beclin BECN1 -2.5 C
sc-
10086
Santa
Cruz
Goat
Dimethyl-Histone H3
(Lys4)
HISTH
3
-2.4 V 07-030 Millipore Rabbit
beta Catenin
CTNN
B1
-2.3 V 9562 CST Rabbit
p27/KIP 1 (phospho
T198)
CDKN
1B
-2.1 V ab64949 Abcam Rabbit
p21
CDKN
1A
-2.0 V sc-397
Santa
Cruz
Rabbit
PDK1 PDPK1 -1.9 V 3062 CST Rabbit
Androgen Receptor AR -1.7 V ab52615 Abcam Rabbit
Shc (phospho Y317) SHC1 -1.7 V 2431 CST Rabbit
Fatty Acid Synthase FASN -1.6 V 3180 CST Rabbit
Granzyme B GZMB
-1.6 V
4275 CST Rabbit
RPA32 (Phospho S4/S8) RPA32
-1.6 C
A300-
245A Bethyl Rabbit
Acetyl CoA Carboxylase
1
ACAC
A
-1.6 C ab45174 Abcam Rabbit
IRS1 IRS1 -1.5 V 06-248 Millipore Rabbit
Dimethyl-K9 Histone H3
H3K9M
E2
-1.5 C ab32521 Abcam Rabbit
FRA-1 FRA1 -1.5 C sc-605
Santa
Cruz
Rabbit
mTOR (phospho S2448) MTOR -1.5 C 2971 CST Rabbit
Claudin 7 CLDN7 -1.4 V
NB100-
91714
Novus
Biologicals
Rabbit
TUFM TUFM
-1.4 V
ab17330
0 Abcam Rabbit
81
Bim
BCL2L
11
-1.4 V ab32158 Abcam Rabbit
CDK1 CDK1 -1.4 C ab32384 Abcam Rabbit
Stat3 STAT3 -1.3 C 4904 CST Rabbit
Estrogen Receptor alpha
(Phospho S118)
ESR1 -1.3 V ab32396 Abcam Rabbit
PRAS40 (phospho T246)
AKT1S
1
-1.3 V
441100
G
Life
Technolog
ies
Rabbit
NAPSIN A
NAPS
A
-1.3 C
ab12918
9
Abcam Rabbit
Merlin/NF2 NF2 -1.3 C
2271000
2
Novus
Biologicals
Rabbit
p70 S6 Kinase (phospho
T389)
RPS6K
B1
-1.2 V 9205 CST Rabbit
Insulin Receptor beta INSRB
-1.2 C
3025 CST Rabbit
Lck LCK -1.2 V 2752 CST Rabbit
Caspase-7 (cleaved
D198)
CASP7 -1.2 C 9491 CST Rabbit
A-Raf ARAF -1.2 V 4432 CST Rabbit
Caspase-3 active CASP3 -1.2 C ab32042 Abcam Rabbit
Caveolin-1 CAV1 -1.2 V 3238 CST Rabbit
PI3 Kinase p110 alpha
PIK3C
A
-1.2 C 4255 CST Rabbit
RSK
RPS6K
A1
RPS6K
A2
RPS6K
A3
-1.2 C 9347 CST Rabbit
PARK7/DJ1 PARK7 -1.2 V ab76008 Abcam Rabbit
GCLM GCLM
-1.2 C
ab12482
7 Abcam Rabbit
Smad3
SMAD
3
-1.2 V ab40854 Abcam Rabbit
B-Raf BRAF -1.2 V ab33899 Abcam Rabbit
Wee1 WEE1
-1.2 C
4936 CST Rabbit
CD26 CD26 -1.2 V ab28340 Abcam Rabbit
c-Abl ABL -1.2 V 2862 CST Rabbit
Bcl2A1
BCL2A
1
-1.2 V
PAB852
8
Abnova Rabbit
SOD2 SOD2 -1.2 V 13141 CST Rabbit
PTEN PTEN 1.2 V 9552 CST Rabbit
CREB CREB1
1.2 C
9197 CST Rabbit
Cyclin B1
CCNB
1
1.2 V 1495-1 Epitomics Rabbit
Rictor
RICTO
R
1.2 C 2114 CST Rabbit
82
AKT
AKT1,2
,3
1.2 V 4691 CST Rabbit
PAICS PAICS
1.2 C
HPA035
895
Sigma-
Aldrich Rabbit
LRP6 (phospho S1490) LRP6
1.2 V
2568 CST Rabbit
PEA-15 PEA15 1.2 V 2780 CST Rabbit
TFAM TFAM 1.2 V 7495 CST Rabbit
P-Cadherin CDH3 1.2 C 2130 CST Rabbit
Rab25 RAB25 1.2 V 4314 CST Rabbit
Monocarboxylic Acid
Transporter 4
SLC16
A4
1.2 V
AB3314
P
Millipore Rabbit
PLK1 PLK1 1.2 C 4513 CST Rabbit
NDRG1 (phospho T346)
NDRG
1
1.2 V 3217 CST Rabbit
MMP2 MMP2 1.2 V 4022 CST Rabbit
Transferrin Receptor TFRC 1.2 V
2250000
2
Novus
Biologicals
Rabbit
SDHA SDHA 1.2 V 11998 CST Rabbit
S6 (phospho S235/S236) RPS6 1.2 V 2211 CST Rabbit
p38 MAPK (phospho
T180/Y182)
MAPK
14
1.2 V 9211 CST Rabbit
Rb (phospho S807/S811) RB1 1.2 V 9308 CST Rabbit
PAR PAR 1.2 C
4336-
BPC-
100
Trevigen Rabbit
MEK1
MAP2
K1
1.3 V 1235-1 Epitomics Rabbit
14-3-3 zeta
YWHA
Z
1.3 V sc-1019
Santa
Cruz
Rabbit
eIF4G
EIF4G
1
1.3 C 2498 CST Rabbit
AKT (phospho S473)
AKT1,2
,3
1.3 V 9271 CST Rabbit
CDK1 CDK1 1.3 C ab32384 Abcam Rabbit
Tuberin TSC2 1.3 V ab32554 Abcam Rabbit
Axl AXL 1.3 V 8661 CST Rabbit
CDKN2A/p16INK4a
CDKN
2A
1.3 V ab81278 Abcam Rabbit
PMS2 PMS2 1.3 V
2251000
2
Novus
Biologicals
Rabbit
FoxO3a
FOXO
3
1.4 C 2497 CST Rabbit
Gab2 GAB2 1.4 V 3239 CST Rabbit
ErbB2/HER2 (phospho
Y1248)
ERBB2 1.4 C AF1768
R&D
Systems
Rabbit
Progesterone Repector PGR 1.4 V ab32085 Abcam Rabbit
DUSP4/MKP2 DUSP4 1.5 V 5149 CST Rabbit
83
Myosin heavy chain 11 MYH11 1.5 V
2137000
2
Novus
Biologicals
Rabbit
84
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Abstract (if available)
Abstract
Claudins are a family of transmembrane proteins integral to the structure and function of tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important features of invasive and metastatic cancer cells. We recently observed that aged Cldn18-/- mice have increased propensity to develop lung adenocarcinoma (LuAd), suggesting a tumor suppressor role for CLDN18. CLDN18.1 is the lung-specific isoform of CLDN18 while the other isoform, CLDN18.2, is normally expressed in the stomach. The goal of this study was to further explore the tumor suppressor role of CLDN18.1 in LuAd as well as to investigate the underlying mechanisms. We found that CLDN18.1 expression is markedly decreased in LuAd patients in a stage-dependent manner and its expression also correlates inversely with promoter methylation, suggesting that CLDN18.1 expression is regulated by DNA methylation in LuAd. Analyses of LuAd patient cohorts further shows that CLDN18.1 expression correlates with patient survival, suggesting a protective role of CLDN18.1 in lung cancer. Furthermore, when restored in LuAd cells that have lost expression, CLDN18.1 significantly attenuates malignant properties including xenograft tumor growth in vivo as well as cell proliferation, migration, invasion and anchorage-independent colony formation in vitro. Based on high throughput analyses of Cldn18-/- murine lung alveolar epithelial type II cells, as well as CLDN18.1-repleted human LuAd cells, we hypothesized, and confirmed by Western analysis, inhibition of insulin-like growth factor 1 receptor (IGF-1R) and AKT phosphorylation by CLDN18.1. Additionally, consistent with recent data in the Cldn18-/- murine model, re- expression of CLDN18.1 suppressed YAP/TAZ target genes in human LuAd cells in vitro, potentially contributing to its tumor suppressor activity. Analysis of LuAd cell line, in which YAP and/or TAZ are silenced with siRNA, demonstrates that inhibition of TAZ, and possibly YAP, may be involved in CLN18.1-mediated AKT inactivation. Taken together, these data indicate a tumor suppressor role for CLDN18.1 in LuAd, mediated by a regulatory network that encompasses YAP/TAZ, IGF-1R and AKT signaling.
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Asset Metadata
Creator
Luo, Jiao (Jane)
(author)
Core Title
Tight junction protein CLDN18.1 attenuates malignant properties and related signaling pathways of human lung adenocarcinoma in vivo and in vitro
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
08/08/2018
Defense Date
03/14/2018
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
CLDN18.1,IGF-1R/AKT,lung adenocarcinoma,OAI-PMH Harvest,tumor suppressor,YAP/TAZ
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application/pdf
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English
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Electronically uploaded by the author
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Frenkel, Baruch (
committee chair
), Borok, Zea (
committee member
), Offringa, Ite (
committee member
)
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jiaoluo@usc.edu,jiaoluo68@gmail.com
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https://doi.org/10.25549/usctheses-c89-60416
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etd-LuoJiaoJan-6697.pdf (filename),usctheses-c89-60416 (legacy record id)
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Luo, Jiao (Jane)
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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
CLDN18.1
IGF-1R/AKT
lung adenocarcinoma
tumor suppressor
YAP/TAZ