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Molecular role of EZH2 overexpression in Colorectal Cancer progression

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Molecular role of EZH2 overexpression in Colorectal Cancer progression

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i

MOLECULAR ROLE OF EZH2 OVEREXPRESSION IN COLORECTAL CANCER
PROGRESSION
by

Tiffany Mays

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(STEM CELL BIOLOGY AND REGENERATIVE MEDICINE)

August 2022

Copyright 2022 Tiffany Mays

ii

Acknowledgements
I acknowledge and thank all members of the Jadhav lab at USC Stem Cell including
Unmesh Jadhav, Alireza Lorzadeh, Sweta Sharma, Eric Sanchez, and Maile-Romero Wolf for
assisting with my MS thesis project. I would also like to thank all members of my thesis
committee including Unmesh Jadhav, my committee chair, Francesca Mariani, and Senta
Georgia. Lastly, I would like to thank my family and friends for their continued support in the
pursuit of this degree, and my ongoing goals in life.

iii

Table of Contents

Acknowledgements ….………………………………………………………………….………….…. ii

List of Tables ..….………………………………………………………………….………………..… v

List of Figures. ...……………………………………………………………………….…………..… vi

Abbreviations……………..…….……………………………………………………….…………. vii

Abstract……..………….………………………………………………………………………………. ix

1. Chapter 1: Introduction
1.1. Colorectal Cancer Overview…………………………………………………………….
1.2. Genetics of Colorectal Cancer………………………………………………………….
1.3. Epigenetics of colorectal cancer………………………………………………………..
1.4. Gastrointestinal Epithelium and Stem Cells…………………………………………..
1.5. Wnt/ ß-Catenin Pathway………………………………………………………………… .
1.5.1. Localization of ß-catenin…………………………………………………………
1.6. PCR2 complex and its role in Colorectal cancer………………………………………
.
2. Chapter 2: Materials and Methods ….
2.1. Intestinal Tissue Processing for frozen and Paraffin sectioning……………………..
2.2. Tissue Embedding in Paraffin and Sectioning…………………………………………
2.3. Tissue Embedding in OCT and Frozen Sectioning……………………………………
2.4. Immunohistochemistry……………………………………………………………………
2.4.1. Day 1……………………………………………………………………………….
2.4.2. Day 2: Immunofluorescence…………………………………………………….
2.4.3. Day 2: DAB Staining……………………………………………………………..
2.5. H&E staining……………………………………………………………………………..
2.6. Alcian Blue Assay………………………………………………………………………..
2.7. Endogenous Phosphatase Assay……………………………………………………...
2.8. Mouse models and tumor cell purification……………………………………………..
2.9. RNA-seq analysis………………………………………………………………………...
2.10..CHIP-seq analysis………………………………………………………………………
2.11..Computational Analysis………………………………………………………………...

3. Chapter 3 Results
3.1. Specific Aim 1……………………………………………………………………………..
3.1.1. Tumor induction and tissue processing…………………………………………
3.1.2. Effect of gain or loss of PRC2 action on gross numbers, morphology of
tumorigenic tissue……………………………………………………………………
3.1.3. Effect of gain or loss of PRC2 action on gross numbers, morphology of
….tumorigenic tissue………………………………………………………………...
3.1.4. Effect of gain or loss of PRC2 action on tumor cell proliferation……………..
3.1.5. Effect of gain or loss of PRC2 action on stem cell abundance……………….
3.1.6. Effect of gain or loss of PRC2 action on stem cell differentiation…………….
3.2. Specific Aim 2
3.2.1. Effect of Ezh2 overexpession and Eed deletion on PRC2 complex stability and
complex stability and H3k27me3……………………………………………………
1
1
2
3
4
5
6
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vii

ix
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8
9
9
9
9
10
10
11
11
12
12
12
13
14
14

14

15
15
16
16

16

iv
3.2.2. Understanding the role of PRC2 action in ß-catenin and.Bcl9 localization…...
3.2.3. Understanding the gene expression and epigenetic H3K27me3 change.
upon altered activity of PRC2 in tumors……………………………………………
4. Chapter 4 Discussion
4.1. Interplay of genetic, signaling, and epigenetic change in colorectal cancer………..
4.2. Presence and requirement of PRC2 action and H3K27me3 in intestinal epithelium
and tumors………………………………………………………………………………….
4.3. Alteration to stem cell properties and cellular proliferation in intestinal tumors an
effect of PRC2 modulation………………………………………………………………..
4.4. Influence of Apc loss and PRC2 modulation on Wnt signaling……………………….

References…………………………………………………………………………………………..

Appendices………………………………………………………………………………………….
Appendix A: Tables and Figures

17

18
19

20

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23

26

30
30

v
List of Tables

Table 1. Primary and secondary antibodies used for immunofluorescence and DAB based
staining of tissues in this thesis…………………………………………………………………….
Table 2. The effect of gain or loss of PRC2 action on tumor cell proliferation………………..
Table 3. The effect of gain or loss of PRC2 action on stem cell abundance………………….
Table 4. The effect of gain or loss of PRC2 action on stem cell differentiation……………….
Table 5. The effect of gain or loss of PRC2 action on ß-catenin+ nuclei………………………
Table 6. The effect of gain or loss of PRC2 action on BCL9 nuclei…………………………….

30

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31

31

31

31

vi
List of Figures

Figure 1. Common genetic changes and progression of colorectal cancer…………………..
Figure 2. (A is Modified from https://www.sciencelearn.org) Structure of the intestinal
epithelium and stem cell differentiation……………………………………………………………
Figure 3. (Modified from Ref. 36) The Wnt/β-catenin pathway…………………………………
Figure 4.

(Modified from Ref. 38) Cellular mechanisms regulating nuclear localization of β-
catenin………………………………………………………………………………………………..
Figure 5. (Modified from Ref. 39) Nuclear translocation of β-catenin by BCL9-2……………
Figure 6. (Modified from Ref. 42) Polycomb repressive complex 2 (PRC2) based gene
Repression………………………………………………………………………………………….
Figure 7. Mouse models for CRC progression………………………………………………….
Figure 8. H&E staining showing tissue and tumor morphology……………………………….
Figure 9. Tumor cell proliferation using BrdU staining…………………………………………
Figure 10. Stem cell abundance in intestinal tumors…………………………………………..
Figure 11. Alcian Blue Assay……………………………………………………………………..
Figure 12. Endogenous Phosphatase Assay…………………………………………………...
Figure 13. EZH2 Immunofluorescence Staining………………………………………………..
Figure 14. H3K27me3 Immunofluorescence Staining………………………………………….
Figure 15. ß-catenin Staining……………………………………………………………………..
Figure 16. BCL9 Staining………………………………………………………………………….
Figure 17. Gene expression changes in tumor stem cells…………………………………….
Figure 18. H3K27me3 presence at Bcl9 gene………………………………………………….

32

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vii
Abbreviations

CRC colorectal cancer
APC Adenomatous polyposis coli
Kras KRAS Proto-Oncogene
SMAD4 SMAD family member 4
TP53 tumor protein p53
DCC Netrin receptor DCC
CBC Crypt base columnar
ISC Intestinal stem cell
TA Transit amplifying
WNT3 Winglesss/ Integrated 3
EGF Epidermal growth factor
DLL1 Delta like canonical notch ligand 1
DLL4 Delta like canonical notch ligand 4
CAR-T Chimeric antigen receptor T
TCR-T T cell receptor T
GSK-3 ß Glycogen synthase kinase-3 ß
CKI- α Casein kinase α
TCF/LEF Transcription factor/ Lymphoid enhancer
binding factor
LRP Low density lipoprotein receptor-related protein
MUC-1 Mucin 1, cell surface associated
TCF4 Transcription factor 4
FOXM1 Forkhead Box M1
LEF1 Lymphoid Enhancer Binding Factor 1
Imp- α Importin- α
Imp- ß Importin- ß
RanGTP GTP binding nuclear protein Ran
Nup Nucleoporin
CRM1 Chromosomal maintenance 1
BCL9 B-cell lymphoma 9
Tyr-P Tyrosinase
Lgs Legless
dTCF/Pangolin d T cell receptor/ Pangolin
Colo320 Colorectal carcinoma cell line 320
HCT116 Human colorectal carcinoma cell line 116
PRC2 Polycomb repressive complex 2
EZH2 Enhancer of zeste homolog 2
EED Embryonic ectoderm development
SUZ12 Suppressor of zeste 12 protein homolog
RBAP46 RB Binding Protein 46
RBAP48 RB Binding Protein 46
JARID2 Jumonji and AT-Rich Interaction Domain Containing 2
AEBP2 AE binding protein 2
EZH2i Enhancer of zeste homolog 2 inhibitor

viii
DLBCL Diffuse large B -cell lymphoma
H3K27me3 Trimethylation of lysine 27 on histone H3 protein subunit
AML Acute myeloid leukemia
MDS Myelodysplastic syndrome
CPG 5’-C-phosphate-G-3’
CIMP CpG Island Methylator Phenotype
DAB 3, 3'-diaminobenzidine
NTM Nontuberculous mycobacteria
NBT/BCIP Nitro blue tetrazolium chloride/ 5-bromo-4-chloro-3-indolyl
phosphate
TAM Tumor-associated macrophage
H&E Hematoxylin and Eosin
MDCK Madin-Darby canine kidney

ix
Abstract
Colorectal cancer (CRC) is one of the leading causes of cancer related deaths that
resulted in more than 860,000 deaths worldwide in 2018
1
. CRC results from well-known genetic
changes in APC, KRAS, and TP53, however, epigenetic changes in CRC progression need to
be further established
1
. Polycomb Repressive Complex 2 (PRC2) mediated histone methylation
(H3K27me3) and DNA methylation are coregulatory epigenetic modifications that play important
roles in gastrointestinal tissue homeostasis and cancer
2,3
. Overexpression of EZH2, the
enzymatic subunit of PRC2 complex
4
,has been associated with CRC and other solid tumors
5-7
.
Gastrointestinal Lgr5
+ve
stem cells (ISCs) are well established cell of origin of CRC and Wnt
signaling is critical for ISC for proliferation and maintenance. Wnt induction causes cytoplasmic
ß-Catenin to relocate to nucleus where it activates many Wnt-target genes linked to cell
proliferation, while in absence of the Wnt signal, Apc containing protein complex marks beta
catenin for degradation in cytoplasm
8-10
. Apc mutations are prevalent in CRC and cause nuclear
accumulation of ß-Catenin resulting in constitutive Wnt signaling and hyperproliferation of ISCs,
leading to adenomas
11
. Currently, there is little understanding of molecular events, particularly
epigenetic controls that modulate Wnt based stem cell control, particularly in early cancer
development.
In order to understand the impact of altered PRC2 activity in CRC, our lab has
developed mouse models that allow inducible deletion of Apc in Lgr5
+ve
ISCs, which produces
adenomas in 2-3 weeks. Additionally, parallel models allow over expression of Ezh2 or deletion
of PRC2 component Eed which eliminates PRC2 activity, along with Apc deletion. Using these
established mouse models, I proposed to study the impact of Ezh2 overexpression on early
gastrointestinal tumorigenesis, specifically on 1) stem cell behavior and 2) Wnt signaling.

1
Chapter 1: Introduction
1.1 Colorectal cancer
Colorectal cancer is the third most common cause of cancer related mortality with 1.85
million cases and 850,00 deaths across the world annually
12
. Statistically, 70% to 75% of
patients survive beyond 1 year, 30% to 35% survive beyond 3 years, and fewer than 20%
survive beyond 5 years from diagnosis
12,13
. However, molecular subtype largely influences the
median survival time and treatment options
12
. Currently, the standard therapies are surgical
resection, chemotherapy, and radiotherapy
7
. While Metastatic CRC remains uncurable, the
primary treatment for unresectable metastatic CRC is systemic therapy, which includes
cytotoxic chemotherapy, biologic therapy, immunotherapy, and combinations of those therapy
options
12,14,15
. There is urgent need for new therapeutic options and additional research is
necessary to understand tumor genetics and epigenetics in order to identify new molecular
targets while simultaneously reducing toxicity to otherwise healthy tissue.

1.2 Genetics of colorectal cancer
CRC results from the stepwise accumulation of genetic alternations in oncogenes and
tumor suppressor genes. Genetic mutations in APC, KRAS, TP53, and SMAD4 are well known
in CRC (Fig.1.)
16,17
. While there is no definitive order of these mutations in CRC progression,
characteristic phases are associated with each mutation. Mutational inactivation's in APC cause
hyperproliferation in the epithelial lining of the intestine
16
. The Ras gene mutation, which is
typically Kras, occurs in early adenomas in a cell that goes through clonal expansion and
creates dysplastic tumors which result in intermediate adenomas
16
. The p53 gene is a cellular
protein located on 17p13.1, mutation of which, leads to carcinoma after late adenoma formation
16,18
. Allele loss at chromosome 18q21.1 is associated with mutations of DCC and SMAD4
tumor suppressor genes
17,19
. SMAD4 is a downstream regulator of the TGF-beta signaling
pathway that accounts for 50-60% of 18q21 allele loss in CRC while DCC, which is also located

2
on 18q, encodes a cell surface protein associated with aggressive CRCs
17,19
. Loss of DCC
typically occurs after intermediate adenoma formation leading towards the late adenoma stage
16
. The discovery of these molecular targets over the past few decades has spurred interest in
targeted therapies for CRC, such as T-cell based immunotherapies for adoptive cell transfer
(CAR-T and TCR-T) and immune checkpoint inhibitors, but most established therapeutic options
are largely ineffective, especially for metastatic CRC
15,20
. This lack of effective targeted
therapeutic options for CRC demonstrates the need for further studies into the molecular and
cellular changes in cancer progression.

1.3 Epigenetics of colorectal cancer
Epigenetic variation is associated with several mutational changes in CRC as CRCs
show large-scale changes in DNA methylation, histone modification, as well as topological
structure
1
. In CRC, DNA methylation changes are the most well-understood. Human CRCs
show global DNA hypomethylation, coupled with excess DNA methylation at CGIs, known as
the CpG island methylator phenotype (CIMP). Even though the hypermethylation at gene
promoters is thought to suppress gene activity, not all hypermethylated genes are deactivated.
Additionally, role of the global hypomethylation in gene regulation or chromosomal instability
remains largely unexplored. These findings demonstrate the lack of knowledge regarding the
functional impact of epigenetic modulation on tumor cell behavior. Additionally, in metastatic
CRCs, metastasized cells in distant organs escape the primary tumors early during cancer
progression and show few to no new mutations as compared to the primary tumors
21-23
. These
findings suggest a significant modulation of epigenetic features in early tumorigenesis impacting
cellular properties.

3
1.4 Gastrointestinal epithelium and stem cells
The inner lining of the gastrointestinal track is arranged as crypt and villus structures
(Fig. 2A). There are multiple invaginations called crypts inside of the intestinal epithelial lining
surrounded by multiple projections into the intestinal lumen called villi. Each villus is covered by
post mitotic epithelial lining that provides nutrient uptake and functions as a protective barrier for
the small intestine, while capillaries and lymph vessels underneath this epithelium absorb
nutrients. The length of the villus is longest within the duodenum, while colon lacks the villi. The
crypt is composed of crypt based columnar cells (CBCs) interspersed by secretory cells known
as Paneth cells, which nurture and provide protection to adjacent CBC cells (Fig. 2B). The
antimicrobial products produced by these Paneth aid in CBC protection, and they also produce
WNT3, EGF, and Notch ligands DLL1 and DLL4, which help maintain the CBCs.
There are about 15 Lgr5
+
CBCs located at the base of each crypt that play the role of
intestinal stem cells (ISCs)
10
. ISCs express Lgr5, respond to Wnt signals, and are the cell of
origin of gastrointestinal tumors. The intestinal stem cell niche is composed of epithelial
component, which is provided by Paneth cells, and a mesenchymal component, which is
composed of myofibroblast, fibroblast, pericytes, endothelial cells, neural cells, and smooth
muscle cells, that aid in EGF signaling, BMP inhibition, and activation of the WNT pathway.
ISCs replicate to produce transit amplifying (TA) progenitor cells that divide about 4-6 times
before producing secretory and absorptive cells
10
. Notch signaling is responsible for
maintenance of the ratio between the secretory and absorptive cells through lateral inhibition as
the progenitor cells reach the upper region of the intestinal crypt. Goblet, Paneth,
enteroendocrine, and tuft cells are secretory cell types that each serve specific roles within the
intestine (Fig. 2C). Enteroendocrine cells aid in metabolic control though production of
hormones, tufts cells help with immune regulation, and goblet cells are responsible for coating
the entire intestinal epithelium in a protective mucosal lining. The absorptive cell lineage is
composed of enterocytes and M cells. Enterocytes absorb ions, water, sugar, and peptides,

4
while M cells lie on top of Peyer’s patches and are responsible for transporting antigens to
lymphoid cells under the intestinal epithelium. Mature epithelial cells shed off from the villus tip
and are quickly replaced by other cells
10
. Complex signaling mechanisms in the intestinal crypt-
villus axis control differentiation of ISCs into distinct mature cell types
10
. Although more than
4,000 genes change expression when ISCs differentiate into their progeny, there is little to no
epigenetic change found in ISCs and their differentiated progeny, with DNA and histone
modifications remaining uniform
24-29
. The cell state transitions in ISC differentiation are instead
controlled largely by signaling molecules such as Wnt, Notch, Egf, and Bmp
10, 30-34
. The cellular
sources of the signaling molecules and how the signaling gradients are created, remain under
study.

1.5 Wnt/ ß-catenin pathway
The Wnt/ß-catenin pathway controls proliferation of the intestinal epithelium. In the
canonical Wnt signaling, when supply of Wnt is absent, the ß-catenin destruction complex,
composed of Dvl, Axin, CK1, GSK3, and APC remains in the cytoplasm. ß-catenin that
accumulates in the cytoplasm is bound by Beta-TrcP, which phosphorylates the “degron” motif
in ß-catenin (Fig. 3A)
35,36
. ß-catenin is then targeted for ubiquitination and degraded through
ubiquitin-mediated proteolysis. When the wnt/ß-catenin pathway is on, Wnt binds to frizzled
receptor and induces frizzled to attach to LRP in the cell membrane (Fig. 3B)
36

35
. As a result,
the ß-catenin destruction complex is ineffective in phosphorylating ß-catenin, which detaches
from the destruction complex and translocate into the nucleus where it binds to TCF/LEF to form
an active transcription-factor complex
37
. The TCF-ß-catenin complex turns on gene transcription
for important proto-oncogenes in humans and causes proliferaton
35,37
. In cancer, loss of Apc
action causes the wnt-ß-catenin pathway to be held in this constant on state which in turn
causes excess ß-catenin accumulation in the cytoplasm and nucleus that causes
hyperproliferation through abnormal TCF- ß-catenin complex activity
35
. Given this central role of

5
this signaling on cell proliferation, the Wnt/ß-catenin pathway and particularly the TCF-ß-catenin
interactions have been targeting of structure based drug design
37
. Such a drug would need to
be highly specific in order to prevent undesirable consequences on binding equilibria of ß-
catenin to its other ligands
37

1.5.1 Localization of ß-catenin
ß-catenin localization differs between normal and malignant tissue. While the mechanisms
controlling this are largely unknown, beta catenin must interact with several other proteins in
order to perform its function
38
(Fig. 4). The cellular mechanisms that regulate nuclear
localization of beta catenin are controlled by a multitude of interactors such as MUC-1, TCF4,
LEF1, FoxM1, Pygo, BCL9, Imp-alpha, Imp-beta, Nup, Axin APC, anGTP, CRM1, RanGTP,
RanBP-3, Tyr-P, alpha-catenin, and cadherin
38
. However, one of the most interesting interaction
of ß-catenin with Pygo and BCL9 occurs during nuclear retention
39

38
(Fig. 4,5).
BCL-9 binds with Pygo to form a complex that promotes ß-catenin nuclear localization
and retention
38,39
(Fig. 4,5). Nuclear Pygo binds to Armadillo, the homolog of ß-catenin, using the
adaptor Lgs, the homolog of BCL9
38
. The armadillo domain aids in binding ß-catenin to
cadherins, APC, Axin, and TCF in a competitive manner
38,40
. The competitive nature of this
binding is thought to be because they come into contact with the same surface area on ß-
catenin
40
. The formation of this trimolecular complex composed of beta catenin, BCL9 and Pygo
could bind dTCF/Pangolin, a homolog of human TCF4, in order to activate the Wnt targets gene
transcription
38,40

Outside of the role of BCL-9 in the trimolecular complex, it has been shown that Bcl9-2
can assist in the switch between ß-catenin’s adhesive and transcriptional functions essential to
phosphorylate ß-catenin in the nucleus and retain it there in MDCK cells
39
. Given that BCL-9 is
also overexpressed in colorectal cancer cell lines, it reinforces Wnt pathway activity and is
required for ß-catenin mediated transcription in human cells. Despite these findings, the

6
molecular mechanisms that regulate the switch between BCL-9’s adhesive and transcriptional
functions are not fully understood
39
. Using Mass Spectrometry, 67 ß-catenin binding partner
proteins have been identified in a recent study, which aids in our understanding of ß-catenin
localization and activation of Wnt target genes
41
.

1.6 PRC2 complex and its role in colorectal cancer
The PRC2 complex consists of many proteins responsible for regulation of transcription
and cancer
42
. The four main components of the PRC2 complex are Ezh2, Suz12, and Rbap46
or Rbap48, and Eed. Ezh2 is the catalytic subunit that adds the repressive H3K27me3
modification on histones, particularly around gene promoters (Fig. 6)
42
, while the other proteins
play role in recruiting the complex to target gene DNA as well as stabilization of the complex.
The canonical gene repressive role of the PRC2 complex also influences cancer as both an
oncogenic and suppressive regulator gene can be modulated through transcriptional
repression
42
. EZH2 can have gain of function alterations that result in its overexpression in
several types of cancers. This overexpression promotes invasion and metastasis in many solid
tumors including colorectal, prostate, and breast
43
. However, some cancers have deactivating
Ezh2 mutations exhibiting the tumor suppressive role of EZH2
42,43
. Two instances of this
suppressive role of EZH2 are seen in myeloid leukemia (AML) and myelodysplastic syndrome,
where loss of chromosome 7 causes loss of EZH2 function
44
. These findings have prompted the
development of EZH2 inhibitor drugs.
While there are a wide variety of EZH2 inhibitors in clinical trials, FDA has approved
tazemetostat (EPZ6438) for treatment of epithelioid sarcomas, which cannot be surgically
resected
45
. On the other hand, another EZH2 inhibitor GSK126 failed in clinical trials to treat
patients with DLBCL because it caused decrease in anti-tumor immunity
45
. These results
highlight the lack of understanding of the direct effects of EZH2 levels on H3K27me3 and gene
activity, as well as the non-H3K27me3 based effects of EZH2 in cancer.

7

Based on these observations, I hypothesize that Ezh2 overexpression affects early
gastrointestinal tumorigenesis by impacting stem cell behavior and Wnt signaling. For my first
specific aim, I have studied the effects of PRC2 modulation on tumor cell proliferation, stem cell
abundance, and stem cell differentiation. For my second specific aim, I have studied the
molecular effects of PRC2 modulation in CRC by looking at the impact on gene expression
through mRNAseq, the impact on Wnt signaling, and the involvement of altered histone
modification H3K27me3 in specific gene regulation.

8
Chapter 2: Materials and Methods
2.1 Intestinal Tissue Processing for Frozen and Paraffin Sectioning
Mice were euthanized using CO2 inhalation in a CO2 chamber. After euthanizing the
mouse, the intestine was harvested by gently pulling on the proximal intestine and cutting the
vascular fat. The intestine was put in a petri dish with cold PBS on ice and the intestine was
flushed with cold PBS to remove the food and other debris. In order to start tissue fixation, the
intestine or parts of it were flushed with cold 4% Paraformaldehyde (PFA). The intestine was cut
along the length and opened on a bibulous paper and left to fix for 10 minutes followed by rolling
with a pair of forceps in form of a ‘Swiss-roll’. The rolled tissue was placed on a needle and put
in cold 4% PFA in 15ml tubes and left overnight at 4
O
C. PFA was washed with cold PBS five
times and the tissues were then transferred into cold 30% sucrose in Swiss-roll format followed
by incubation overnight at 4
O
C. These tissues were used for embedding in OCT. Alternatively,
fixed tissues were transferred to 70% ethanol in Swiss-roll format and incubated at 4
O
C O/N and
used for embedding in paraffin.

2.2 Tissue Embedding in Paraffin and Sectioning
After overnight incubation in 70% ethanol, tissues were processed using Sakura Finetek
tissue processor that exposes the tissue to increasing concentration of ethanol, xylene, and
paraffin wax. After this processing, the tissues were embedded in a paraffin wax using plastic
cassettes and the tissue blocks were kept in 4
O
C till sectioning. The paraffin blocks were chilled
on ice before tissue sectioning. Tissue embedding was done on the Tissue-Tek 5 embedding
module 5101, 5 μm tissue sections generated by a the Leica microtome were floated on DI
water at 40
O
C and picked on to slides. The slides were dried overnight on a 60
O
C heating block.

9
2.3 Tissue Embedding in OCT and Frozen Sectioning
After overnight incubation in 30% sucrose, tissues were embedded in OCT inside a plastic
block and frozen using dry ice and ethanol bath. The layers of Swiss-rolled tissue were
separated to ensure that there is OCT in between the layers. The blocks were kept frozen in -
80
O
C till sectioning. 7 μm sections were cut using cryostat set at -20
O
C and the slides were
stored at -20
O
C in a dark and moisture protected container to preserve fluorescence.

2.4 Immunohistochemistry
2.4.1 Day 1
Slides with Paraffin tissue sections were passed through the following solutions in series
(5 minutes each): xylene, xylene, followed by ethanol concentrations of 100%, 100%, 95%,
90%, 80%, 70%, water. For antigen retrieval, the slides were put in 10mM sodium citrate buffer,
pH 6.0 in a pressure cooker and exposed to high pressure antigen retrieval for 6 min for all
antibody stains except ß-catenin. For ß-catenin, the slides were put in high pressure for 12
minutes for antigen retrieval. Slides were cooled on ice for 15 minutes and washed with water
for 5 minutes while shaking. Slides were then Incubated in 0.5% H202 in methanol for 20
minutes to inhibit endogenous peroxidases (on shaker) and washed with water in coplin jar for 5
minutes on shaker. Tissues were blocked with 2% BSA blocking buffer at RT for 30 min.
Primary antibodies were prepared in 1% BSA blocking buffer. A table of all antibodies is
provided as a Table in (Table. 1). The primary antibody was added on the tissue sections at
given concentrations and slides were incubated in the 4
O
C overnight.

2.4.2 Day 2: Immunofluorescence
Primary antibody was removed, and the slides were washed in PBS for 7 minutes twice on
shaker. Excess PBS was wiped off and add secondary antibody was added at appropriate

10
dilution (Table. 1). The incubation happened in humidified light protected chamber in cold room
for 2 hours. After 2hr incubation, the secondary antibody was dumped, and slides were washed
2x with PBS for 7 minutes each while shaking. The slides were mounted with mounting media
containing DAPI. The slides were stored at 4
O
C or used for imaging.

2.4.3 Day 2: DAB staining
After removing the primary antibody, the slides were washed 2x in PBS for 7 minutes each
on shaker. The extra PBS was wiped off and the secondary antibody was added to each
slide. The incubation happened in humidified light protected chamber in cold room for 1 hour.
The ABC solution was prepared (1 drop of Reagent A and 1 drop of Reagent B in 2.5 ml of
PBS) and let sit at RT for 30 min. After 1hr incubation with secondary antibody, the secondary
antibody was removed, and slides were washed 2x with PBS for 7 minutes each. The ABC
solution was added on slides and incubated for 30 min. The slides were washed in 1X PBS for 5
min. The DAB reagent was prepared using 15 µL of DAB liquid chromogen solution and 1 ml of
DAB liquid buffer. DAB was added to the slides and observed under microscope. The slides
were counterstained with hematoxylin if necessary. The slides were dehydrated by incubating in
water, followed by ethanol with concentration of 70%, 80%, 90% 95%,100%,100%, followed by
xylene for 5 minutes in each solution. The extra xylene was removed using paper towel and the
slides were mounted with clear mounting media.

2.5 H&E Staining
Slides were de-paraffinized using two changes of xylene for total of 7-10 minutes. Then,
slides were rehydrated in series of ethanol dilutions (5 minutes each) 100%, 95%, 90%,80%,
70%, and water. Slides were then placed inside a coplin jar with Hematoxylin. Periodically the
slide was checked under the microscope for staining after being washed in water. To get rid of
excessive staining, the slides were washed with acid alcohol followed by wash in sodium

11
bicarbonate. After a final wash in water, slides were dehydrated for 5 minutes each through
70%, 80%, 90%, 100% ethanol series, and put in xylene for 5 minutes 2x. Finally, slides were
mounted with mounting media and coverslip.

2.6 Alcian Blue Assay
Slides were de-paraffinized with two changes of xylene, total of 7-10 minutes. Then,
slides were rehydrated in ethanol series for 5 minutes each 100%, 95%, 90%,80%, 70%, and
water. Alcian Blue solution was made by mixing 1g Alcian Blue (8GX, Sigma) and 100ml of 3%
Acetic Acid pH to 2.5. Slides were placed inside a coplin jar containing alcian blue solution while
checking periodically under microscope for the staining intensity. Slides were rinsed with water
and counterstained with nuclear fast red for 6 minutes. After a final wash in water, tissues were
rehydrated for 5 minutes each through 70%, 80%, 90%, 100% ethanol series, and put through
xylene for 5 minutes 2x. Slides were mounted with mounting media and coverslip.

2.7 Endogenous Phosphatase Assay
Tissues were de-paraffinized with two changes of xylene, total of 7-10 minutes followed
by rehydration in ethanol series for 5 minutes each 100%, 95%, 90%,80%, 70%, water. NTM
solution was made by mixing 200 microliters 5M NaCl, 5 ml Tris-HCL pH 9.5, 2.5 mL 1MgCl2,
and 4.3 mL water. Slides were incubated in NTM solution for 5 minutes and developed in
NBT/BCIP solution (Roche, dissolved in dH2O) with repeated observation under microscope for
staining intensity. Tissues were counterstained with Nuclear fast red, washed in water, followed
by rehydration for 5 minutes each through 70%, 80%, 90%, 100% ethanol series, and put
through xylene for 5 minutes 2x. Slides were mounted with mounting media and coverslip.

12
2.8 Mouse models and tumor cell purification
Genetically engineered mouse models were generated on C57/BL6 or 129/SVJ background
(Fig. 7a). Adult mice of both sexes, aged 2-8 months, were used for experiments. Deletion of
Apc gene in Lgr5+ ISCs was induced by two tamoxifen (TAM) injections (2mg intraperitoneal –
IP in sunflower oil on consecutive days) in Apc
Fl/Fl
; Lgr5
EGFP-IRES-CreER
; Rosa26
LsL-tdTomato
, (referred
to as ApcKO) (Fig. 7b). 14 days after the induction, hundreds of adenomas can be seen in
intestine as well as colon of these mice (Fig. 7c). In order to up- or down-regulate PRC2 activity,
we overexpressed Ezh2 using Apc
Fl/Fl
;Col1a
LsL-Ezh2
;Lgr5
EGFP-CreER-T2
;Rosa26
LsL-tdTomato
(referred to
as ApcKO-Ezh2OE) or deleted Eed using Apc
Fl/Fl
;Eed
Fl/Fl
;Lgr5
EGFP-CreER
;Rosa26
LsL-tdTomato

(referred to as ApcKO-EedKO) mice. I also used Col1a
LsL-Ezh2
;Lgr5
EGFP-CreER-T2
;Rosa26
LsL-tdTomato

(referred to as Ezh2OE) mice to study effect of Ezh2 overexpression in the native tissue. For
purification of stem cells or non-stem tumor cells, intestinal tissues were washed and epithelial
cells were isolated by shaking the proximal intestinal tissue in ice-cold 5 mM EDTA for 40 min.
Single-cell suspension was generated by treating the epithelia with 4X TrypLE solution at 37
O
C
for 15 minutes. Stem cells (Lgr5+, green and red) or non-stem cells (red) were purified using
FACS and used for gene expression and chromatin assays (Fig. 7D).

2.9 RNA-seq analysis
About 100,000 FACS purified Lgr5+ stem cells or non-stem cells were collected in Trizol
(ThermoFisher) reagent and RNA was extracted using standard manufacturer protocol. RNA
isolates were treated with DNase to remove contaminating DNA and total RNA (500 ng) was
used to prepare libraries with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) following
the manufacturer’s protocol.
2.10 CHIP-seq analysis
For H3K27me3 ChIP-seq, FACS purified Lgr5+ stem cells or non-stem cells were
collected in PBS. About 50,000 cells were treated with 0.2 U micrococcal nuclease (Sigma) in

13
buffer containing 50 mM Tris-HCl (pH7.6), 1 mM CaCl2, 0.2% Triton X-100, protease inhibitors
(Roche), and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF) at 37°C for 6 min, followed by
dialysis against RIPA buffer (50 mM HEPES (pH 7.6), 1 mM EDTA, 0.7% Na deoxycholate, 1%
NP-40, 0.5 M LiCl) for 3 hr at 4°C. Chromatin was isolated by centrifugation and incubated
overnight at 4°C with well-validated ChIP-grade Ab against H3K27me3 (Diagenode). Chromatin-
antibody complexes were capture with magnetic beads (Dynal) that we washed 4 times in RIPA
buffer and twice in 1 mM EDTA in 10 mM Tris-HCL, pH 8. Cross-links were reversed using 1%
SDS and 0.1 M NaHCO3 for 6 hr at 65°C. DNA was purified using a kit (QIAGEN) or by isolating
the mononucleosome fraction in 2% E-gels (Invitrogen). Libraries were prepared using
ThruPLEX kits (Rubicon), and DNA size distribution was confirmed using High-sensitivity Qubit
dsDNA Assay Kit (ThermoFisher). All libraries (RNA-seq, ATAC-seq, and ChIP-seq) were
sequenced on a NOVO seq 500 instrument (Illumina) to obtain 150 bp paired-end reads.

2.11 Computational analyses
Raw reads from mRNA- and ChIP-seq were aligned to the mouse genome (Mm10,
Genome Reference Consortium GRCm38) using TopHat v2.0.6
46
or Bowtie2
47
. For RNA-seq,
transcript levels were expressed as read counts using HTSeq
48
. Data were normalized and
sample variability assessed by principal component analysis in DEseq2
49
. Differential
expression was defined using the indicated fold-changes and false-discovery rate (FDR) 0.05
using DEseq2. For ChIP-seq, aligned signals in raw (bam) files were filtered to remove PCR
duplicates and reads that aligned to multiple locations. Raw signals from individual samples or
highly correlated replicates from a given cell type were converted to signal files (bigWig) using
DeepTools v2.1.0
50
and visualized using integrated genomic viewer (IGV, Broad Institute) (Fig.
13).

14
Chapter 3: Results
3.1 Specific Aim 1: To quantify the effects of Ezh2 overexpression on tumor cell
proliferation, stem cell abundance, and stem cell differentiation.

3.1.1 Tumor induction and tissue processing
In order to induce tumors in various mouse models (WT, Ezh2OE, ApcKO,
ApcKO-Ezh2OE, ApcKO-EedKO) (Fig. 7A), adult mice were injected with 2 doses of
TAM on consecutive days and intestinal and colon tissues were procured on day 14 (Fig.
7B). Tissues were processed and preserved for frozen as well as paraffin sections and
histological analysis was conducted for understanding the impact of gain or loss of
PRC2 activity on tumor stem cells, cell proliferation, and cell differentiation.

3.1.2 Effect of gain or loss of PRC2 action on gross numbers, morphology of
tumorigenic tissue
I conducted H&E staining on 5um sections of the entire intestinal tissue
embedded in paraffin blocks as ‘Swiss-roles’ and observed the changes in gross
morphology of the tissue, particularly focusing on the tumorigenic areas (Fig. 8). ApcKO
showed adenoma formation particularly towards the proximal end of the tissue, where
tumor and normal tissue were clearly visible side by side (Fig. 8A,B), while distal part of
the tissue had fewer tumor-like areas (Fig. 8 B,E). This is consistent with the highest
number of Lgr5+ stem cells that express the EGFP-Cre allele in the proximal part of the
intestine and decreasing number of such cells in the distal intestine. Given the pervasive
tumor formation in the proximal intestinal tissue, I could not count the exact tumor
numbers or differences in the PRC2 modulated samples. However, in comparison to
ApcKO tissue, PRC2 modulated tissues (ApcKO-Ezh2OE, ApcKO-EedKO) showed no

15
significant differences in tumor spread or morphology by gross microscopic observations
(Fig. 8 C,D,F,G).

3.1.3 Effect of gain or loss of PRC2 action on tumor cell proliferation
I conducted an immunofluorescence co-staining for BrdU as well as tdTomato on 5um
sections of proximal intestine (duodenum) as well as mid-section of intestine (jejunum)
embedded in paraffin (Fig. 9) for various genotypes of mice (ApcKO, ApcKO-EedKO, and
ApcKO-Ezh2OE). BrdU represents proliferative cells in the S phase of cell cycle, while
tdTomato stains for all tumor cells. In order to quantify the proliferative cell fraction in various
tumor types, I counted minimum 1,500 tumor (TdTomato+) as well as the BrdU + cells within the
tumor cells (Fig. 9). After comparing the number of BrdU + cells within Tdtom+ cells across the
different genotypes, there was higher percentage of BrdU + cells among Tdtom+ cells in PRC2
modulated tumors (Table 2), however only ApcKO-EedKO condition showed difference in
proliferating cells compared to ApcKO tumors that was close to being significant (p=0.06).

3.1.4. Effect of gain or loss of PRC2 action on stem cell abundance
I conducted an immunofluorescence co-staining of GFP that stains the Lgr5+ cells with
Cre activity (cell of tumor origin), along with tdTomato which stains all tumor cells, using 5 µm
sections of jejunum and duodenum tissue embedded in paraffin (Fig. 10). The various
genotypes used for this staining are (ApcKO, ApcKO-EedKO, and ApcKO-Ezh2OE). After
comparing the number and percentage of GFP+ cells within Tdtom+ cells across mouse
genotypes (minimum 1,500 tumor cells), I did not find significant differences in the number of
stem cells as a result of PRC2 modulation (Table 3).

16
3.1.5. Effect of gain or loss of PRC2 action on stem cell differentiation.
I conducted alcian blue and endogenous phosphatase assays on 5 µm sections of jejunum
and duodenum tissue embedded in paraffin. The various genotypes used for this staining are
(Apc-KO, ApcKO-EedKO, and ApcKO-Ezh2OE.). The alcian blue assay was used to stain for
the goblet cell protein, Muc2 (Fig. 11 A,B,C, D, E, and F) and the endogenous phosphatase
assay was used to stain for the enterocyte brush border protein encoded by gene alkaline
phosphatase gene Alpi. In both conditions nuclear fast red was used to stain the nuclei (Fig. 12
A,B, and C). Upon counting minimum of 2,000 cells, I found that the percentage of Alcian blue
positive goblet cells were decreased in PRC2 overactivated condition (ApcKO-Ezh2OE) in
comparison to ApcKO genotype (p=0.04), while loss of PRC2 action (ApcKO-EedKO) did not
cause a significant change in goblet cell numbers (Table 4). Enterocyte cells in all tumor
samples were located only on the edge of the tumor periphery (Fig. 12) and given the non-
uniform distribution of tumor surface as well as limited capture of the tumor surface on slide
sections, no conclusions could be drawn on differences in number of these cells across the
three genotypes.

3.2 Specific Aim 2: To understand the molecular role of PRC2 modulation in CRC on
tumor cell gene expression, Wnt singling, and underlying role of H3K27me3
changes.

3.2.1 Effect of Ezh2 overexpression and Eed deletion on PRC2 complex stability and
H3K27me3.
I first looked into the consequences of Ezh2 overexpression and upon Eed deletion on
stability of PRC2 complex and its canonical function of adding H3K27me3. To do this, I
conducted immunohistochemical analysis for Ezh2 and H3K27me3 (Fig. 13 A-L and Fig.15 A-L)
using intestinal tissues from WT, ApcKO, ApcKO-EedKO, and ApcKO-Ezh2OE. As noted before

17
in previous studies
28
, I saw relatively more Ezh2 staining in crypt cells as compared to villus
cells, while H3K27me3 was lower in crypt cells and increased in differentiated villus cells (Fig.
13C and Fig. 14 C.). While tumors from all ApcKO and ApcKO-Ezh2OE mice showed positivity
and high levels of Ezh2 as well as H3K27me3 in tumors, ApcKO-EedKO tumors showed loss of
H3K27me3, confirming loss of PRC2 activity in absence of Eed protein.

3.2.2 Understanding the role of PRC2 action in ß-catenin and Bcl9 localization
I conduced immunohistochemical stains of ß-catenin and Bcl9 on 5 µm sections of jejunum
and duodenum tissue embedded in paraffin (Fig. 15 A-F and Fig. 16 A-F). The various
genotypes used for this staining are (ApcKO, ApcKO-EedKO, and ApcKO-Ezh2OE). ß-catenin
is a core component of the wnt/ ß-catenin signaling pathway that controls cell proliferation
through translocation to nucleus upon Wnt stimulation. Thus, understanding ß-catenin
localization in tumor cells can reveal possible mechanisms in tumors that allow
hyperproliferation of cells. BCL9 is a ß-catenin interacting proteins that can influences its
localization and retention inside the cell nucleus. To understand this molecular interplay
between ß-catenin and BCL9 and to see if this is modulated in PRC2 up- or down-regulation, I
analyzed the localization (nuclear vs cytoplasmic) of these two proteins in minimum 2,000 cells
in all genotypes. There is a higher percentage of ß-catenin positive punctate nuclei in ApcKO
tumors (91.7%), while this number drops to only 52% cells in ApcKO-Ezh2OE tumors as many
cells show diffuse staining over nucleus as well as cytoplasm (Fig 16 C, D) (Table 5). Despite
this big difference, the statistical significance for this change is low (p=0.1) as I only have 2
samples and because of the tumor heterogeneity (as discussed in the next section). ApcKO-
EedKO also showed some loss in nuclear ß-catenin (79.9%, p=0.08).
BCL9 staining showed prevalence of the protein in nucleus of ApcKO tumor cells (94%, Fig. 16
A, B,). While there was almost no change upon loss of PRC2 (ApcKO-EedKO), I saw about
15% reduction in the nuclear BCL9 upon overexpression of Ezh2, with diffuse staining across

18
nuclei and cytoplasm (79%, Fig. 16 C, D) (Table 6).

3.2.3 Understanding the gene expression and epigenetic H3K27me3 change upon altered
activity of PRC2 in tumors
To understand the gene expression changes in stem cells in various genotypes (WT,
ApcKO, ApcKO-EedKO, and ApcKO-Ezh2OE), I conduced mRNAseq using RNA purified from
the stem cells in each condition. Differential gene expression was determined using the
DeSeq2 program (q<0.01 and with 2 fold change in expression, Fig. 17). Replicates from each
of the genotype showed overlap, and the principal component analysis (PCA) results showed
that the majority of gene expression changes happen upon Apc deletion, while PRC2
modulation has relatively less impact on gene expression (Fig. 17A). The results also showed
that there are 2,613 genes upregulated in ApcKO stem cells and 1,691 genes are
downregulated (volcano plot in Fig. 17B). In comparison, PRC2 modulation cause very few
additional gene expression changes beyond ApcKO. Interestingly, Bcl9, the ß-catenin interactor,
showed increased expression in ApcKO suggesting a possible role in modulation of ß-catenin
localization in tumor cells. To understand if this upregulation of Bcl9 (and up- or down-
regulation of other genes) is controlled by epigenetic mechanisms, I looked into histone
modifications in WT cells and all tumor genotypes. As PRC2 modulation may have direct or
indirect effects through H3K27me3 gains or losses (resulting from EzhOE or EedKO,
respectively), I conducted CHIP-seq analysis in purified stem cells from all conditions (WT,
ApcKO, ApcKO-EedKO, and ApcKO-Ezh2OE) to look at the distribution of H3K27me3.
H3K27me3 CHIP-seq signal showed no changes at the Bcl9 locus, suggesting that Bcl9 gene
expression is not controlled by PRC2 mediated H3K27me3 across all different tumor types (Fig.
18).

19
Chapter 4: Discussion
4.1 Interplay of genetic, signaling, and epigenetic change in colorectal cancer
Colorectal cancer evolves through accumulation of multiple successive mutations; Apc loss
is the founding mutation in more than 70% CRC cases. As a key regulatory molecule in the Wnt
pathway (Fig. 3), Apc loss results in increased accumulation of ß-catenin, a Wnt effector
molecule that translocated to the nucleus and activates target genes, resulting in constitutive
proliferation of cells, which is a hallmark of cancer. ß-catenin localization in normal cells is tightly
controlled by multiple interacting proteins that control its amount inside the nucleus, thus
affecting the cellular response to Wnt activation. Cancer-specific utilization of ß-catenin
interacting proteins to alter Wnt singling is part of ongoing studies, but targeting such
interactions is thought to be a therapeutic strategy. In this regard, identifying how ß-catenin
interactors control their localization in cancer is important for development of novel therapies. In
my work, I address how ß-catenin localization is influenced in early tumorigenesis upon loss of
Apc, how BCL9, one of the ß-catenin interactors, may influence its localization, as well as how
this molecular interplay may be influenced by epigenetic modulation.
In addition to genetic mutations and their impact on signaling pathways, cell behavior
and gastrointestinal tumor evolution also depends on epigenetic change. Indeed, DNA
methylation and changes in chromatin structure are already discovered in CRCs
2,3
. Additionally,
gain of function mutations in epigenetic modulator Ezh2, the catalytic subunit of PRC2 complex,
are also present in some CRCs
4,6
. Surprisingly, inactivating mutations in Ezh2 are also found in
other cancers
4,7
, which highlights the diverse role that PRC2 action may play in CRC
tumorigenesis as well as the complications in considering Ezh2 inhibition as a therapeutic
strategy against cancer. More broadly, two important limitations in using epigenetic modifiers as
the targets for chemical inhibition in therapy are: a) limited mechanistic details of how various
epigenetic modifiers function in the context of tumorigenesis, and b) missing information on
when during tumor development, epigenetic changes occur. This information is critical to

20
determine the timing of chromatin modifier inhibition for effective therapy as well as to
understand the expected specific and nonspecific effects of such therapy. In this light, PRC2
based gene repression through H3K27me3 modification and heterochromatin formation is one
of the most important chromatin controls in many cellular processes. H3K27me3 is known to
turn-off hundreds of developmental genes during tissue maturation, while it is unaltered in most
adult stem cell differentiation processes. In this light, even though H3K27me3 changes are
prevalent in CRC, how this modification or broadly the PRC2 action may influence tumor
development remains unknown. In my thesis, I have used mouse models of Apc deletion
(representing earliest unknown changes in CRC) and Ezh2 overexpression or Eed deletion
(representing PRC2 hyperactivity or loss of action) to understand the role of PRC2 based
control of cellular signaling, stem cell states, and cell proliferation in early tumorigenesis.

4.2 Prescence and requirement of PRC2 action and H3K27me3 in intestinal
epithelium and tumors
In native intestinal crypt-villus axis, as ISCs differentiate into fast cycling TA cells and then
into post-mitotic villus cells, PRC2 action (Ezh2) is highest in the cycling cells. Accordingly,
immunohistochemical staining demonstrates that the proliferating cells in crypt show higher
amounts of Ezh2 (PRC2 action) and the amount reduces in the villus cells (Fig. 13C), while
H3K27me3 is present at a lower level in crypts cells and increases in villus cells (Fig. 14C).
Higher amount of Ezh2 in the dividing cells of crypts represents the consistent need to add
H3K27me3 to all the repressed genes after every cell division in order to maintain the gene
repression. However, in the villus, the cells are non-dividing, thus reducing the requirement of
PRC2 action in order to maintain the gene repression. During early tumorigenesis (ApcKO),
these trends of PRC2 and H3K27me3 were changed, as tumor cells showed consistently high
Ezh2 and H3K27me3 in majority of cells (Fig. 13E, F, Fig. 14E, F). In ApcKO-Ezh2OE mice, the
change in Ezh2 levels is undetectable in immunohistochemical analysis as the Ezh2OE allele is

21
only designed to increase the gene expression by 1.2-1.4 fold. On the other hand, in ApcKO-
EedKO tumors, lack of H3K27me3 staining (Fig. 14K, L) confirms the uniform loss of PRC2
action. With the PRC2 modulation in these two mouse models, we do not see any major
influence on spread or reduction of tumorigenic areas or tumor sizes by looking at gross
morphology of the tissue after dissection or after sectioning and H&E staining of the entire
tissue (Fig. 8). This suggests that change in PRC2 action does not have impact on the tumor
morphology and growth at this early stage of adenoma formation. It is possible that choosing an
earlier timepoint (day 7 or day 10, as opposed to day 14) may reveal any effects of PRC2
modulation on tumor formation that are overshadowed by the cellular over proliferation by day
14.

4.3 Alteration to stem cell properties and cellular proliferation in intestinal tumors and
effect of PRC2 modulation
Cellular hyperproliferation is a hallmark of cancer. As gastrointestinal epithelium is one of
the most proliferative mammalian tissues, chances of genetic and epigenetic deregulation
leading to loss of control in proliferative pathways is also very high. Epithelial proliferation is
normally controlled through tight regulation by signaling pathways and Wnt signaling plays a
significant role in maintaining stem and TA cells proliferation in GI track. Apc loss, which
constitutively activates Wnt signaling, causes hyperproliferation leading to early adenomas in
the intestinal as well as colonic tissue.
Using BrdU incorporation (IP injection 1 hr before mouse euthanasia), followed by
immunostaining, I could quantify proliferating (BrdU+) cells in mouse intestinal adenomas
(tdTom+) from ApcKO as well as the mice with PRC2 hyperactivity or loss of action.
Surprisingly, both the hyperactivity and loss of activity of PRC2 show increase in proliferative
cells. Although I see clear gains in percent of proliferating cells in PRC2 modulated tumors (from
15% in ApcKO upto 30% in ApcKO-EedKO), these findings do not show high statistical

22
significance. This is because of two factors: a) we have been able to add two samples to these
and most other counts and b) many tumors have heterogeneity of cellular changes, which is
inherent to tumor behavior and may be resolved by analysis of larger number of samples.
Additionally, this may represent impact on cell properties caused by the epigenetic change
(Ezh2 overexpression, in this case), which is more apparent in the case of other studies
presented here and discussed below.
One prominent way that H3K27me3 based PRC2 action controls the cell proliferation
is through the tumor suppressor gene Cdkn2a. In native intestinal epithelium this gene is
constitutively repressed with H3K27me3, while loss of PRC2 action or modulation of
H3K27me3, as part of many cancers, allows de-repression/activation of this gene causing cell
cycle arrest
28
. Interestingly, in context of Apc loss induced early tumorigenesis (ApcKO), upon
loss of Eed and H3K27me3 (Fig. 14K, L), I do not see Cdkn2a upregulation (Fig. 17B).
Instead, I observe loss of Eed and PRC2 action (H3K27me3 staining) causes increased
proliferation in the ApcKO-EedKO tumors (15% gain of BRDU+ cells). This suggests that
tumor cells, upon mutations that may restrict their capacity to grow and survive, may adopt to
alternative molecular pathways to overcome blocks to cellular hyperproliferation. Thus, Eed
deletion, which causes proliferation block through Cdkn2a activation in native tissue, is
triggering an alternate way to keep Cdkn2a gene suppressed in absence of H3K27me3 in
cancer. To identify such novel phenomenon, future investigations may focus on additional
repressive epigenetic features being gained around Cdkn2a gene, which may include
repressive histone marks such as H3K9me3 and gain of DNA methylation. These data also
demonstrate the complexity in design of effective PRC2 inhibitors and how cancer may evade
treatments using such inhibitors.
Surprisingly, my studies do not show significant change in the number of tumor stem
cells upon PRC2 modulation (Fig.11). This suggests that while stem cell hyperproliferation is
key to early tumor formation upon Apc loss, PRC2 modulation does not impact the stem cell

23
expansion. In this context, the increased proliferation observed in PRC2 modulated tumors
(25% in ApcKO-Ezh2OE and 30% in ApcKO-EedKO as compared to 15% in ApcKO) would
represent altered properties of non-stem cells, which further suggests an impact on cell
differentiation. Indeed, I see decrease in goblet cell numbers upon Ezh2 overexpression in
tumors (from 3.8% in ApcKO to 1.6% in ApcKO-Ezh2OE, Fig.12). Previous studies have
shown that loss of PRC2 action and H3K27me3 can lead to gain of secretory lineage cells
including goblet cells
52
, while in my studies the increased Ezh2 activity shows loss of goblet
cell differentiation at the cost of tumor cell proliferation.

4.4 Influence of Apc loss and PRC2 modulation on Wnt signaling
ß-catenin localization to nucleus is essential for canonical Wnt signaling as ß-catenin
interacts with TCF/LEF family transcription factors in the nucleus to activate Wnt target genes.
This Wnt signaling is required for stem cell proliferation as well as maintenance of stem cell
specific genes in gastrointestinal epithelium. Thus, hyperproliferation in ApcKO adenomas may
be directly linked to ß-catenin localization, while change in ß-catenin localization may be
involved in alteration of tumor cell properties upon additional mutations or epigenetic change.
My immunohistochemical analysis showed that almost all cells in ApcKO adenomas had nuclear
ß-catenin (91%, Fig. 15). While loss of PRC2 action (ApcKO-EedKO) had some impact on the
ß-catenin localization (79% ß-catenin+ cells), many cells in ApcKO-Ezh2OE adenomas (48%)
had diffuse ß-catenin staining, which did not match the punctate staining in the other two
genotypes (Fig. 15 C, D). One possibility is that the Ezh2 overexpression causes reduced
expression of ß-catenin, thus reducing nuclear accumulation of the factor. However, ß-catenin
expression is unchanged upon PRC2 modulation (Fig. 17). An alternative possibility is that ß-
catenin interacting proteins change expression or localization upon Ezh2 overexpression, in turn
influencing ß-catenin localization. To address this possibility, I first looked at gene expression of
more than 60 known ß-catenin interacting proteins
41
in ApcKO adenomas with or without PRC2

24
modulation using RNA-seq data (Fig. 17). Among only 4 of these genes upregulated in various
genotypes of adenomas, I focused on understanding the role of BCL9. As BCL9 is known to aid
nuclear localization and retention of ß-catenin, this may provide possible mechanism of driving
ß-catenin based canonical Wnt signaling and cell hyperproliferation in tumors. Interestingly,
while ApcKO-Ezh2OE tumors show almost 40% reduction in nuclear ß-catenin (Fig. 15), only
these tumors also showed about 15% reduction nuclear BCL9 with diffuse staining across the
cell body (Fig. 16), suggesting that Ezh2 overexpression may decrease BCL9 nuclear
localization, which in turn might limit ß-catenin nuclear localization through direct interaction. To
test this further, co-immunoprecipitation assays could be performed using cytoplasmic and
nuclear extracts from ApcKO and ApcKO-Ezh2OE tumors, where reduced presence and
interaction of ß-catenin and BCL9 in ApcKO-Ezh2OE nuclear extracts (in comparison to ApcKO)
will indicate BCL9 mediated control of ß-catenin localization.
Additionally, both ß-catenin and BCL9 show non-uniform nuclear distribution within and
across ApcKO-Ezh2OE tumors, as some cells have punctate nuclear staining while others show
diffuse staining across cell body (Fig. 15 C, D, Fig. 16 C, D). This suggests diverse Wnt action
and its impact on cells within individual tumors as well as different tumors, further indicating
distinct evolution of molecular and cellular properties in tumors. Interestingly, in many ApcKO-
Ezh2OE tumors, ß-catenin and BCL9 show nuclear staining in cells towards the crypt regions
(near to the muscle layer in the tissue), while the diffuse staining is more prevalent in cells
farther away from the crypt zone. This suggests, in addition to the constitutive Wnt signaling
(caused by Apc loss), the cells are influenced by the signaling environment that is geared to
induce differentiation as they move away from the crypt region (e.g. higher Bmp and Egf
signaling). This highlights the importance of future studies in understanding the impact of
mesenchymal signaling on epithelial cell behavior in cancer.
In summary, my studies provide detailed and mechanistic understanding of alterations in
stem cell properties, cellular proliferation and differentiation, as well as signaling in the earliest

25
stages of gastrointestinal tumorigenesis. Using parallel genetically engineered animal models, I
have studied the molecular impact of modulation in PRC2 action on tumor epigenome, gene
expression, and cell singling essential for tumor growth. These studies will be instrumental in
future identification of molecular pathways underlying cancer progression and targets for novel
epigenetic therapies.

26
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Appendices

30
Appendix A: Tables and Figures
Table 1. Primary and secondary antibodies used for immunofluorescence and DAB based
staining of tissues in this thesis.
Primary Antibody Catalog# Dilution Secondary Antibody Catalog# Dilution
Abcam
Anti-BrdU antibody
[BU1/75 (ICR1)] -
Proliferation Marker
(ab6326)
ab6326

1: 300 Invitrogen Donkey
anti-Rat IgG (H+L)
Highly Cross-
Adsorbed Secondary
Antibody, Alexa Fluor
488
A21208 1:1000
Cell Signaling
Tri-Methyl-Histone
H3 (Lys27) (C36B11)
Rabbit mAb

9733

1:750 Invitrogen Goat anti-
Rabbit IgG (H+L)
Cross-Adsorbed
Secondary Antibody,
Alexa Fluor 488
A11008 1:1000
Arigo
biolaboratories
Anti- tdtomato
antibody
ARG55724

1:50 Invitrogen Donkey
anti-Goat IgG (H+L)
Cross-Adsorbed
Secondary Antibody,
Alexa Fluor 594
A11058 1:1000
Cell Signaling Ezh2
(D2C9)
XP Rabbit mAb

5246 1:100 Invitrogen Goat anti-
Rabbit IgG (H+L)
Cross-Adsorbed
Secondary Antibody,
Alexa Fluor 488
A11008 1:1000
Santa Cruz Anti-GFP
Antibody (B-2)

sc-9996 1:50 Invitrogen Donkey
anti-Mouse IgG (H+L)
Highly Cross-
Adsorbed Secondary
Antibody, Alexa Fluor
488
A21202 1:1000
Cell Signaling
β-Catenin (D10A8)
XP® Rabbit mAb

8480S

1:25 AffiniPure Alpaca anti-
rabbit IgG (H+L)
Biotin SP-long spacer
conjugate

611-065-
215

1:1000
Thermofisher BCL9
Polyclonal Antibody
rabbit
22947-1-
AP

1:25 AffiniPure Alpaca anti-
rabbit IgG (H+L)
Biotin SP-longspacer
conjugate

611-065-
215

1:1000

31
Table 2. The effect of gain or loss of PRC2 action on tumor cell proliferation.

Number of
Tdtom+ cells
Number of BrdU+
cells
Percentage of BrdU +
cells out of Tdtom+ cells
ApcKO 3,869 597 15.43
ApcKO-EzhOE 1,827 454 24.85
ApcKO-EedKO 2,409 729 30.26

Table 3. The effect of gain or loss of PRC2 action on stem cell abundance.

Number of
Tdtom+ cells
Number of GFP+
cells
Percentage of GFP+
cells out of Tdtom+ cells
ApcKO 1,719 282 16.40
ApcKO-EzhOE 1,892 282 14.90
ApcKO-EedKO 2,584 341 13.20

Table 4. The effect of gain or loss of PRC2 action on stem cell differentiation.

Number of all nuclei Number of Alcian
blue+
Percentage of
Alcian Blue+ cells
ApcKO 2,826 107 3.79
ApcKO-EzhOE 2,404 39 1.62
ApcKO-EedKO 2,203 64 2.91

Table 5. The effect of gain or loss of PRC2 action on ß-catenin+ nuclei

Number of all nuclei
counted
Number of ß-
catenin+ nuclei
Percentage of ß-
catenin+ nuclei
ApcKO 3,688 3,385 91.78
ApcKO-EzhOE 2,053 1,068 52.02
ApcKO-EedKO 3,380 2,701 79.91

Table 6. The effect of gain or loss of PRC2 action on BCL9 nuclei

Number of all nuclei
counted
Number of BCL9+
nuclei
Percentage of BCL9+
nuclei
ApcKO 3,410 3,214 94.25
ApcKO-EzhOE 2,656 2,116 79.67
ApcKO-EedKO 2,470 2,299 93.08

32

Figure 1. Common genetic changes and progression of colorectal cancer. Normal
gastrointestinal epithelium becomes hyperproliferative upon deactivating Apc tumor suppressor
gene mutation, forming early benign polyps. Mutations in K-ras oncogene and DCC tumor
suppressor cause formation of intermediate and late benign polyps, while a p53 mutation
causes these polyps to turn into carcinoma. Mutations in these four genes is enough to cause
metastatic CRC, although it is usually associated with additional mutations.

33

Figure 2. (A is Modified from https://www.sciencelearn.org) Structure of the intestinal
epithelium and stem cell differentiation. A. Inner lining of the intestine showing crypt and
finger like projections into the lumen that are villi. B. Intestinal epithelial crypt and villus
structures showing intestinal stem cells (ISC, Lgr5+) in green color at the bottom of the crypts.
Grey cells are stem cell daughters that form the transit amplifying (TA) cells. TA cells further
divide and differentiate into functional cells on villus. C. ISCs form progenitors or TA cells of
secretory or absorptive type in the crypt, which further form mature cells of those lineages:
Goblet, Paneth, and Enteroendocrine cells (secretory cells), Enterocytes (absorptive cells).

34

Figure 3. (Modified from Ref. 36) The Wnt/β-catenin pathway. A. In absence of extracellular
Wnt, the pathway is off. β-catenin destruction complex consisting of Apc floats freely within the
cytoplasm and β-catenin is constantly phosphorylated by CK1 and GSK3-β in this complex. β-
catenin is further ubiquitinated by β-Trcp and degraded by proteasomes. B. Upon binding of
Wnt ligand to the frizzled receptor, it becomes a part of the frizzled receptor complex with Lrp5.
This further causes deactivation of kinases in the β-catenin destruction complex further allowing
accumulation of β-catenin, which translocates into the nucleus to turn on target genes along
with Pygo, LGS, and TCF factors.
A B

35

Figure 4.

(Modified from Ref. 38) Cellular mechanisms regulating nuclear localization of β-
catenin. Six major mechanisms that regulate import of β-catenin into the nucleus, its retention
in the nucleus and its export outside the nucleus are represented. In this thesis, we particularly
consider how β-catenin localization is influenced by the interactions with BCL9 to aid its nuclear
retention.

36

Figure 5. (Modified from Ref. 39) Nuclear translocation of β-catenin by BCL9-2.
Immunofluorescence based staining of Kidney Epithelia Cells (MDCK) shows cytoplasmic β-
catenin (A) and BCL-9 (B) signal; C shows merged signals. Upon hepatocyte growth factor
(HGF) based induction of epithelial-mesenchymal transition, β-catenin and its binding fragment
of BCL9-2 (amino acids 387-530) are localized to the nucleus instead of the cytoplasm (D-F).

A
B C
D E F
Beta–catenin
binding
fragment of
BCL9-2

37

Figure 6. (Modified from Ref. 42) Polycomb repressive complex 2 (PRC2) based gene
repression. Canonical PRC2 is composed of the catalytic subunits EZH1 or EZH2, and
structural subunits EED, SUZ12, and RBAP46 or RBAP48. Accessory subunits JARID2 and
AEBP2 aid the targeting of PRC2 complex to specific genomic loci consisting of unmethylated
CpG islands. PRC2 catalyzes the trimethylation of histone H3 at lysine 27, which promotes
genomic compaction, heterochromatin formation, and gene repression.

38

Figure 7. Mouse models for CRC progression. A. Genotypes of various genetically modified
mouse model used in this study. B. In all mouse models, ISCs in about 30% of crypts in
proximal duodenum express EGFP as well as tamoxifen inducible Cre from the Lgr5 gene locus
(Lgr5
EGFP-CreER
). Two injections of tamoxifen on consecutive days (D1 and D2) cause activation
of the causing deletion of floxed alleles including Apc and also trigger constitutive expression of
tdTomato from the Rosa26 locus (Rosa26
LsL-tdTomato
) causing all stem cells with Cre activity as
well as their daughters to be tdTOmato+ allowing lineage tracing. In mice with Apc loss,
hundreds of adenomas are formed in intestine as well as colon within weeks. We harvest the
tissue 14 days after tamoxifen induction. C Gross morphology of the proximal intestinal tissue
showing adenoma formation. Immunofluorescence images show of GFP labeled Lgr5+
intestinal stem cells (ISC) that are located within the TdTomato+ cancer cells. We can purify
these cell types using FACS use for gene expression (RNA-seq) or epigenetic assays (CHIP-
seq).

C

B
D
A

B
B

39

Figure 8. H&E staining showing tissue and tumor morphology. A shows the normal crypt
villus structures (to the right) besides a tumor (to the left) in ApcKO tissue. B, C and D shows
the comparison of H&E for ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO tissues. Arrows
indicate tumorigenic areas in the tissue. E, G, and F show individual tumorigenic areas of the
tissue. (Scale bar= 1600 µm)

40
Figure 9. Tumor cell proliferation using BrdU staining. Representative panels from all tumor
conditions (ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO) showing immunohistochemical
staining of BRDU and TdTomato using 5 µm paraffin embedded tissue sections. Dapi staining
marks all cellular nuclei, Tdtomato marks all cells with Apc deletion (tumor cells), and BrdU is
used to mark proliferating cells in the S phase of the cell cycle. The images were taken at a 20x
magnification. (Scale bar = 400 µm) (The bar plot shows percent of BrdU+ cells among tdTom+
(adenoma) cells)

41

Figure 10. Stem cell abundance in intestinal tumors. Representative panels from all tumor
conditions (ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO) showing immunohistochemical
staining of GFP and TdTomato using 5 µm paraffin embedded tissue sections. Dapi staining
marks all cellular nuclei, Tdtomato marks all cells with Apc deletion (tumor cells), and GFP
staining marks tumor stem cells. The images were taken at a 20x magnification. (Scale bar =
400 µm) (The bar plot shows percent of GFP+ cells among tdTom+ (adenoma) cells)

42

Figure 11. Alcian Blue Assay The figure shows alcian blue assay for ApcKO, ApcKO-Ezh2OE,
and ApcKO-EedKO genotypes (A, C, and E) (Scale bar = 100 µm). Dotted rectangles show
zoomed in areas in B, D, and F on the right. The bar plot shows percent of Alcian blue+ cells
indicative of goblet cells among nuclear fast red stained nuclei.

43

Figure 12. Endogenous Phosphatase Assay The figure shows endogenous phosphatase
assay for ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO genotypes, which stains enterocytes
brush borders in blue. Nuclear fast red stains the nuclei. (Scale bar = 100 µm)

44

Figure 13. EZH2 Immunofluorescence Staining The figure shows immunofluorescence
staining for EZH2 in WT, ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO intestinal tissue. Dapi is
used to show the nuclei in A, D, G, and J in blue. EZH2 staining is in B, E, H, and K in green.
The images were taken at 10X magnification. (Scale bar = 400 µm, dotted rectangles show
zoomed in areas of C, F, I, and L on the right).

45

Figure 14. H3K27me3 Immunofluorescence Staining The figure shows immunofluorescence
staining for H3K27me3 in WT, ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO genotypes in
intestinal tissue. Dapi is used to show the nuclei in A, D, G, and J in blue. H327me3 staining is
in B, E, H, and K in green. The images were taken at 10X magnification. (Scale bar = 400 µm,
dotted rectangles show zoomed in areas of C, F, I, and L on the right).

46

Figure 15. ß-catenin Staining The figure shows a DAB staining for ß-catenin. Intestinal tissues
with ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO genotypes are shown. The images were
taken at 10X magnification. (Scale bar = 100 µm, dotted rectangles show zoomed in areas of B,
D and F on the right). The bar plot shows percent of ß-catenin+ nuclei among non-punctate
nuclei.

47

Figure 16. BCL9 Staining The figure shows a DAB staining for BCL9 in ApcKO, ApcKO-
Ezh2OE, and ApcKO-EedKO genotypes in intestinal tissue. The images were taken at 10X
magnification. (Scale bar = 100 µm, dotted rectangles show zoomed in areas of B, D and F on
the right). The bar plot shows percent of BCL9+ nuclei among non-punctate nuclei.

48

Figure 17. Gene expression changes in tumor stem cells. A shows high correlation of stem
cell gene expression (mRNA-seq replicates from individual mice) among various genotypes as
multiple samples from each genotype cluster together in principle component analysis (PCA)
plot. mRNA-seq data are analyzed using DeSeq2 program. Majority of gene expression change
in stem cell occurs upon Apc loss, while PRC2 modulation causes lesser amount of gene
expression change. B Volcano plots showing significant gene expression changes in
comparison of all genotypes. Three known β-catenin binding partners are downregulated upon
loss of Apc: Hspe1, Nme2, and Rpl23a, while four β-catenin binding partners are upregulated:
Aldoa, Dbn1, Myl9, and Bcl9.

49
Figure 18. H3K27me3 presence at Bcl9 gene. Integrative genomic viewer (IGV) tracks
showing H3K27me3 signal in WT, ApcKO, ApcKO-Ezh2OE, and ApcKO-EedKO stem cells. The
lack of significant H3K27me3 signal around Bcl9 gene suggests that Bcl9 expression is not
directly influenced by canonical PRC2 action in tumor cells.

Abstract (if available)

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer related deaths that resulted in more than 860,000 deaths worldwide in 20181. CRC results from well-known genetic changes in APC, KRAS, and TP53, however, epigenetic changes in CRC progression need to be further established1. Polycomb Repressive Complex 2 (PRC2) mediated histone methylation (H3K27me3) and DNA methylation are coregulatory epigenetic modifications that play important roles in gastrointestinal tissue homeostasis and cancer2,3. Overexpression of EZH2, the enzymatic subunit of PRC2 complex4,has been associated with CRC and other solid tumors5-7. Gastrointestinal Lgr5+ve stem cells (ISCs) are well established cell of origin of CRC and Wnt signaling is critical for ISC for proliferation and maintenance. Wnt induction causes cytoplasmic ß-Catenin to relocate to nucleus where it activates many Wnt-target genes linked to cell proliferation, while in absence of the Wnt signal, Apc containing protein complex marks beta catenin for degradation in cytoplasm8-10. Apc mutations are prevalent in CRC and cause nuclear accumulation of ß-Catenin resulting in constitutive Wnt signaling and hyperproliferation of ISCs, leading to adenomas11. Currently, there is little understanding of molecular events, particularly epigenetic controls that modulate Wnt based stem cell control, particularly in early cancer development.
In order to understand the impact of altered PRC2 activity in CRC, our lab has developed mouse models that allow inducible deletion of Apc in Lgr5+ve ISCs, which produces adenomas in 2-3 weeks. Additionally, parallel models allow over expression of Ezh2 or deletion of PRC2 component Eed which eliminates PRC2 activity, along with Apc deletion. Using these established mouse models, I proposed to study the impact of Ezh2 overexpression on early gastrointestinal tumorigenesis, specifically on 1) stem cell behavior and 2) Wnt signaling.

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Asset Metadata

Creator Mays, Tiffany Michelle (author)

Core Title Molecular role of EZH2 overexpression in Colorectal Cancer progression

School Keck School of Medicine

Degree Master of Science

Degree Program Stem Cell Biology and Regenerative Medicine

Degree Conferral Date 2022-08

Publication Date 08/02/2022

Defense Date 06/10/2022

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

Tag colorectal cancer,epigenetics,EZH2,genetics,ISC,LGR5,OAI-PMH Harvest,PRC2,stem cell,Wnt/Beta-catenin pathway

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Jadhav, Unmesh (committee chair), Georgia, Senta (committee member), Mariani, Francesca (committee member)

Creator Email tiffany.mays@northwestern.edu,tiffanymichellemays@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111375994

Unique identifier UC111375994

Legacy Identifier etd-MaysTiffan-11071

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Rights Mays, Tiffany Michelle

Type texts

Source 20220803-usctheses-batch-967 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu