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Notch Signaling Mediates Tumor-CAF

Crosstalk in Basal-like Breast Cancer

By

Jifeng Song

A thesis submitted in conformity with the requirements for

the degree of Master of Science

Graduate Department of Medical Biophysics

University of Toronto

©Copyright by Jifeng Song (2014)

ii

Notch Signaling Mediates Tumor-CAF Crosstalk

in Basal-like Breast Cancer

Jifeng Song

Master of Science

Department of Medical Biophysics

University of Toronto

2014

Abstract

Increasing evidence indicates the importance of the tumor microenvironment in

cancer progression. Cancer-associated fibroblasts (CAFs), in particular, have been

reported to support tumorigenesis by promoting cell proliferation, invasion and

angiogenesis. However, the mechanisms by which CAFs and tumor cells, specifically

the basal-like subtype of breast cancer (BLBC), interact with each other, remain

unclear. Interestingly, BLBC tumors are characterized by activation of the Notch

pathway. Here, we performed co-cultures of BLBC cells with CAF-like fibroblasts and

investigated the role of Notch in these interactions. We showed CAF-derived TGFβ

drives tumor Notch signaling and expression of the Notch target uPA. Notch

activation also drives expression of c-MET which is required for uPA expression.

Supporting functional assays suggested CAF-like cells promote invasion of

MDA-MB231, in a fashion that depends upon TGFβR1, c-MET, uPA and Notch.

Therefore, Notch participates in tumor-CAF crosstalk in BLBC cells, and may

represent an important target for cancer therapy.

iii

Acknowledgements

One day, when I was chatting with an old friend over coffee, she said: Jeff, do

you still remember in elementary school that you always wanted to do science?

Suddenly, I realized that my dream did come true. Although this 3-year journey was

filled with some failed experiments, long holiday lab hours, and stress before exams, I

found it worthy since I experienced so much personal growth, both in terms of

knowledge and in abilities such as scientific critical thinking and public speaking. First

thing first, I would like to take this chance to say thank-you to all the supportive

people around me.

I want to start my notes of thanks with a special acknowledgement to my

supervisor Dr. Michael Reedijk. Mike, your guidance, constructive criticism and

patience really motivated me and helped me moving forward. As opposed to a “boss”

role, you are taking a “father” role since you are the only supervisor I know who is

willing to spend so much time and effort on a student. I am truly grateful to be a part

of your lab.

I am deeply grateful to my unofficial co-supervisor, Dr. Qiang Shen for his

continuous support with both the project and my life. His unconditional help with

experimental design and trouble-shooting greatly facilitated the progress of my

project. Qiang, I will miss the times where we chatted about everything during the

breaks.

I would like to acknowledge and thank my committee members Dr. Pam Ohashi

and Dr. Laurie Ailles. Their advice and support were critical for the development of

my project. I also thank them for giving me an easy time on those committee

meetings.

I owe special thanks to my present/former lab mates in Dr. Reedijk’s laboratory,

Brenda Cohen, Julia Izrailit, Dr. Sarah Lamorte and Dr. Mamiko Shimizu, for their

critical and informative discussions. Brenda, thank you for taking care of every single

detail of the experiments and making my life much easier.

Outside of the academic world, first I would like to thank my family, especially

iv

my mother who supports me all the time, both spiritually and financially. I have also

received tons of help from my dearest friends. Thank you Vivian, Yan, Yang, Robert,

Zoe, Nikki, Anna, Yingying, Roy, Jimmy, Linus, Donald, Jing and two other Michaels.

Your inspiration and encouragement allowed me to overcome fears, stay focused,

and conquer whatever obstacles I face. There is still a long way to go but we will do it

together.

Last but not least, I want to say thank-you to my little puppy Kiwi (or you should

thank me first for raising you up). Your consistent enthusiasm and cuteness

transformed every bad day I had. Let us keep growing.

v

Table of Contents

Abstract……………………………………..……………………………………………………………………..……..ii

Acknowledgements…………………….…………………………………………………………………..………iii

Table of Contents…………………………………………………………………………………………….....……v

List of Figures………………………………………………………………………………………………..…….….vii

List of Tables…………………….……………………………………………………………………………..……viii

CHAPTER 1: Introduction………………………………………………………………………………..………..1

1.1. The mammary gland.....................................................................................2

1.1.1. Mammary gland structure and physiology……………………………………….2

1.1.2. Mammary gland development…………………………………………………………2

1.2. Breast cancer……………………………………………………………………………………………..4

1.2.1. Epidemiology…………………………………………………………………………………..4

1.2.2. Breast cancer subtypes…………………………………………………………………….5

1.3. Tumor microenvironment…………………………………………………………………………..7

1.3.1. Structure of the tumor microenvironment…………...……………………….7

1.3.2. Cancer-associated fibroblast……….…………………………………………………..8

1.3.3. Tumor-promoting roles of CAFs........................................................10

1.4. The Notch signaling pathway.......................................................................11

1.4.1. Notch signaling in development........................................................11

1.4.2. The mechanism of Notch signaling...................................................13

1.4.3. The structure of Notch ligands and receptors...................................14

1.5. Notch signaling in breast cancer...................................................................16

1.5.1. Evidence for oncogenic Notch in breast cancer development..........16

1.5.2. Notch activation and the BLBC subtype………....................................17

1.5.3. Mechanisms of Notch activation in breast cancer.............................18

1.6. The urokinase-type plasminogen activator system......................................20

1.7. The TGFβ signaling pathway.........................................................................21

1.7.1. The mechanism of TGFβ signaling.....................................................21

1.7.2. TGFβ signaling and cancer.................................................................21

1.7.3. TGFβ and CAF.....................................................................................23

1.8. The HGF-MET signaling pathway..................................................................23

1.8.1. The mechanism of HGF-MET signaling..............................................23

1.8.2. HGF-MET signaling and cancer...........................................................24

1.8.3. HGF and CAF......................................................................................25

1.9. Hypothesis and Aims....................................................................................25

CHAPTER 2: Materials and Methods…………………………………………………….……….....……27

2.1. Cell culture……………………………………………………………………………………………….28

2.2. RNA interference………………………………………………………………………………………29

2.3. RT-qPCR: RNA preparation, reverse transcription (RT), and quantitative

real-time PCR (qPCR)……………………………………………………………………………………….30

vi

2.4. Co-culture conditions……………………………………………………………………………….31

2.5. Conditioned media and whole cell lysate preparations, Western blotting,

and antibodies………………………………………………………………………………………………..31

2.6. Cell counting…………………………………………………………………………………………….33

2.7. Invasion assay…………………………………………………………………………………………..34

CHAPTER 3: Results………..…………………………………………………………………………...…………35

3.1. Fibroblast-derived TGFβ promotes JAG1/Notch-mediated uPA expression in

BLBC cells………………………………………………………………………………………...……………..36

3.1.1. Fibroblast-derived TGFβ induces uPA expression in BLBC cells.........36

3.1.2. Notch signaling is required for fibroblast TGFβ-mediated uPA

up-regulation in MDA-MB231 and HCC1143 BLBC cell lines................……..40

3.2. CAF-like cells promote Notch-dependent uPA secretion in BLBC cells……….44

3.3. TGFβR1 and c-MET receptors are required for CAF-induced uPA expression

in BLBC cells…………………....................………………………………………………………………48

3.4. CAF-like cells promote uPA, Notch, TGFβR1 and c-MET–dependent invasion

of MDA-MB231 cells……………………………………………………………………………………….52

3.5. c-MET is a downstream target of Notch in BLBC cell lines………………………...54

CHAPTER 4: Discussion and Future Directions……………………………….………………………57

4.1. TGFβ in the tumor microenvironment…..………………………………………………..58

4.2. Exp-CAF2: a model cell line for CAFs……………………………………………………….59

4.3. The HGF/c-MET signaling axis and its crosstalk with Notch in BLBC

cells………………………………………….……………………………………………………………………..60

4.4. The roles of uPA in promoting cell invasion and in growth factor

activation ……………………………………………………………………………………………………….62

4.5. Co-culture systems……………………………………………………………………………………62

4.6. The clinical significance of the work presented in this thesis…………………….63

REFERENCES……………………………………………………………………………………………………………65

vii

List of Figures

Figure 1.1: Stages of postnatal mammary gland development.……………………………......3

Figure 1.2: The tumor microenvironment.....................................................................8 Figure 1.3: Canonical Notch signaling in mammals.....................................................14 Figure 1.4: Notch ligands and their receptors in mammals........................................15 Figure 1.5: A schematic representation of simplified canonical TGFβ signaling

pathway.......................................................................................................................22 Figure 1.6: A schematic representation of the HGF-MET signaling pathway…………...24 Figure 3.1.1: Characterization of the cell lines used in co-culture experiments.........37

Figure 3.1.2: Fibroblast-derived TGFβ promotes uPA expression in MDA-MB231 and

HCC1143 cell lines……..................................................................................................39 Figure 3.1.3: Fibroblast TGFβ-induced uPA expression is tumor cell Notch-dependent

.....................................................................................................................................43

Figure 3.2.1: Experimentally-generated Exp-CAF2 cells share myofibroblastic traits

with CAFs.....................................................................................................................45 Figure 3.2.2: Exp-CAF2 cells promote JAG1/Notch-mediated uPA expression in BLBC

cells..............................................................................................................................47 Figure 3.3.1: TGFβR1 and c-MET receptors are required for uPA expression in BLBC

cells…………………………………………….…………………………………………………………………..........50 Figure 3.4.1: Exp-CAF2 cells promote uPA, Notch, TGFβR1 and c-MET–dependent

invasion of breast cancer cells………………………………………………………………………..……….53 Figure 3.5.1: Notch potentiates c-MET signaling between BLBC cells and CAFs.........55

viii

List of Tables

Table 2.1: siRNAs used in reverse transfection………………………………………………………..29

Table 2.2: DNA sequences of the primers used for mRNA quantification by RT-

qPCR……………………………………………………………………………………………………………………....30

Table 2.3: Serum-free media used in co-culture conditions…………………………………….31

Table 2.4: Primary antibodies used in Western blot analyses………………………………….33

Table 2.5: Secondary antibodies used in Western blot analyses………………………………33

1

CHAPTER 1: Introduction

2

1.1. The mammary gland

1.1.1. Mammary gland structure and physiology

The mammary glands are complex secretory organs that distinguish mammals

from all other animals. Their unique anatomical structure enables the secretion of

milk and thus the nourishment of the newborn.

The mature mammary gland is comprised of an epithelial ductal system and

surrounding stromal components. The mammary epithelium consists of two major

cell types: luminal and basal. Luminal epithelial cells form ducts and secretory alveoli,

which become milk-secreting lobules during lactation. On the basal side,

myoepithelial (basal) cells produce and attach to, the basement membrane (Figure

1.1). Altogether, this bi-layered structure allows lactation when the outer

myoepithelial cells contract to squeeze milk produced by the inner alveolar luminal

cells1,2

.

The stromal compartment includes extracellular matrix (ECM) and numerous

stromal cell types such as endothelial and immune cells, fibroblasts, and adipocytes.

Many stromal cell types, and several ECM molecules, play critical roles in mammary

duct morphogenesis and tissue homeostasis3.

1.1.2. Mammary gland development

There are three major stages of mammary gland development-embryonic,

pubertal, and reproductive. The mammary gland undergoes significant structural and

functional changes directed by both signals from the mesenchyme (during the

embryonic stage) and circulating hormones (during puberty and in adulthood). With

the onset of puberty, epithelial ducts at the nipple start expansive growth that fills

the fat pad with the epithelial mammary tree. This growth is regulated by growth

hormone and estrogen, and insulin-like growth factor-1 (IGF-1). During pregnancy,

the combined actions of progesterone and prolactin initiate alveologenesis, where

proliferating epithelial cells become milk-secreting lobules. The lack of demand for

milk at weaning triggers the process of involution that can be further divided into

two phases: phase one is reversible, and is accompanied by apoptosis, alveolar cell

detachment and accumulation of shed cells into the lumen; phase two includes

Figure 1.1: Stages of postnatal mammary g

ducts (solid lines) at the nipple grow allometrically until puberty. Puberty initiates a process

called ductal morphogenesis that fills the fat pad with a ductal tree. Terminal end buds (TEBs)

are club-shaped structures at the tips of growing ducts that penetrate the fat pad.

(expanded) is composed of body cells that differentiate into luminal epithelial cells

cells that generate myoepithelial cells. Alveologenesis occurs

induction of prolactin, which together with progesterone, fuels the growth of alveolar cells

(white oval) and thus milk production.

process of involution that removes milk

tree back to its original adult architecture

tages of postnatal mammary gland development. At birth, the rudimentary

at the nipple grow allometrically until puberty. Puberty initiates a process

that fills the fat pad with a ductal tree. Terminal end buds (TEBs)

uctures at the tips of growing ducts that penetrate the fat pad.

) is composed of body cells that differentiate into luminal epithelial cells

cells that generate myoepithelial cells. Alveologenesis occurs during pregnancy with the


(white oval) and thus milk production. Lack of demand for milk at weaning initiates the

process of involution that removes milk-producing epithelial cells and remodels the ductal

tree back to its original adult architecture.

3

At birth, the rudimentary

at the nipple grow allometrically until puberty. Puberty initiates a process

that fills the fat pad with a ductal tree. Terminal end buds (TEBs)

uctures at the tips of growing ducts that penetrate the fat pad. The TEB

) is composed of body cells that differentiate into luminal epithelial cells, and cap

during pregnancy with the


Lack of demand for milk at weaning initiates the

odels the ductal

4

apoptosis, ECM breakdown and protease activation, and is irreversible. The

mammary gland is then remodelled back to its pre-pregnancy state 1,4

.

Both myoepithelial and luminal epithelial cells arise from a common progenitor,

the mammary stem cell (MaSC), which is characterized by the ability to self-renew

(i.e. go through cycles of cell division while maintaining their undifferentiated state)

as well as by the ability to differentiate and generate all the cell types in mammary

tissue (multipotency)5. The existence of MaSCs in mammary tissue is suggested by

the observation that small fragments of the rodent duct when transplanted into

cleared mammary fat pads could develop an entire and functional mammary tree6,7

.

Current markers to identify the MaSC population include Lin-/CD44

+/CD24

low/- and

increased aldehyde dehydrogenase activity8,9

.

1.2. Breast cancer

1.2.1. Epidemiology

Excluding cancers of the skin, breast cancer is the most common cancer among

women, accounting for nearly 1 in 3 cancers diagnosed in US women10

. The lifetime

risk of developing invasive breast cancer for US woman is 1 in 811

. Today, breast

cancer is the second leading cause of deaths from cancer (after lung cancer) in US

women, with an estimated 39,520 new deaths in 201110

. From 1990 to 2007, the

mortality rate decreased 2.2% per year due to both improvements in breast cancer

treatment and early detection12

. Breast cancer in men only accounts for

approximately 1% of breast cancer cases in the US10

. Risk factors for breast cancer

include female gender, advanced age, family history, early menarche, late

menopause, hormone replacement therapy, high breast tissue density and radiation

to the chest area (mantle field radiation therapy).

5

1.2.2. Breast cancer subtypes

Breast cancer is a heterogeneous disease that has been traditionally classified

according to the site of origin of the cancer. The majority are ductal carcinomas,

which originate in the ducts that move milk from the breast to the nipple. Lobular

carcinomas, in contrast, start in the lobular compartment of the breast. Breast cancer

can start in other areas of the breast in rare cases. Although these classification

methods are providing useful information about disease treatment and outcome,

recent studies reveal that they have limitations and that tumors of the same

histological subtype (e.g. ductal carcinoma) may have distinct outcomes and

dramatically different responses to therapies13

. The introduction of high-throughput

technologies that survey thousands of genes at once has opened up new

opportunities for classifying breast cancer based on gene expression patterns14

. In

2000, a pioneer study provided the basis for an improved molecular taxonomy of

breast cancers using complementary DNA microarrays15

. Hierarchical clustering of

microarray data has revealed at least four distinct breast cancer subgroups:

luminal/ER+, normal breast-like, ERBB2+ (HER2+) and basal epithelial-like. This

classification was further refined by separating the luminal/ER+ group into three

subgroups, termed luminal subtype A, B and C16

. Most impressively, survival analysis

showed significantly different outcomes for patients belonging to the various

subgroups16

.

Most breast cancers are luminal/ER+ tumors. This subtype is characterized by

the relatively high expression of many genes expressed by normal luminal epithelial

cells15

. Due to the expression of estrogen receptor (ER), luminal tumors are sensitive

to hormone therapy such as tamoxifen and/or aromatase inhibitors. Luminal subtype

A demonstrates the highest expression of the ERα gene, and has the best prognosis

among all the subtypes17,18

. Luminal subtype B/C tumors have higher proliferative

activity and a significantly worse patient outcome19

.

Normal breast-like tumors represent roughly six to ten percent of all breast

cancer cases17

. Their gene expression pattern is typified by the high expression of

genes characteristic of basal epithelial cells and adipocytes, and the low expression

6

of genes characteristic of luminal epithelial cells15

. Their clinical and pathological

significance is yet to be determined.

ERBB2+ (HER2+) tumors are characterized by high expression of several genes in

the ERBB2 amplicon on chromosome 17 including human epidermal growth factor

receptor-2 (Her2/neu) and growth factor receptor-bound protein 7 (GRB7)15

.

Representing about twenty percent of all breast cancer cases, this subtype is usually

negative for ER/PR expression and associated with poor prognosis18

. The

over-expressed oncogene Her2 can be clinically targeted by the use of Trastuzumab

(Herceptin) or Lapatinib. Chemotherapy plus trastuzumab cuts the risk of breast

cancer recurrence in half, compared to chemotherapy alone among women with

HER2+ tumors20,21

.

About ten to twenty percent of breast tumors fall into the basal epithelial-like or

basal-like breast cancer (BLBC) subtype, based on gene expression patterns15

. BLBC

tumors are characterized by high expression of genes that are usually expressed in

normal breast basal myoepithelial cells. Classic basal epithelial markers such as

cytokeratin 5/6, cytokeratin 17 and epidermal growth factor receptor (EGFR) are

often overexpressed in BLBC tumors22

. The majority of, though not all, BLBC tumors

lack the expression of ER, PR and HER2 (ER-, PR-, HER2-; triple-negative; TN). These

negatives imply that the growth of triple-negative tumors is not fuelled by estrogen

or progesterone, nor by the overexpression of HER2. BLBC and triple-negative breast

cancers have been shown to possess a more aggressive clinical behavior including

higher tumor grade23

, lower five-year survival rate24

and a higher recurrence rate

compared with other molecular subtypes. Therapies against BLBC and triple-negative

breast cancers are limited due to the lack of tailored therapies and the heterogeneity

within this group23

. Currently, therapeutic strategies such as inhibitors of the poly

ADP-ribose polymerase (PARP) enzyme are being investigated in clinical trials25

, in

hope to improve prognosis of BLBC and triple-negative breast tumors.

7

1.3. Tumor microenvironment

1.3.1. Structure of the tumor microenvironment

The tumor microenvironment (TME)/tumor stroma is defined as a collection of

all the non-transformed components in the vicinity of tumor26

(Figure 1.2). Since the

TME is being recognized as an important participant of tumor progression and

response to treatment, more and more research is shifting away from tumor

cell-centric approaches and focuses on stromal components and their interactions

with tumor cells2. The TME is composed of various nonimmune cells such as

fibroblasts, endothelial cells, pericytes, and immune cells27

. Most of the components

have been implicated in promoting tumor progression. For example,

tumor-associated macrophages (TAMs) facilitate angiogenesis, ECM degradation and

tumor invasion28,29

. Matrix metalloproteases (MMPs), which are synthesized

predominantly by fibroblasts, can also activate cytokines, adhesion molecules, and

growth factors, which contribute to tumor progression by increasing tumor

proliferation or promoting angiogenesis2,30

. Thus, the development of stromal-based

therapeutic approaches is currently one of the most important subjects in

translational oncology31

.

Figure 1.2: The tumor microenvironment.

microenvironment which includes

endothelial cells of the blood and lymphatic vessels, pericytes, stromal fibroblasts,

multiple bone marrow-derived cells (BMDCs) such as tumor

myeloid-derived suppressor cells (MDSCs), TIE2

mesenchymal stem cells (MSCs)

1.3.2. Cancer-associated fibroblast

Human carcinomas often exhibit a

stroma” or “reactive stroma

numbers of stromal cells, deposition of ECM proteins

Cancer-associated fibroblasts (CAFs) are among the predominant cell types within

the reactive stroma. CAF populations are frequently observed within the stromal

compartment of various solid tumors, including those of the

lung and pancreas34,35

. The majority of fibr

acquired a modified (activated) phenotype, similar to fibroblasts associated with

umor microenvironment. Tumor cells are surrounded by a complex

microenvironment which includes the extracellular matrix (ECM) and various cell types:

endothelial cells of the blood and lymphatic vessels, pericytes, stromal fibroblasts,

derived cells (BMDCs) such as tumor-associated macrophages (TAMs),

derived suppressor cells (MDSCs), TIE2-expressing monocytes (TEMs) and

mesenchymal stem cells (MSCs)32

(adapted and modified image from Qiang Shen).

associated fibroblasts

Human carcinomas often exhibit a significant stromal phenotype-“desmoplastic

reactive stroma”, which is characterized by the presence of large

numbers of stromal cells, deposition of ECM proteins and capillaries

associated fibroblasts (CAFs) are among the predominant cell types within

. CAF populations are frequently observed within the stromal

compartment of various solid tumors, including those of the breast, prostate,

The majority of fibroblasts within the tumor stroma

a modified (activated) phenotype, similar to fibroblasts associated with

8

Tumor cells are surrounded by a complex

and various cell types:

endothelial cells of the blood and lymphatic vessels, pericytes, stromal fibroblasts, and

associated macrophages (TAMs),

expressing monocytes (TEMs) and

desmoplastic

, which is characterized by the presence of large

capillaries33

.

associated fibroblasts (CAFs) are among the predominant cell types within

. CAF populations are frequently observed within the stromal

breast, prostate, colon,

oblasts within the tumor stroma have

a modified (activated) phenotype, similar to fibroblasts associated with

9

wound-repair or fibrosis, although non-activated fibroblasts are also present36

. In

breast cancer, around 80% of stromal fibroblasts are thought to acquire this activated

phenotype37

. Activated fibroblasts (which are also known as myofibroblasts) are

featured by the expression of stress fibres, elevated secretion of ECM molecules

including type I collagen and tenascin-C (TN-C), MMPs, growth factors such as

insulin-like growth factor (IGF) and epidermal growth factor (EGF), as well as having a

faster proliferative profile compared with normal (non-activated) fibroblasts33,38,39

.

Recent studies have reported on the identification of markers for activated

myofibroblasts. Several different markers, such as α-smooth muscle actin (α-SMA)40

,

Fibroblast-activation protein (FAP)33

, and TN-C41

, may be useful for detecting the

activated myofibroblast population in CAFs. However, those molecular markers are

not exclusive to activated myofibroblasts as they are also found in other inhabitants

of the stroma26,33

.

In general, CAFs display vast heterogeneity manifested by differences in gene

expression patterns reflecting different cells of origin. A pioneer study by Allinen and

colleagues analyzed gene expression profiles of isolated stromal cells using serial

analysis of gene expression (SAGE)42

. Their results demonstrated differential gene

expression across different CAF samples sets. Moreover, CAFs show distinct gene

expression profiles compared to their corresponding normal fibroblast

counterparts42

.

CAFs are proposed to originate from heterogeneous cell types including

pre-existing fibroblasts, preadipocytes, smooth muscle cells, endothelial cells and

bone marrow-derived progenitors43–46

. Bone marrow-derived cells are a significant

source of CAFs, based on the observation that labelled bone marrow cells introduced

into tumor-bearing mice constitute nearly 25% of the stromal fibroblasts in the

vicinity of the tumor47

. Cancer cells that undergo epithelial-to-mesenchymal

transition (EMT) may also serve as an additional source of CAFs33

. Due to the

apparent heterogeneity among CAF cells, Madar’s group proposed “CAFs” as a cell

state rather than a specific cell type26

.

Despite the dramatic gene expression changes in CAF populations, clonal

10

somatic genetic alterations (chromosomal abnormalities, loss of heterozygosity,

mutations) are very rarely detected in CAFs42,48

. Other mechanisms, such as DNA

methylation within the genome of CAFs, may give rise to their altered gene

expression profiles. Hu and colleagues investigated epigenetic modifications in CAFs

using methylation-specific digital karyotyping49

. Significant methylation changes were

identified during tumor progression, suggesting that epigenetic modifications are at

least partly responsible for the altered phenotype of CAFs.

1.3.3. Tumor-promoting roles of CAFs

Increased numbers of stromal myofibroblasts (CAFs) are associated with

higher-grade malignancies and poor outcome in humans31,50

. The gold standard for

identifying CAFs is their capacity to promote tumor progression in vivo, using a

co-implantation xenograft model in which tumor cells are implanted into

immunodeficient mice together with CAFs or normal fibroblasts26,36

. Human prostatic

CAFs, but not normal fibroblasts, stimulate tumor growth of co-injected simian virus

40 (SV-40)-transformed (initiated) prostatic epithelial cells51

. Orimo and colleagues

demonstrated that CAFs extracted from human breast carcinomas promote the

growth of admixed breast cancer cells (MCF-7-ras) with greater efficiency than do

normal mammary fibroblasts35

. This effect is mediated in part by stromal cell-derived

factor 1 (SDF-1/CXCL12), which is highly expressed by CAFs. CAF-secreted SDF-1 not

only boosts neoangiogenesis by recruiting endothelial progenitor cells into the tumor

mass, but also enhances tumor growth directly through CXCR4 (the receptor for

SDF-1) on cancer cells35

. Hepatocyte growth factor (HGF) is another CAF-derived

factor that has been implicated in promoting colony formation of breast cancer cells

in soft agar52

. Moreover, CAFs extracted from human colon adenocarcinomas show

up-regulation of TN-C and HGF, both of which co-operate to promote the

invasiveness of colon carcinoma cells41

. Collectively, these findings highlight the

important role of CAFs in promoting tumor growth, invasion and neoangiogenesis

through various secreted factors.

In addition to their primary tumor-promoting role, CAFs have been reported to

contribute to stemness of cancer cells, which is assessed by the self-renewal ability

11

and the expression of acknowledged cancer stem cell (CSC) markers within the tumor

cell population53

. CAF-derived HGF acts via the c-Met receptor on nearby colon

carcinoma cells, activating the Wnt signaling pathway and subsequently CSC

phenotype54

. Another study indicated that CAF-secreted chemokine (C-C motif)

ligand 2 (CCL2) can confer the CSC phenotype on breast cancer cells by activating

Notch signaling55

.

1.4. The Notch signaling pathway

1.4.1. Notch signaling in development

Notch is an evolutionarily conserved signaling pathway that participates in

metazoan development and adult tissue homeostasis. Morgan et al. first described a

Notch mutant in Drosophila, based on its dominant wing-notching phenotype56,57

.

The authors found this phenotype to be X-chromosome-linked and passed from

parent to progeny in a Mendelian fashion. The critical role of Notch in Drosophila

development was first demonstrated by Poulson et al. who showed the hallmark

phenotype of dying homozygous null Notch mutant embryos58

. These embryos

displayed a classic "neurogenic" phenotype, featured by hypertrophy of the nervous

system at the expense of the epidermis. Later in 1983, the cloning of the Notch locus

initiated the molecular era of Notch and led to the identification of its gene product

in Drosophila59

, C.elegans60

, and in the vertebrate Xenopus61

.

Since its discovery, Notch has been shown to be involved in a variety of cellular

processes including cell division, cell fate specification, differentiation, apoptosis,

migration, invasion, adhesion, epithelial cell polarity, stem cell maintenance, and

angiogenesis. The role Notch plays in developmental processes, especially in

vertebrate CNS development, has been closely investigated in the past 15 years62

. As

described above, lack of Notch function in Drosophila leads to an embryonic lethal

phenotype with hypertrophy of neural tissue (neurogenic phenotype)58

. In

vertebrates, Notch maintains a neural progenitor pool by inhibiting premature

differentiation62

. In the mouse, targeted mutation of Notch1 results in precocious

neuronal differentiation, indicated by expanded domains of expression of early

differentiation markers NeuroD, Math4A, and NSCL-163

. To overcome the early

12

lethality of Notch1 mutants, conditional deletion of Notch1 has been carried out in a

mouse model and demonstrates precocious neuronal differentiation64

, earlier neural

progenitor pool depletion65

, and reduced neural progenitor frequency (assayed as

neurospheres) in vitro66

.

Notch has been found to participate in binary cell-fate decisions via a

mechanism termed lateral inhibition, where individual cells adopt different fates

according to the states of their immediate neighbours67

. Within a group of equivalent

progenitor cells, those with the highest Delta ligand expression level will become

neurons by signaling to neighbouring Notch receptor-expressing cells, preventing

them from differentiating prematurely into neurons. Ultimately, cells within the

progenitor pool exclusively express the Notch ligand or the receptor, thereby

adopting distinct developmental pathways68

.

In addition to its role in CNS development, Notch has been shown to regulate

developmental processes of other organs/tissues including heart, mammary gland,

pancreas, gastrointestinal tract and bone.62

In cardiovascular development, Notch

signaling is required for arterial specification and patterning, as opposed to venous

fate specification69,70

. Notch signaling also promotes commitment of bipotent

mammary progenitor cells along the luminal lineage during mammary

development71,72

and pregnancy73

.

The involvement of Notch signaling in self-renewal of stem cells has been

described by several groups. By virtue of an in vitro non-adherent mammosphere

system, Dontu et al. demonstrated that Notch can act on mammary stem cell (MaSC)

to promote self-renewal, as indicated by an increase in secondary mammosphere

formation upon addition of a Notch-activating DSL peptide74

. In contrast, inactivated

Notch in a MaSC-enriched population results in elevated stem cell activity in vivo,

suggesting a role for endogenous Notch in restricting MaSC expansion71

. This

apparent contradiction may be caused by the different assaying methods used.

Nevertheless, Notch apparently plays an essential role in development by

maintaining stem cell populations and by directing binary fate decisions.

13

1.4.2. The mechanism of Notch signaling

Notch receptors are synthesized as single precursor proteins that undergo

cleavage in the trans-Golgi network by the protease furin at site 1 (S1)75

. The

resulting two non-covalently linked domains form a Notch heterodimer on the cell

surface76

. Notch signaling is initiated by the binding of Notch ligand on one cell to the

extracellular domain of a Notch receptor on a neighboring cell77

(Figure 1.3). Upon

ligand-receptor binding, the ligand becomes ubiquitinated by ubiquitin ligases such

as mind bomb78

or neuralized79,80

and thus internalized. Ligand endocytosis results in

a conformational change in Notch that triggers two consecutive proteolytic events at

the receptors. The first cleavage event is catalyzed by the ADAM (disintegrin and

metalloprotease)/TACE (TNF-α-converting -enzyme) family of proteases at site 2 (S2)

on the extracellular side81

. The second cleavage occurs within the transmembrane

domain at site 3 (S3) and is mediated by the γ-secretase complex which is composed

of four integral membrane proteins: presenilin, Nicastrin, Aph-1 and Pen-282–84

. This

results in the release of the Notch intracellular domain (NICD) into the cytoplasm and

its subsequent translocation into the nucleus, where it binds to the transcription

factor CSL (CBF-1/RBPJκ in vertebrates, Suppressor of Hairless in Drosophila, LAG in

C.elegans)77

. In the absence of Notch signaling, CSL acts as a constitutive

transcriptional repressor by binding to promoters of its target genes and recruiting

histone deacetylase85

and corepressors86

. After NICD binding, CSL becomes a

transcriptional activator by replacing the corepressor complex with coactivators such

as Mastermind-Like 1 (MAML)87

and histone acetyltransferase88

. This leads to

transcriptional activation of downstream target genes including Hairy enhancer of

split (Hes) genes and the Hes-related (Hey) family of basic helix-loop-helix (bHLH)

transcription factors89,90

.

Figure 1.3: Canonical Notch signaling in mammals. The interaction between Notch ligands

(DLL1,3,4 and JAG1,2) and the heterodimeric Notch receptor (N

sequential proteolytic cleavage events. The first is mediated by the ADAM/TACE family of

proteases and occurs approximately 12 amino acids outside the

The resulting truncation is then further cleaved by

domain, liberating NICD into the cytoplasm

CBF-1/RBPJκ, causing transactiva

image from Qiang Shen).

1.4.3. The structure of Notch

Mammals possess five Notch ligands: Jagged (JAG) 1&2,

to Drosophila Serrate, and Delta

(Figure 1.4). Notch ligands are expressed as membrane

surface. They share an extracellular Delta/Serrate/LAG

binding91

, followed by a variable number of EGF

family also harbour a cysteine

ligands92

. The intracellular portion contains regions th

signaling activities, interaction with the cytoskeleton, and ubiquitination

In mammals, the four Notch receptors (N

heterodimers, harbouring a large extracellular domain involved in ligand binding,

Canonical Notch signaling in mammals. The interaction between Notch ligands

(DLL1,3,4 and JAG1,2) and the heterodimeric Notch receptor (NOTCH 1-4) initiates two

cleavage events. The first is mediated by the ADAM/TACE family of

proteases and occurs approximately 12 amino acids outside the transmembrane domain

The resulting truncation is then further cleaved by γ-secretase within the transmembrane

erating NICD into the cytoplasm (S3). NICD translocates to the nucleus

, causing transactivation of downstream target genes (adapted and modified

1.4.3. The structure of Notch ligands and receptors

possess five Notch ligands: Jagged (JAG) 1&2, which are homologous

Serrate, and Delta-like (DLL) 1,3,4, homologous to Drosophila

. Notch ligands are expressed as membrane-bound proteins on the cell

tracellular Delta/Serrate/LAG-2 (DSL) domain for receptor

, followed by a variable number of EGF-like repeats. Ligands of the JAG

family also harbour a cysteine-rich (CR) domain which is missing from the DLL

The intracellular portion contains regions that are required for ligand

signaling activities, interaction with the cytoskeleton, and ubiquitination93

.

he four Notch receptors (NOTCH1-4) are transmembrane

, harbouring a large extracellular domain involved in ligand binding,

14

Canonical Notch signaling in mammals. The interaction between Notch ligands

4) initiates two

cleavage events. The first is mediated by the ADAM/TACE family of

domain (S2).

secretase within the transmembrane

nucleus and binds

(adapted and modified

which are homologous

Drosophila Delta

bound proteins on the cell

2 (DSL) domain for receptor

like repeats. Ligands of the JAG

R) domain which is missing from the DLL

at are required for ligand

4) are transmembrane

, harbouring a large extracellular domain involved in ligand binding, and

a cytoplasmic domain involved in signal transduction.

consists of 29-36 epidermal growth factor (EGF)

binding interactions94

, followed by three cysteine

that prevent ligand-independent signali

The intracellular portion contains

direct interaction between NICD and CSL

in protein-protein interactions in the coactivator complex

signals (NLS); a transcription

four receptors97

; and a C-terminal

(PEST) that regulates protein stability through ubiquitinati

Figure 1.4: Notch ligands and their

the Notch ligands (JAG1&2, DLL1,3,4)

DSL domain (blue oval), followed by multiple EGF

additional CR domain (black) that is not found in DLL ligands.

mammalian NOTCH1-4. The extracellular domain contains a variable number of EGF

repeats (orange), followed by a negative regulatory region (NRR) containing a LNR domain

(grey) and a HD domain. NRR encloses the S2 cleavage site and functions to prevent Notch

activation in the absence of ligand binding. The cytoplasmic part of the receptor is composed

of a RAM23 domain (light blue), six ANK repeats (red), two nuclear localization

(blue), a TAD domain (yellow), and a PEST sequence (green)

a cytoplasmic domain involved in signal transduction. The extracellular portion

epidermal growth factor (EGF)-like repeats that are critical for

, followed by three cysteine-rich LIN12/Notch repeats (LNR)

independent signaling, and a heterodimerization (HD)

The intracellular portion contains a RBPJκ-association module (RAM23) that mediates

direct interaction between NICD and CSL95

; six ankyrin/cdc10 (ANK) repeats

protein interactions in the coactivator complex96

; two nuclear localization

signals (NLS); a transcriptional transactivation domain (TAD) that differs among the

terminal proline/glutamic acid/serine/threonine-rich motif

(PEST) that regulates protein stability through ubiquitination98

.

ligands and their receptors in mammals. A) Schematic representation of

(JAG1&2, DLL1,3,4). On the extracellular side they have an amino

DSL domain (blue oval), followed by multiple EGF-like repeats (orange). JAG ligands have an

) that is not found in DLL ligands. B) Schematic representation of

4. The extracellular domain contains a variable number of EGF


and a HD domain. NRR encloses the S2 cleavage site and functions to prevent Notch


of a RAM23 domain (light blue), six ANK repeats (red), two nuclear localization signals (NLS)

(blue), a TAD domain (yellow), and a PEST sequence (green). See text for details.

15

The extracellular portion

like repeats that are critical for

LIN12/Notch repeats (LNR)

(HD) domain.

) that mediates

; six ankyrin/cdc10 (ANK) repeats involved

; two nuclear localization

al transactivation domain (TAD) that differs among the

rich motif

Schematic representation of

. On the extracellular side they have an amino-terminal

. JAG ligands have an

Schematic representation of

4. The extracellular domain contains a variable number of EGF-like


and a HD domain. NRR encloses the S2 cleavage site and functions to prevent Notch


signals (NLS)

16

1.5. Notch signaling in breast cancer

1.5.1. Evidence for oncogenic Notch in breast cancer development

The role of Notch signaling in breast cancer was first suggested by studies

designed to understand the mechanism(s) of mouse mammary tumor virus

(MMTV)-induced mammary adenocarcinoma99

. The int-3 (Notch4) locus was

identified as a proviral integration site in these tumors. MMTV insertion was found to

initiate transcription of the previously-silent Notch4 gene, leading to expression of a

constitutively activated NOTCH protein that lacks most of its extracellular portion and

contains transmembrane and intracellular domains (ICD). This truncation resembles

activated N4-ICD100

. Similarly, the Notch1 gene was found to be another target for

MMTV provirus insertional activation101

.

To determine the in vivo consequence of activated N1, N3 or N4 in mammary

gland, transgenic mice harbouring Notch-ICD transgenes were generated.

MMTV/N4-ICD transgenic animals demonstrate a dual phenotype with arrested

mammary gland development and poorly differentiated mammary

adenocarcinomas102

. MMTV/N1-ICD and MMTV/N3-ICD mice exhibit a very similar

phenotype to N4-ICD animals103

. These studies clearly demonstrate that Notch

hyperactivation in the mouse mammary gland disrupts normal developmental events

and promotes tumorigenesis.

Mounting evidence from human cancer cell line studies suggest the oncogenic

potential of Notch in vitro. The expression of a 1.8kb NOTCH4 mRNA species in the

immortalized human mammary epithelial cell line MCF10A confers these cells with

the ability to grow in soft agar104

. This transcript encodes a truncated and activated

portion of N4-ICD. Additionally, N1-ICD overexpression is sufficient to transform

normal breast epithelial cells105

. Notch activation, evidenced by N1-ICD accumulation

and Hey1 overexpression, is observed in most breast cancer cell lines105

.

Weijzen et al. provided the first clue that Notch is activated in human primary

breast ductal carcinomas106

. Increased expression of NOTCH1 was seen in four breast

tumors that over-expressed H-Ras. NOTCH1 downregulation in Ras-transformed cells

leads to a significant reduction in cell proliferation both in vitro and in vivo. These

17

findings suggested that NOTCH1 is a downstream effector of oncogenic Ras and is

necessary to maintain the neoplastic phenotype of Ras-transformed cells106

.

Elevated expression of JAG1 and/or NOTCH1 was observed in human breast

cancer and was associated with poor overall survival107

. Patients with tumors

expressing high levels of JAG1 or NOTCH1 have significantly lower 5-year survival rate

and shorter medium survival time. Remarkably, high-level coexpression of JAG1 and

NOTCH1 is associated with a further reduction in overall survival.

1.5.2. Notch activation and the BLBC subtype

Several lines of evidence support a role for Notch activation in BLBC tumors. Our

group has demonstrated a statistically significant association between elevated

expression of the Notch ligands and receptors and the BLBC subtype in specimens

from breast cancer patients107,108

. Tumors expressing JAG1 and/or NOTCH1 in the

highest quartile of the expression range have defining features of poor-prognosis

breast cancer, specifically the BLBC subtype107

. In a microarray analysis of 46 breast

cancer cell lines, JAG1 mRNA is significantly overexpressed in BLBC/TN cells109

. More

evidence for the importance of Notch signaling in the BLBC subtype came from a

study examining the proliferative-dependency of HER2- and HER2+ breast cancers on

the expression of NOTCH receptors110

. Results from this study indicate the functional

importance of NOTCH3 in HER2-, but not HER2+, breast cancer cells. Remarkably, in

this study, four out of five HER2- cell lines with established NOTCH3-dependency are

of the BLBC subtype. Additionally, BRCA1-mutant breast tumors, which are

predominantly BLBC, are associated with high JAG1 expression compared to their

predominantly luminal BRCA2 counterparts111

. Interestingly, Notch activity may

compensate for the loss of ER or HER2 signaling and lead to development of

resistance to endocrine and targeted therapies112,113

. Taken together, these findings

suggest that aberrant Notch activation can provide cells with compensatory

growth-promoting signals in the absence of key growth stimulatory pathways such as

ER and HER2.

18

1.5.3. Mechanisms of Notch activation in breast cancer

Gain-of-function mutations in Notch genes were found to be partly responsible

for the pathogenesis of human T-cell acute lymphoblastic leukemias (T-ALL). In 2004

Weng et al. reported that more than 50% of all T-ALL patients harbour activating

mutations in two key domains within the NOTCH1 receptor114

. Mutations occurred

within the extracellular heterodimerization (HD) domain (13% of cases) and resulted

in ligand-independent activation, or they occurred within the C-terminal PEST

domain (26% of cases), leading to increased stability of N1-ICD. In 18% of cases both

domains contained activating mutations. Prompted by this discovery, Lee et al.

analyzed 48 breast carcinomas for mutations in the Notch1-4 loci115

. Surprisingly,

only one Notch2 activating mutation was found in breast cancers, indicating that

Notch genes are rarely mutated in solid tumors115,116

. In a study investigating the role

of NOTCH3 in breast cancer, the frequency of Notch3 gene amplification in primary

tumors was found to be less than 1%110

. Genomic hybridization array data has shown

that Notch4 gene is amplified in 34% of TN breast cancers but concurrent NOTCH4

overexpression has not been observed in these cases117

. Therefore, activating

mutation or gene amplification in the Notch loci is unlikely to be responsible for

aberrant Notch activation in breast cancer.

Current evidence supports the notion that Notch activation in breast cancer

occurs primarily through up-regulation of Notch ligand and/or receptor expression

rather than through mutations/amplifications of Notch loci. Elevated levels of JAG1

and NOTCH1 were noted in a subset of tumors with poor prognosis pathologic

features such as the BLBC subtype and high grade107

. Both transcriptional and

post-translational mechanisms have been found to mediate the up-regulation of

Notch ligands and receptors. For instance, breast cancer cells exposed to a hypoxic

environment induce the expression of Notch3 and Jagged1 through up-regulation of

the 66 kDa isoform of the SHC gene (p66Shc)118

. In another study, the activation of

the Wingless-type (Wnt) signaling in human mammary epithelial cells, as achieved by

ectopic Wnt-1 expression, up-regulates Notch ligands of the Delta family (DLL1,3,4)

as well as NOTCH3 and NOTCH4 receptors119

. Oncogenic Ras not only induces the

19

expression of NOTCH1 and DLL1, but also facilitates Notch activation by up-regulating

the γ-secretase component presenilin-1, resulting in increased activation of

NOTCH1106

. NUMB was previously described as an antagonist of the Notch signaling

pathway by promoting ubiquitination and subsequent degradation of Notch

receptors120

. Pece et al. showed that NUMB-mediated negative control on Notch

signaling is lost in 50% of breast cancers121

. The prolyl-isomerase Pin1 was found to

interact with NOTCH1 and potentiates its cleavage by γ-secretase, leading to

increased release of N1-ICD and enhanced NOTCH1 transcriptional and tumorigenic

activity122

. Remarkably, Pin1 is also a direct target of NOTCH1, thereby generating a

positive loop. More recently, our group has identified a pseudokinase TRB3 as a

master regulator of JAG1 expression in breast cancer through its control of

MAPK-ERK and TGFβ/SMAD4 signaling axes123

.

In summary, aberrant activation of Notch in breast cancer is likely due to the

activation of pathways that enhance the expression or activity of Notch signaling

components, rather than through mutation or amplification of Notch ligand/receptor

loci.

20

1.6. The urokinase-type plasminogen activator system

Several direct transcriptional targets of Notch have been identified in breast

cancer, including c-Myc124

, Slug125

, CCND1 (cyclin D1 gene)109

,Survivin126

and Pin1122

.

Our lab has identified uPA as a direct transcriptional target of JAG1-mediated Notch

signaling in human breast cancer127

.

The urokinase-type plasminogen activator (uPA) system is involved in multiple

physiological and pathologic processes including embryogenesis, wound healing, cell

migration, cell invasion and cancer metastasis. The serine protease uPA cleaves the

inactive form of plasminogen, converting it to active plasmin which has an activity

that is at least several hundred-fold higher than that of plasminogen. uPA receptor

(uPAR) on the cell surface facilitates uPA-catalyzed plasminogen activation. Plasmin,

either directly or indirectly through MMPs, can degrade components of the ECM or

basement membrane (BM), contributing to cancer cell migration and invasion128

. In

addition, uPAR complexes with other membrane proteins for signal transduction,

modulating cell adhesion and migration by mechanisms not involving plasmin

generation129

.

Considerable evidence strongly suggests that uPA-catalyzed plasminogen

activation is rate-limiting for tumor invasion and metastasis. For example, either an

anti-catalytic antibody to uPA or an anti-uPAR antibody inhibits invasion of

MDA-MB231 cells into Matrigel in a dose-dependent manner130

. Using a model of

dissemination of human tumors in nude mice, Quax et al. reported a correlation

between cancer cell uPA expression and lung metastasis in several human melanoma

cell lines131

. Clinically, in human breast cancer, high levels of uPA enzyme activity are

found to correlate with shorter disease-free interval and poor outcome132,133

. Breast

cancer patients with high uPAR expression also show shorter overall survival134

.

21

1.7. The TGFβ signaling pathway

1.7.1. The mechanism of TGFβ signaling

The transforming growth factor-β (TGFβ) pathway has been established as

essential for development, tissue homeostasis and cancer progression135

. This

pathway is initiated by the binding of ligands (TGFβ1, TGFβ2 and TGFβ3) to the type 2

TGFβ receptor (TGFβR2) dimer, which is a serine/threonine receptor kinase (Figure

1.5). The activated type 2 receptor recruits and phosphorylates a type 1 TGFβ

receptor (TGFβR1), forming a hetero-tetrameric complex with the ligand136

. The

carboxyl-end serine residues of SMAD proteins, SMAD2 or SMAD3, are

phosphorylated by the activated receptors. Phosphorylation induces a

conformational change in SMAD2/3 and their subsequent association with SMAD4137

.

The SMAD complex then enters the nucleus where it binds transcription cofactors

and initiates transcriptional activation/repression of several downstream genes138

.

The TGFβ pathway can also operate in a SMAD-independent way, which involves

activation of the PI3K-AKT, RHOA and MAPK pathways by activated hetero-tetrameric

receptors138

.

All three TGFβ ligands are synthesized as high molecular-weight precursors

(pre-pro- TGFβ) containing a signal peptide and a latency associated peptide (LAP).

After furin cleavage, the TGFβ homodimer remains associated with LAP in a

non-covalent way. This complex is not secreted into the cytoplasm unless it is bound

by another protein called latent TGFβ-binding protein (LTBP), forming a larger

complex named large latent complex (LLC)139

. In order to release the active TGFβ

homodimer, the LLC is fixed to the ECM by transglutaminase and further processed

by proteases, reactive oxygen species (ROS), and thrombospondin-1 (TSP-1)140

.

1.7.2. TGFβ signaling and cancer

TGFβs are multi-functional cytokines that have context-dependent dual effects

on tumor progression. Studies elucidating the tumor suppressor role of TGFβ provide

evidence that the loss of TGFβ signaling components is associated with cancer

occurrence and progression141,142

. TGFβR2 inactivation in mouse fibroblasts results in

intra-epithelial neoplasia in the prostate and invasive squamous cell carcinoma of

22

TGFβR2

TGFβR1 Cytoplasm

TGFβ

PSMAD

2/3P

SMAD

2/3P

SMAD4

Nucleus

Transcription

Figure 1.5: A schematic representation of simplified canonical TGFβ signaling pathway. Upon

activation, the ligand binds to the type 2 TGFβ receptor (TGFβR2), which causes recruitment

and phosphorylation of TGFβR1. The activated hetero-tetrameric receptor complex then

phosphorylates SMAD2 or SMAD3, rendering them high affinity for SMAD4. Through

interactions with a variety of transcriptional cofactors, the nuclear-localized SMAD complex

regulates transcription of hundreds of target genes.

the forestomach143

. Early in tumor development, TGFβ signaling adopts a

tumor-suppressive role through inhibition of cell proliferation138

. However, mouse

models suggest that TGFβ can switch from a tumor suppressor in early stage

mammary tumors to a tumor promoter in late stage tumors, and may also increase

the risk of metastases144

. In an orthotopic xenograft model to reconstruct the human

mammary gland, Kuperwasser et al. found that overexpressing TGFβ in fibroblasts

could induce the initiation of breast cancer from the normal human epithelium145

.

The pro-tumorigenic roles of TGFβ are thought to be mediated by the induction of

cancer cell EMT and of cellular migration and invasion138,146

. This is consistent with

the observation that higher expression of TGFβ1 in the tumor stroma is associated

with later stage or recurrent breast cancers147–149

and with a faster rate of disease

progression150

.

23

1.7.3. TGFβ and CAFs

In vitro studies have suggested TGFβ as one of the key mediators of fibroblast

trans-differentiation into myofibroblasts36

. The maintenance of myofibroblast

identity is also driven by an autocrine TGFβ signaling loop151

. Sources of TGFβ in

tumors vary and include the cancer cells themselves as well as stromal

components135

. Studies in prostate cancer152,153

and breast cancer151

have implied

that CAFs may be a source of tumoral TGFβ since they produce elevated levels of

TGFβ1 compared to fibroblasts from normal tissue.

1.8. The HGF-MET signaling pathway

1.8.1. The mechanism of HGF-MET signaling

c-MET is a receptor tyrosine kinase (RTK) expressed in epithelial cells and serves

as a high-affinity receptor for Its ligand, Hepatocyte growth factor/scatter factor

(HGF/SF), which is secreted by mesenchymal cells as precursor (pro-HGF) and is later

converted to the active form by extracellular proteases154

(Figure 1.6). The binding of

HGF to c-MET initiates receptor homodimerization and phosphorylation of two

tyrosine residues (Y1234 and Y1235) within the kinase domain155

. Subsequently,

tyrosines in the C-terminal multifunctional docking site domain become

phosphorylated, recruiting downstream signaling proteins. Examples of effectors

include the adaptor proteins Growth factor receptor-bound protein 2 (Grb2)156

and

GRB2-associated binding protein 1 (Gab-1)157

, phosphatidylinositol 3-kinase (PI3K),

phospholipase Cγ (PLCγ)158

, Src homology domain-containing 5’ inositol phosphatase

(Shp2)159

, and components involved in the Mitogen activated protein kinase (MAPK)

cascade such as Son of sevenless (Sos) and Ras160

. The activation of HGF-MET

signaling regulates distinct cytoplasmic signaling pathways that trigger the “invasive

growth” program through promoting cell proliferation, survival, migration and

invasion156,160–163

.

Figure 1.6: A schematic representation of

receptor is an α/β heterodimer that contains an intracellular tyrosine kinase domain (grey

rectangle) and a C-terminal multifunctional docking site domain. Upon HGF/SF binding,

c-MET receptors undergo homodimerization and phosphorylation of two tyrosine residues

(Y1234 and Y1235) within the kinase domain. Subsequently, tyrosine 1349 and 1356

phosphorylated, recruiting downstream signaling effectors (see text for details) that enhance

the execution of the “invasive growth

migration and invasion.

1.8.2. HGF-MET signaling and cancer

Deregulated MET signaling has been associated with the malignant progression

of tumors. c-MET was originally identified as an oncogene in a human osteosarcoma

cell line harbouring a chromosome rearrangement that fused the tyrosine kinase

domain of c-MET to an upstream translocating promoter

rearrangement results in constitutive dimerization and thus activation of c

receptors165

. Expression of TPR

epithelial-derived tumors in transgenic mice

kinase domain are found in both sporadic and inherited forms of human renal

papillary carcinomas167,168

. Amplification of the

overexpression, has been observed in a number

gastric and oesophageal carcinomas

A schematic representation of the HGF-MET signaling pathway. The

erodimer that contains an intracellular tyrosine kinase domain (grey

terminal multifunctional docking site domain. Upon HGF/SF binding,

homodimerization and phosphorylation of two tyrosine residues

5) within the kinase domain. Subsequently, tyrosine 1349 and 1356


invasive growth” program by promoting cell proliferation, survival,

MET signaling and cancer


was originally identified as an oncogene in a human osteosarcoma

a chromosome rearrangement that fused the tyrosine kinase

to an upstream translocating promoter region (TPR)

rearrangement results in constitutive dimerization and thus activation of c

. Expression of TPR-MET leads to the development of multiple

derived tumors in transgenic mice166

. Activating mutations in the c


Amplification of the c-MET gene, with consequent protein

overexpression, has been observed in a number of human primary tumors including

carcinomas169,170

and medulloblastomas171

. In the absence

24

The c-MET

erodimer that contains an intracellular tyrosine kinase domain (grey

terminal multifunctional docking site domain. Upon HGF/SF binding,

homodimerization and phosphorylation of two tyrosine residues

5) within the kinase domain. Subsequently, tyrosine 1349 and 1356 become


program by promoting cell proliferation, survival,


was originally identified as an oncogene in a human osteosarcoma

a chromosome rearrangement that fused the tyrosine kinase

TPR)164

. This

rearrangement results in constitutive dimerization and thus activation of c-MET

the development of multiple

Activating mutations in the c-MET


gene, with consequent protein

of human primary tumors including

In the absence

25

of gene amplification, transcriptional up-regulation of c-MET has been documented

in a variety of cancers such as colorectal172

, ovarian173

, pancreatic174

and breast154,175

.

For instance, hypoxia has been shown to activate the c-MET promoter via the

transcription factor hypoxia inducible factor 1α (HIF1α) in pancreatic cancer176

.

Another mechanism of MET signaling activation is through the establishment of

ligand-dependent autocrine or paracrine loops. The ligand, HGF/SF, is frequently

overexpressed in the reactive stroma of primary tumors including lung177

,

gastric178

,and breast179–181

. High expression of HGF in the stroma of these tumors is

correlated with a more aggressive phenotype and poor prognosis in patients.

1.8.3. HGF and CAFs

Several groups have established a link between the tumor-promoting ability of

CAFs and elevated HGF expression levels. In the hepatocellular carcinoma

microenvironment, CAFs promote the proliferation of carcinoma cells both in vitro

and in vivo by releasing elevated levels of HGF182

. CAF-derived HGF is responsible for

increased tumor growth and colony-forming ability in breast tumorigenesis52

. CAFs

foster the ability of transformed esophageal epithelial cells to invade into the ECM

through HGF secretion183

. More functional roles of CAF-derived HGF are emerging,

such as promoting the CSC phenotype54

and inducing drug resistance in tumor

cells184

. Thus, HGF from a CAF origin plays a definitive role in the cancer progression

process and acts as a mediator of tumor-stromal interactions.

1.9. Hypothesis and Aims

As mentioned above, the BLBC subtype is featured by elevated expression of the

Notch ligands/receptors, and Notch pathway activation. Interestingly, compared to

the luminal subtype, BLBC is associated with unique stromal-epithelial interactions as

suggested by the characteristic set of secreted factors and gene expression pattern

when co-cultured with fibroblasts185

. Based on these associations, I aimed to

determine the role of Notch in tumor cell-CAF crosstalk and in tumor progression,

specifically in BLBC tumors.

Preliminary data from our lab suggest that tumor-associated macrophage

(TAM)-derived TGFβ activates BLBC cell Notch signaling by up-regulating JAG1

26

expression. This results in uPA overexpression and secretion, promoting tumor cell

migration, invasion and intravasation. Notch signaling in tumor cells also drives the

expression of the TGFβ receptor TGFβR1, and uPA-dependent maturation of

TAM-derived TGFβ, forming a positive paracrine feedback loop (unpublished data by

Shen et al., manuscript in preparation).

CAFs in the tumor microenvironment can be an additional source of TGFβ. I

hypothesize that Notch signaling plays a critical role in carcinoma-CAF crosstalk of

BLBC, and that CAFs promote tumor cell invasion through Notch- and TGFβ-

dependent mechanisms. The aims of my work are:

Specific Aim 1: To assess the effect of fibroblast-derived TGFβ on Notch activation in

BLBC cell lines.

Specific Aim 2: To use CAF-like fibroblast cell lines and a co-culture system to evaluate

the role of Notch signaling in cancer-CAF crosstalk.

Specific Aim 3: To evaluate the functional role of Notch signaling in cancer-CAF

crosstalk by using in vitro invasion assays.

27

CHAPTER 2: Materials and Methods

28

2.1. Cell culture

The human BLBC cell lines, MDA-MB231 and HCC1143, were purchased from the

American Type Culture Collection (ATCC) and grown according to their specifications.

MDA-MB231 cells were grown at 37°C, 0% CO2 in ATCC-formulated Leibovitz’s L-15

medium supplemented with 10% fetal bovine serum (FBS, Wisent) and 1% penicillin

and streptomycin (Pen-Strep, Life Technologies). HCC1143 cells were cultured in

ATCC-formulated RPMI-1640 supplemented with 10% FBS and 1% Pen-Strep, and

grown at 37°C, 5% CO2. Fibroblasts generated from human reduction mammoplasties

and immortalized with hTERT (human telomerase reverse transcriptase) and

expressing GFP (RMF/EG) were obtained from Dr. Laurie Allies (Department of

Medical Biophysics, University of Toronto) and were originally generated by Dr.

Charlotte Kuperwasser (Tufts University, Boston, MA, USA)145

. A variant of this line

expressing exogenous TGFβ1 (RMF/EG-TGFβ) were obtained at the same time. Both

RMF lines were cultured in ATCC-formulated Iscove’s Modified Dulbecco’s Medium

(IMDM) supplemented with 10% FBS and 1% Pen-Strep, and grown at 37°C, 5% CO2.

The Exp-CAF2 and Ctl2 cell lines were generated experimentally from reduction

mammoplasty fibroblasts using a co-implantation breast tumor xenograft model and

were a generous gift from Dr. Akira Orimo (Juntendo University, Tokyo, Japan), whose

group has previously described the methodology in detail151

. Both Exp-CAF2 and Ctl2

cells were cultured in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (DMEM)

supplemented with 10% FBS and 1% Pen Strep, and grown at 37°C, 5% CO2.

29

2.2. RNA interference

All reverse transfections were carried out in 6-well tissue culture plates. Small

interfering RNA (siRNA) (amount indicated in Table 2.1) was diluted in 500μl

Opti-MEM (Life Technologies) inside the tissue culture plate wells. Next, 5μl

LipofectamineRNAiMAX (Invitrogen) was added to each well. The mixture was

incubated at room temperature for 20 minutes to allow the siRNA-liposome complex

to form. After being diluted with their corresponding complete growth media, 3X105

cancer cells (MDA-MB231, HCC1143) or 2X105 fibroblasts were added to each well

and incubated for 48 hours. In experiments where secreted uPA levels were

investigated at 48 hours post-transfection, the siRNA-media was replaced by fresh

serum-free media at 24 hours post-transfection.

siRNA Company and

catalogue #

pmol/well Sequence

Scrambled Dharmacon;

D-001810-10-20

100 5’-UGGUUUACAUGUCGACUAA-3’

5’-UGGUUUACAUGUUGUGUGA-3’

5’-UGGUUUACAUGUUUUCUGA-3’

5’-UGGUUUACAUGUUUUCCUA-3’

uPA Santa Cruz;

sc-36779

100 5’-CCACACACUGCUUCAUUGAtt-3’

5’-CCCAUGGUUGAGAAAUGAAtt-3’

5’-GUCUGAUUGUUAAGUCUAAtt-3’

NOTCH1 Santa Cruz;

sc-36095-A

70 5’-CACCAGUUUGAAUGGUCAAtt-3’

NOTCH3 Santa Cruz;

sc-37135

70 5’-GUCAGAAUUGUGAAGUGAAtt-3’

5’-CUCGUCAGUUCUUAGAUCUtt-3’

5’-CCUCUCAUUUCCUUACACUtt-3’

JAG1 Dharmacon;

M-011060-02

100 (cancer cells);

150 (fibroblasts)

5’- CGAAUGGAGUACAUCGUAU-3’

5’-CACCAGGUCUUACUACGGA-3’

5’-CGACAAGGCUGCAGUCCUA-3’

5’-GAAGAAUGUUUCCGCUGAA-3’

TGFβR1 Santa Cruz;

sc-40222

100 5’-UAUUCAAACAUGACCAUGCtt-3’

5’-UAGAAGUCCAGCACUCUUGtt-3’

5’-UGUAACUCAAAGGUUCUAGtt-3’

c-MET Santa Cruz;

sc-29397

100 5’-GGUACCACUUGAUUUCAUAtt-3’

5’-CCACUCAUUUAGAAUUCUAtt-3’

5’-GCAAGCAAUUGGAAACAAAtt-3’

Table 2.1: siRNAs used in reverse transfection.

30

2.3. RT-qPCR: RNA preparation, reverse transcription (RT), and quantitative

real-time PCR (qPCR)

Primer sequences were designed using Primer-BLAST (National Center for

Biotechnology information) and are listed in Table 2.2. Total RNA was extracted using

RNeasyPlus Mini Kit (Qiagen) according to the manufacturer’s protocol at room

temperature. cDNA was prepared from 1μg of RNA using the iScript cDNA synthesis

kit (Bio-Rad) and subjected to quantitative real-time PCR using the default PCR cycle

on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Amplified DNA

products were detected and quantified by SYBR Green using Power SYBR Green PCR

Master Mix (Applied Biosystems). Dissociation curve analysis was also performed to

ensure the absence of non-specific amplification. Each well on the qPCR plate

contained a 10μl mixture: 5μl SYBR Green PCR Master Mix, 1μl of each primer

(0.5μM final), 2μl of ddH2O, and 1μl of template cDNA. Each sample was tested in

triplicate and a negative water control was included for each primer set.

Quantitative

PCR

Forward Primer Reverse Primer

NOTCH1

cDNA

5’-CCTGCCTGTCTGAGGTCAAT-3’ 5’-GGGTCACAGTCGCACTTGTA-3’

NOTCH3

cDNA

5’-CCTGCGATCAGGACATCAA-3’ 5’-GCAGGAGCAGGAAAAGGAG-3’

JAG1 cDNA 5’-TGACCAGAATGGCAACAAAA-3’ 5’-CTCATTACAGATGCCGTGGA-3’

TGFβ1 cDNA 5’-ACAATTCCTGGCGATACCTCAGC

A-3’

5’-CGCTAAGGCGAAAGCCCTCAATTT-3’

HGF cDNA 5’-CGAACACAGCTATCGGGGTA-3’ 5’-AACTCTCCCCATTGCAGGTC-3’

β-actin

cDNA

5’-CCACACTGTGCCCATCTACG-3’ 5’-AGGATCTTCATGAGGTAGTCAGTCAG-

3’

Table 2.2: DNA sequences of the primers used for mRNA quantification by RT-qPCR.

Real-time qPCR analysis yielded a CT (cycle threshold) value for each PCR product in

each reaction. The CT is defined as the number of cycles required for the fluorescent

signal to cross the threshold. Results were normalized to β-actin expression. The fold

change was calculated according to the following equation:

31

Fold change= 2^{[(Mean CT of gene X in treatment group)/(Mean CT of β-actin in

treatment group)]-[(Mean CT of gene X in control group)/(Mean CT of β-actin in

control group)]}

2.4. Co-culture conditions

Cancer cells and fibroblasts were grown in the appropriate serum-free cancer cell

media (Table 2.3) for 72 hours in 6-well plates at 37°C, 5% CO2. 3X105 cancer cells and

1.5X105 fibroblasts (ratio 2:1) were seeded into each well, together with 2.5ml

serum-free media. If siRNA treatment was involved prior to seeding into co-culture,

cells were detached using 0.05% trypsin-EDTA (Life technologies) and then

suspended in complete growth media, followed by centrifugation and resuspension

in serum-free media. Mono-culture was also performed in parallel by seeding the

same number of cells as in co-culture conditions.

MDA-MB231 HCC1143

RMF/EG or RMF/EG-TGFβ DMEM RPMI-1640

Exp-CAF2 / Ctl2 DMEM RPMI-1640

Table 2.3: Serum-free media used in co-culture conditions.

2.5. Conditioned media and whole cell lysate preparations, Western blotting, and

antibodies

Confluent monolayers of adherent cells were washed with ice-cold 1X

phosphate-buffered saline (PBS) and lysed in 150μl RIPA Lysis Buffer (25mM Tris pH

7.6, 150mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) supplemented with

PhosSTOP (Roche) and Protease inhibitor cocktail tablets (Roche). Lysates were

sonicated briefly using the Sonic Dismembrator Model 100 (Fisher Scientific)

followed by centrifugation at 4°C for 10 minutes to remove cellular debris. Protein

concentration was determined using the DC Protein Assay kit (Bio-Rad) and the

EL800 Absorbance Microplate Reader (BioTek). For co-culture experiments, both

whole cell lysates and conditioned media were harvested after 72 hours’ incubation.

Whole cell lysates were prepared by adding 150μl RIPA directly to the wells and by

32

processing lysates as described above. An Equal volume of conditioned media from

each well was collected and concentrated to a smaller volume (120μl) using Ultra-4

Centrifugal Filter Devices (Millipore). All protein samples were mixed with Sample

Buffer (2X) (125mM Tris pH 6.8, 20% Glycerol, 4% SDS, 0.005% bromophenol blue). In

most cases, DTT (100mM) was added and the sample was denatured at 95°C for 5

minutes (Table 2.4). Samples were then loaded onto SDS-polyacrylamide gels along

with PiNK Plus Prestained Protein Ladder (GeneDireX). To detect secreted proteins in

the conditioned media, equal volumes (20μl) of sample were loaded, together with

equal volumes (10μl) of whole cell lysates to detect β-actin (loading control). For

non-secreted proteins, 5-20μg of total protein was loaded. Electrophoresis was

performed in 1X Running Buffer (25mM Tris, 192mM Glycine, 0.1% SDS) at 180V

using Mini PROTEAN Tetra Cell (Bio-Rad). Proteins were transferred to PVDF

membrane (Bio-Rad) using Mini Trans-Blot Cell (Bio-Rad) at 350mA for 1 hour in 1X

Transfer Buffer (25mM Tris, 192mM Glycine, 10% methanol). Protein membranes

were blocked with Blocking Solution (10mM Tris, 150mM NaCl, 0.05% Tween-20, 5%

non-fat milk) at 4°C overnight. Primary antibodies were added and membranes were

incubated according to Table 2.4. After washing membranes 3 times for 5 minutes

each in 1% milk (10mM Tris, 150mM NaCl, 0.05% Tween-20, 1% non-fat milk),

membranes were then incubated with HRP-conjugated secondary antibodies at room

temperature for 1-2 hours (Table 2.5). After 3 additional washes, proteins were

detected with ECL or ECL prime Western Blotting Detection Reagents (GE Healthcare)

according to manufacturer’s instructions. Blots were exposed to HyBlot CL films

(Denville Scientific, Inc.).

33

Antibody

Detecting

protein in

DTT and heat-

denaturation

applied

Gel

%

Company

and Cat.#

Dilution Incubation

time

Detection

reagent

uPA conditioned

media

No 10% Millipore;

MAB7776

1/2000 3-4 hours ECL

TGFβ conditioned

media

yes 10% Cell

Signaling;

3711S


JAG1 whole cell

lysate

yes 8% Santa Cruz;

H-114

1/1000 overnight ECL

NOTCH1 whole cell

lysate

yes 8% Santa Cruz;

C-20


NOTCH3 whole cell

lysate

yes 8% Santa Cruz;

M-134


N1-ICD whole cell

lysate

yes 8% Cell

Signaling;

Val1744

1/300 3-4 hours ECL prime

αSMA

(1A4)

whole cell

lysate

yes 10% Abcam;

Ab7817

1/300 3-4 hours ECL

c-MET

(3D4)

whole cell

lysate

yes 8% Invitrogen;

37-0100


TGFβR1 whole cell

lysate

yes 10% Santa Cruz;

V-22

1/300 overnight ECL

β-actin,

HRP conj-

ugated

whole cell

lysate

yes 10% Santa Cruz;

C-11


Table 2.4: Primary antibodies used in Western blot analyses.

Antibody Company and Cat.# Dilution

Donkey anti-rabbit IgG-HRP Santa Cruz; sc-2313 1/5000

Goat anti-mouse IgG-HRP Santa Cruz; sc-2005 1/5000

Table 2.5: Secondary antibodies used in Western blot analyses.

2.6. Cell counting

Cell numbers were determined after 72 hours’ incubation for both mono-culture and

co-culture conditions. Cells were detached using 0.05% trypsin-EDTA (Life

Technologies), washed and examined by loading into Bright-line hemacytometer

(Hausser Scientific) under a fluorescence microscope (Leica DMIRB). Total cell

34

number was calculated based on the average number of cells in 4 corner squares.

The number of stromal cells was then determined by counting GFP+ cells in the same

field.

2.7. Invasion assay

Invasion assays were performed using 24-well BioCoat Matrigel Invasion Chambers

(Corning). Calcein-AM was added to untreated/siRNA transfected MDA-MB231 cells

to a final concentration of 5μg/ml for 45 minutes in order to stain the live cell

population. 4X104 stained cancer cells were then trypsinized, spin-washed twice with

serum-free DMEM, seeded into the insert together with 2X104 unstained stromal

cells in 500μl serum-free DMEM. The bottom well contained 1ml DMEM

supplemented with 2% FBS as chemoattractant. After 22 hours’ incubation at 37°C, 5%

CO2, non-invading cells on the upper side of the membrane were scraped off using

pre-wet cotton tipped swabs. The membranes were fixed in 3.7% formaldehyde for 5

minutes, washed in PBS, and then mounted onto a microscope slide with mounting

medium for fluorescence (Vector Laboratories, Inc.). Green fluorescent cells were

counted with a Leica DMIRB at 20X magnification. Four fields were counted per

membrane. All invasion assays were replicated 3 times.

35

CHAPTER 3: Results

36

3.1. Fibroblast-derived TGFβ promotes JAG1/Notch-mediated uPA expression in

BLBC cells

3.1.1. Fibroblast-derived TGFβ induces uPA expression in BLBC cells

Since Notch signaling promotes progression of the BLBC subtype, a subtype

highlighted by its unique interations with fibroblasts (see section 1.9), the possibility

of a causal link between CAFs and Notch activation was raised. In order to explore

this link in BLBC, I employed an in vitro co-culture system in which BLBC cell lines

(MDA-MB231 or HCC1143) were cultured together with fibroblast cell lines. These

studies were initially undertaken with a reduction mammary fibroblast (RMF/EG) cell

line 145

and a variant of this line expressing high levels of TGFβ (RMF/EG-TGFβ). While

these fibroblast cell lines are not CAFs, they were selected to explore the role of

TGFβ, a hallmark growth factor of CAFs, in tumor cell-CAF crosstalk. To prepare for

the co-culture experiments, a survey of the expression levels of Notch components

and TGFβ in the aforementioned cell lines was performed. I verified that the

RMF/EG-TGFβ cell line expressed high levels of TGFβ mRNA (Figure 3.1.1 A) and

protein, secreted in both latent (52kDa) and mature (14kDa) forms (Figure 3.1.1 B),

and that both fibroblast cell lines showed no or low Notch activation as evidenced by

the absence of N1-ICD (Figure 3.1.1 B). As previously reported, the BLBC cell lines

expressed JAG1, NOTCH1 and NOTCH3 (HCC1143) and demonstrated Notch

activation (Figure 3.1.1 B).

Next, co-culture experiments were undertaken (the experimental design is

shown schematically in Figure 3.1.2 A). Breast cancer cells and fibroblasts were

mixed in a ratio of 2:1 and cultured in serum-free media for 72 hours. As a control,

mono-culture of each cell line was performed under the same culture conditions. As

a readout for Notch activation127

, urokinase-type plasminogen activator (uPA) was

quantified in the conditioned media at 72 hours. While MDA-MB231 (Figure 3.1.2 B,

lane 1) and HCC1143 (Figure 3.1.2 C, lane 1) expressed uPA in mono-culture, in

co-culture with RMF/EG-TGFβ there was a large increase of uPA (Figure 3.1.2 B and C,

lane 5). In addition, while RMF/EG-TGFβ in mono-culture produced both latent and

mature forms of TGFβ, co-culture with MDA-MB231 cells resulted in a decrease in

A.

Figure 3.1.1: Characterization of the cell lines used in co

of TGFβ1 mRNA in the cell lines. RT

expression and are expressed relative to the value of RMF/EG. Experiments were done in

biological triplicate; bars represent standard error of the means (SEM) (*, p<0.05).

expression of JAG1, NOTCH1/3, N1

and HCC1143) and fibroblast cell

loading control. Mw markers are shown in kDa.

B.

Characterization of the cell lines used in co-culture experiments. A)

1 mRNA in the cell lines. RT-qPCR mRNA levels are normalized according to


represent standard error of the means (SEM) (*, p<0.05).

expression of JAG1, NOTCH1/3, N1-ICD and TGFβ (secreted) in BLBC cell lines (MDA

cell lines (RMF/EG and RMF/EG-TGFβ). β-actin is included as a

markers are shown in kDa.

37

Expression

qPCR mRNA levels are normalized according to β-actin


represent standard error of the means (SEM) (*, p<0.05). B) Protein

ell lines (MDA-MB231

actin is included as a

38

latent TGFβ in the conditioned media (Figure 3.1.2 B, compare lanes 4 and 5). This is

likely due to MDA-MB231 uPA-mediated conversion of latent TGFβ to its active form,

as previously described186

. Interestingly, the presence of RMF/EG cells also increased

uPA expression, despite the fact that TGFβ was not detected under these conditions

(Figure 3.1.2 B, lane 3; C, lane 4), suggesting that signaling pathways other than those

mediated by TGFβ may be supporting uPA expression in these cell lines.

To eliminate the possibility that increased uPA production was simply caused by

cancer cell proliferation resulting from the co-culture conditions, live cell counting in

mono- and co-culture conditions was performed. Since the RMF/EG lines are

GFP-positive (see Materials and Methods), breast cancer cells can easily be

differentiated from fibroblasts by GFP fluorescence. The addition of RMF/EG-TGFβ

cells did not significantly influence the number of cancer cells after 72 hours’

incubation (Figure 3.1.2 D and E), indicating that the differences in the total uPA

levels were not due to differences in cell numbers. Taken together, these results

suggest that fibroblast-derived TGFβ induces uPA expression in these BLBC cell lines.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Via

ble

ce

ll n

um

be

rs a

fte

r se

rum

-

fre

e i

ncu

ba

tio

n(M

on

o/C

o-c

ult

ure

)

A.

B.

D.

Figure 3.1.2: Fibroblast-derived TGF

HCC1143 cell lines. A) Schematic

siRNA-transfected breast cancer cell lines

and cultured in serum-free media for 72 hours.

analyses of uPA and TGFβ (latent and mature forms)

72 hours’ mono- or co-culture.

loaded and probed for β-actin.

viable cells were counted after

and the ratios were calculated. Data are me

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Via

ble

ce

ll n

um

be

rs a

fte

r se

rum

-

fre

e i

ncu

ba

tio

n(M

on

o/C

o-c

ult

ure

)

C.

E.

derived TGFβ promotes uPA expression in MDA-MB231 and

Schematic of cancer cell-fibroblast co-culture. Untreated or

breast cancer cell lines and fibroblast cell lines were mixed in a ratio of 2:1

free media for 72 hours. See text for details. B and C) Western blot

(latent and mature forms) expression in conditioned media

culture. An equal proportion of whole cell lysate from all samples was

actin. Mw markers are shown in kDa. D and E) For each cell type,

viable cells were counted after the culture period in both mono- and co-culture conditions

calculated. Data are mean ± SEM (D: n=5; E: n=3).

39

MB231 and

Untreated or

were mixed in a ratio of 2:1

Western blot

expression in conditioned media after

samples was

For each cell type,

culture conditions

40

3.1.2. Notch signaling is required for fibroblast TGFβ-mediated uPA up-regulation in

MDA-MB231 and HCC1143 BLBC cell lines

Since uPA is a direct transcriptional target of Notch signaling in human breast

cancer127

, I wished to determine whether the TGFβ-induced uPA expression was

dependent upon Notch signaling. Both BLBC cells and fibroblasts were subjected to

siRNA-mediated knockdown of uPA or Notch components, followed by co-culture

experiments. Based on previous studies demonstrating that BLBC cells depend upon

active NOTCH1 and NOTCH3 for uPA expression127

, NOTCH1 and NOTCH3 siRNAs

were combined into a single knockdown condition.

As a first step, I confirmed that the siRNA treatments could effectively reduce

the expression of their target genes in MDA-MB231 or RMF/EG-TGFβ cells (Figure

3.1.3 A-D). As previously reported, NOTCH1/3 siRNA in MDA-MB231 achieved

efficient target knockdown and a corresponding reduction in uPA expression (Figure

3.1.3 B). In contrast, JAG1 siRNA only resulted in a partial reduction of uPA expression.

This is likely due to the redundancy of Notch ligands in driving Notch activation and

subsequent uPA expression. Although JAG1 silencing reduced NOTCH1 mRNA

expression in MDA-MB231 cells (Figure 3.1.3 A), this was not seen at the protein

level (Figure 3.1.3 B). In RMF/EG-TGFβ cells, NOTCH1/3 siRNA treatment led to an

efficient knockdown of NOTCH3 (Figure 3.1.3 C) and a partial decrease in NOTCH1

expression (Figure 3.1.3 C and D). In both cell lines tested, NOTCH3 was detectable

only by RT-qPCR due to its low expression levels (Figure 3.1.3 A and C).

Next, co-culture experiments were performed to determine whether Notch

signaling plays a role in TGFβ-induced uPA expression. In co-culture with

RMF/EG-TGFβ both BLBC cell lines displayed a large increase in uPA expression

(Figure 3.1.3 E and G, lane 2). RMF/EG-TGFβ-induced uPA overexpression was

abolished when uPA, NOTCH1/3 or JAG1 were knocked down in MDA-MB231 or

HCC1143 cells (Figure 3.1.3 E and G, lanes 3-5), suggesting that secreted uPA was

produced exclusively by BLBC cells in a Notch-dependent way. In contrast, knocking

down uPA or Notch components in RMF/EG-TGFβ cells had minimal effect on uPA

production (Figure 3.1.3 F, lanes 3-5). To summarize these experiments, Notch

41

signaling within BLBC cell lines, but not within fibroblasts, is required for fibroblast

TGFβ-mediated uPA expression.

A.

C.

E.

G.

B.

D.

. F.

42

43

Figure 3.1.3: Fibroblast TGFβ-induced uPA expression is tumor cell Notch-dependent. A and

C) RT-qPCR analysis of NOTCH1, NOTCH3 and JAG1 mRNA levels in MDA-MB231 (A) and

RMF/EG-TGFβ (C) cells after 48 hours treatment with Scr, uPA, NOTCH1/3 or JAG1 siRNAs.

Data are normalized according to β-actin expression and are presented as the average of 3

biological replicates. Bars represent SEM (*, p<0.05 compared with Scr). B and D) Western

blot of JAG1, NOTCH1 or uPA in MDA-MB231 (B) or RMF/EG-TGFβ (D) treated with siScr or

siRNA targeting uPA, NOTCH1/3 or JAG1 for 48 hours. β-actin was probed as loading controls

(see Materials and Methods). E and F) uPA expression in conditioned media from

MDA-MB231 cells (black bar) and RMF/EG-TGFβ (red bar), treated with either scrambled (Scr)

siRNA or siRNA targeting uPA, NOTCH1 and 3, or JAG1 and cultured either alone or in

co-culture. G) uPA expression in conditioned media from HCC1143 cells (black bar) and

RMF/EG-TGFβ (red bar), treated with either scrambled (Scr) siRNA or siRNA targeting uPA,

NOTCH1 and 3, or JAG1 and cultured either alone or in co-culture. Mw markers are shown in

kDa.

44

3.2. CAF-like cells promote Notch-dependent uPA secretion in BLBC cells

While RMF-TGFβ allowed exploration of the specific role of TGFβ in

fibroblast-BLBC cell interactions, the next objective was to determine whether these

findings were applicable to CAFs. For these experiments the Exp-CAF2 line was used.

Exp-CAF2 was originally generated by Kojima et al. according to the following

procedures151

: immortalized reduction mammoplasty fibroblasts expressing GFP and

the puromycin-resistance gene were mixed with MCF-7-ras breast carcinoma cells

and then co-injected into immunodeficient nude mice. The tumor xenograft that

formed was resected at day 42 post-implantation and dissociated into a single-cell

suspension. These cells were then cultured in vitro and selected in the presence of

puromycin. Puromycin-resistant cells were again mixed with MCF-7-ras cells and

allowed to grow in host mice for another 200 days. These experimental generation 2

cancer-associated fibroblast (Exp-CAF2) cells (total 242 days in an MCF-7 xenograft)

subsequently underwent biochemical and functional analyses. These studies

revealed that compared to control (Ctl2) fibroblasts (see below), Exp-CAF2 cells

display the traits of CAF myofibroblast populations extracted from human invasive

carcinomas, including the expression of high levels of activated myofibroblast

markers α-SMA and TN-C151

. Typical of CAFs, Exp-CAF2 can significantly promote

tumor growth in a tumor xenograft assay, resulting in high tumor volume and

micro-vascular density151

.

To characterize Exp-CAF2 cells, the expression of CAF markers and Notch

signaling components were examined. As a control, Ctl2 cells were tested. The Ctl2

line was generated by Kojima et al. by injecting GFP-labeled, puromycin-resistant,

immortalized human mammary stromal fibroblasts into nude mice as pure cultures

without MCF-7-ras cells151

. These cells were handled and isolated in the same way as

Exp-CAF2 cells, and called control fibroblast-2 (Ctl2) cells. Exp-CAF2 cells were

confirmed to express α-SMA (Figure 3.2.1 A). They also featured higher expression of

TGFβ and HGF, hallmark growth factors of CAFs, relative to Ctl2 cells (Figure 3.2.1 B

and C), confirming their myofibroblastic identity. Interestingly, although Notch

receptor (NOTCH1) and ligand (JAG1) were both expressed by Exp-CAF2, Notch

A.

B.

D.

Figure 3.2.1: Experimentally-generated Exp

CAFs. A) Western blotting of

Expression of TGFβ1 mRNA in

Exp-CAF2, MDA-MB231 and HCC1143 cells. RT

to β-actin expression and are expr

in biological triplicate; bars represent standard error of the means (SEM) (*, p<0.05).

Protein expression of Notch components JAG1, NOTCH1 and N1

MDA-MB231 lysate was loaded as a comparison.

markers are shown in kDa.

. C.

generated Exp-CAF2 cells share myofibroblastic traits with

g of α-SMA protein expression in Ctl2 or Exp-CAF2 cells.

Ctl2 and Exp-CAF2 cells. C) Expression of HGF mRNA in Ctl2,

MB231 and HCC1143 cells. RT-qPCR mRNA levels are normalized according

actin expression and are expressed relative to the value of Ctl2. Experiments were done

represent standard error of the means (SEM) (*, p<0.05).

Protein expression of Notch components JAG1, NOTCH1 and N1-ICD in Ctl2 or Exp

oaded as a comparison. β-actin is included as a loading control.

45

CAF2 cells share myofibroblastic traits with

CAF2 cells. B)

Expression of HGF mRNA in Ctl2,

qPCR mRNA levels are normalized according

essed relative to the value of Ctl2. Experiments were done

represent standard error of the means (SEM) (*, p<0.05). D)

in Ctl2 or Exp-CAF2.

actin is included as a loading control. Mw

46

activation was not detected, as indicated by the absence of N1-ICD (Figure 3.2.1 D).

As was seen with the RMF/EG-TGFβ line, in co-culture with Exp-CAF2 cells there

was a significant increase in uPA expression in BLBC cell lines (Figure 3.2.2 A and B,

lane 5). This up-regulation of uPA could be reversed with knockdown of NOTCH1/3 or

JAG1 in MDA-MB231 cells, demonstrating Notch dependence (Figure 3.2.2 C, lanes

5-6). Both Ctl2 and Exp-CAF2 cell lines showed undetectable levels of uPA secretion

in mono-culture conditions (Figure 3.2.2 A, lanes 2 and 4; B, lanes 2 and 3). As was

seen with RMF/EG cells, changes in the total uPA levels were not caused by

CAF-induced cancer cell proliferation (Figure 3.2.2 D). In contrast, the presence of

Ctl2 cells had little/no impact on uPA levels in the conditioned media (Figure 3.2.2 A,

lane 3; B, lane 4). These data suggest that like RMF/EG-TGFβ cells, CAF-like cells

(Exp-CAF2) but not normal fibroblasts (Ctl2), can promote Notch-mediated uPA

secretion in co-cultured BLBC cells.

A.

C.

Figure 3.2.2: Exp-CAF2 cells promote JAG1

and B) Western blot analyses of uPA expression in

or co-culture. C) uPA expression in conditioned media from MDA

Exp-CAF2 (red bar), treated with either scrambled (Scr) siRNA or siRNA targeting uPA,

NOTCH1 and 3, or JAG1 and cultured either alone or in co

kDa. D) For each cell type, viable cells were counted after

and co-culture conditions and the ratio

0

0.2

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0.6

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1

1.2

1.4

1.6

1.8

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n(M

on

o/C

o-c

ult

ure

)

B.

D.

promote JAG1/Notch-mediated uPA expression in BLBC

Western blot analyses of uPA expression in conditioned media after 72 hours

uPA expression in conditioned media from MDA-MB231 cells (black bar) and

CAF2 (red bar), treated with either scrambled (Scr) siRNA or siRNA targeting uPA,

NOTCH1 and 3, or JAG1 and cultured either alone or in co-culture. Mw markers are shown in

For each cell type, viable cells were counted after the culture period in both mono

culture conditions and the ratios were calculated. Data are mean ± SEM (n=3).

47

BLBC cells. A

conditioned media after 72 hours’ mono-

MB231 cells (black bar) and

CAF2 (red bar), treated with either scrambled (Scr) siRNA or siRNA targeting uPA,

markers are shown in

period in both mono-

SEM (n=3).

48

3.3. TGFβR1 and c-MET receptors are required for CAF-induced uPA expression in

BLBC cells

The next objective was to clarify the underlying mechanism by which CAF-like

cells were able to induce uPA expression in BLBC cells. Based on experiments in the

RMF/EG-TGFβ line, and that TGFβ was overexpressed from Exp-CAF2 (Figure 3.2.1 B)

or patient-derived CAFs151–153

, one mechanism was predicted to involve TGFβ. In

addition, since RMF/EG (Figure 3.1.2 B and C) or Ctl2 cells (Figure 3.2.2 B) could

trigger uPA up-regulation even with no/low levels of TGFβ expression, the possibility

of involvement of factors other than TGFβ was raised. Several CAF-derived signaling

molecules have been suggested to mediate the crosstalk between tumor and the

surrounding CAFs, including SDF-135

, HGF41,52

, CCL255

and platelet-derived growth

factor C (PDGF-C)187

.

In addition to TGFβ, HGF was selected for further investigation since its receptor,

c-MET, was previously shown by our group to be a target of Notch activation in BLBC

cells188

. Furthermore, Exp-CAF2 cells (Figure 3.2.1 C) or patient-derived CAFs52

express elevated levels of HGF, and HGF stimulation initiates the “invasive growth”

program by affecting uPA expression in prostate cancer cells189

. To undertake these

experiments, first, a survey of the expression level of TGFβR1 and c-MET was taken in

BLBC cell lines. Comparable levels of TGFβR1 and c-MET proteins were observed in

MDA-MB231 and HCC1143 cells (Figure 3.3.1 A), confirming the potential of these

cells to respond to TGFβ and HGF. To prepare for the mono-/co-culture experiments,

TGFβR1 and c-MET siRNA treatments were shown to effectively reduce protein

expression of their targets (Figure 3.3.1 B and C).

In mono-culture conditions, either TGFβR1 or c-MET knockdown in BLBC cells

resulted in a reduction of uPA secretion (Figure 3.3.1 D and E, lanes 1-3), indicating

that both TGFβR1 and c-MET-mediated signaling pathways are required for uPA

expression. Indeed, both TGFβ and HGF are expressed in BLBC cells (Figure 3.1.1 A;

Figure 3.2.1 C), suggesting autocrine activation of these pathways in mono-culture,

resulting in uPA expression. In co-culture with Exp-CAF2 cells there was a significant

increase in uPA expression (Figure 3.3.1 D, lane 5). Knockdown of either TGFβR1 or

49

c-MET led to reduced uPA expression (Figure 3.3.1 D, lanes 6-7), consistent with

Exp-CAF2 production of these ligands. To further verify the importance of TGFβR1

and c-MET in mediating fibroblast-BLBC cell interactions, additional co-culture

experiments were performed using HCC1143 and the RMF/EG (-TGFβ) cell lines

(Figure 3.3.1 E). Similar uPA expression patterns were observed, suggesting that

fibroblast-induced uPA expression in HCC1143 cells was dependent upon the

expression of TGFβR1 or c-MET proteins. Overall, these results suggest that TGFβR1

and c-MET receptors are required for uPA expression in BLBC cells, and that their

ligands, produced by Exp-CAF2 or RMF/EG (-TGFβ) cells, contribute to uPA expression

in BLBC cell lines.

A.

D.

E.

B.

C.

50

51

Figure 3.3.1: TGFβR1 and c-MET receptors are required for uPA expression in BLBC cells. A)

Western blot of c-MET or TGFβR1 in BLBC cell lines. B) TGFβR1 and C) c-MET protein

expression in MDA-MB231 cells treated with Scr, TGFβR1 or c-MET siRNAs, analyzed by

western blot 48 hours post-treatment. β-actin is included as a loading control. D) uPA

expression in serum-free media from MDA-MB231 cells (black bar) or Exp-CAF2 (red bar)

cultured either alone or in co-culture. MDA-MB231 cells were treated with either scrambled

(Scr) siRNA or siRNA targeting TGFβR1 or c-MET. E) Protein expression of uPA and both forms

of TGFβ (latent and mature) in conditioned media from HCC1143 (black bar), RMF/EG (blue

bar) or RMF/EG-TGFβ (red bar) cultured either alone or in co-culture. MDA-MB231 cells were

treated with either siScr or siRNA targeting TGFβR1 or c-MET. Mw markers are shown in kDa.

52

3.4. CAF-like cells promote uPA, Notch, TGFβR1 and c-MET–dependent invasion of

MDA-MB231 cells

To determine whether Exp-CAF2-induced uPA expression in BLBC cells promotes

cell invasion, in vitro Matrigel invasion assays (the experimental design is shown

schematically in Figure 3.4.1 A) were undertaken. Co-culture with Exp-CAF2 cells

increased the number of invasive MDA-MB231 cells by 117.6%, whereas co-culture

with Ctl2 cells enhanced cell invasion up to 34.1% (Figure 3.4.1 B). siRNA-mediated

knockdown of uPA, Notch components, TGFβR1 or c-MET in the co-cultures made the

cells invade as poorly as mono-cultured cells (Figure 3.4.1 C), indicating that these

knockdowns nullify the effect of co-culture. Thus, Exp-CAF2-induced invasion is

dependent upon uPA, Notch signaling, TGFβR1 and c-MET within the breast cancer

cells, consistent with the previous biochemical data demonstrating that CAF-like cells

promoted Notch-, TGFβR1- and c-MET-dependent uPA expression in tumor cells.

A.

C.

Figure 3.4.1: Exp-CAF2 cells promote

of breast cancer cells. A) Schematic representation of

invasion analysis. Arrow indicates direction of invasion.

MDA-MB231 cells cultured either alone or in co

numbers are expressed relative to

analysis of MDA-MB231 cells cultured either alone o

MDA-MB231 cells were treated with either siScr or siRNA targeting uPA, NOTCH1/3, JAG1,

TGFβR1 or c-MET. Assays were done in biological triplicate;

p<0.001).

B.

CAF2 cells promote uPA, Notch, TGFβR1 and c-MET–dependent invasion

Schematic representation of in vitro transwell chamber assay for

Arrow indicates direction of invasion. B) Comparison of invasion of

MB231 cells cultured either alone or in co-culture with Ctl2 and Exp-CAF2. Invaded cell

expressed relative to the value of MDA-MB231 in mono-culture. C)

MB231 cells cultured either alone or in co-culture with Exp

MB231 cells were treated with either siScr or siRNA targeting uPA, NOTCH1/3, JAG1,

were done in biological triplicate; bars represent SEM (*, p<0.05

53

dependent invasion

ranswell chamber assay for

Comparison of invasion of

Invaded cell

C) Invasion

culture with Exp-CAF2.

MB231 cells were treated with either siScr or siRNA targeting uPA, NOTCH1/3, JAG1,

(*, p<0.05; **,

54

3.5. c-MET is a downstream target of Notch in BLBC cell lines

Since both c-MET and Notch signaling are required for CAF-induced uPA

overexpression, the possibility that c-MET regulates uPA expression by activating

Notch signaling in BLBC cells, was explored. While c-MET siRNA treatment knocked

down its intended target, it had no effect on JAG1, NOTCH1, or Notch activation as

evidenced by N1-ICD protein levels in MDA-MB231 or HCC1143 (Figure 3.5.1 A and B).

The collective observations that c-MET knockdown resulted in a reduction of uPA

expression (Figure 3.3.1 D and E, lane 3) without altering Notch signaling in BLBC cell

mono-culture conditions suggest that c-MET regulates uPA expression in a

Notch-independent fashion.

Previously, our lab has identified c-MET as a Notch target gene in BLBC cell

lines188

. c-MET promoter activity was found to be dependent on NOTCH1 receptor

expression. Consistent with this, NOTCH1/3 combined knockdown resulted in a

reduction of c-MET expression in both MDA-MB231 and HCC1143 cells (Figure 3.5.1

C), confirming c-MET as a downstream target of Notch in BLBC cells.

Taken together, these data support a model where CAFs promote BLBC tumor

uPA expression through at least two secreted factors: TGFβ and HGF. Both

TGFβ/TGFβR1 signaling and HGF/c-MET signaling contribute to uPA production via a

Notch-dependent mechanism. Notch drives tumor cell expression of TGFβR1 and

c-MET receptors, and uPA-dependent maturation of TGFβ (see section 1.9), closing a

paracrine activation loop (Figure 3.5.1 D). Therefore, BLBC cell Notch signaling

potentiates tumor-CAF crosstalk and may represent an important target for cancer

therapy.

A.

C.

D.

B.

55

56

Figure 3.5.1: Notch potentiates c-MET signaling between BLBC cells and CAFs. A and B)

Western blot analyses of c-MET, NOTCH1, JAG1 and N1-ICD protein expression in

MDA-MB231 (A) and HCC1143 (B) cells after a 48 hours treatment with Scr or c-MET siRNAs.

C) Protein expression of c-MET in MDA-MB231 and HCC1143 cells treated with scrambled

(Scr), c-MET or NOTCH1/3 siRNAs for 48 hours. Expression of β-actin is included as a loading

control. Mw markers are shown in kDa. D) Model: CAFs promote tumor uPA expression

through secreted TGFβ and HGF, both of which contribute to uPA production via a

Notch-dependent mechanism. Notch in turn drives tumor cell expression of TGFβR1 and

c-MET receptors, and uPA-dependent maturation of TGFβ, closing a paracrine loop.

57

CHAPTER 4: Discussion and Future Directions

58

The microenvironment features that are unique to BLBC have not been

well-characterized. It is apparent that stromal cells in the TME make a significant

contribution to the progressive growth and metastatic spread of cancer cells32

. A

tumor in vivo is more than the sum of its parts and represents the product of

reciprocal interactions. Due to the aggressive nature and the lack of targeted therapy

in BLBC, knowledge about stromal-epithelial interactions can be valuable as it may

provide solutions to therapeutically retard tumor growth or reduce metastases.

Recent studies have documented the tumor-promoting ability of CAFs using a variety

of xenograft mouse models35,51

. Herein I describe mechanisms responsible for BLBC

cell-CAF interactions and identify Notch as a central player.

To explore the role of Notch in BLBC cell-CAF crosstalk, co-culture experiments

were performed using BLBC cell lines together with fibroblast cell lines.

Fibroblast-derived TGFβ was found to stimulate tumor uPA secretion and invasion in

a Notch-dependent way. CAF-like cells could influence tumor uPA expression via

additional growth factors such as HGF, whose receptor c-MET has recently been

identified by our group as a Notch target in BLBC cells188

. These results support a

model where Notch potentiates tumor cell-CAF interactions by driving tumor cell

expression of TGFβR1, c-MET and uPA. In turn, uPA facilitates maturation of TGFβ

derived from stromal cells. Together, these interactions promote tumor invasion as

measured in in vitro assays. Therefore, Notch signaling is a key player in BLBC

tumor-CAF crosstalk and may represent an important target for cancer therapy.

4.1. TGFβ in the tumor microenvironment

Initially, based on evidence that TGFβ is a defining cytokine expressed by CAFs

(see section 1.7.3), co-culture experiments were undertaken specifically to focus on

the potential role of this growth factor in mediating BLBC tumor cell-stroma

interactions. Interestingly, co-culture experiments comparing RMF/EG to

RMF/EG-TGFβ (Figure 3.1) demonstrated the importance of TGFβ in uPA expression

in BLBC cells. However, these findings did not preclude the possibility that TGFβ

could function in an autocrine and indirect fashion to drive the expression of

additional cytokines/growth factors within RMF/EG that were responsible for the

59

observed effect on uPA expression in BLBC cells. Therefore, knockdown experiments

were performed (Figure 3.3.1) and clearly demonstrated the importance of TGFβR1

in BLBC cells, suggesting that TGFβ plays a direct and key role in driving uPA

expression in tumor cells.

As TGFβ is frequently found to be overexpressed in the stroma of human breast

cancer, multiple sources of TGFβ have been identified, including the cancer cells

themselves as well as various cells of the tumor stroma such as tumor-associated

macrophages (TAMs)190

, CAFs and myeloid precursor cells135,191

. The present

observations are consistent with previous reports that CAFs express 2-4 times more

TGFβ than normal fibroblasts (Figure 3.2.1 B)151–153

. Interestingly, Mono-culture

experiments (Figure 3.3.1) suggested that tumor-derived TGFβ could facilitate uPA

production in an autocrine fashion. TGFβ released by carcinoma cells could be

functionally important during the initial stages of tumor progression, as it may trigger

myofibroblast differentiation in surrounding fibroblasts36

, resulting in increased TGFβ

accumulation.

Recently, Ganapathy et al. determined the functional significance of the TGFβ

pathway in human BLBC metastasis192

. Applying TGFβ inhibitors to MDA-MB231

sublines resulted in reduced metastatic burden to either lung or bones in vivo. The

current model provides a possible molecular mechanism where CAF-derived TGFβ

induces BLBC cell uPA expression in a JAG1/Notch-dependent way.

4.2. Exp-CAF2: a model cell line for CAFs

Although RMF-TGFβ allowed the exploration of the role of TGFβ in BLBC uPA

expression, this line is not an ideal representative of CAFs since it does not express

α-SMA, the hallmark feature of CAFs (data not shown). Therefore, Exp-CAF2 cells

were used to model CAF behavior in the co-culture system for the following reasons:

1) they express elevated levels of CAF markers α-SMA and TN-C151

; 2) like CAFs, the

expression of α-SMA in Exp-CAF2 cells is stably maintained during in vitro

propagation without the ongoing interaction with carcinoma cells35,151

; 3) Similar to

the tumor-promoting roles of CAFs, Exp-CAF2 can significantly promote tumor

growth in a tumor xenograft assay, resulting in high tumor volume and

60

micro-vascular density151

; 4) Exp-CAF2 cells are easily cultured and propagated in

vitro, as opposed to primary cells which have limited life span and are hard to

culture193

.

Our experiments using the Exp-CAF2 cell line revealed that CAF-like cells send

signals to co-cultured BLBC cells and drive Notch activation in these cells (Figure

3.2.2), implying a critical role of Notch in BLBC cell-CAF crosstalk. Interestingly,

studies using patient-derived CAFs have shown that CAFs can promote tumor growth,

invasion, neoangiogenesis and stemness of cancer cells (see section 1.3.3), which

might be explained by Notch activation within tumor cells. Further experiments that

address both the involvement and the significance of Notch signaling are required to

better understand its role in cancer cell-CAF crosstalk in breast tumors.

4.3. The HGF/c-MET signaling axis and its crosstalk with Notch in BLBC cells

In addition to TGFβ/TGFβR1 signaling, the importance of c-MET in uPA

expression in BLBC cells was unraveled by co-culture experiments with Exp-CAF2 cells

(Figure 3.3). HGF, the only known natural ligand for c-MET181

, is primarily expressed

and secreted from fibroblasts, although other stromal components such as

endothelial cells, neutrophils and macrophages can be additional cellular sources of

HGF39,181,194,195

. Consistently, CAF-like Exp-CAF2 cells express more HGF mRNA than

tumor cells or normal fibroblasts (Figure 3.2.1 C). In both BLBC cell lines tested,

evidence for autocrine activation of c-MET existed in the mono-culture setting

(Figure 3.3.1), which may contribute to the aggressive nature of BLBC cells.

The present study shows that c-MET is necessary for CAF-induced uPA

expression in BLBC cells in co-culture. However, the requirement of CAF-derived HGF

in this context has not been directly addressed. In future, there are several

approaches that should be taken. First, the co-culture experiments should be

repeated using CAF-like cells where HGF production has been silenced by siRNAs or

shRNAs. Secondly, using an HGF neutralizing antibody in the co-culture may confirm

the importance of the HGF/c-MET signaling axis in tumor cell-CAF crosstalk.

The mono-culture experiments suggested that c-MET knockdown in BLBC cells

results in a reduction of uPA expression without affecting JAG1, NOTCH1 or Notch

61

activation levels, implying a Notch-independent regulatory pathway on uPA through

c-MET (Figure 3.5.1). To further confirm that c-MET regulates uPA independent of

Notch, a rescue experiment could be performed in BLBC cell lines where c-MET is

expressed from an exogenous promoter. Notch knockdown in these cells is predicted

to have less effect on uPA expression.

The current data do not preclude the possibility that HGF-induced c-MET

activation could lead to Notch activation and thus uPA up-regulation, especially when

high level of HGF is provided by CAFs in co-culture conditions. Evidence exists in the

literature to support the notion that HGF stimulation activates Notch by modulating

Notch ligand expression levels. HGF stimulation leads to activation of the Notch

pathway by up-regulating its ligands JAG1 and DLL4196

, and uPA overexpression in

prostate cancer DU145 cells189

. Another study revealed that c-MET activation results

in transcriptional induction of DLL1/4 and the Notch effector Hes-1197

. Whether HGF

stimulation is associated with Notch ligand up-regulation, Notch activation or uPA

overproduction could be explored biochemically in BLBC cells.

Our lab has previously reported c-MET as a NOTCH1 target gene in BLBC cells188

.

The present work also confirmed that NOTCH1 positively regulates c-MET expression

in both MDA-MB231 and HCC1143 cells (Figure 3.5.1). Whether or not c-MET is a

direct Notch target remains to be determined, since neither of the two high-affinity

CBF-1 binding sites with the c-MET promoter was found to form a complex with

CBF-1188

. Multiple hypothetical models have been suggested, including one where

NOTCH1 indirectly regulates c-MET expression via an as of yet, unidentified

intermediate188

.

An increasing body of work indicates that c-MET is preferentially expressed in

BLBC cells compared to other breast cancer subtypes198

. In addition, a recent study

identified an HGF signature that is strongly correlated with the BLBC subtype180

.

Therefore, consistent with the previous notion that HGF/c-MET signaling triggers the

“invasive growth” program, the present findings establish its importance in BLBC

cell-CAF crosstalk in terms of facilitating uPA production and tumor invasion in a

Notch-dependent fashion.

62

4.4. The roles of uPA in promoting cell invasion and growth factor activation

Activation of the uPA system has been implicated as a rate-limiting step for

tumor cell migration/invasion (see section 1.6). The current biochemical/functional

data are in line with this idea: tumor cells exhibit a greater ability to invade into

Matrigel when more uPA is detected in the corresponding mono-/co-culture

conditions (Figure 3.4.1). Interestingly, Ctl2 cells can significantly (p<0.05) enhance

MDA-MB231 invasion without affecting uPA secretion (Figure 3.2.2). Other

fibroblast-derived factors (for example, MMPs) could potentially account for this

increase in tumor invasion.

Odekon et al. first established the requirement for uPAR-bound uPA in

plasmin-dependent cellular conversion of latent TGFβ to mature TGFβ186

. Indeed, in

my experiments an association between elevated uPA production and less latent

TGFβ was observed (Figure 3.1.2 and 3.3.1). Interestingly, uPA is also documented in

the process of HGF activation199,200

. HGF bears a structural similarity to plasminogen,

the main substrate of uPA. Being synthesized and secreted as inactive single-chain

pro-form, HGF is converted to an active two-chain, disulfide-linked form by uPA

enzymatic activity. The involvement of uPA-driven HGF activation is yet to be

confirmed in the current cell lines and co-culture systems. Taken together, not only

does secreted uPA facilitates cell invasion, it also promotes tumor cell-CAF crosstalk

by providing mature growth factors such as TGFβ and possibly HGF, forming a

positive paracrine feedback loop.

4.5. Co-culture systems

In vitro co-culture systems are a convenient way to recapitulate tumor-stromal

interactions, but these models may not accurately reflect interplays that occur in a

primary tumor. 1) Unlike primary tumor, in co-culture the cell density and

tumor:stromal cell ratio are empirically chosen. 2) In co-culture cells are evenly

distributed within the wells in 2D, rather than having the three-dimensional

structures found in a primary tumor. 3) In co-culture the interactions between two

cell types are studied, ignoring the potential compounding effect that multiple

different cell types could present. In addition, direct co-culture systems have limited

63

ability to reveal detailed molecular mechanisms/dynamics governing tumor-stromal

crosstalk. For example, human breast cancer MDA-MB468 cells could “educate”

surrounding normal fibroblasts to secrete HGF to support their own progression

through paracrine signaling52

. Carcinoma-derived interleukin-6 (IL-6) induces

fibroblast activation, which in turn drives tumor EMT through secretion of MMPs53

.

These ways of mutual interplay between carcinoma cells and CAFs are hard to

capture by the direct co-culture method. Furthermore, direct co-culture systems fail

to address the requirement of direct cell-cell contact in tumor cell-CAF crosstalk. It

would be helpful to conduct interaction transwell cultures where both cells are

grown separated by a membrane but can communicate via soluble factors185

.

Nevertheless, these precursor co-culture experiments have established the

foundation for future in vivo mouse work to investigate the role of Notch in BLBC

tumor-CAF crosstalk.

4.6. The clinical significance of the work presented in this thesis

As our knowledge about the tumor microenvironment begins to accumulate,

the interplay between tumor and its stroma has become a fertile ground for novel

treatment discovery. The BLBC subtype is associated with early recurrence, poor

prognosis and few effective treatment options. It has recently been appreciated that

BLBC cells, in contrast to the luminal subtype, display unique interactions with

stromal components185

. Therefore, a deeper understanding of the molecular and

cellular mechanisms by which BLBC tumor and stromal cells cooperate in malignancy,

may lead to novel cancer therapies designed to neutralize the tumor-promoting

effects of the tumor microenvironment.

Deregulated Notch signaling is associated with BLBC tumors, correlates with a

more aggressive phenotype, and is linked to poor prognosis. The TGFβ/TGFβR and

HGF/c-MET signaling axes have been implicated in multiple aspects of cancer

progression, and are thought to play important roles in BLBC. The present work

suggests for the first time, that paracrine signaling between CAF and BLBC tumor

cells is mediated by CAF-derived TGFβ and HGF in a fashion that depends on Notch.

Therefore, therapeutic inhibition of Notch in BLBC tumors may lead to attenuated

64

responsiveness to stromal TGFβ or HGF, as well as reduced levels of uPA/invasion and

metastasis. Furthermore, our model provides a rationale to target CAF populations,

or to assess the potential of CAF enrichment as a predictive biomarker in

Notch-activated breast cancer.

The oncogenic tune of Notch signaling, together with the central role Notch

plays in tumor cell-CAF crosstalk, predict a benefit by combining conventional

therapies with Notch inhibitors. The efficacy of γ-secretase inhibitors (GSIs) to treat

Notch-activated breast cancer is being evaluated in clinical trials201

. Due to the

unselective nature of GSIs, severe gastrointestinal tract cytotoxicity is observed,

which may limit their therapeutic use. Despite the problems with GSIs, it is proposed

that Notch inhibition can be achieved on multiple levels. Currently, monoclonal

antibody that targets the NRR region of individual Notch receptors is being

investigated as a promising therapeutic agent202

. This will allow for a more specific

treatment approach, reduced side effects and elucidation of discrete functions of

individual Notch receptors. Further clinical studies designed to attenuate Notch

signaling in breast cancer will have to assess the influence on TGFβR1 and c-MET

expression in those tumors.

Targeting the tumor-promoting stromal components (CAF population in this

study) is considered to be essential for the development of new and effective cancer

therapies203,204

. Signaling pathways mediating interactions of CAFs with tumor cells

are believed to hold promise as therapeutic targets. The disruption of TGFβ/TGFβR or

HGF/c-MET signaling using ligand/receptor antagonists, neutralizing antibodies, or

kinase inhibitors has shown promising anti-cancer effects in different experimental

mouse tumor models205–207

. However, attenuating a single signaling pathway may

have limited benefit due to multiple factors/signaling pathways contributing to the

tumor-promoting ability of CAFs (see section 1.3.3). Thus, CAF-directed therapy that

aims to “normalize” CAFs themselves might be of great value33

. The cell-surface

serine protease known as fibroblast-activation protein (FAP) emerges as a candidate

for specifically targeting CAFs. In a recent study, a monoclonal anti-FAP antibody was

covalently linked with DM1, an anti-mitosis agent. This antibody substantially

65

attenuated the growth of stromal-rich tumor xenografts with no evidence of

toxicity208

. The FAP expression-based CAF targeting strategy may be further

combined with anti-Notch/anti-TGFβ/anti-HGF approaches in breast cancer patients

with reactive stroma and/or Notch hyperactivation.

In summary, this study contributes to our ever expanding knowledge of

tumor-stromal crosstalk in BLBC by uncovering Notch as a central player. Further

work is required to elucidate the molecular mechanisms and significance of this

interplay in mouse models and in human patients, which may facilitate the

development of targeted therapeutics in Notch-activated breast cancers.

66

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