University of Cincinnati

UNIVERSITY OF CINCINNATI

Date: 21-Sep-2009

I, Sara Meyer , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Cell & Molecular Biology It is entitled: The Ron Receptor Tyrosine Kinase in Tissue Morphogenesis

Student Signature: Sara Meyer

This work and its defense approved by: Committee Chair: Susan Waltz, PhD Susan Waltz, PhD

Kathleen Goss, PhD Kathleen Goss, PhD

Christopher Wylie, PhD Christopher Wylie, PhD

Nelson Horseman, PhD Nelson Horseman, PhD

Sohaib Khan, PhD Sohaib Khan, PhD

11/18/2009 174

The Ron Receptor Tyrosine Kinase in Tissue Morphogenesis

A dissertation submitted to the Graduate School at the University of Cincinnati in partial

fulfillment of the requirements for the degree of

Doctor of Philosophy

In the Department of Cancer and Cell Biology of the College of Medicine

Sara Elizabeth Meyer

B.S. Ohio University

September 21, 2009

Committee Chair, Co-Advisor: Susan E. Waltz, Ph.D.

Co-Advisor: Kathleen H. Goss, Ph.D.

Abstract

The Ron receptor tyrosine kinase is overexpressed in many human cancers

including colorectal and breast, and studies have established Ron as a predictor of

disease outcome and as a therapeutic target. Ron overexpression and constitutive

activation contributes to the tumorigenic properties of human colon cancer cells.

Moreover, metastatic dissemination of colon cancer cells from primary orthotopic tumors

in mice can be reduced upon Ron knockdown. The majority of hereditary and sporadic

colorectal cancers harbor aberrant Apc/!-catenin signaling, however, the relationship

between Ron, Apc, and !-catenin signaling in intestinal tumorigenesis is not well

understood. We sought to test the requirement of Ron tyrosine kinase signaling for

initiation of intestinal tumors in vivo using a well-characterized mouse model of mutant

Apc-driven intestinal tumorigenesis. By generating ApcMin/+ mice with a targeted

deletion of the tyrosine kinase domain of Ron (RonTK-/-), we found that Ron is not required for intestinal adenoma formation, and that Ron loss increases tumor burden in a large fraction of mice. Unexpectedly, the loss of Ron in non-transformed intestinal epithelium significantly increases crypt cell proliferation, which may lead to an increased susceptibility to tumor initiation in this model. !-catenin localization and target gene expression were not significantly altered in ApcMin/+;RonTK-/- mouse tumors or normal intestine compared to controls, suggesting that Ron is not required for !-catenin signaling in this model. Like in colon cancer, Ron overexpression has also been observed in approximately half of human breast cancers. Mammary-specific overexpression of Ron in mice results in mammary carcinomas in 100% of mice that metastasize to the lungs and liver, supporting the conclusion that Ron overexpression is

a causal oncogenic factor in breast cancer. Interestingly, mammary glands from virgin

mice with aberrant Ron expression have dilated mammary ducts and sparse ductal

branches. Based on these observations, and that molecules deregulated in breast

cancers often have important roles in development, we hypothesized that Ron is a novel

regulator of mammary gland morphogenesis. To study Ron in mammary development,

RonTK-/- mice were utilized. We found that Ron tyrosine kinase domain-deficient mice

had enhanced ductal morphogenesis during puberty, which was also evident when the

mice were ovariectomized. Increased ductal elongation and earlier regression of

terminal end buds was also observed in RonTK-/- mammary glands. Interestingly, accelerated pubertal mammary development was accompanied by increased phosphorylated MAPK, which was necessary for enhanced RonTK-/- epithelial branching morphogenesis in vitro. Together, these studies identified novel roles for Ron tyrosine

kinase in the regulation of normal intestinal tissue homeostasis and normal mammary

gland development. Interestingly, these studies identified important new roles for the

Ron receptor in normal tissues. These studies not only further our knowledge of Ron

receptor biology, but also provide meaningful insight into the potential consequences of

Ron loss that might result from a cancer therapy directed at this receptor.

iii

Chapter 2 contains published original research; citation:

Meyer, S.E. et al., The Ron receptor tyrosine kinase is not required for adenoma

formation in ApcMin/+ mice, Mol. Carcinog. 2009. doi:10.1002/mc.20551

PMID: 19452510

" 2009 Wiley-Liss, Inc.

Chapter 3 contains published original research; citation:

Meyer, S.E., et al., The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis, Dev. Biol. 2009; 333(1): 173-85. doi:10.1016/j.ydbio.2009.06.028

PMID: 19576199

" 2009 Elsevier Inc.

Acknowledgements

I would like to first acknowledge my graduate mentors Dr. Kathleen Goss and Dr.

Susan Waltz who were instrumental in my progress as a scientist. Their guidance and mentoring of experimental design, grant writing, manuscript writing, presentation skills, and helpful discussions have really developed, matured, and nurtured my scientific career. I also thank them for their continuous support of my goals and aspirations as a young researcher and for being excellent role models. I also thank Dr. Waltz for serving as Chair of my thesis committee.

I would like to thank my thesis committee Dr. Nelson Horseman, Dr. Erik

Knudsen, Dr. Christopher Wylie, and Dr. Sohaib Khan. This committee has helped me make critical decisions with regards to experimental design and data interpretation that have not only kept me on track to graduate, but was important for my development as a scientist. They have also generated many scientific questions and ideas with respect to my thesis that has helped my research evolve into a more comprehensive body of work.

I would also like to thank all of the past and present members of the Goss and

Waltz laboratories including Dr. Robert Holdcraft, Kimberly Becher, Tara Willson, Dr.

Bryon Boulton, Dr. Jennifer Prosperi-Sullivan, Dr. Glendon Zinser, William Stuart,

Megan Thobe, Dr. Rebecca McClaine, Purnima Wagh, Jerilyn Gray, Devikala

Gurusamy, and Dr. Nikolaos Nikolaidis. All of these people have not only been instrumental in my progression and development as a scientist, but also have become my friends.

I acknowledge the National Institutes of Health (NIH) for the funding award T32-

CA59268 to Dr. Sohaib Khan at the University of Cincinnati, and would like to thank the selection committee for granting me a position on this training grant.

I would like to give special thanks to the Legislative Ambassador volunteers of the American Cancer Society (ACS) in Ohio, particularly Grace Tompos a colon cancer survivor. They have all really been a large inspiration in my life and career.

Lastly, I would like to thank my friends and family for their never-ending support, encouragement, and understanding especially my parents William and Jane Maxfield, and my close friends Emily Stipanovich and Kristin Dick. I thank them for always being there to listen and remind me to have some fun. Finally, I would like to express my utmost thankfulness for my best friend and husband John Meyer whose unwavering love, support, encouragement, and patience have given me the confidence and drive to keep moving forward.

Preface

This thesis dissertation is manuscript-based consisting of two independent research hypotheses, which resulted in two first author publications. There are seven chapters herein:

1 – Introduction and literature review

2 – Manuscript one: preface, abstract, introduction, materials and methods, results, and discussion

3 – Manuscript two: preface, abstract, introduction, materials and methods, results, and discussion

4 – Discussion

5 – Bibliography

6 – Declaration of original research

vii

Table of Contents

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

Acknowledgements……………………………………………………………………….….....v

Preface…………………………………………………………………………..………………vii

Table of Contents……………………………………………………………………………...viii

List of Figures……………...………………………………………………………………..…..xi

List of Tables…………...………………………………………………………………………xiii

Chapter 1 – Literature Review

Introduction………………………………………………………..…………………………..1-2

Ron Receptor Tyrosine Kinase Structure and Expression……………………….………1-5

HGFL is the Ligand for the Ron Receptor Tyrosine Kinase……………………………...1-9

Ron Receptor Signaling…………………………………………………………………….1-10

Mouse Models of the Ron Receptor…………………...………………………………….1-13

Intestinal Architecture and Homeostasis………………………….…...... ….…………...1-16

From Intestinal Epithelial Homeostasis to Intestinal Tumorigenesis: The Importance of

APC/!-catenin Signaling……………………………………...... ……...... …..1-19

Human Colorectal Cancer and Mouse Models of Mutant Apc-Mediated Intestinal

Tumorigenesis……………………………………………………………………………….1-21

Ron Receptor Signaling in Colon Cancer…………………………………...... 1-24

Mammary Gland Development………………….…………………………………..……..1-27

Mammary Gland Branching Morphogenesis……………………………………………..1-31

viii

Ron Receptor Signaling in Breast Cancer………………………………………………..1-33

Abbreviations………………………………………………………….……………………..1-36

Chapter 2 – Meyer et al., Molecular Carcinogenesis 2009.

Preface……………………………………………………………………………….………..2-2

Abstract……………………………………………………………………………..………….2-3

Abbreviations…………………………………………………………………….……………2-4

Introduction……………………………………………………………………..……………..2-5

Results………………………………………………………………………..………………..2-7

Figures and Tables……………………………………………………………………...…..2-11

Discussion.………………………..….………...……………………….…………………...2-22

Materials and Methods……………………………………………………………………...2-27

Acknowledgements………………………………………………….………………………2-31

Chapter 3 – Meyer et al., Developmental Biology 2009.

Preface……………………………………………………………………….………………..3-2

Abstract……………………………………………………………………..……………...….3-3

Abbreviations………………………………………………………………………………….3-4

Introduction………………………………………………………………..…………………..3-5

Results…………………………………………………………………..……………………..3-9

Figures and Tables……………………..………………………………………...…………3-18

Discussion………………………...…….…………...…………………………….………...3-63

Materials and Methods……………………………………………………………………...3-71

Acknowledgements………………………………………………………….………………3-80

Chapter 4 – Overall Discussion

The Ron receptor regulates tissue morphogenesis ……………………………….……..4-2

Expression of the Ron tyrosine kinase domain influences crypt proliferation but is not

required for adenoma formation in Apc-mutant mice……………………………………..4-5

Future research questions derived from the study of Ron in ApcMin/+ intestinal tumorigenesis………………………………………………………………………………….4-6

Ron regulates pubertal mammary gland development………………………………….4-13

Future research questions derived from the study of Ron in pubertal mouse mammary gland development………………………………………………………………………….4-15

Clinical implications of Ron receptor loss in the intestine and breast………………….4-23

Summary……………………………………………………………………………………..4-24

Abbreviations………………………………………………………………………………...4-26

Chapter 5 – Bibliography

References..…………………….……………………………………………………………..5-2

Chapter 6 – Declaration of Original Research

Declaration of original research……………………………………………………………..6-2

List of Figures

Chapter 1 – Literature Review

Figure 1 – The human and mouse Ron gene and protein structures…………………...1-7

Figure 2 – General summary of Ron receptor signaling targets and molecular

crosstalk………………………………………………………………………………………1-11

Figure 3 – Structure, segments, and cell types of the intestine………………………..1-18

Figure 4 – A simplified illustration of the Canonical Wnt signaling pathway…...……..1-20

Figure 5 – Schematic of the developmental stages of the mammary gland..….……..1-28

Figure 6 – Histological appearance and morphology of mammary ducts and terminal end buds……………………………………………………………………………………...1-30

Chapter 2 – Meyer et al., Molecular Carcinogenesis, 2009

Figure 1 – Immunohistochemical analysis of Ron expression in ApcMin/+ small intestinal

and colon normal tissue and tumors………………………………………………………2-11

Figure 2 – Analysis of overall tumor burden and spatial distribution of tumors in ApcMin/+

mice with and without Ron………………………………………………………………….2-12

Figure 3 – Histological comparison of hematoxylin and eosin stained ApcMin/+ tumors

and surrounding normal tissue with and without Ron...... 2-14

Figure 4 – Immunohistochemical detection and quantification of PCNA-positive tumor

and normal crypt cells as a measure of proliferation…………………………………….2-16

Figure 5 – Immunohistochemical detection and quantification of cells undergoing cell

death in tumors and normal crypt cells……………………………………………………2-18

Min/+ Figure 6 – !-catenin expression in Apc tumors and surrounding normal tissue from

mice with and without Ron…………………………………………………………………2-20

Chapter 3 – Meyer et al., Developmental Biology, 2009

Figure 1 – The Ron receptor is expressed throughout post-natal mouse mammary gland development, predominantly in the epithelium.………………………………………..…3-18

Figure 2 – RonTK-/- mice have significantly accelerated pubertal mammary gland

development.……………………………………………………………………………..….3-21

Figure 3 – RonTK-/- mammary glands display significantly increased branching after

ovariectomization……………………………………………………………………………3-23

Figure 4 – Histological appearances and rates of proliferation and cell death of

RonTK+/+ and RonTK-/- mammary epithelium……………………………………….….3-25

Figure 5 – RonTK-/- mouse mammary glands contain significantly increased

phosphorylated Akt and MAPK…………………………………………………………….3-27

Figure 6 – RonTK-/- primary mammary epithelial organoids display significantly advanced branching in vitro that is reduced upon MAPK inhibition……………..…….3-28

Figure 7 – RonTK-/- mammary glands have significantly altered gene expression patterns compared to RonTK+/+ controls during development…………………...….…3-30

Chapter 4 – Discussion

Figure 1 – A working model of the role of Ron in the normal intestine………………….4-8

Figure 2 – A working model of the role of Ron in mammary gland development…….4-17

xii

List of Tables

Chapter 1 – Literature Review

Table 1 – In vivo models of Ron receptor tyrosine kinase signaling…………………..1-15

Table 2 – Mouse models of mutant Apc-mediated intestinal tumorigenesis……….…1-22

Table 3 – Summary of well-established regulators of pubertal mouse mammary morphogenesis………………………………………………………………………………1-32

Chapter 2 – Meyer et al., Molecular Carcinogenesis, 2009

Table 1 – Real-time PCR analysis of !-catenin target gene expression in ApcMin/+ mice small intestine and colon tissue with and without Ron…………………………………..2-21

Chapter 3 – Meyer et al., Developmental Biology, 2009

Table 1 – Validation of select microarray gene expression by real-time PCR………..3-32

Supplementary Table S1 – 393 genes differentially expressed in RonTK-/- mammary

glands compared to wild-type over time…………………………………………….…….3-33

Supplementary Table S2 – Genes differentially expressed in RonTK-/- mammary glands

that are uniquely different from wild-type at 5 weeks of age……………………………3-44

Supplementary Table S3 – Genes differentially expressed in RonTK-/- mammary glands

that are uniquely different from wild-type at 6 weeks of age……………………………3-46

Supplementary Table S4 – Genes differentially expressed in RonTK-/- mammary glands

that are uniquely different from wild-type at 7 weeks of age……………………………3-50

Supplementary Table S5 – Genes differentially expressed in RonTK-/- mammary glands

compared to wild-type at all time points…………………………………………………..3-52

xiii

Supplementary Table S6 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the developmental process…………………………………...3-53

Supplementary Table S7 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the process of transcription…………………………………...3-55

Supplementary Table S8 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the process of morphogenesis……………………………….3-57

Supplementary Table S9 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the cell differentiation process………………………………..3-59

Supplementary Table S10 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the process of kinase activity………………………...3-60

Supplementary Table S11 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the process of cell motility……………………………3-61

Supplementary Table S12 – Genes differentially expressed in RonTK-/- mammary glands compared to wild-type in the process of cell adhesion…………………………3-62

Chapter 4 – Discussion

Table 1 – Molecules important for intestinal homeostasis and tumorigenesis…..……..4-3

Table 2 – Molecules important for mammary gland development and tumorigenesis…..……………………………………………………………………………..4-4

xiv

Chapter 1

Literature Review

Introduction

Since the year 2000 when the American Cancer Society began reporting surveillance data, colorectal cancer in both men and women, and breast cancer, in women, have consistently been some of the most frequently diagnosed types of cancer in the United States, and also account for the greatest number of cancer-related deaths, with the exception of lung cancer (ACS, 2000; ACS, 2009). Since the discovery of the

Ron receptor tyrosine kinase in 1993, numerous studies have shown that Ron is overexpressed in a variety of human cancers including breast, prostate, pancreas, kidney, colon, lung, ovary, bladder, liver, thyroid, uterus, brain, and stomach [1-7].

Specifically, approximately half of human colon and breast cancers overexpress Ron [8-

11]. Over the last two decades, the development of targeted molecular therapies directed against receptor tyrosine kinases have transitioned to routine clinical use for the treatment of cancer [12,13]. Recent progress has been made toward establishing the Ron receptor as a valid therapeutic target in a variety of cancers; however, not much is known about Ron function in early-stage tumorigenesis or in the development and homeostasis of normal tissues [1,14,15]. Receptor tyrosine kinases often play disparate and complex roles in tumorigenesis and development in a cell-type and context specific manner [16]. Therefore, it is important to study Ron receptor function in tumorigenesis and in normal tissue to better predict and understand the consequences of Ron expression or inhibition in cancer. Thus, this thesis is focused on addressing the role of the Ron receptor in breast tissue development and homeostasis and early tumorigenesis of the intestine. I have generated two independent hypotheses to further the understanding of Ron receptor biology both in cancer and in normal tissue. Both

1-2

hypotheses presented in this thesis were tested using a previously generated model in

which mice harboring a germline deletion of the tyrosine kinase domain (TK) of Ron

(RonTK-/-) are compared to mice expressing endogenous Ron (RonTK+/+) [17].

Project 1: Role of the Ron receptor tyrosine kinase in ApcMin/+ mouse intestinal tumorigenesis. Studies have demonstrated that Ron overexpression and tyrosine phosphorylation occurs in approximately half of human colon cancers [9,10]. The majority of sporadic colon cancers, colon cancer cell lines, and the hereditary form of colon cancer familial adenomatous polyposis (FAP) acquire loss of function of the tumor suppressor adenomatous polyposis coli (APC) or aberrant activation of the transcriptional co-activator !-catenin [18-20]. It was demonstrated that Ron

overexpression contributes to the tumorigenic properties proliferation, anchorage-

independent cell growth, and survival of the colon cancer cell lines HT-29, HCT116, and

SW620, which also have aberrant APC/!-catenin signaling [8,11,21]. It is well

documented that deregulation of !-catenin activity occurs in the majority of human

colorectal cancers through several mechanisms including inactivating APC mutations,

stabilizing !-catenin mutations, and stabilizing !-catenin modifications like tyrosine

phosphorylation [22,23]. It has been shown in cell lines in vitro that Ron may modulate

!-catenin stability [24-26]. This is one potential mechanism through which Ron might

regulate colon tumorigenesis, either coordinately with mutant APC, or independent of

APC status. In addition, the requirement of Ron expression for intestinal tumor

formation, and effect of Ron loss on the normal intestinal epithelium in vivo were not

known. To test the hypothesis that the Ron receptor tyrosine kinase is important for

1-3

mutant Apc-mediated intestinal tumorigenesis, I utilized mice that are heterozygous

for a mutation in Apc (ApcMin/+), and upon loss of the wild-type allele, develop intestinal adenomas with 100% incidence [20,27]. These mice were crossed with RonTK-/- mice,

which do not develop spontaneous intestinal tumors, to generate ApcMin/+;RonTK+/+,

ApcMin/+;RonTK+/-, and ApcMin/+;RonTK-/- mice. These mice were analyzed and compared for tumor number, location, size, and histological appearance. Mice were also examined for !-catenin localization, target gene expression, PCNA and Ki67 proliferative indexes, and cell death [28]. The results of this study are described in

Chapter 2.

Project 2: Role of the Ron receptor tyrosine kinase in mammary gland growth

and morphogenesis. Immunohistochemical and protein expression analyses from our laboratory and others have demonstrated that Ron is overexpressed in approximately

50% of breast cancers [1,29]. In addition, our laboratory has shown that mammary-

specific overexpression of Ron in mice is sufficient to induce mammary tumor formation

with 100% incidence and metastasis in about 90% of mice [30]. Many of the same

molecules that are deregulated in breast cancers are also important regulators of

mammary gland development [31]. Previously, it was shown by Northern analysis that

Ron is expressed postnatally with increasing expression during pubertal mammary gland development in mice (5-10 weeks of age), and likewise, Ron expression was observed in normal human breast tissue by Western analysis [29,32]. Interestingly,

overexpression of Ron in adult virgin mouse mammary glands results in altered

appearance even prior to tumor formation with dilated mammary ducts and fewer ductal

1-4

side branches than wild-type controls [30]. Further, the Met receptor, a related family

member of Ron, has been shown to regulate mammary gland development [33]. Ron

has also plays a role in liver and kidney morphogenesis [34,35]. Together, these data

lead to the hypothesis that the Ron receptor tyrosine kinase regulates mammary

gland growth and morphogenesis. To test this hypothesis, wild-type (RonTK+/+) and

RonTK-/- mice containing a germline deletion of the tyrosine kinase domain of Ron were

used. RNA and protein assays illustrated the spatial and temporal expression pattern of

Ron in mouse mammary glands. Mammary gland development was assessed in virgin

female mice ages 5-15 weeks by whole mount analyses. Ovarian hormone signaling,

mammary epithelial cell proliferation, cell death, ex vivo branching morphogenesis, Akt

and Mapk activation, and gene expression profiles were also examined to help elucidate

the mechanisms and intracellular signaling that contributes to Ron regulation of

mammary development [36]. These results are shown in Chapter 3.

Ron Receptor Tyrosine Kinase Structure and Expression

In 1993, a novel protein named RON (for Recepteur d’Origine Nantaise) was first

identified when screening a cDNA library from human tumors using probes homologous

to known protein tyrosine kinase sequences [2]. Subsequently, a human foreskin

keratinocyte cDNA library was screened to obtain the full-length 4.5 kb Ron cDNA [2].

Figure 1A shows the alignment of the human and mouse Ron genes, which share

73.6% identity [37,38]. To date there are 45 putative Ron orthologs, of which several

have been confirmed including human [2], monkey [2], mouse [39], rat [40], chicken

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[41], fugu fish [42], zebrafish [43], and xenopus [44]. This evidence shows that Ron is highly conserved throughout evolution and suggests that it may be an essential gene.

The Ron protein is synthesized as a glycosylated single chain polypeptide precursor (Figure 1B and C - upper) that is proteolytically cleaved to generate #- and !- chains [2,45-47]. The linkage of these two chains by disulfide bonds yields a functional

#! heterodimeric protein (Figure 1B and C- lower). Together, Ron’s heterodimeric structure, neighboring tyrosine residues in the kinase domain, and multifunctional docking site with the capacity to bind SH2 domain containing proteins classified Ron as a receptor tyrosine kinase [2,45]. The amino acid sequence in the intracellular region of the Ron receptor is 63% identical to the Met receptor exemplifying the greatest degree of homology between Ron and any other receptor tyrosine kinase. Therefore, Ron is classified as a member of the Met receptor tyrosine kinase family [2]. Ron and Met also have similar c-terminal multifunctional docking site regions with two tyrosine residues, a feature that is unique to this receptor family [2].

1-6

1-7

Figure 1. The human and mouse RON gene and protein structures. A) The human

RON gene is 16.9 kb in length, encodes 20 exons, and is located on chromosome 3.

The mouse Ron gene is 13.3 kb in length, encodes 19 exons, and is located on chromosome 9. The major difference between the human and mouse Ron genes is that exon 13 of the human RON gene that encodes a portion of the juxtamembrane region

(highlighted in pink) is absent from the mouse Ron gene [38,48]. B and C) Schematic representation of the locations of conserved domains, disulfide bonds after proteolytic cleavage, and key tyrosine residues in the kinase domain and c-terminal multifunctional

docking site within the human (B) and mouse (C) Ron receptor proteins [37,45]. The

color-coded regions in A, B, and C correspond to the domains listed in the legend.

In humans, Ron is expressed in normal esophagus, small intestine, colon, skin,

lung, hepatocytes, Kupffer cells, monocytes, granulocytes, splenic macrophages, and

bone marrow tissues [5,21,45]. Ron is overexpressed in a significant fraction of human

cancers including breast, prostate, pancreas, kidney, colon, lung, ovary, bladder, liver,

thyroid, uterus, brain, and stomach [1-7]. Studies have also demonstrated Ron

expression in human epithelial cell lines derived from normal breast, gastric, pancreatic,

colorectal, hepatocellular, and mammary carcinomas, hematopoietic cells, and

megakaryocytes [5,8,21,45,49]. In mouse embryos, Ron expression was detected as

early as day E12.5 in developing neural structures (postoptic area, diencephalic vesicle)

and in the liver. At E16.5, Ron expression was detected in the spinal and cranial nerve

ganglia, ossification centers, adrenal gland, skin, and lung. Ron expression was also

found in the developing mouse gut epithelium as early as E16.5, with continued

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expression observed at E18.5, at birth, and in the epithelium of adult small intestine and

colon [46,50]. In adult mice, Ron expression has been observed in peritoneal

macrophages, brain, adrenal glands, gastrointestinal tract (including stomach, small

intestine, and colon), testis, kidney, placenta, uterus, epididymis, and mammary glands

[32,38,46,47,51].

HGFL is the Ligand for the Ron Receptor Tyrosine Kinase

The Hepatocyte Growth Factor-like Protein (HGFL), also known as Macrophage-

Stimulating Protein (MSP), is the ligand for the Ron receptor tyrosine kinase [39,45,52].

MSP was crudely separated from human serum in 1978 as the molecule in serum that induced migration, spreading, and phagocytosis of purified primary mouse peritoneal macrophages, and its stability suggested it was a protein [52-55]. In 1991, another group independently discovered by sequence analysis a gene that was 50% identical to

Hepatocyte Growth Factor (HGF), the ligand for the Met receptor, so they named it

Hepatocyte Growth Factor-like protein (HGFL) [56]. It was realized two years later that

MSP and HGFL are the same protein, and it will be referred to as HGFL in the remainder of this thesis [57,58].

HGFL is in a large family of kringle domain-containing serine-proteases of plasminogen-related growth factors, which includes hepatocyte growth factor (HGF), the ligand for Met; however, neither protein has protease activity [56,59-63]. HGFL is synthesized as a single chain polypeptide precursor, pro-HGFL, primarily by the liver and secreted into the blood circulation. There is evidence that the kidney, pancreas, and lung also produce HGFL [60,64,65]. Pro-HGFL is cleaved extracellularly by

1-9

proteases into a disulfide-linked 50 kDa # and 30 kDa ! heterodimer [63,66-68]. Pro-

HGFL has been shown to be inactive and it does not bind to Ron; however, cleaved

HGFL binds to Ron via the ! chain of HGFL [69,70].

Studies have shown that HGFL, and not HGF, stimulates tyrosine

phosphorylation of Ron, and that only the Ron ! chain was tyrosine phosphorylated

upon HGFL treatment [45]. Conversely, HGF, but not HGFL, stimulated tyrosine

phosphorylation of Met [45]. Despite the fact that HGF and HGFL share 45% sequence

homology, the extracellular regions of Ron and Met are only 25% homologous, which

may account for the specificity of HGFL for Ron and HGF for Met [2,45,55,65].

Exogenous expression of the extracellular sema domain of Ron was sufficient to block

ligand binding and reduce kinase activity of the full-length Ron receptor, thus this

domain of Ron may contain the ligand-binding region [71,72]. These studies, however, are only correlative and fail to show the exact mechanism and location of interaction between Ron and its ligand HGFL.

Ron Receptor Signaling

Numerous studies have investigated the effects of Ron signaling using a variety

of cell types with results that are cell type- and context-dependent, and are summarized

in Figure 2 [7,73-78]. The complexities of Ron signaling may be achieved in a variety of

ways including: 1) the use of the multifunctional docking site and tyrosine kinase

domain, 2) crosstalk with other cell surface molecules and intracellular signaling

pathways, and 3) ligand-dependent and ligand-independent mechanisms of receptor

activation. Examples of such types of signaling mechanisms are reviewed below.

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Figure 2. General summary of Ron receptor signaling targets and molecular crosstalk.

The illustration is collective representation of Ron signaling reported in macrophages, epithelial cells, and tumor cells.

One of the mechanisms that accounts for such signaling diversity displayed by

Ron is the multifunctional docking site in the C-terminus [79,80]. Upon HGFL activation,

Ron can associate with the SH2 domains in such proteins as the p85 subunit of PI3K,

1-11

Shc, and Grb2 via the tyrosines 1330 and 1337 found in Ron’s multifunctional docking

site [73,79,81]. The activation of these SH2 domain-containing proteins by Ron then

leads to other intracellular signaling events, including regulation of iNOS expression,

NO production, Akt activity, FAK activation, Mapk activity, NF$B inhibition, Ras

activation, and IL-6 production [78,82-84]. In addition to the tyrosine residues in the

Ron multifunctional docking site, phosphorylated serine 1394, in this region has also been shown to play a role in Ron-mediated activation of intracellular signaling pathways including Akt, protein phosphatase 1 (PP1), and 14-3-3 scaffolding proteins [85].

Another mechanism for Ron activation and its diverse intracellular signaling is through interaction with other cell surface molecules. Interestingly, interactions between

Ron and other cell surface receptors including Met, EGFR, integrins, and B type Plexins occur using both the tyrosine kinase domain and multifunctional docking site [86-89]. In addition to transactivation of the Ron kinase domain by these receptors in the absence of HGFL, HGFL-mediated Ron activity can transactivate Met and EGFR in the absence of their respective ligands [87,88]. Plexin B1 can also induce Ron activation in the absence of HGFL to confer invasive growth properties [89]. In addition, a study recently showed that in MCF10A human mammary epithelial cells Ron-mediated proliferation and migration was HGFL-dependent, but Ron expression promotes cell survival, increases cell spreading, and further enhances cell migration independently of HGFL

[49]. Together, these findings illustrate the complexities of Ron receptor signaling that occurs through differential usage of the tyrosine kinase and C-terminal domains, and that Ron activation can be achieved in the presence and absence of HGFL.

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Mouse Models of the Ron Receptor

The use of gene knock-out and gene overexpression studies in mice have greatly

aided in our understanding of the physiological roles of the Ron receptor during

embryogenesis, tissue development, injury, inflammation, and tumorigenesis. The

following is a brief summary of some of the more innovative mouse models. In addition,

a full list of Ron mouse models can be found in Table 1.

In mice, Ron is also known as stem cell-derived kinase (Stk). Therefore, in order

to remain consistent with the published details of the Ron animal models both names,

Ron and Stk, will be used in this section. The first mouse model generated to test the

biological significance of the Ron receptor in mice was the Stk-/- mouse model wherein the !-galactosidase gene was knocked-in to the first exon of mouse Ron to disrupt gene expression [90]. These mice are viable. In contrast, however, another group reported that a larger deletion from 300 bases 5’ to the transcription start site through exon 15 of

Ron in mice (Ron-/-) was embryonic lethal by day E7.5 due to the inability of Ron-/-

trophectoderm to properly implant in the uterus [51,91]. Macrophages from Stk-/- and

Ron+/- mouse models showed reduced activation and survival upon lipopolysaccharide

(LPS) challenge indicating that Ron is important for macrophage activity [90,91]. The

apparent discrepancy in the viability of these two alleles may be due to internal

transcription initiation in the remainder of the Ron gene in the Stk-/- model that only removed exon 1, or the possibility of disruption of additional genes around the Ron locus based on the large deletion in Ron-/- model. Although not listed in Table 1, HGFL-

/- mice are viable, fertile, and have no overt phenotypic abnormalities except

development of lipid containing vacuoles in hepatocytes [92].

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Since the germline knockout of the large region of Ron lead to embryonic lethality, another model was developed to study the physiological role of the Ron tyrosine kinase domain (TK; exons 13-18) in tissue development, injury, inflammation, and tumorigenesis. Cre-mediated excision of exons 12-18 of mouse Ron results in a

premature stop codon just three codons in to exon 19 [17]. This germline deletion of the

RonTK domain leads to viable mice, and the expression of a truncated Ron ! chain of

approximately 105 kDa in RonTK-/- primary mouse macrophages [17]. In general, the

histological appearance of most tissues from RonTK-/- mice appeared overtly normal compared to RonTK+/+ mice [17]. The ovaries, however, were smaller, had fewer

corpora lutea (CL), and increased inducible nitric oxide synthase (iNOS) and nitric oxide

(NO), but RonTK-/- mice are fertile and deliver normal sized litters [17,93]. In agreement with the Stk-/- and Ron+/- models, RonTK-/- macrophages exhibited reduced activation, decreased survival, and were unable to block NO production when challenged with LPS and INF% [17]. RonTK-/- mice also displayed exaggerated edema and inflammation of

the skin compared to RonTK+/+ mice when challenged with an irritant or allergen [17].

As illustrated by Table 1, RonTK-/- mice have been a valuable model for studying Ron signaling in tissue development, injury, inflammation, and tumorigenesis. Importantly, the RonTK-/- mice were utilized in this thesis project to test the hypothesis that Ron

tyrosine kinase signaling is important for Apc-mutant intestinal tissue homeostasis and

tumorigenesis (Chapter 2), and in mammary gland development (Chapter 3) in mice.

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Table 1. In vivo models of Ron receptor tyrosine kinase signaling.

Model Name Model Details Phenotype Reference Ron loss: Stk-/- Germline deletion of mouse Increased macrophage NO [90] Ron exon 1; viable production, increased inflammation in vivo upon IFN%/LPS treatment

Ron-/- Germline deletion of mouse +/- macrophages-increased NO [91] Ron promoter-exon 10; +/- mice-earlier death by LPS -/- lethal E6.5-E7.5 +/- viable

RonTK-/- Germline deletion of mouse Decreased ovary size and CLs [17] Ron TK domain; viable

LPS challenge Increased macrophage NO, decreased mouse survival

Irritant/allergy challenge Increased inflammation, delayed recovery

Listeria Listeria monocytogenes Increased bacterial burden and [94] Stk-/- exposure decreased survival

LPS/GaLN LPS/GaLN-induced Less liver damage, decreased [95] RonTK-/- liver injury liver apoptosis and necrosis, early increased TNF#

Ni Nickel-induced acute Increased inflammatory [96] RonTK-/- lung injury cytokine production, increased NO, and decreased survival

EAE EAE induced early Increased susceptibility to MS [97] RonTK-/- multiple sclerosis

PyMT RonTK-/- MMTV-PyMT induced Delayed tumor formation, [98] mammary tumorigenesis decreased microvessel density, increased apoptosis, decreased tumor proliferation

TPA Ras o/e TPA-induced skin Decreased skin papilloma size [99] RonTK-/- carcinogenesis model and number, decreased proliferation, decreased malignant conversion

Intranasal LPS LPS-induced lung Increased TNF#, NO [100] -/- RonTK inflammation and injury production, NF$B activation More severe injury

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Cecal Cecal ligation/puncture- Decreased survival, increased [101] ligation/puncture induced bacterial bacterial infection, liver RonTK-/- peritonitis and sepsis necrosis, reduced neutrophil migration Decreased macrophage production of cytokines/chemokines

siRON HCT116 Cecal injection orthotopic Reduced metastasis [102] colon cancer; siRNA- mediated knockdown of human RON in HCT116 cells

Ron overexpression: D1232V NIH3T3 xenograft, tail Tumor formation and lung [103] M1254T vein injection (human RON) metastases

G1209H NIH3T3 xenograft, tail Tumor formation; [104] M1231T vein injection (human RON) Mutants formed lung L1176V metastases RON (o/e)

RON (o/e) Tg. Surfactant protein C Lung adenocarcinomas [105] promoter driven human RON overexpression

MMTV-Ron Tg. Mammary specific 100% mammary [30] Mouse Ron overexpression adenocarcinoma formation, ~ 90% lung and liver metastases

FL-Ron Tg. expression of full-length Increased INF%, more severe [106] mouse Ron (resistant to liver damage, decreased mRNA splicing); survival Concanavalin A-induced acute liver injury

Stk, stem cell-derived kinase refers to mouse Ron; IFN%, interferon gamma; NIH3T3, fibroblast cells; NO, nitric oxide; LPS lipopolysaccharide; o/e, overexpressed; TK, tyrosine kinase domain; Tg., transgenic; CL, corpora lutea; GaLN, galactosamine; TNF, tumor necrosis factor; Ni, nickel; EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MMTV, mouse mammary tumor virus; PyMT, polyoma middle T antigen; TPA, 12-O-tetradecanoylphorbol-13-acetate; NF-$B, nuclear factor $B; FL, full-length; siRON, siRNA-mediated human RON knockdown.

Intestinal Architecture and Homeostasis

The gastrointestinal tract serves multiple purposes including food digestion, nutrient absorption, and serves as a structural barrier from pathogens. The intestines

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can be subdivided into several segments, including the duodenum, jejunum, ileum, and

proximal and distal colon based on the structure (Figure 3A and 3B) and function of

each segment. The major histological differences between the small intestine and colon

are that the small intestine consists of crypts and villi composed mostly of enterocytes,

whereas the colon contains only crypts composed mostly of goblet cells (Figure 3A and

3B). As depicted in Figure 3C, there are four major types of terminally differentiated

epithelial cells in the intestine: enterocytes (absorptive), goblet cells (mucus-producing),

Paneth cells (lysozyme-producing), and endocrine cells (hormone-producing). All the cell types of the small intestine and colon both differentiated and progenitor cells can be generated from the clonal population of multi-potent stem cells located at the base of

intestinal crypts [107,108]. Notice in Figure 3C that the terminally differentiated cells migrate out of the stem cell niche and up the crypt/villus axis toward the intestinal lumen, except Paneth cells which remain in the crypts. Over time, as new cells are produced, the older cells slough off into the lumen keeping the crypt/villus height constant. This continuous regenerative process is subject to regulatory mechanisms in order to maintain a balance, or homeostasis, of many factors including epithelial cell

number, type, distribution along the crypt/villus axis, and organization into crypt/villus

structures. All the epithelial cells of the crypt/villus can be replaced with new epithelial

cells in approximately 3 days in mice, and in a manner where new cell production is

equaled by cell loss so that overall cell numbers are static within any given normal

crypt/villus structure [109-111].

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Figure 3. Structure, segments, and cell types of the small intestine and colon. A)

Hematoxylin and Eosin staining of paraffin-embedded tissue harvested from each segment of the mouse small and large intestine. Black bars denote villus height, red

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ovals encompass crypts, and black arrows point to representative goblet cells. Note the

entire axis of the colon is a crypt. Scale bar; 100µm. B) Villus height decreases from proximal duodenum to distal ileum, and the colon is comprised entirely of crypts. The colonic crypts contain the greatest number of goblet cells, which is also visible in the colon H&E panel in A. C) Schematic illustration of an intestinal villus and crypt. The location and types of epithelial cells found throughout the entire intestine are indicated.

The directional migration of differentiated epithelial cells, produced by the crypt stem cells, toward the villus tip is also indicated. While C represents a schematic of the small intestine, a similar scenario exists in the colon, with the exceptions noted in B.

From Intestinal Epithelial Homeostasis to Intestinal Tumorigenesis: The

Importance of APC/!-catenin Signaling

Some of the major signaling pathways that regulate intestinal homeostasis

include Wnt, Notch, BMP, and PI3K [107]. Specifically, the canonical Wnt signaling

pathway (Figure 4) is essential for the maintenance of the stem cell niche and intestinal

homeostasis [107]. The adenomatous polyposis coli (APC) protein functions as part of

the destruction complex, which also contains Axin and GSK3!, targets !-catenin for

proteasomal degradation [112,113]. APC acts in this destruction complex in normal

epithelial cells in the absence of Wnt ligand to keep the Wnt pathway off. In the

presence of Wnts or mutational inactivation of APC, !-catenin is freed from the

destruction complex, allowed to translocate to the nucleus where it binds to the Tcf/Lef

family of transcription factors to mediate transcription of such target genes as cyclin-D1

and c-myc [114,115]. !-catenin is also required to localize E-cadherin to the plasma

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membrane and tether it to the actin cytoskeleton to form epithelial cell adherens

junctions.

Figure 4. A simplified illustration of the Canonical Wnt signaling pathway. In the

absence of Wnt ligand (left), !-catenin is targeted for degradation by the destruction

complex consisting of APC, axin, and GSK3!. In the presence of Wnt ligand (right), !-

catenin is free to accumulate in the cytoplasm and translocate into the nucleus where it

activates transcription of Tcf/Lef target genes. !-catenin links membrane bound E- cadherin to the actin cytoskeleton (middle).

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APC is expressed in the small intestine and colonic epithelium in adult mice and

humans in an increasing gradient pattern from low expression in the crypts to high

expression at the crypt/villus junction and in the villi [116]. This gradient of APC

expression correlates with !-catenin localization. In the undifferentiated crypt cells !- catenin is localized to the cytoplasm and nucleus, however in the villus, !-catenin is localized to the cell-cell junctions [117,118]. Indeed, as the expression pattern and localization of APC and !-catenin suggest, APC/!-catenin signaling is crucial to the maintenance of the stem cell compartment and intestinal homeostasis [116,119-121].

Upon deletion of either of the transcriptional effectors of the Wnt pathway !-catenin or

Tcf, there is a loss of the proliferative crypt compartment, differentiation of stem cells, increased villus cell apoptosis, and detachment of enterocytes leading to death of the mice [122-124]. Conversely, mutational inactivation of the tumor suppressor APC results in nuclear accumulation of !-catenin, aberrant expression of !-catenin/Tcf target genes, expansion of the proliferative crypt compartment, increased crypt fission, and ultimately intestinal tumorigenesis [117,125,126].

Human Colorectal Cancer and Mouse Models of Mutant Apc-Mediated

Intestinal Tumorigenesis

Nearly 80% of all sporadic colorectal tumors exhibit inactivating mutations in

APC, or stabilizing mutations in !-catenin that prevent its binding with the destruction

complex [22,127]. Patients with the autosomal dominant inherited syndrome familial

adenomatous polyposis (FAP) develop hundreds to thousands of colon adenomas at a

young age (<30 years) that progress to adenocarcinomas without surgical resection

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[128,129]. FAP patients also develop microadenomas in the duodenum [130]. The underlying genetic alterations for this condition are mutations within the mutation cluster region (MCR) of the APC gene on chromosome 5, and result in a truncated protein

[19,131-133]. There are numerous mouse models of Apc-mediated intestinal tumorigenesis (Table 2) that have afforded researchers the ability to determine the requirement of other signaling molecules and modifiers of intestinal tumorigenesis, and have provided useful models for testing cancer therapeutics [134].

Table 2. Mouse models of mutant Apc-mediated intestinal tumorigenesis. Tumor Tumor Intestine Model Mutation Reference Burden Histopathology Histopathology ApcMin/+ Truncated at ~30 - >100 Adenoma Cystic crypts, no [135] codon 850 colonic ACF Apc"716/+ Truncated at ~300 Adenoma No colonic ACF [136] codon 716

Apc1638N/+ Truncated at <10 Differentiated Colonic ACF [137-139] codon 1638 (no adenocarcinoma; protein expression Desmoid tumors detected)

Apc1638T Truncated at codon - No intestinal - [140] 1638 tumorigenesis, nipple cysts Apc1309/+ Frameshift at ~35 Intestine and colon [141] codon 1309 polyps

Apc"14/+ Frameshift at ~65 Adenomas and Colon ACF [142] codon 580 invasive carcinomas

Apc580D Inducible frameshift ~6 Adenomas [143] at codon 580

Apc"580 K14-Cre frameshift ~120 Adenomas and Colon ACF [144] at 580 and invasive carcinomas truncation at codon 605

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Apc"474/+ Duplicated exons 7, >100 Sessile polyps Intestinal gland [145] 8, 9, and 10; hyperproliferation frameshift at codon 474 ApcneoR Hypomorphic <1 [146] ApcneoF alleles decreased Apc by 80-90% Apc1572T/+ Truncated at codon - Multifocal mammary - [147] 1572 adenocarcinomas, no intestinal tumorigenesis

Apc1322T >Min Dysplastic [148] adenomas Adapted from McCart et al., 2008 [134]. Apc, adenomatous polyposis coli; Min, multiple intestinal neoplasias; ACF, aberrant crypt foci; K14, keratin 14 promoter; Cre, Cre recombinase ; neoR/F, neomycin gene reverse/forward.

The most well characterized and utilized model of intestinal tumorigenesis is the

ApcMin/+ mouse. ApcMin/+ mice were established through random ENU-induced

mutagenesis of C57BL/6 mice that resulted in multiple intestinal neoplasias (Min) [135].

This phenotype was a consequence of a thymine (T) to alanine (A) transversion in the

Apc gene at nucleotide 2549 generating a nonsense mutation that results in truncation

of Apc [149]. ApcMin/Min mice are not viable, as breeding of ApcMin/+ mice heterozygous for mutant Apc results only in ApcMin/+ and Apc+/+ mice [135]. ApcMin/+ mice develop aberrant crypt foci (ACF), that more resemble dysplastic crypts and are denoted

ACF(Min) that progress to adenomas upon LOH for the wild-type Apc allele in both the small intestine and colon [20,150-152]. All ApcMin/+ mice become anemic with grossly

visible adenomas by 2 months of age [135]. ApcMin/+ mice die of intestinal tumor burden

as early as 3 months of age on the C57BL/6 background with reports of tumor numbers

between 30 to greater than 100 adenomas per mouse [135]. Together, this evidence

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demonstrates that ApcMin/+ mice closely mimic the human disease; however, the

adenomas in ApcMin/+ mice do not progress to a malignant state and most tumors develop in the small intestine rather than the colon. It is believed that the lack of tumor progression in ApcMin/+ mice is due, at least in part, to their short lifespan [153].

Therefore, it is traditionally accepted that additional chemical or genetic insults are necessary to drive intestinal tumor progression in ApcMin/+ mice. The ApcMin/+ genotype

has been introduced into many other genetic models that have resulted in decreased

adenoma formation and/or size (EGFRWa2, MMP7-/-, MMP9-/-), increased adenoma

formation/size (villin-VEGF, Atm-/-), or induced progression (Smad3-/-, Pten+/-) [154-160].

Ron Receptor Signaling in Colon Cancer

Ron was found to be overexpressed by immunohistochemical analyses in 51%

(23/45) of colon cancers [6]. Interestingly, Ron expression by RT-PCR and

immunohistochemical staining in the colorectal mucosal crypts of ulcerative colitis

patients was increased compared to normal mucosa, suggesting that Ron may also play

roles in other intestinal disorders, which are linked to tumorigenesis [161]. These

studies highlight the potential prognostic significance of Ron expression levels as well

as suggest a therapeutic benefit to blocking Ron receptor expression and function in

cancer.

Genetic mutations and protein variants of the Ron receptor have been identified

in human cancers including lung and colorectal; however, the penetrance of these

mutations and variants within the human population is either unknown or extremely rare.

In addition, in vitro studies showed that not all of these alterations are advantageous for

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tumor growth and survival such that their relevance in human disease is poorly

understood [162,163]. The Ron variant Ron&165 was identified in a human gastric

cancer cell line and in primary human normal and malignant colon tissues; yet this

variant was non-transforming in vitro [9]. Another variant, RonDelta160 (Ron&160), is produced by alternative exon splicing of 109 amino acids resulting in an in-frame deletion of two exons in the extracellular region of the Ron ! chain mRNA, and was found in HT-29 colon cancer cells in addition to expression of the full-length Ron receptor [8]. The protein produced by this splicing event is 160 kDa with a normal sized

# chain, but a shortened 125 kDa !-chain [8,164]. Exogenous expression of Ron&160 in SV/40-transformed colon epithelial cells and MDCK cells that have low endogenous

Ron expression, as well as T47D breast cancer cells that have high endogenous Ron

expression, leads to increased cell motility and invasion in vitro [75,164]. Furthermore,

introduction of either Ron&160, or another Ron variant Ron&155, also a product of

splicing (removal of exons 5, 6, and 11) in the extracellular !-chain, in NIH3T3 cells

resulted in tumor formation in nude mice [163]. No mutations in the tyrosine kinase

domain of Ron have been identified to date in human cancers although, experimental

mutation analyses showed that the tyrosine residues in the kinase domain and docking

site of Ron are important for the tumorigenic properties of overexpressed Ron [163].

Another Ron variant, Ron&170, originally identified in a lung tumor cell line, lacks part of

the tyrosine kinase domain and subsequent C-terminus due to a stop codon generated

by alternative splicing of exon 18, and yields a Ron variant that acts as a dominant-

negative to wild-type Ron [165]. The prevalence of this variant has never been clearly

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reported and remains unstudied in most cancers. Lastly, another Ron variant, short

form Ron (sfRon), which lacks the # chain and most of the extracellular domain of the !

chain of Ron, but retains the transmembrane, tyrosine kinase domain, and C-terminal multifunctional docking site of Ron, was identified in human lung, ovary, and intestinal tissue and in human cancers [166]. sfRon confers an invasive phenotype to epithelial cells in vitro including loss of E-cadherin, increased growth, increased motility, and

anchorage-independent growth [166].

In adult human digestive system specifically, Ron expression in normal tissues

by immunohistochemical staining was observed in epithelia of the esophagus, small

intestine, colon, hepatocytes, Kupffer cells, and splenic macrophages [6,21]. Ron

expression has also been observed in mouse small intestine and colon [46,47]. Given

the wide distribution of Ron expression throughout out the GI tract, whether Ron may be

important in proper tissue development, homeostasis, and in the events during

tumorigenesis in these tissues remains an important question. Ron receptor

overexpression was found in several human colon cancer cell lines including Colo201,

DLD-1, HCT116, HT-29, SW620, and SW837 [8]. In addition to receptor

overexpression, constitutive receptor tyrosine phosphorylation and kinase activity could

not be further induced upon HGFL treatment in Colo201, SW837, and HCT116 cells [8].

In colon epithelial cells, exogenous Ron expression increased cell migration in response

to HGFL stimulation. In the same study, Ron expression also enhanced anchorage-

independent cell growth in soft agar in the presence of serum containing media that was

dependent upon the ability of Ron to activate MAPK, but not Akt signaling [24]. siRNA-

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mediated knockdown of Ron expression in the human colon adenocarcinoma cell lines

HT-29, HCT116, and SW620 resulted in decreased proliferation, increased cell death, reduced HGFL-induced cell migration, and decreased colon formation in soft agar [24].

Stable knockdown of Ron expression in SW620 cells increased tumor latency and decreased tumor size in xenografts compared to the parental cell line [24]. Additionally, knockdown of Ron in HCT116 cells reduced metastasis compared to parental cells in an orthotopic model of intestinal tumorigenesis in immune compromised mice [102]. Lastly, it was found that knockdown of Ron expression was associated with loss of the transcriptional co-activator !-catenin, a downstream Wnt signaling target that is routinely deregulated in sporadic and hereditary human colon cancers [24]. While these studies illustrate that the expression levels and functional importance of Ron overexpression in colon cancer cells lines in vitro and in xenograft models in vivo, virtually nothing is known about Ron function in the normal intestine, nor its role in Apc/!-catenin mediated intestinal tumorigenesis. Thus the first goal of my thesis that is described in Chapter 2 was to test the requirement of Ron tyrosine kinase in ApcMin/+ intestinal tumorigenesis.

Mammary Gland Development

Mammary gland development can be divided into several stages including embryonic, puberty, pregnancy, lactation, and involution (Figure 5). The mammary gland consists of two major compartments epithelial (ducts, end buds, alveoli) and stromal (fibroblasts, inflammatory cells, adipocytes, muscle cells). The main role of the epithelial compartment is to produce milk and form the canals through which milk passes out of the body. The stromal compartment supports the epithelial structures and

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consists mainly of fibroblasts, muscle cells, macrophages, and adipocytes. Throughout all stages of mammary gland development, crosstalk between these compartments is tightly regulated to produce a functionally normal mammary gland.

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Figure 5. A schematic of the developmental stages of the mouse mammary gland. At

birth, mice are born with a rudimentary ductal tree connected to the nipple. Puberty

begins with the onset of cyclical ovarian hormone production around 5 weeks of age

and pubertal mammary development ends around 10 weeks of age, with the

differentiation of terminal end buds as seen in the adult resting gland. In humans,

lobules form during puberty, persist through adulthood, and produce milk during

lactation. In mice lobules do not form until pregnancy and lactation. After lactation,

mammary glands involute (epithelial apoptosis and matrix remodeling) to the resting

adult glandular appearance.

Mammary ducts are formed by a single layer of columnar epithelium, called

luminal epithelial cells that line the lumen of the ducts where milk will flow through

during lactation (Figure 6A and 6C). During puberty, pregnancy, and lactation there are

specialized epithelial structures that develop off of the ducts in response to hormonal

and growth factor cues. During puberty the tips of the mammary rudimentary ducts

rapidly proliferate at the forefront to form multi-layered epithelial buds, called terminal end buds (TEB) (Figure 6B and 6C). As the TEBs invade the surrounding stroma, the centermost body cells undergo apoptosis to form an empty lumen surrounded by a

differentiated bilayered (luminal and myoepithelium) epithelium in a process known as

ductal elongation [167]. Branching morphogenesis occurs simultaneously with ductal

elongation during puberty, and is reviewed in greater detail in Chapter 1 page 1-31. At

the end of puberty, the TEBs regress upon reaching the edges of the fat pad resulting in

a robust mammary ductal network that has completely filled the mammary fat pad in

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adult mice [167,168]. During pregnancy, alveolar buds develop off of the ducts, and are composed of secretory cuboidal epithelial cells, which secrete milk proteins into the lumen during lactation. A clustering of many alveoli together is called a lobule [169].

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Figure 6. Histological appearance and morphology of mammary ducts and terminal

end buds. Hematoxylin and eosin histochemical staining of paraffin-embedded inguinal

mammary gland from a 6 week-old wild-type mouse showing two mammary ducts (A),

which are indicated by asterisks (*), and a terminal end bud (B). The main cell types

that comprise mammary ducts and terminal end buds are indicated in A and B. C)

Illustration of the cell types found in the mammary gland by cross-section through the

transition from multi-layered terminal end bud (TEB) to single-layer duct. Adapted from

[168]. D) Close-up images of a bifurcating terminal end bud (black arrowhead) and a

lateral side branch (white arrowhead) in a 6 week-old mouse mammary gland prepared

by whole mount staining.

Mammary Gland Branching Morphogenesis

There are two developmental stages, puberty and pregnancy, in which mammary

branching morphogenesis occurs in mice and humans. During puberty, there are two

types of branching morphogenesis that occur in the mammary gland including end bud

bifurcation, also known as dichotomous branching, and lateral side branching (Figure

6D). The asymmetric branching morphogenesis during puberty occurs simultaneously

with ductal invasion to extend the newly formed branches into the fat pad, and these

ducts/branches will serve as the passageway for milk to flow from alveoli to the nipple

during lactation. The branches formed during puberty provide the surface area for

pregnancy-induced lobuloalveolar branching morphogenesis, which forms the milk- producing structures for lactation [168].

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Branching morphogenesis is a combined result of multiple processes including epithelial cell proliferation, migration, invasion, and apoptosis [170]. Important signaling pathways that mediate these processes include nuclear hormone receptor signaling

(ER#, PR, VDR, GH), growth factor receptor signaling (EGF, IGF, FGF, TGF!), cell adhesion components (Integrin #2!1, E-cadherin, !-catenin, DDR1), matrix remodeling enzymes (MMP-2, -3, -14, ADAM17), and transcription regulators (p27, AP-1).

Examples of autocrine (TGF!), paracrine (FGF/FGFR2, IGF/IGF-IR), and endocrine

(estrogen/ER) signaling have all been found to play an important role in the regulation of mammary morphogenesis [171,172]. Some of these well-established regulators are summarized in more detail in Table 3, however, these and additional regulators of mammary branching morphogenesis have been extensively reviewed in the literature

[168,171,173-175].

Table 3. Summary of well-established regulators of pubertal mouse mammary morphogenesis. Gene Expression* Model Phenotype Reference ER# epithelia and MMTV-Cre/ER#fl/fl Ablated pubertal mammary gland [176] stroma development

PR luminal PRKO Reduced lateral branching in virgin [177,178] epithelia adults transplanted with PRKO epithelium

VDR epithelia and VDRKO Accelerated mammary ductal [179] stroma elongation and increased branching

GHR epithelia and GHRKO Reduced ductal extension and [180,181] stroma branching

PTHrPR stroma PTHrPR-/- Impaired ductal growth and branching [182]

EGFR epithelia and Egfr-/- Impaired ductal growth and branching [183] stroma

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IGF-1R epithelia IGF-1R-/- Impaired ductal growth and branching [184]

EphA2 epithelia EphA2-/- Impaired ductal growth and branching [185]

TGF!RII epithelia Tgfbr2MGKO Enhanced virgin branching [186] and morphogenesis stroma Tgfbr2FspKO Impaired ductal growth and branching [187]

MMP-3 stroma MMP-3 -/- Reduced lateral side branching [188]

MMP-2 epithelia and MMP-2 -/- Retarded ductal extension, enhanced [188] stroma lateral side branching

#2!1- epithelia and #2 -/- Reduced ductal branching [189] integrin stroma

*The cellular compartment, if known, is bolded to indicate the source of gene expression that is necessary for proper mammary development. Adapted from Howlin et al., 2006 and Sternlicht, 2006 [168,173]. ER, estrogen receptor; MMTV, mouse mammary tumor virus; PR, progesterone receptor; GHR, growth hormone receptor; VDR, vitamin D receptor; PTHrPR, parathyroid hormone related protein receptor; EGFR, epidermal growth factor receptor; IGF-IR, insulin growth factor receptor; EphA2, ephrin receptor; TGF!RII, transforming growth factor ! type II receptor; MMP, matrix metalloproteinase; MGKO, mammary gland knockout; FspKO, fibroblast specific knockout.

Ron Receptor Signaling in Breast Cancer

Ron is overexpressed in 50% (35/74) to 56% (25/45) of breast cancers [1,29].

Importantly, gene expression measured by oligonucleotide arrays for Ron (MST1R),

HGFL (MST1), and the protease MT-SP1, which cleaves inactive pro-HGFL to active

HGFL, demonstrated that overexpression of these three genes occurred in 14.6%

(43/295) of breast cancers examined and was a strong independent indicator of both metastasis and death [190]. Co-expression of the Ron and Met receptors in human

breast tumors also correlates with a poorer 10-year survival rate of 11.8% compared to

those patients with breast tumors that are Ron and Met negative (79.3%) [191]. These

studies highlight the potential prognostic significance of Ron expression levels as well

1-33

as suggest a therapeutic benefit to blocking Ron receptor expression and function in

cancer.

Only one splice variant of the Ron receptor, &Ron, has been identified in primary

human breast cancer specimens, and of the 16 samples tested 12 were positive for

&Ron. This variant was also found in colon cancers, but was not prevalent in normal

tissues [192]. &Ron is a constitutively active alternatively spliced form of Ron that lacks exon 11 [192,193]. It was recently communicated that three other Ron variants may

exist in breast cancers, although, the details of this finding are still unpublished [162].

Studies have shown that Ron is expressed at low levels in normal adult human

breast tissue by immunohistochemical staining of human tissue arrays and mouse

mammary glands by northern analysis [6,32], in addition to Ron overexpression in

approximately 50% of primary breast cancers and many breast cancer cell lines

[1,29,194]. Interestingly, Ron receptor expression is increased in the polyoma middle-T antigen mouse (MMTV-PyMT) mammary tumors wherein loss of Ron receptor function leads to a delay in tumor kinetics and growth [98]. Recently our laboratory has

demonstrated that overexpression of the full-length wild-type Ron receptor specifically in

the mouse mammary gland (MMTV-Ron) is sufficient to induce mammary tumorigenesis

with 100% penetrance, and metastasis to the lung and liver in approximately 90% of

mice [30]. This study indicates that aberrant Ron receptor overexpression is sufficient

to cause mammary tumorigenesis. Some studies have investigated the mechanism of

Ron gene regulation in human breast cancer cell lines and have found that the

transcription factors Sp1 and the p65 subunit of NF-$B positively regulate Ron gene

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expression, both of which are downstream of signaling pathways deregulated in cancers

[194,195]. Despite the overwhelming number of studies showing that Ron is important in breast tumorigenesis, virtually nothing is known about the role of endogenous Ron expression in normal breast tissue development. Therefore, the goal of my second thesis project was to investigate the consequence of Ron loss on mammary gland development, and is described in Chapter 3.

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Abbreviations

ACF aberrant crypt foci

ACS American Cancer Society

ADAM17 also known as tumor necrosis factor # converting enzyme (TACE)

Akt also known as protein kinase B (PKB)

AP-1 activator protein-1 transcription factor

APC adenomatous polyposis coli

Atm ataxia telangiectasia mutated

BMP bone morphogenic protein

CL corpora luteua

Cox2 cyclooxygenase 2

Cre Cre recombinase

DDR1 discoidin domain receptor

Dvl Dishevelled

ECM extracellular matrix

EGF epidermal growth factor

EGFR epidermal growth factor receptor

ENU N-ethyl-N-nitrosourea

ER estrogen receptor

ES embryonic stem cells

FAK focal adhesion kinase

FAP familial adenomatous polyposis

FGF fibroblast growth factor

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FGFR fibroblast growth factor receptor

Gab1 Grb2-associated binding protein 1

GH growth hormone

GHR growth hormone receptor

Grb2 growth factor receptor-bound protein 2

GSK3! glycogen synthase kinase 3!

H&E hematoxylin and eosin

HGF hepatocyte growth factor

HGFL hepatocyte growth factor-like protein, same as MSP

IGF insulin growth factor

IGF-IR insulin growth factor type I receptor iNos inducible nitric oxide synthase

INF% interferon %

IPT immunoglobulin-like fold shared by plexins and transcription factors

domain

JNK c-Jun N-terminal kinase

Ki67 protein marker of proliferation

KO knockout

Lef lymphoid enhancer factor

LOH loss of heterozygosity

LPS lipopolysaccharide

Mapk mitogen activated protein kinase, same as Erk

MCR mutation cluster region

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Min multiple intestinal neoplasia

MMP matrix metalloproteinase

MMTV mouse mammary tumor virus

MSP macrophage stimulating protein, same as HGFL

MST1 macrophage stimulating protein 1, gene name for HGFL

MST1R macrophage stimulating protein 1 receptor, gene name for Ron

MT-SP1 multidomain serine proteinase, matriptase, cleaves pro-HGFL

NF$B nuclear factor kappa-light-chain-enhancer of activated B cells

NO nitric oxide

o/e overexpressed

PCNA proliferating cell nuclear antigen

PI3K phosphoinositide 3-kinase

PLC% phospholipase C %

PP1 protein phosphatase 1

PR progesterone receptor

PSI plexin semaphorins integrins domain

Pten phosphatase and tensin homolog

PTHrPR parathyroid hormone related protein receptor

PyMT polyoma virus middle T-antigen

RON Recepteur d’Origine Nantaise

SH2 Src homology 2 domain

SOS Son of Sevenless, guanine nucleotide exchange factor

STAT signal transducer and activator of transcription

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Stk Stem cell-derived tyrosine kinase, refers to mouse Ron

SV-40 simian virus - 40

TEB terminal end bud

Tg transgenic

TGF! transforming growth factor !

TK tyrosine kinase domain

VDR vitamin D receptor

VEGF vascular endothelial growth factor

1-39

Chapter 2

The Ron Receptor Tyrosine Kinase is Not Required for

Adenoma Formation in ApcMin/+ Mice

Sara E. Meyer, Susan E. Waltz, and Kathleen H. Goss. 2009. The Ron Receptor Tyrosine Kinase is Not Required for Adenoma Formation in ApcMin/+ Mice. Molecular Carcinogenesis. doi:10.1002/mc.20551 PMID: 19452510 " 2009 Wiley-Liss, Inc.

Meyer et al., Molecular Carcinogenesis, 2009.

Preface

This chapter includes original research data generated by Sara Meyer while

under the mentorship of graduate advisor Dr. Kathleen Goss in collaboration with Dr.

Susan Waltz. The goal of this work was to test the importance of Ron receptor

signaling in intestinal tumorigenesis in an in vivo scenario, using the well-established

ApcMin/+ mouse model. The results of this work were accepted for publication in

Molecular Carcinogenesis in May, 2009 [28].

2-2 Meyer et al., Molecular Carcinogenesis, 2009.

Abstract

The Ron receptor tyrosine kinase is overexpressed in approximately half of all human

colon cancers. Increased Ron expression positively correlates with tumor progression,

and reduction of Ron levels in human colon adenocarcinoma cells reverses their

tumorigenic properties. Nearly all colon tumors demonstrate loss of the adenomatous

polyposis coli (APC) tumor suppressor, an early initiating event, subsequently leading to

!-catenin stabilization. To understand the role of Ron in early-stage intestinal

tumorigenesis, we generated Apc-mutant (ApcMin/+) mice with and without Ron signaling. Interestingly, we report here that significantly more ApcMin/+ Ron-deficient mice developed higher tumor burden than ApcMin/+ mice with wild-type Ron. Even

though baseline levels of intestinal crypt proliferation were increased in the ApcMin/+

Ron-deficient mice, loss of Ron did not influence tumor size or histological appearance

of the ApcMin/+ adenomas, nor was !-catenin localization changed compared to ApcMin/+

mice with Ron. Together, these data suggest that Ron may be important in normal

intestinal tissue homeostasis, but that the expression of this receptor is not required for

the formation and growth of adenomas in ApcMin/+ mice.

2-3 Meyer et al., Molecular Carcinogenesis, 2009.

Abbreviations

Apc adenomatous polyposis coli

Cox2 cyclooxygenase-2

EGFR epidermal growth factor receptor

Fak focal adhesion kinase

FAP familial adenomatous polyposis

HGFL hepatocyte growth factor-like protein iNOS inducible nitric oxide synthase

MAPK mitogen-activated protein kinase

Min multiple intestinal neoplasia

MMP7 matrix metalloproteinase 7

PCNA proliferating cell nuclear antigen

PI3K phosphatidylinositol-3-kinase

TK tyrosine kinase domain

TUNEL terminal deoxynucleotidyl transfer mediated dUTP nick end labeling

2-4 Meyer et al., Molecular Carcinogenesis, 2009.

Introduction

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the

majority of sporadic human colon cancers [18]. Patients with familial adenomatous

polyposis (FAP) inherit a germline mutation in APC that causes the development of

hundreds to thousands of colon polyps at a young age, some of which will progress into

carcinomas if not removed [18,19]. The ApcMin/+ mouse serves as a useful model of

FAP; germline mutation of the mouse Apc gene results in multiple spontaneous

adenomatous polyps that demonstrate loss of heterozygosity of the wild-type Apc allele

[20,27]. One important difference between FAP and the ApcMin/+ model is that the

adenomas in ApcMin/+ mice do not progress to carcinomas without additional chemical insults or genetic alterations, perhaps due to the short lifespan of the animals as a result of the intestinal obstruction and anemia from tumor burden [27,155,196]. At the

molecular level, ApcMin/+ adenomas, as well as inherited and sporadic human colorectal tumors, demonstrate cytosolic and nuclear accumulation of the transcription co-activator

!-catenin. !-catenin is a tightly regulated effector of APC, whose activity and the expression of its transcriptional targets contributes significantly to tumor development when Apc is inactivated [23,197,198]. The ApcMin/+ model has been useful in testing the importance of a variety of signaling pathways and tumor markers, such as EGFR,

MMP7, and iNOS in intestinal tumorigenesis using genetic and pharmacological approaches [158,199,200].

Recently, studies have shown that the heterodimeric transmembrane receptor tyrosine kinase Ron, a member of the Met tyrosine kinase family, may be important in human

2-5 Meyer et al., Molecular Carcinogenesis, 2009.

colon cancer [8,9]. Ron is overexpressed, constitutively phosphorylated, and activated

in many cancer types, including primary human sporadic colon cancer and in the colon

cancer cell lines Colo201, HT-29, and HCT116 [8-11,201]. Activation of the Ron

receptor occurs upon binding of the ligand, hepatocyte growth factor-like protein

(HGFL), which induces receptor tyrosine phosphorylation and activation [2,202]. The

downstream effects of Ron activation are vast and include cell proliferation, cell cycle

progression, cell motility, angiogenesis, cell survival, apoptosis, cellular transformation,

tumor progression, and metastasis [203]. The PI3K, MAPK, Ras, Src, Fak, and !-

catenin signaling pathways have all been implicated in mediating the signal from Ron to

exert these effects [8,10,11,24,30,98,163,201]. These features make Ron an attractive

therapeutic target for cancer; however, very little is known about its role in colorectal

tumorigenesis in vivo. Therefore, we sought to determine the in vivo role of Ron in

intestinal tumorigenesis using the well-characterized ApcMin/+ mouse model, since APC loss is an early event in sporadic colon cancer [18,20,204,205]. In this report, we demonstrate that Ron is expressed in the non-transformed intestinal epithelium and adenomas in the ApcMin/+ mice. By generating ApcMin/+ mice that lack the tyrosine

kinase domain of the Ron receptor (RonTK-/-), we show that Ron is not required for

intestinal tumor initiation, and its loss leads to an increase in the number of animals

exhibiting higher tumor numbers compared to control ApcMin/+ mice, perhaps due to enhanced proliferation in the morphologically normal intestine.

2-6 Meyer et al., Molecular Carcinogenesis, 2009.

Results

Ron is Expressed in the Intestine of Apc-mutant Mice. In the human adult small intestine and colon, Ron expression is localized to the crypt cells with a granular cytosolic subcellular localization [21]. To determine the expression pattern of Ron in the non-transformed intestine and adenomas from ApcMin/+ mice, immunohistochemistry was performed with an anti-Ron antibody (Figure 1). Ron was homogeneously expressed in epithelial cells of the small intestine. In addition, Ron was also localized to the crypt cells in the colon, but was expressed at a low level in other areas. In adenomas from the ApcMin/+ mice, Ron expression was diffuse. Quantitative real-time

PCR analysis of RNA isolated from ApcMin/+ tumors compared to Apc+/+ normal intestinal tissue showed similar Ron expression levels (data not shown). From these data, we conclude that Ron is expressed in normal intestinal epithelium, particularly in areas (i.e. crypts) thought to give rise to tumors [151,152,206], and its expression is maintained in intestinal adenomas from ApcMin/+ mice.

Tumor Formation in ApcMin/+ Mice Lacking the Ron Receptor Tyrosine Kinase.

Given that Ron is expressed in normal and transformed intestinal tissue from ApcMin/+ mice, we next sought to determine the impact of Ron on intestinal tumor formation in these mice. Germline deletion of the tyrosine kinase domain of Ron in mice was reported previously, and results in the production of a truncated, non-functional receptor when stimulated with HGFL [17]. The Ron tyrosine kinase domain null (RonTK-/-) mice do not have any overt intestinal defects, nor do they develop spontaneous intestinal tumors. RonTK-/- and ApcMin/+ mice, both on the C57BL/6J background, were bred to

2-7 Meyer et al., Molecular Carcinogenesis, 2009.

generate mice that carried both mutant alleles. Three month-old ApcMin/+;RonTK-/- and

ApcMin/+;RonTK+/+ controls were euthanized and analyzed for intestinal tumorigenesis.

Even though there was not a significant difference in the mean number of tumors along the entire gastrointestinal tract or in individual regions in ApcMin/+ mice with and without

Ron (Figure 2A), we found that significantly more ApcMin/+;RonTK-/- mice (11/13) had

higher tumor numbers than the median (29 tumors) for the ApcMin/+;RonTK+/+ control mice (p=0.0017 by binomial test; Figure 2B). Mean tumor size was also unchanged between ApcMin/+;RonTK-/- (1.54 ± 0.04 mm) and ApcMin/+;RonTK+/+ (1.49 ± 0.04 mm)

mice. Histopathological examination of tumor and surrounding normal tissue from

ApcMin/+;RonTK-/- and ApcMin/+;RonTK+/+ mice revealed that the tumors were

indistinguishable from one another and considered benign adenomas without prominent

inflammation in either the tumor or surrounding non-transformed tissue (Figure 3).

Furthermore, there were no significant differences in the tumor number, size, or

histopathology of ApcMin/+;RonTK+/- mice compared to the ApcMin/+;RonTK+/+ and

ApcMin/+;RonTK-/- animals (data not shown). Together, these results demonstrate that

the Ron receptor is not required for formation of intestinal adenomas in ApcMin/+ mice.

ApcMin/+ Ron-deficient Mice Exhibit Increased Proliferation of the Non-transformed

Intestinal Epithelium. Ron is a multifunctional protein that has been shown to regulate

proliferation and cell death in several in vitro and in vivo models [24,25,79,95,98,207].

To determine if Ron inactivation affected these processes in ApcMin/+ intestinal tissues, proliferation and cell death were quantified in intestinal tissues from ApcMin/+;RonTK+/+

and ApcMin/+;RonTK-/- using the immunohistochemical detection of proliferating cell

2-8 Meyer et al., Molecular Carcinogenesis, 2009.

nuclear antigen (PCNA), and the TUNEL assay (Figures 4 and 5). The ApcMin/+;RonTK-

/- mice exhibited significantly more PCNA-positive cells in the non-transformed crypt

epithelium of the small intestine and colon than ApcMin/+;RonTK+/+ tissues (Figure 4A), although analysis of adenomas from these mice showed no significant differences

(Figure 4B). Ki67 immunohistochemistry was also performed on the same tissues and verified this finding (data not shown). In contrast, there were no significant differences in the number of TUNEL-positive cells in neither the non-transformed crypt epithelium nor the adenomas from ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice (Figures 5A and

5B). Together, these results suggest that Ron loss does not affect the growth of

ApcMin/+ adenomas, despite an increase in proliferation of the non-transformed epithelium in ApcMin/+;RonTK-/- mice.

Ron Deficiency Does Not Alter !-Catenin Localization or Target Gene Expression in ApcMin/+ Mice. Loss of Apc is associated with increased !-catenin transcriptional activity that is sufficient for tumorigenesis in the mouse gut [197,208]. Stabilization and nuclear accumulation of !-catenin is a very common feature of adenomas from ApcMin/+

mice [209], and moreover, tyrosine phosphorylation and nuclear localization of !-catenin has been observed in mouse mammary tumors driven by Ron overexpression [30].

Therefore, one possible mechanism by which Ron loss may influence adenoma formation in ApcMin/+ mice is by alteration of !-catenin expression or activity. To determine if Ron inactivation overtly alters the localization of !-catenin in this model, we performed immunohistochemistry with an anti-!-catenin antibody. There was abundant cytosolic and nuclear !-catenin in adenomas from ApcMin/+;RonTK+/+ mice and ApcMin/+

2-9 Meyer et al., Molecular Carcinogenesis, 2009.

Ron-deficient mice (Figure 6). Prominent staining at cell-cell contacts was observed in the non-transformed intestinal epithelium of ApcMin/+ mice with and without Ron signaling (Figure 6). Moreover, no significant changes in the expression of several !- catenin/Tcf target genes associated with intestinal tumorigenesis, including cyclin D1, c- myc, and Cox-2, were observed (Table 1). These data suggest that the mechanism by which Ron loss influences adenoma formation in this model is not likely to be through further modulation of !-catenin localization and transcriptional activity.

2-10 Meyer et al., Molecular Carcinogenesis, 2009.

Figures and Tables

Meyer et al. Figure 1

Figure 1. Ron is expressed in the non-transformed intestinal epithelium and adenomas

of ApcMin/+ mice. Immunohistochemistry on tissue sections from the small intestine (A

and B) and colon (C and D) (n=4 per group) of three month-old ApcMin/+ mice was performed using an anti-Ron antibody to detect Ron expression. No staining was observed using an isotype-matched IgG control (data not shown). Non-transformed epithelial and tumor areas are designated by N (non-transformed) and T (tumor). 200X

Magnification. Scale bar; 50 µm.

2-11 Meyer et al., Molecular Carcinogenesis, 2009.

Meyer et al. Figure 2

Figure 2. Ron deletion significantly increases the number of ApcMin/+ mice with high tumor burden, but does not impact mean tumor number or distribution. ApcMin/+ mice were crossed with RonTK+/- mice to generate ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/-

mice. Intestinal tumors were analyzed at three months of age. A) The mean number of

tumors per mouse and within each segment of the gastrointestinal tract (duodenum,

jejunum, ileum, and colon) per genotype (n=13). p>0.05 for all comparisons between

genotypes, Wilcoxon rank-sum test. B) The mean number of tumors per mouse is plotted per genotype (n=13). Each data point represents a single mouse; the median

2-12 Meyer et al., Molecular Carcinogenesis, 2009.

tumor number in ApcMin/+;RonTK+/+ controls is denoted (29, black line). p=0.0017, binomial test.

2-13 Meyer et al., Molecular Carcinogenesis, 2009.

Meyer et al. Figure 3

Figure 3. Adenomas from ApcMin/+;RonTK-/- mice are histologically similar to those from

ApcMin/+;RonTK+/+ mice. Tumor and surrounding non-transformed small intestine and

colon tissues from three-month old ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice were stained with hematoxylin and eosin and examined histologically. At least two tumors and surrounding normal tissue per gastrointestinal segment (duodenum, jejunum, ileum,

2-14 Meyer et al., Molecular Carcinogenesis, 2009. and colon) per mouse (n=4 per genotype) were examined histologically. No differences were detected between genotypes in the appearance of non-transformed (N) and tumor

(T) tissues in the small intestine and colon. 100X magnification. Scale bar; 25 µm.

2-15 Meyer et al., Molecular Carcinogenesis, 2009.

Meyer et al. Figure 4

2-16 Meyer et al., Molecular Carcinogenesis, 2009.

Figure 4. Proliferation in the ApcMin/+;RonTK-/- non-transformed intestinal epithelium is significantly increased compared to ApcMin/+;RonTK+/+ mice. Tissues from three month- old ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice were analyzed for proliferation by staining with an anti-PCNA antibody. Proliferating cell nuclei are dark brown (black arrows). 200X magnification. Scale bar; 50 µm. Quantification of PCNA staining is graphed as the mean number of PCNA-positive cells per crypt ± SDV in the normal small intestine (n=4) and colon (n=4) (A) and the percentage ± SDV of proliferating cells in adenomas (B). *p<0.02, Student t-test.

2-17 Meyer et al., Molecular Carcinogenesis, 2009.

Meyer et al. Figure 5

2-18 Meyer et al., Molecular Carcinogenesis, 2009.

Figure 5. Cell death in ApcMin/+;RonTK-/- mice is not altered compared to

ApcMin/+;RonTK+/+ mice. Tissues from three month-old ApcMin/+;RonTK+/+ and

ApcMin/+;RonTK-/- mice were analyzed for cell death using the TUNEL assay. TUNEL- positive cells are dark brown and have been magnified to show their histological appearance (black box). 400X magnification. Scale bar; 50 µm. Quantification of

TUNEL staining is graphed as the mean number of TUNEL-positive cells/crypt ± SDV in

the normal small intestine (n=4) and colon (n=4) (A) and as the percentage ± SDV of

TUNEL-positive cells in adenomas of the small intestine and colon (B). p>0.05, Student

t-test.

2-19 Meyer et al., Molecular Carcinogenesis, 2009.

Meyer et al. Figure 6

Figure 6. !-catenin localization is similar in intestinal tissue from ApcMin/+;RonTK+/+ and

ApcMin/+;RonTK-/- mice. Tissues from three month-old ApcMin/+;RonTK+/+ and

ApcMin/+;RonTK-/- mice (n=4 per genotype) were analyzed for !-catenin localization by

immunohistochemistry using an anti-!-catenin antibody (brown). Non-transformed

epithelium demonstrates predominantly membranous staining (arrows), and adenomas

exhibit intense nuclear (arrows) and cytosolic staining in the small intestine and colon

from both genotypes. 400X magnification. Scale bar; 50 µm

2-20 Meyer et al., Molecular Carcinogenesis, 2009.

Table 1. Fold-change of !-catenin/Tcf target gene mRNA expression in RonTK-/- intestines relative to RonTK+/+ controls ± SEM.

Cyclin D1 c-Myc Cox2 Small Intestine 1.09 ± 0.13 1.08 ± 0.35 1.12 ± 0.19 Colon 1.23 ± 0.15 1.44 ± 0.54 1.41 ± 0.29 p > 0.05 for all genes by Student t-test.

2-21 Meyer et al., Molecular Carcinogenesis, 2009.

Discussion

Despite the numerous reports describing Ron overexpression and activation in various

cancers including breast, lung, pancreas, and colon, little is known about the

cooperation of Ron with other pathways deregulated in cancer [8-10]. Ron is a valid

therapeutic target for cancer as antibody inhibition can reduce tumor cell growth in

xenograft models [1,14]. Therefore, it is important to understand the consequence of

Ron inactivation in normal tissues as well as during tumorigenesis in vivo. The ApcMin/+

model of murine intestinal tumorigenesis resembles the majority of human inherited and

sporadic colon cancers, which contain aberrant APC/!-catenin signaling

[149,205,206,210,211]. Using this paradigm for intestinal tumorigenesis, we show here

that the loss of Ron in ApcMin/+ mice is not required for intestinal tumorigenesis.

Moreover, our results show that germline loss of Ron in this model allows for an increase in the number of ApcMin/+ mice with high tumor burden compared to control

ApcMin/+ mice with wild-type Ron, but there was no overall affect on tumor progression.

While the rates of cell death in both non-transformed and adenoma tissues and the localization of !-catenin remained constant, Ron loss increased proliferation in the normal intestinal crypt epithelium. This study is significant as it indicates that Ron expression in ApcMin/+ mice is not required for tumorigenic transformation or the

progression to adenomas.

Our studies demonstrate that Ron is expressed in the normal small intestine and colonic

epithelium and adenomas of ApcMin/+ mice. Specifically, expression of Ron in the colonic crypt epithelium correlates with the location in which adenomas arise in the

2-22 Meyer et al., Molecular Carcinogenesis, 2009.

intestine of these animals [151,152,206]. Previously, Boon et al., 2006 [212] published

immunohistochemical staining of Ron in ApcMin/+ mice suggesting that Ron was expressed in the non-transformed epithelium. In this report, the investigators were unable to detect Ron expression at appreciable levels in the ApcMin/+ adenomas using non-quantitative methods [212]; however, this does not preclude the validity of our data, which demonstrates that Ron expression is maintained in the ApcMin/+ adenomas. Our results are also consistent with the findings in human tissue showing that Ron is expressed in the normal intestinal epithelium with continued expression, or overexpression, observed in colorectal cancer [9,11,21]. Thus, our studies extend upon those in the literature and show that Ron is expressed in the small intestine and colon of non-transformed epithelium and adenomas of ApcMin/+ mice.

To determine the functional significance of Ron expression in the gut, we monitored adenoma formation in Ron tyrosine kinase-proficient and -deficient ApcMin/+ mice.

Interestingly, we found that significantly more ApcMin/+;RonTK-/- mice developed a greater number of tumors than ApcMin/+;RonTK+/+ mice, although these tumors were not

different in size or histology. These data indicate that Ron signaling is not required for

intestinal tumorigenesis in the presence of an Apc mutation. These results were

unexpected given that Ron overexpression has been shown to promote the tumorigenic

phenotype in vitro [10,74,201]. In contrast to other studies wherein silencing of Ron

overexpression in immortalized human colon carcinoma cell lines reversed their

tumorigenic properties and had dramatic consequences with respect to !-catenin

activation, our study shows that basal Ron expression is not required for cellular

2-23 Meyer et al., Molecular Carcinogenesis, 2009.

transformation or adenoma formation in the presence of an Apc mutation in vivo

[6,11,24]. Our data do not, preclude, however, the importance of Ron overexpression

observed in human colorectal cancer, where studies have suggested that inhibiting this

overexpressed receptor may have important anti-tumor effects [1,10,14,74,201]. In

addition to focusing on early-stage tumor progression, unique to our studies is that we

have examined the effect of de novo Ron loss in all cell types on intestinal

tumorigenesis in vivo, where other factors, such as the tumor microenvironment, play a

role. It is also possible that there are species differences and differences in the

absolute levels of Ron produced in these models that might contribute to these

apparently conflicting data. However, our result is consistent with a previous study that

showed stable knockdown of Ron in metastatic SW620 colon cancer cells did not

prevent tumor formation in nude mice [24], and another study that observed increased

benign tumor formation in an in vivo skin tumorigenesis model in mice deficient for Ron

[99]. There is also precedent for other tumor-associated molecules, such as

telomerase, to have different functions in normal tissue and at various stages of

tumorigenesis [213]. It is noteworthy that the variability in mean tumor number per

animal was greater than what we have observed and published using the ApcMin/+ model previously [158,214,215]. Given that the RonTK-/- mice were backcrossed 8

generations onto the C57BL/6J genetic background prior to mating with ApcMin/+ mice,

and that no identified ApcMin/+ modifying loci are on the same chromosome as Ron

(chromosome 9), we have no evidence to suggest that an ApcMin/+ modifying locus is

present in our model [27,216-218]. However, we cannot discount the possibility that a

novel modifier might be present in the region directly around the Ron gene.

2-24 Meyer et al., Molecular Carcinogenesis, 2009.

Using markers of proliferation, we found that Ron inactivation in ApcMin/+ mice leads to significantly greater numbers of intestinal crypt cells undergoing proliferation, which may make the cells more susceptible to Apc loss of heterozygosity and transformation. It is interesting, however, that the tumors themselves failed to show enhanced proliferation in spite of Ron loss, which is consistent with the in vitro studies described above [24]. It

is well documented that 100% of intestinal adenomas demonstrate loss of the wild-type

copy of Apc [20], and we confirmed that Apc was similarly lost in adenomas from the

ApcMin/+;RonTK-/- mice, suggesting that Apc inactivation is likely still required for driving

adenoma formation (S.M., K.G., unpublished data). As RonTK-/- mice do not develop spontaneous gastrointestinal tumors, it is clear that Ron loss is not sufficient to initiate tumor formation. Finally, although Ron loss did not significantly affect apoptosis in the non-tumor or adenoma tissues, we cannot rule out a role for Ron in tumor cell survival as has been shown in other tumor types [219].

We investigated whether modulation of !-catenin signaling may be a mechanism by which Ron loss increased tumor load in ApcMin/+ mice. Our results suggest that Ron loss does not profoundly influence !-catenin nuclear localization in this model given that there were no differences in !-catenin localization between ApcMin/+;RonTK-/- and

ApcMin/+;RonTK+/+ control mice in normal and adenoma small intestine and colon tissues. Additionally, quantitative real-time PCR analyses on normal intestinal tissue from RonTK+/+ and RonTK-/- mice showed that transcript levels of !-catenin target genes

c-myc and cyclin-D1 [114,115] were unchanged with respect to Ron status.

2-25 Meyer et al., Molecular Carcinogenesis, 2009.

Nevertheless, it remains possible, that Ron may alter !-catenin activity in a way that

was not measured in our experiments or captured in the time frame of our analyses.

As a second potential mechanism, we hypothesized that Ron may protect against tumor

formation through modulating stromal-epithelium interactions in the intestine, for

example, by regulating inflammation through cytokine production or the production of

free oxygen radicals [17,90]. However, there were no apparent differences in

inflammatory cell recruitment in the intestines of ApcMin/+;RonTK-/- tissues by

histopathological analysis compared to controls. Cox-2, also considered a !-catenin transcriptional target, was examined because Ron inactivation can inhibit its expression in macrophages [220-223], and pharmacological or genetic Cox-2 inhibition can

significantly decrease tumor numbers in ApcMin/+ mice [224]. However, we did not find a statistically significant change in Cox-2 expression in RonTK-/- intestinal tissues compared to wild-type controls. While Cox-2 may not be the major molecular target of

Ron in this model, it is possible that other factors may modulate inflammation in the gut downstream of Ron that could impact intestinal tumor formation.

In summary, our studies demonstrate that Ron is not required for intestinal tumorigenesis in the ApcMin/+ model. Interestingly, our studies also suggest that while

Ron targeted therapies may be effective for the treatment of advance-stage cancers

that exhibit overexpression of this receptor [1,14], blocking Ron in normal tissue may

have an unfavorable outcome in the context of other alterations that promote tumor

formation.

2-26 Meyer et al., Molecular Carcinogenesis, 2009.

Materials and Methods

Animals: ApcMin/+ mice on a C57BL/6J background (The Jackson Laboratory) were crossed with Ron receptor tyrosine kinase domain knockout mice (RonTK-/-) [17].

RonTK-/- mice were generated on a Black Swiss background and backcrossed 8

generations onto the C57BL/6J background prior to mating with the ApcMin/+ mice,

therefore only approximately 0.4% of the original Black Swiss background was

introduced into the initial cross. ApcMin/+;RonTK+/+, ApcMin/+;RonTK+/-, and

ApcMin/+;RonTK-/- mice (n=13 each) were euthanized at three months of age. At this

point, their intestines were harvested, fixed in 4% paraformaldehyde overnight, and then

transferred to 70% ethanol. Tumor number, size, and location along the gastrointestinal

tract were determined using an Olympus dissection microscope as described previously

[158]. For histopathological and immunohistochemical analyses, a minimum of 2

tumors per gastrointestinal segment (duodenum, jejunum, ileum, and colon) from each

mouse (at least 4 mice per genotype) were excised and embedded in paraffin. For all analyses, ApcMin/+;RonTK+/+ mice served as controls while ApcMin/+;RonTK+/- and

ApcMin/+;RonTK-/- mice were the experimental animals.

Histopathological Analysis: 4-µm sections were deparaffinized, hydrated through

graded ethanol washes, and stained with Mayer’s hematoxylin and eosin. The sections

were dehydrated, incubated in Citrisolv, and mounted with permount. N=4 per

genotype, at least two tumors and surrounding normal tissue were analyzed per

gastrointestinal segment per mouse. Certified pathologists analyzed tumor grade and

2-27 Meyer et al., Molecular Carcinogenesis, 2009.

inflammation without previous knowledge of the genotype and according to published

guidelines [225].

Immunohistochemistry: 4-µm-thick sections were deparaffinized and hydrated as

above. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide

and non-specific immunoreactivity was blocked using 5% goat serum in phosphate-

buffered saline. The ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- small intestine (n=4 per genotype) and colon (n=4 per genotype) tumor and surrounding normal tissue sections were incubated with a rabbit polyclonal anti-Ron ! C-20 antibody (Santa Cruz), rabbit

polyclonal anti-!-catenin antibody (NeoMarkers), or equivalent concentration of rabbit

IgG (Sigma) at a concentration of 2µg/ml. Primary antibodies were detected using

0.75µg/ml goat anti-rabbit biotinylated secondary antibody (Vector Labs). Antibody

immunoreactivity was amplified using the VECTASTAIN ABC kit (Vector Labs), and

visualized using DAB substrate (Vector Labs). The sections were counterstained in

hematoxylin, dehydrated, incubated with Citrisolv, and mounted. All images were

captured using a Nikon FX-35DX camera attached to the Nikon Microphot microscope

and Spotcam Advanced software (Nikon).

Proliferation Assay: Immunohistochemistry was performed on small intestine (n=4 per

genotype) and colon tumor (n=4 per genotype) and surrounding normal tissue sections

using an anti-PCNA mouse monoclonal antibody (BD Biosciences; 1 µg/ml), and goat

anti-mouse biotinylated secondary antibody (Vector Labs; 0.75 µg/ml) as described

above. The proliferation index was measured by quantification of PCNA-positive nuclei.

2-28 Meyer et al., Molecular Carcinogenesis, 2009.

Three fields of intact crypts per section were counted on a Nikon Microphot-FXA EP1-

FL3 microscope at 400X magnification. Quantification of PCNA-positive cells in

intestinal crypts from four different ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice were used to calculate the average number of proliferating cells per crypt. In the size- matched adenomas, the percentage of proliferating cells was determined by quantifying the number of PCNA-positive cells out of at least 600 total cells per section at 400X magnification from four different ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice.

Apoptosis Assay: Small intestine and colon tumor and surrounding normal tissue sections from ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice (n=4 per genotype) were

analyzed for apoptosis using the ApopTag Plus Peroxidase In Situ Apoptosis Detection

Kit (Chemicon International) according to the manufacturer’s instructions. Apoptosis in

the normal small intestine and colon was quantified by counting the number of TUNEL-

positive nuclei in intact crypts at 400X magnification in tissues from three different

ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- mice. In the size-matched adenomas, the

number of TUNEL-positive nuclei out of at least 600 total nuclei was quantified per

tumor at 400X magnification from three different ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/-

mice.

Real-time PCR Analyses: Duodenal and colon tissues were harvested from 3 month-

old C57BL/6J wild-type or RonTK-/- mice on the same background (n=6 each), snap

frozen in liquid nitrogen, and stored at -80°C. RNA was isolated from frozen tissues

using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA

2-29 Meyer et al., Molecular Carcinogenesis, 2009.

was generated from 2 !g of total RNA using the High Capacity cDNA Reverse

Transcription Kit (Applied Biosystems). Quantitative real-time PCR analysis was then

performed using the Applied Biosystems 7300 Real Time PCR System and Sequence

Detection Software Version 1.3.1 (Applied Biosystems). Each cDNA sample was run in

duplicate and amplified by Power SYBR Green PCR Master Mix (Applied Biosystems)

using the following primers: cyclin D1 forward 5’-ccatgaactacctggaccg-3’ and reverse

5’-cacaaacctctgtgcatgc-3’; c-myc forward 5’-ttccacggccttctctcctt-3’ and reverse 5’-

tcaatttcttcctcatcttcttgct-3’; Cox-2 forward 5’-cctgccccacagcaaact-3’ and reverse 5’-

ccttcctcccgtagcagatg-3’; Gapdh forward 5’-aatggtgaaggtcggtgtg-3’ and reverse 5’-

gaagatggtgatgggcttcc-3’. Table 1 presents the data as the average relative gene

expression normalized to Gapdh for each gene and shown as a fold change relative to

the wild-type control tissues. Each value represents 6 tissues per genotype from

individual mice analyzed in duplicate from one experiment; three experimental

replicates were performed with similar results.

Statistical Analyses: To assess tumor burden, a comparison of the mean tumor

numbers of ApcMin/+;RonTK+/+ and ApcMin/+;RonTK-/- was performed using the non- parametric Wilcoxon rank-sum test. Evaluation of the number of animals with tumor multiplicities greater than the median (29 tumors) of the ApcMin/+;RonTK+/+ mice was

performed using a binomial test. Statistical analyses of proliferation, apoptosis, and

real-time PCR analyses were performed using a Student t-test.

2-30 Meyer et al., Molecular Carcinogenesis, 2009.

Acknowledgements

The authors thank Drs. Laura James from the Department of Surgery at the University of Cincinnati and Dingcai Cao from the Department of Surgery at the University of

Chicago for statistical analyses. We also thank Dr. Gregory Boivin from the Department of Pathology at the University of Cincinnati for histopathological evaluation and Rita

Angel from the Department of Pathology at the University of Cincinnati for Ki67 immunohistochemistry.

2-31

Chapter 3

The Ron Receptor Tyrosine Kinase Negatively Regulates

Mammary Gland Branching Morphogenesis

Sara E. Meyer, Glendon M. Zinser, William D. Stuart, Peterson Pathrose, and Susan E. Waltz. 2009. The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis. Developmental Biology. Sep. 1; 333(1): 173-185. doi:10.1016/j.ydbio.2009.06.028 PMID: 19576199 " 2009 Elsevier Inc.

Meyer et al., Developmental Biology, 2009.

Preface

This chapter includes original research performed by Sara Meyer while under the

mentorship of graduate advisor Dr. Susan Waltz. S.E.M. partially contributed to Figures

1 and 2, contributed all in Figures 3, 4, 5, 6, 7, and Table 1; exact contributions of the

authors can be found in Chapter 6. The goal of this work was to determine the role of

the Ron receptor tyrosine kinase in pubertal mammary growth and morphogenesis

using wild-type and Ron tyrosine kinase domain-deficient mice. The results of this work were published in Developmental Biology Volume 333 Issue 1, pages: 173-185 on

September 1, 2009 [36].

3-2 Meyer et al., Developmental Biology, 2009.

Abstract

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is

overexpressed in approximately 50% of human breast cancers. Despite the recent

studies on Ron in breast cancer, nothing is known about the importance of this protein

during breast development. To investigate the functional significance of Ron in the

normal mammary gland, we compared mammary gland development in wild-type mice

to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of

Ron (TK-/-). Mammary glands from RonTK-/- mice exhibited accelerated pubertal

development including significantly increased ductal extension and branching

morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of

epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK,

which predominantly localized to the epithelium, were associated with increased

branching morphogenesis. Additionally, purified RonTK-/- epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK-/- glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.

3-3 Meyer et al., Developmental Biology, 2009.

Abbreviations

BrdU bromodeoxyuridine

DAVID database for annotation, visualization and integrated discovery

Fak focal adhesion kinase

HGFL hepatocyte growth factor-like protein

HMEC human mammary epithelial cells

HPAC human primary adipocytes cells

LN lymph node

LY294002 phosphatidylinositol-3-kinase inhibitor

MAPK mitogen-activated protein kinase, also known as Erk

PD98059 mitogen-activated protein kinase kinase (MEK1) inhibitor

PI3K phosphatidylinositol-3-kinase

PyMT polyoma middle T-antigen

TEB terminal end bud

TGF! transforming growth factor beta

TK tyrosine kinase domain

TUNEL terminal deoxynucleotidyl transfer mediated dUTP nick end labeling

3-4 Meyer et al., Developmental Biology, 2009.

Introduction

Mammary gland development is a highly regulated, intricate, and continuous process

throughout the life of an animal beginning in the embryo, and continuing postnatally

during puberty, pregnancy, lactation, and involution [173,175]. Importantly, studies

have shown that many of the factors necessary for proper mammary gland development

are also deregulated during breast cancer. Therefore, it is imperative to continue to

study novel regulators of mammary development in order to gain further insight and

understanding of breast tumorigenesis [31,226].

During normal pubertal mammary gland development in mice (5-10 weeks of age),

generation of the mammary ductal tree occurs through two simultaneous morphological

processes- ductal elongation and branch formation [227,228]. The mammary

epithelium elongates into the fat pad by proliferation of the terminal end buds (TEB) and

simultaneous hollowing out of the end buds by apoptosis to form ducts to create the

primary ductal network [228]. There are two types of branching that can occur in a pubertal mouse mammary gland, TEB bifurcation and lateral side branching from existing ducts [227,229]. While the exact mechanisms that regulate the length, placement, and number of ducts is not fully understood, proper ductal elongation and branching morphogenesis during puberty is necessary to provide enough surface area for alveoli to form during pregnancy and lactation to supply an adequate amount of milk to nurse young pups.

3-5 Meyer et al., Developmental Biology, 2009.

Branching morphogenesis is subjected to complex positive and negative regulatory

signals, generated from the surrounding stroma, serum, and crosstalk between these

components and the epithelium that orchestrate the growth of the mammary epithelium

during pubertal development [230]. The ovarian hormones estrogen and progesterone and their receptors induce ductal elongation and side branching, respectively, and are required for proper mammary gland development [231]. In addition, growing evidence supports that growth factor and receptor tyrosine kinase signaling are essential for proper mammary development and branching morphogenesis [16]. Receptor tyrosine kinases function to transduce extracellular signals inside the cell, as well as crosstalk with other cell surface molecules [232]. It has been shown that receptor tyrosine kinases play a key role in the communication between the mammary epithelium and surrounding mammary gland environment, including stroma and sera, to contribute both positive and negative regulatory signals during mammary gland development [171].

Previous studies have shown that the molecules that regulate pubertal mammary gland development are frequently deregulated or overexpressed during mammary tumorigenesis [16,31]. Receptor tyrosine kinases are often overexpressed in human breast cancers and are a desirable target for cancer therapeutics [233]. In addition, mammary-specific overexpression of several receptor tyrosine kinases has been shown to drive mammary tumorigenesis in mice and, in more rare circumstances, metastasis

[234,235].

3-6 Meyer et al., Developmental Biology, 2009.

The membrane spanning receptor tyrosine kinase Ron, a member of the Met family, has

recently been shown to be overexpressed in a variety of human cancers including

breast cancer [7,29,45,74]. Importantly, mammary-specific overexpression of Ron in the mouse gives rise to tumors with 100% incidence that progress to mammary carcinomas that metastasize with high frequency [30]. The Ron receptor consists of a

35 kDa alpha chain, with ligand binding capacity, joined by disulfide bonds to a 150 kDa beta chain containing the transmembrane and intracellular tyrosine kinase domains

[236]. In humans and mice, Ron is expressed in many tissues including the mammary gland [29,32]. Hepatocyte growth factor-like protein (HGFL), also known as macrophage stimulating protein, is the ligand for Ron and is present in the circulation

[39,45]. Upon binding of HGFL to Ron, receptor dimerization and tyrosine autophosphorylation occurs for activation. Downstream signaling targets of Ron activity include PI3K, Src, FAK, Akt, and MAPK that can lead to proliferation, cell survival, cell motility, cell shape change, and invasion [78,203].

Ron mRNA is increasingly expressed throughout pubertal mammary gland development in mice [32]; however, nothing is known about the morphological impact Ron signaling has on pubertal mammary gland development. Based on our previous studies indicating that Ron signaling is sufficient to induce mammary tumorigenesis, we hypothesized that Ron receptor signaling would impact postnatal mouse mammary gland development. To test this hypothesis we compared pubertal mammary gland development (5-10 weeks) in wild-type (RonTK+/+) and Ron tyrosine kinase domain null mice (RonTK-/-). We are the first to report that Ron signaling profoundly impacts

3-7 Meyer et al., Developmental Biology, 2009. pubertal mouse mammary gland development. Surprisingly, we found that in the absence of Ron signaling, RonTK-/- mice exhibited significantly increased ductal extension and branching morphogenesis without significant changes in epithelial cell turnover. Furthermore, using ovariectomized mice we show that mammary glands from

RonTK-/- mice also displayed excessive branching morphogenesis, compared to wild- type controls. In conjunction with increased branching, we also observed elevated phosphorylation of Akt and MAPK in RonTK-/- mammary glands compared to controls.

Additionally, isolated primary RonTK-/- mammary ductal epithelial fragments

(organoids) demonstrated a significant increase in branching morphogenesis, ex vivo, that was blocked by MAPK inhibition. By microarray analysis, deletion of the Ron tyrosine kinase domain significantly altered the genetic profile of pubertal mammary glands in comparison to wild-type control glands with many of the genes grouped into developmental and morphological categories. Taken together, these results demonstrate that the Ron receptor tyrosine kinase is a novel and important regulator of pubertal mouse mammary gland development.

3-8 Meyer et al., Developmental Biology, 2009.

Results

The Ron receptor is expressed in the mouse mammary gland at specific phases

of glandular development. Previous studies on intact mammary glands have shown

that Ron mRNA expression increases progressively during ductal morphogenesis, is

down regulated at the onset of pregnancy, and remains low throughout the remainder of

postnatal mammary development [32]. Since the ligand for Ron, HGFL, is a motility

factor that promotes epithelial cell migration [237], and since Ron is a member of the c-

Met family of receptor tyrosine kinases of which Met and its ligand, hepatocyte growth

factor (HGF), have been shown to induce branching morphogenesis [238,239], we

hypothesized that Ron may contribute to the rapid epithelial migration and branching

characteristic of mammary ductal morphogenesis. Our first objective was to define the

cellular compartment in which Ron is expressed in the mammary gland. Prior studies

had only demonstrated Ron mRNA expression from whole gland homogenates. Using

primers that span the beginning of the tyrosine kinase domain, which is deleted in the

RonTK-/- mice [17], we found that Ron mRNA is expressed in wild-type virgin mouse

mammary glands at 5-8 weeks of age, but undetectable at 10 weeks of age (Figure 1A).

Interestingly, Ron expression was also observed in later phases of mammary

development including pregnancy (timed mating day 16), lactation, and involution day 4,

but was undetectable by involution day 10 (Figure 1A). We did not detect a PCR

product in the RonTK-/- mammary glands using these primers as predicted (Figure 1A).

However, utilizing a set of primers directed to a region upstream of the TK deletion did

not amplify a Ron product in the RonTK-/- glands (data not shown). This is consistent

with previously published data characterizing the RonTK-/- mice [17]. Correspondingly,

3-9 Meyer et al., Developmental Biology, 2009.

Ron protein expression was observed during mammary development in wild-type virgin mouse mammary glands 5-8 weeks of age, pregnancy, and lactation (Figure 1B). Ron protein expression was undetectable in mammary glands harvested on involution day

10 and in mammary fat pads devoid of epithelium (Figure 1B). Further, Ron protein expression was also observed in the normal mouse mammary epithelial cell line Eph4, and the normal human mammary epithelial cell lines MCF10A and HMEC (Figure 1B and 1C-right). Despite the scant Ron mRNA expression in the human primary adipocyte cell line (HPAC) by real-time PCR (Figure 1C, left), Ron protein was undetectable in these cells by Western analysis (Figure 1C, right).

Immunohistochemical detection of Ron in 6 week-old virgin wild-type mammary glands showed an intense staining pattern of Ron in the mammary epithelium of both ducts and terminal end buds (Figure 1D). Taken together, these data suggest that Ron is expressed during specific time frames throughout mouse mammary gland development, primarily in the epithelial compartment in both humans and mice with low to undetectable levels of Ron expression in adipose tissue.

Deletion of the Ron tyrosine kinase domain accelerates pubertal mammary gland development. To examine the functional contribution of Ron during mammary development, a temporal analysis of glandular architecture was undertaken in wild-type mice (RonTK+/+) and mice with homozygous deletion of the tyrosine kinase domain of

Ron (RonTK-/-). Developmental analysis of whole mount preparations from 5, 6, 7, 8, and 10 week-old virgin female RonTK-/- mouse mammary glands were compared to age and weight-matched RonTK+/+ control glands. Surprisingly, the RonTK-/-

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mammary glands displayed accelerated branching morphogenesis as evident by the

denser mammary ductal tree compared to RonTK+/+ controls (Figure 2A). To evaluate

the developmental progress of the RonTK+/+ and RonTK-/- mammary glands, terminal

end bud (TEB) number, ductal elongation, and secondary and tertiary branch points

were quantified. Ductal outgrowth, measuring from the center of the lymph node to the

furthest TEB, was significantly increased in RonTK-/- mammary glands at 6 and 7

weeks of age as compared to RonTK+/+ controls (Figure 2B). We also saw a trend

toward an increase in TEB number at 5-8 weeks in the RonTK-/- glands, but these

differences did not reach statistical significance in this study (Figure 2C). At 10 weeks,

TEB number significantly decreases in RonTK-/- glands compared to RonTK+/+ controls (Figure 2C), correlating with the completion of ductal morphogenesis in the

RonTK-/- glands compared to controls. Most strikingly, secondary and tertiary branch points were significantly increased in mammary glands from 6, 7, and 8 week-old

RonTK-/- mice as compared to RonTK+/+ controls (Figure 2D and 2E). In spite of the significant developmental acceleration displayed in the mammary glands from RonTK-/- mice throughout 6-8 weeks of age, ductal extension and branching morphogenesis equalize with the RonTK+/+ mice by 15 weeks of age. Moreover, the RonTK-/- mice are overtly normal with respect to fertility, pregnancy, and lactation.

The Ron receptor regulates mammary branching morphogenesis in ovariectomized mice. Estrogen, a nuclear hormone, responsible for mammary epithelial growth and ductal elongation, and progesterone, also a nuclear hormone, predominately responsible for ductal side branching are both essential for normal

3-11 Meyer et al., Developmental Biology, 2009. pubertal mammary gland development [176,240,241]. To test if the RonTK-/- mice may have accelerated branching morphogenesis due to an alteration in serum hormone levels, we analyzed serum that was isolated from 5, 6, and 7 week-old virgin female

RonTK+/+ and RonTK-/- mice and found no significant differences in circulating estrogen or progesterone levels (Figure 3A). To further examine the role of Ron in branching morphogenesis in the absence of circulating hormones, RonTK+/+ and

RonTK-/- mice were ovariectomized at 3 weeks of age prior to pubertal onset.

Following ovariectomy the mammary glands were allowed to develop for three weeks prior to isolation at 6 weeks of age and analyzed for ductal elongation and secondary and tertiary branch events. Examination of ovariectomized mice at the time of sacrifice confirmed that all ovaries were removed from the mice in this study. Interestingly, mammary glands from ovariectomized RonTK-/- mice demonstrated significantly increased secondary and tertiary branching events compared to mammary glands from ovariectomized RonTK+/+ controls (Figure 3B and C). Of note, all end buds in the ovariectomized mice regardless of genotype were considerably smaller than in mice with intact ovaries. Although ovariectomized RonTK+/+ mice appear to have similar average numbers of secondary branches as RonTK+/+ mice with intact ovaries

(compare Figure 2D and 3B), the ovariectomized mice had significantly fewer secondary branches overall (and correspondingly less ductal morphogenesis) compared to their wild-type counterparts, and the lengths of these secondary branches were significantly blunted. In total, these data suggest that Ron signaling may regulate mammary branching morphogenesis, at least in part, independent of ovarian hormone stimulation.

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RonTK-/- mammary cell turnover is similar to RonTK+/+ controls. We

hypothesized that a potential mechanism by which Ron might regulate mammary gland

development is by modulation of mammary epithelial cell homeostasis (i.e. through

changes in cell proliferation and/or cell death). While the histological appearance of

RonTK-/- (Figure 4E-F) ductal structures and terminal end buds were similar to those of the RonTK+/+ control glands (Figure 4A-B), an overall increase in the number of ductal structures in RonTK-/- glands was dramatically apparent (Figure 4E vs. 4A). To examine mammary epithelial cell turnover rates, mammary glands from 5, 6, and 7

week-old RonTK+/+ and RonTK-/- mice were analyzed for proliferation by

bromodeoxyuridine (BrdU) incorporation and for apoptosis by terminal nick end labeling

(TUNEL). Similar percentages of BrdU (Figure 4C and G) and TUNEL staining (Figure

4D and H) epithelial cells were detected in the end buds, as well as ducts (data not

shown), at all time points analyzed suggesting that Ron ablation does not alter

mammary epithelial cell turnover.

Ron signaling in the mammary gland negatively regulates the activation of

signaling pathways critical for enhanced branching morphogenesis. Previous

evidence has shown that Akt and MAPK are important for mammary branching

morphogenesis [16,242]. Given that these are hallmark signaling pathways downstream of the Ron receptor tyrosine kinase, we tested whether the impact of Ron signaling on mammary branching morphogenesis may modulate the regulation of the

Akt and/or MAPK signaling. Western analysis of proteins isolated from whole mammary

3-13 Meyer et al., Developmental Biology, 2009.

glands from 6 week-old RonTK+/+ and RonTK-/- mice revealed significantly increased phosphorylation of Akt and MAPK proteins in RonTK-/- mammary glands (Figure 5A).

Moreover, immunohistochemical analyses of mammary glands show elevated phosphorylation of MAPK (Figure 5B) localized predominantly in the mammary epithelium of RonTK-/- mice, as compared with RonTK+/+ controls. Alternatively, immunohistochemical analysis of Akt phosphorylation showed a more diffuse staining pattern in the mammary stromal fat pad and epithelium, with more staining observed overall in RonTK-/- glands as compared to RonTK+/+ controls (data not shown).

Ron tyrosine kinase and MAPK signaling regulate mammary epithelial branching morphogenesis in ex vivo cultures. To test for the importance of Ron signaling emanating from isolated mammary epithelial cells, branching morphogenesis was analyzed in primary mammary epithelial organoid cultures purified from virgin female

RonTK+/+ and RonTK-/- mice. The isolated organoids were embedded in growth factor reduced Matrigel, cultured for 6 days, and analyzed for branching morphogenesis. No branching morphogenesis was evident on the day of organoid embedding. Figure 6A shows representative images of the predominant phenotypes of RonTK+/+ and RonTK-

/- organoid structures observed which included spheres (Figure 6A), buds (black arrows), and branches (white arrows). The number of organoids displaying buds and branches out of the total number of organoids present were quantified. As shown graphically in Figure 6E, significantly more RonTK-/- (Figure 6B and 6D) mammary epithelial organoids developed buds and branches (71%) compared to the RonTK+/+

(Figure 6A and 6C) organoids (32%), of which the predominant phenotype was sphere

3-14 Meyer et al., Developmental Biology, 2009. formation (Figure 6A). Given that phosphorylated MAPK was primarily localized to the mammary epithelium of the RonTK-/- mice (Figure 5B), we hypothesized that this activity was responsible for the exaggerated branching in the mutant animals. To test whether MAPK activity played a role in branching morphogenesis in the RonTK-/- mammary epithelium, RonTK-/- and RonTK+/+ organoids embedded in Matrigel were treated with or without the MAPK inhibitor PD98059 at a final concentration of 2µM for six days. MAPK inhibition resulted in nearly complete inhibition of branching morphogenesis of RonTK-/- mammary epithelial organoids compared to vehicle treated controls (Figure 6E). At a dose of 5µM, PD98059 completely blocked branching in both

RonTK+/+ and RonTK-/- organoids, however sphere formation was compromised suggesting toxicity associated at this dose. In a similar set of studies, 5µM of the Akt inhibitor LY294002 resulted in a modest 10% decrease in the number of RonTK+/+ mammary epithelial organoids with branches and a 15% decrease in RonTK-/- organoids with branches compared to vehicle treated controls. These differences, however, were not statistically significant. Higher concentrations of LY294002 also compromised sphere formation, again suggesting toxicity effects. Taken together, these data suggest that MAPK activity in the RonTK-/- mammary epithelium is critical for branching morphogenesis ex vivo.

Deletion of the Ron tyrosine kinase domain yields profound effects on gene expression during mammary gland development. To examine the role of Ron during mammary development in more detail, we compared the gene expression profiles of mammary gland mRNA harvested from 5, 6, and 7 week-old RonTK+/+ and

3-15 Meyer et al., Developmental Biology, 2009.

RonTK-/- female mice. The mRNA was subjected to microarray using the murine 430 chip from Affymetrix. The hybridization profiles from three independent RonTK+/+ mice per time point were averaged as baseline controls for comparison to RonTK-/- experimental animals. ANOVA analyses identified 393 genes that were significantly differentially expressed in RonTK-/- mammary glands compared to RonTK+/+ control mice over 5, 6, and 7 weeks of age are illustrated in a heat map (Figure 7A), and are depicted temporally in a Venn diagram in Figure 7B. A complete list of the genes in the heat map and Venn diagram can be found in the Supplementary Tables S1 and S2-S4, respectively. These analyses have provided several types of information. First, a number of genes that change during each developmental window based on Ron expression were identified. Interestingly, the majority of genes (188 genes) are differentially expressed solely at the 6 week time point (Supplemental Table S3).

Second, genes that overlap in any two of the three time points were found

(Supplemental Table S5). Only 7 genes fell into this category including Mrpl3, Mid1,

Cdkal1, Pcdh17, and three other unstudied sequences. Finally, 10 genes were differentially expressed following Ron ablation at all time points including Tmem30a,

Mapk6, Nck1, Acpl2, Srprb, and other unstudied sequences (Supplemental Table S5).

Using DAVID functional annotation analyses on the 686 genotypically differentially expressed genes, a number of biological processes were significantly altered including morphogenesis, cell motility, adhesion, and development (Figure 7C, Supplemental

Tables S6-S12). These results demonstrate that Ron tyrosine kinase receptor signaling significantly affects that genetic profile of the developing mammary gland.

3-16 Meyer et al., Developmental Biology, 2009.

To validate the gene expression changes observed in our microarray analyses, real-

time quantitative PCR was used. Gene expression changes of Ceacam10, Acpl2, and

Pcdh17 in RonTK-/- mammary glands were examined and values were normalized to

18S as loading control and graphed as fold change relative to RonTK+/+ controls (Table

1). Microarray results for these genes suggested an increasing trend in Ceacam10 over

time, a 2-fold increase in Pcdh17, and a 60% reduction in Acpl2 transcripts in RonTK-/-

mammary glands compared to RonTK+/+. Importantly, our real-time PCR recapitulated

the trends observed in gene expression found by microarray analysis for transcripts

tested.

3-17 Meyer et al., Developmental Biology, 2009.

Figures and Tables

Figure 1. The Ron receptor is expressed throughout post-natal mouse mammary gland development, predominantly in the epithelium. A) Real-time PCR analysis for

3-18 Meyer et al., Developmental Biology, 2009.

Ron mRNA expression using RNA isolated from wild-type 5, 6, 8, and 10 week-old virgin, pregnant (timed mating day 16), lactating (day 10), and involuting (days 4 and

10) mouse mammary glands (a minimum of 3 mice per time point were used).

Involuting mammary glands from RonTK-/- mice serve as a negative control with the primer set homologous to sequences spanning the deleted region in the RonTK-/- mice.

Ron expression values were normalized to 18S as an internal control and the average expression level of Ron relative to 18S from two independent experiments ± SEM for each time point is illustrated. B) Western analysis of Ron expression in wild-type 5, 6,

8, and 10 week-old virgin, pregnant, lactating, and involuting (day 4) mouse mammary glands, 3 week-old wild-type female stromal mammary fat pads devoid of epithelium and lymph node (Fat pad), and normal mouse mammary epithelial cell line Eph4. Parp and Actin serve as loading controls. Each lane represents an individual animal; experiments were repeated three times with different animals and cell isolations showing similar results. C) Real-time PCR analysis of Ron mRNA expression in RNA isolated from normal human mammary epithelial immortalized cell line (HMEC) and normal human primary adipocytes (HPAC) (left). Expression values were normalized to

18S as an internal control and the average expression level of Ron in each cell line relative to 18S from three independent experiments ± SEM is illustrated. Western analysis of Ron expression in lysates prepared from the normal human mammary epithelial cells MCF10A and HMEC, and normal human primary adipocytes HPAC is depicted on the right. Actin serves as loading control. Experimetns were repeated three times with different cell isolations showing similar results. D) Mammary gland sections from 6 week-old virgin wild-type mice (n=3) were subjected to

3-19 Meyer et al., Developmental Biology, 2009. immunohistochemistry with an antibody directed against the Ron receptor tyrosine kinase. Ron positive cells appear brown against the blue hematoxylin counterstain.

Ron expression primarily localized to the ductal epithelial cells (a, block arrow) and the terminal end buds (b, block arrow). Staining with an isotype control is shown as a negative control (c & d, block arrows and arrowheads). Experiments were repeated twice with different mice showing similar results. The lymph node (LN) is indicated for orientation.

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Figure 2. RonTK-/- mice have significantly accelerated pubertal mammary gland development. Mammary glands were isolated from 5, 6, 7, 8, and 10 week-old female

RonTK+/+ and RonTK-/- mice (n=10 per genotype) and examined by whole mount analysis. A) Images of representative RonTK+/+ and RonTK-/- age and weight- matched mammary glands illustrate the denser ductal network (arrows indicate representative ductal/branch structures) and accelerated epithelial fat pad penetration in

RonTK-/- mice observed throughout the developmental time course. The mammary lymph node (LN) is indicated for orientation. Scale bar; 5mm. The developmental parameters average ductal elongation (B), terminal end bud number (C), and number of secondary (D) and tertiary branch points (E) were quantified as described in the materials and methods and graphed as averages per group ± SEM. * p < 0.05 compared to the corresponding control group by Student t-test.

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Figure 3. RonTK-/- mammary glands display significantly increased branching after ovariectomization. Circulating estrogen and progesterone levels were measured by ELISA in serum isolated from 5, 6, and 7 week-old RonTK+/+ and RonTK-/- mice

(n=6 per group). The average concentration of estrogen and progesterone ± SEM are illustrated (A). B) RonTK+/+ and RonTK-/- mice (n=3 per genotype) were

3-23 Meyer et al., Developmental Biology, 2009. ovariectomized at 3 weeks of age and sacrificed at 6 weeks of age, at which point whole mounts were generated from their mammary glands and representative images are shown (top). The mammary lymph node (LN) is indicated for orientation. Arrows point to representative branches observed. Scale bar; 2mm. The average number of secondary and tertiary branch points per genotype were quantified and graphed ± SEM

(C). *p < 0.05 compared to the corresponding control group by Student t-test.

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Figure 4. Histological appearances and rates of proliferation and cell death of

RonTK+/+ and RonTK-/- mammary epithelium. 6 week-old RonTK+/+ (A-D) and

RonTK-/- (E-H) mammary glands were paraffin embedded, sectioned, and stained with hematoxylin and eosin. Representative sections are shown demonstrating the histological appearance of the mammary ducts (A and E), terminal end buds (B and F).

Arrows depict the increased number of ductal structures observed in the RonTK-/- glands (E) compared to controls (A). Cellular proliferation and death were analyzed by

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immunohistochemical detection of BrdU incorporation in end buds (C and G) and

TUNEL staining of the end buds (D and H). The percentage ± S.E.M of BrdU and

TUNEL-positive cells out of total cells (n=4 mice per genotype and 4 end buds per

mouse were quantified) is indicated in the upper right hand image corner (C, D, G, and

H). Images are all from representative 6 week-old mouse mammary glands. Scale bar;

100µm.

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Meyer et al., Figure 5

Figure 5. RonTK-/- mouse mammary glands contain significantly increased

phosphorylated Akt and MAPK. A) Western analyses of proteins isolated from

RonTK+/+ and RonTK-/- mammary glands using anti-phospho Akt (pAkt), anti-Akt(pan),

anti-phospho-MAPK (pMAPK), and anti-total MAPK. Actin served as loading control.

Each lane represents an individual mouse, n=4 per genotype. Experiment was

repeated twice with different animals showing similar results. Immunohistochemical

analysis of phosphorylated MAPK localization using an anti-phospho-MAPK antibody

(B) as illustrated by brown staining in the end buds (top panels) and ducts (bottom

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panels) of RonTK+/+ and RonTK-/- mammary glands (n=4 per genotype). Scale bar;

100µm. Meyer et al., Figure 6

Figure 6. RonTK-/- primary mammary epithelial organoids display significantly advanced branching in vitro that is reduced upon MAPK inhibition. Primary mammary epithelial cells (organoids) were purified from female RonTK+/+ (A and C) and RonTK-/- (B and D) mice and cultured in Matrigel for 6 days. A-D) Representative

images of organoids cultured in growth medium described in materials and methods

3-28 Meyer et al., Developmental Biology, 2009. with no protrusions, buds (black arrows), or branches (white arrows). Images represent the extent of branching exhibited in each genotype observed in multiple independent experiments. Scale bar; 100µm. E) RonTK+/+ and RonTK-/- organoids were cultured in Matrigel for 6 days treated with 2µM PD98059 or equivalent volume vehicle (DMSO) control. The number of organoids with branches were quantified in three independent experiments, averaged, and depicted as percentage of branched organoids ± SEM. At least 260 organoids were quantified per group. * p < 0.05 comparing vehicle treated

RonTK+/+ and RonTK-/- organoids by Student t-test. ** p < 0.05 comparing vehicle and

PD98059 treated RonTK-/- organoids by Student t-test.

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Figure 7. RonTK-/- mammary glands have significantly altered gene expression patterns compared to RonTK+/+ controls during development. RNA isolated from the mammary glands of 5, 6, and 7 week-old RonTK+/+ and RonTK-/- female mice was run on a Affymetrix Genechip Array. Probe sets were first filtered based on a minimum raw expression value of 120, then RonTK+/+ probes were averaged as a baseline control for comparison. An ANOVA was performed comparing genes altered in RonTK-

/- mammary glands to the baseline control. A) Gene tree order represents 393 genes altered in RonTK-/- mammary glands according to age, where each gene is represented as a single colored line that corresponds to an expression value. Each vertical column represents a single mouse, and each age group contains three individual mice. B) A

Venn diagram was constructed from the gene list used in A to demonstrate genes altered in RonTK-/- mammary glands compared to controls that are unique to 5, 6, or 7 weeks of age, or are shared in common over two or more age groups. Some of the genes shared in common between 5 and 7 weeks of age are Mrpl3, Mid1, and Cdkal1;

6 and 7 weeks is Pcdh17; 5, 6, and 7 weeks are Mapk6, Nck1, Acpl1, Srprb, and

Synaptotagmin binding. C) DAVID functional annotation software was used to group genes into biological processes that were significantly altered in RonTK-/- mammary glands compared to RonTK+/+ baseline controls. Supplemental tables of the complete gene lists and fold change expression data are on page 3-33 of this Chapter.

3-31 Meyer et al., Developmental Biology, 2009.

Table 1. Real-time PCR validation of mammary gland microarray analysis. From

the microarray analyses, RonTK-/- gene raw expression values are shown as fold-

change relative to the average RonTK+/+ value. For real-time PCR, RonTK-/- relative gene expression values were first normalized to 18S as a loading control, and then expressed as fold-change of RonTK-/- expression relative to RonTK+/+ control.

Fold Change Genes: Microarray Real-Time PCR Ceacam10 5 week 1.44 1.08 6 week 1.94 2.08 7 week 4.76 2.49

Acpl2 5 week 0.31 0.26 6 week 0.29 0.33 7 week 0.36 0.30

Pcdh17 5 week 2.62 2.32 6 week 3.12 2.72 7 week 1.82 1.71

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Supplemental Table S2. 98 genes differentially expressed in 5 week-old RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1435810_at 5730455O13Rik 0.97 1.05 0.00496 1435036_at A530050D06Rik 0.84 1.32 0.00490 1443973_at Dact2 0.99 1.10 0.00475 1436754_at AI839735 0.99 1.10 0.00470 1450564_x_at Ifna1 1.01 1.13 0.00469 1426071_at Tiaf2 0.96 1.10 0.00469 1452891_at 5730568A12Rik 0.98 1.09 0.00464 1443557_at --- 0.96 1.09 0.00453 1419660_at 1600012F09Rik 0.93 1.74 0.00453 1423758_at E430034L04Rik 0.97 1.09 0.00450 1458932_at Pex2 0.99 1.07 0.00440 1460304_a_at Ubtf 1.01 0.88 0.00437 1419400_at Mttp 1.11 0.79 0.00425 1448309_at Ap3m1 0.95 1.06 0.00418 1436255_at D830044I16Rik 0.96 1.08 0.00417 1436004_at Usp27x 1.01 0.83 0.00412 1448796_s_at Tbrg4 0.97 1.11 0.00411 1430378_at 2900011G08Rik 0.97 1.18 0.00408 1424839_a_at 2810405F18Rik 0.98 1.11 0.00408 1423393_at Clic4 1.01 0.85 0.00398 1427904_s_at 2410091C18Rik 0.89 0.97 0.00394 1450606_at Pnmt 0.97 1.05 0.00394 1427418_a_at Hif1a 0.95 1.12 0.00387 1438800_at Nagk 1.01 0.83 0.00387 1425218_a_at Scgb3a2 1.00 1.09 0.00379 1450807_at Ltb4r2 1.03 0.93 0.00370 1438556_a_at Tmod3 0.97 0.77 0.00367 1449557_at 1600012F09Rik 0.99 1.66 0.00365 1443500_at Mllt10 0.90 1.04 0.00363 1449488_at Pitx1 0.99 1.03 0.00361 1456991_at Cobll1 0.98 1.08 0.00351 1423858_a_at Hmgcs2 1.44 0.68 0.00348 1428863_at Ankrd39 0.97 1.06 0.00345 1434773_a_at Slc2a1 0.98 1.17 0.00344 1422768_at Syncrip 0.96 1.20 0.00342 1436753_at Adck5 1.03 0.88 0.00339 1417627_a_at Limk1 1.06 0.86 0.00331 1431905_s_at 4933427G17Rik 1.04 0.89 0.00327 1430078_a_at Ogg1 1.00 1.07 0.00327 1438812_x_at Usp19 1.00 1.03 0.00325 1419609_at Ccr1 1.26 0.81 0.00319 1442307_at --- 1.07 0.73 0.00317 1449014_at Lactb 0.92 1.05 0.00317 1416326_at Crip1 1.09 0.93 0.00309 1431380_at 5730409L17Rik 0.90 1.03 0.00301 1428588_a_at 2810443J12Rik 0.98 1.07 0.00286 1443698_at Fbxo39 1.32 0.87 0.00278

3-44 Meyer et al., Developmental Biology, 2009.

1425986_a_at Dcun1d1 0.95 1.09 0.00264 1427882_at 4930588M11Rik 1.01 0.94 0.00264 1435604_at Trim37 1.00 1.12 0.00264 1453868_at Ccdc11 1.04 0.95 0.00257 1419043_a_at Iigp1 1.05 0.78 0.00244 1428660_s_at Tor3a 1.12 0.90 0.00242 1453885_at Slc24a2 0.98 1.16 0.00235 1424166_at Msh3 0.98 1.10 0.00233 1455511_at Sephs1 0.99 0.84 0.00232 1456100_at Selv 0.99 1.06 0.00228 1450421_at Tgfa 1.00 1.08 0.00226 1448670_at Ube2e3 0.98 1.03 0.00225 1425043_s_at 0610037D15Rik 1.02 0.87 0.00219 1442619_at Klf15 1.00 1.09 0.00216 1448263_a_at Cndp2 1.08 0.93 0.00216 1442789_at --- 0.98 1.06 0.00211 1415848_at Csh1 1.00 1.08 0.00210 1421911_at Stat2 1.15 0.88 0.00201 1430893_at Mup1 0.79 3.71 0.00193 1416742_at Cfdp1 1.00 0.95 0.00189 1456631_at --- 1.00 1.06 0.00185 1431304_a_at 1300007B12Rik 0.95 1.04 0.00177 1455804_x_at Oxct1 0.96 1.35 0.00173 1447667_x_at Map3k4 0.98 1.07 0.00172 1432096_at Snrpn 1.01 1.15 0.00161 1445626_at Lgals3 1.19 0.76 0.00156 1428199_at 4930578F03Rik 1.01 1.10 0.00135 1432475_at 1700014B07Rik 0.99 1.12 0.00128 1431644_a_at Ica1 0.94 1.15 0.00126 1421998_at Tor3a 1.23 0.98 0.00120 1457774_at 2310079N02Rik 0.90 1.02 0.00117 1427650_a_at Runx1 0.99 1.14 0.00113 1460444_at Arrb1 1.00 0.87 0.00111 1420265_x_at Cog8 1.00 1.07 0.00105 1419168_at Mapk6 0.93 1.15 0.00088 1421061_at Guca1a 0.86 0.98 0.00083 1460177_at Cndp2 1.03 0.90 0.00080 1428941_at Zfp198 0.95 1.02 0.00075 1455765_a_at Abcc8 0.87 1.25 0.00069 1450355_a_at Capg 1.08 0.89 0.00067 1426840_at Ythdf3 0.97 1.09 0.00064 1415697_at E430034L04Rik 0.98 1.16 0.00061 1423129_at Shoc2 0.95 1.04 0.00052 1449628_s_at Stard7 1.00 1.13 0.00050 1416655_at C1galt1c1 0.96 1.03 0.00046 1420894_at Tgfbr1 0.98 1.16 0.00033 1453580_at 5630401D24Rik 0.96 1.03 0.00032 1448841_at Pfpl 1.01 0.90 0.00028 1456316_a_at Acbd3 1.11 0.97 0.00018 1442481_at Dusp4 0.95 1.04 0.00002 1426808_at Lgals3 1.21 0.72 0.00001

3-45 Meyer et al., Developmental Biology, 2009.

Supplemental Table S3. 188 genes differentially expressed in 6 week-old RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1425991_a_at Ankrd25 1.07 0.84 0.00499 1425056_s_at Saps2 1.04 0.88 0.00497 1431769_at 2210406O10Rik 1.02 0.96 0.00493 1425813_at Pign 0.98 1.13 0.00492 1422907_at Gnat2 1.04 0.90 0.00491 1434270_at Nptxr 1.04 0.93 0.00489 1455638_at AI225750 0.83 1.00 0.00483 1442934_at C81608 1.11 0.98 0.00479 1454841_at 4921511H13Rik 0.99 1.11 0.00477 1442075_at AI314604 1.09 0.81 0.00471 1434793_at Wdr78 0.96 1.19 0.00470 1453269_at Unc5b 1.06 0.96 0.00469 1434320_at Gtf3c4 0.95 1.09 0.00467 1460589_at Zfp597 0.98 1.41 0.00467 1442541_at Oaz2 1.01 0.91 0.00459 1443551_at Atp2a2 0.82 1.10 0.00457 1435546_a_at 1810013L24Rik 0.92 1.13 0.00454 1449843_at St8sia2 1.08 0.97 0.00454 1452321_at Brwd1 0.90 1.06 0.00453 1457256_x_at Ptch2 1.05 0.89 0.00452 1460500_at 5033421C21Rik 1.06 0.74 0.00449 1428583_at 1110001M19Rik 0.93 1.16 0.00446 1455059_at 9430093I07Rik 0.90 0.97 0.00444 1422490_at Bnip2 0.99 1.09 0.00442 1437892_at Zfp306 0.93 1.53 0.00442 1421667_at Nmur1 1.04 0.93 0.00440 1458655_at Wwtr1 1.02 0.86 0.00440 1421012_at Srprb 1.02 1.32 0.00435 1421409_at Msi1h 1.02 0.86 0.00432 1452435_at Wdr22 1.01 1.10 0.00429 1428666_at Nars 0.99 1.08 0.00425 1419096_at Apom 1.04 0.94 0.00419 1439890_at Fbxl7 1.08 0.95 0.00418 1447471_at --- 1.00 1.09 0.00417 1437969_s_at 0610007P22Rik 0.96 1.08 0.00415 1454470_at 4930404F17Rik 1.08 0.97 0.00409 1433528_at Gtf2a2 1.01 0.94 0.00408 1418978_at MGI:1927479 0.96 1.03 0.00405 1441802_at Spred2 1.02 0.88 0.00394 1442735_at Oaz2 1.02 0.81 0.00394 1433846_s_at C430003P19Rik 0.99 1.04 0.00388 1449585_at Il1rap 1.00 1.16 0.00386 1443787_x_at Casp14 1.02 0.94 0.00383 1424296_at Gclc 1.05 1.63 0.00383 1434742_s_at 2810401C16Rik 1.01 0.92 0.00380 1440432_at Lztfl1 1.00 1.15 0.00375 1425282_at Ibrdc2 0.94 0.70 0.00372

3-46 Meyer et al., Developmental Biology, 2009.

1451070_at Gdi1 1.00 1.12 0.00371 1425884_at Bxdc1 1.11 1.02 0.00368 1437001_at Gsk3b 1.06 0.81 0.00367 1418502_a_at Oxr1 0.99 1.09 0.00364 1447025_at Ube2e2 1.06 0.88 0.00356 1450899_at Nedd1 0.99 1.05 0.00355 1436842_at B230380D07Rik 1.04 0.75 0.00352 1452073_at 6720460F02Rik 1.00 0.80 0.00348 1428814_at Akap8l 0.98 1.04 0.00347 1459177_at AU015741 1.04 0.91 0.00346 1433209_at 2210017G18Rik 1.04 0.86 0.00344 1441682_s_at Xpot 1.01 1.08 0.00339 1439519_at Slc34a3 1.10 0.90 0.00332 1446742_at Nfia 1.06 0.82 0.00330 1434031_at Zfp692 0.91 0.97 0.00328 1447537_at 1500032P08Rik 0.89 1.18 0.00323 1442535_at --- 0.94 1.25 0.00322 1451889_at Notch2 1.04 0.89 0.00321 1425696_at Txnl6 1.02 1.13 0.00315 1457243_at 1110032O16Rik 1.00 1.08 0.00315 1459485_at Neo1 1.03 0.76 0.00315 1431960_at Wwox 1.01 0.90 0.00311 1417249_at Polm 1.01 1.10 0.00310 1417406_at Sertad1 0.83 1.15 0.00306 1437868_at BC023892 0.96 1.53 0.00304 1459420_at --- 1.03 0.82 0.00301 1425855_a_at Crk 0.96 1.04 0.00300 1432610_at 2900024J01Rik 0.99 0.83 0.00294 1422463_a_at Mrpl3 1.03 0.82 0.00291 1447055_at Dnajc11 1.02 0.91 0.00289 1417086_at Pafah1b1 1.00 1.15 0.00287 1428824_at 2310003C23Rik 0.99 1.06 0.00287 1448096_at --- 0.99 1.24 0.00271 1416891_at Numb 0.94 1.11 0.00267 1448535_at Elp4 0.90 1.04 0.00264 1420333_at Txndc8 1.02 0.90 0.00261 1424285_s_at Arl6ip4 0.99 1.06 0.00260 1453808_at 2210416O15Rik 1.05 0.87 0.00257 1418667_at 2410002O22Rik 1.00 1.13 0.00257 1450340_a_at Clcnkb 1.07 0.94 0.00257 1443416_at C79741 1.09 0.99 0.00256 1427013_at Car9 1.04 0.90 0.00254 1421036_at Npas2 1.02 0.93 0.00252 1442840_at D4Ertd669e 1.15 0.97 0.00251 1435636_at 2310051F07Rik 0.95 1.16 0.00246 1416208_at Usp14 0.98 1.09 0.00244 1432131_at 4930404N11Rik 1.01 0.93 0.00243 1436284_s_at AI225750 0.85 1.10 0.00240 1419892_at 1110021J02Rik 1.02 0.92 0.00239 1418777_at Ccl25 0.98 1.10 0.00239 1425818_at 4930520O04Rik 1.00 0.87 0.00236 1459524_at Mcc 1.06 0.91 0.00235 1419883_s_at Atp6v1b2 0.99 1.09 0.00234 1417519_at Plagl2 1.00 1.11 0.00231

3-47 Meyer et al., Developmental Biology, 2009.

1421163_a_at Nfia 1.02 0.87 0.00229 1419420_at St6galnac5 0.90 1.33 0.00226 1420070_a_at LOC277973 1.03 0.88 0.00223 1452462_a_at Banp 0.97 1.28 0.00221 1458074_at Rslcan24 1.03 0.93 0.00219 1441175_at Arx 1.01 0.94 0.00214 1422464_at Mrpl3 1.00 0.84 0.00212 1455649_at Ttc9 1.04 0.82 0.00209 1458269_at Pcdh9 1.02 0.92 0.00205 1451391_at 2700050L05Rik 0.97 1.14 0.00199 1434569_at AA474455 1.00 1.13 0.00197 1430355_a_at Steap3 1.12 0.91 0.00196 1418841_s_at Cdc2l1 0.96 1.12 0.00193 1416899_at Utf1 1.08 0.86 0.00192 1431528_at 5830427D02Rik 1.01 1.13 0.00188 1418184_at 2610019I03Rik 1.01 0.82 0.00185 1439531_at E130311K13 1.06 0.93 0.00183 1419986_at D11Ertd461e 1.05 0.95 0.00181 1416587_a_at Xrcc1 0.99 1.09 0.00178 1450833_at Chrm1 1.06 0.86 0.00178 1445917_at C81269 1.04 0.86 0.00174 1453436_at 4921516I12Rik 1.09 0.93 0.00172 1435678_at 2610017I09Rik 1.03 0.89 0.00167 1420857_at Lancl2 1.01 1.05 0.00163 1433364_at A930036I15Rik 1.07 0.90 0.00161 1450835_a_at Gfra4 1.06 0.86 0.00161 1429725_at Atbf1 1.08 1.00 0.00156 1422965_at Agtrap 1.01 0.88 0.00156 1455624_at --- 1.00 0.92 0.00149 1446411_at --- 1.12 0.98 0.00141 1425371_at Polb 0.98 1.05 0.00141 1431773_at Cbx1 1.01 0.92 0.00137 1445015_at --- 1.03 0.91 0.00133 1450381_a_at Bcl6 1.28 0.87 0.00130 1436540_at D10Bwg1379e 1.01 1.14 0.00128 1429335_at Snapc1 0.97 1.21 0.00127 1418446_at Slc16a2 1.03 0.78 0.00124 1437255_at Zbtb7 1.10 0.99 0.00121 1447187_at Ripk5 0.99 0.90 0.00121 1418577_at Trim8 0.91 1.15 0.00118 1453774_at 2810002O09Rik 1.01 0.88 0.00117 1442400_at Prickle1 1.03 0.89 0.00116 1441627_at Ebf1 1.07 0.85 0.00114 1450581_at Galr3 1.07 0.92 0.00111 1418114_at Rbpsuh 1.00 0.86 0.00111 1453968_at 4833439F03Rik 1.05 0.88 0.00109 1435426_s_at 4933439C20Rik 0.96 1.27 0.00103 1433118_at 2300004M11Rik 1.01 0.84 0.00102 1439826_at Hspa14 1.00 1.07 0.00102 1427322_at Brwd1 0.91 1.07 0.00101 1459431_at Nfia 1.03 0.77 0.00100 1431181_a_at Luc7l 0.97 1.16 0.00099 1417310_at Tob2 1.00 0.77 0.00098 1431951_a_at Usp16 0.99 1.07 0.00096

3-48 Meyer et al., Developmental Biology, 2009.

1457333_at Sdk2 1.04 0.86 0.00095 1452484_at Car7 1.11 0.91 0.00094 1423837_at 2400003C14Rik 0.98 1.12 0.00088 1442351_a_at BC029214 1.02 1.15 0.00088 1437023_at Zfp12 0.95 1.12 0.00088 1442244_at Inadl 0.89 1.10 0.00081 1449678_at Tnpo3 1.13 0.99 0.00075 1444065_at 9330151E16Rik 1.05 0.88 0.00074 1431875_a_at E2f1 0.99 0.93 0.00072 1438476_a_at Chd4 0.97 0.81 0.00072 1452895_at Fbxo45 0.95 1.19 0.00071 1420372_at Sntb2 1.01 0.85 0.00066 1440909_at Irgc1 1.12 0.92 0.00059 1457166_at AA536749 0.99 0.86 0.00057 1453498_x_at Steap3 1.07 0.83 0.00055 1443981_at LOC544892 1.02 0.85 0.00049 1436084_at Scrt1 1.04 0.90 0.00049 1423841_at Bxdc2 0.95 1.20 0.00045 1436516_at 1110065L07Rik 0.98 1.16 0.00044 1420911_a_at Mfge8 1.00 1.18 0.00042 1454284_at Slc25a25 1.07 0.96 0.00031 1417397_at Slc9a1 0.99 1.06 0.00031 1453779_at 1700020L24Rik 1.04 0.94 0.00030 1447523_at Zfp294 1.05 0.91 0.00025 1440973_at LOC552874 0.99 1.26 0.00023 1451434_s_at 5430405G24Rik 0.96 1.08 0.00021 1440187_at Taf3 0.86 1.02 0.00021 1445107_at A530064N14Rik 1.03 0.85 0.00020 1435315_s_at 2900034E22Rik 0.99 1.12 0.00020 1444971_at Rbm5 0.99 1.19 0.00014 1446313_at Zfp30 1.06 0.75 0.00008 1422223_at Grin2b 1.05 0.88 0.00005 1455606_at BC004022 0.92 1.09 0.00001

3-49 Meyer et al., Developmental Biology, 2009.

Supplemental Table S4. 90 genes differentially expressed in 7 week-old RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1433908_a_at Cttn 1.07 1.17 0.00495 1439945_at Zfp449 0.98 1.36 0.00489 1416774_at Wee1 1.09 0.72 0.00488 1448609_at Tst 1.01 1.64 0.00488 1430476_at Srp54 0.93 0.80 0.00485 1429217_at Zfp655 0.84 1.00 0.00482 1443321_at 8430438D04Rik 1.04 1.35 0.00480 1423299_at Txnl1 1.10 1.00 0.00473 1434001_at 2210019E14Rik 1.03 0.90 0.00469 1456765_at 6430511F03 0.97 1.17 0.00468 1458769_at D7Ertd558e 0.97 0.82 0.00464 1434386_at Atp2c1 1.17 1.07 0.00460 1450431_a_at Nedd4 1.03 1.11 0.00444 1418870_at 4930579J09Rik 1.00 1.07 0.00443 1437692_x_at Anxa2 0.99 1.04 0.00440 1426072_at Cmklr1 0.86 1.47 0.00436 1430546_at Cryzl1 0.93 1.11 0.00426 1433850_at Ppp4r2 1.02 0.90 0.00425 1417941_at Hdhd4 1.06 0.83 0.00414 1457878_at C430042M11Rik 1.16 1.05 0.00410 1448628_at Scg3 1.27 2.35 0.00403 1448883_at Lgmn 0.96 1.12 0.00398 1422021_at Spry4 0.90 1.00 0.00394 1456445_at 4930563D23Rik 0.98 0.85 0.00390 1416773_at Wee1 1.14 0.65 0.00389 1441348_at Zfp422-rs1 0.99 0.81 0.00368 1436265_at 6330405H19 0.92 1.39 0.00360 1419091_a_at Anxa2 0.98 1.25 0.00358 1453382_at Fbxo42 1.07 0.90 0.00355 1454171_x_at 9530053H05Rik 1.00 0.94 0.00347 1440781_at B830007D08Rik 1.00 0.55 0.00329 1415981_at 5031400M07Rik 0.90 0.99 0.00320 1446967_at Fbxl7 1.08 0.83 0.00319 1456945_at Nudt6 1.01 1.22 0.00318 1432156_a_at Rnf32 0.97 0.79 0.00303 1421365_at Fst 0.90 1.31 0.00301 1452406_x_at Erdr1 1.08 1.61 0.00300 1446294_at Tcf12 0.97 1.06 0.00295 1419482_at C3ar1 0.86 1.18 0.00276 1453368_at 2310003H01Rik 1.03 0.96 0.00272 1447371_at 9430037G07Rik 0.95 1.47 0.00266 1459454_at Hcngp 0.97 1.07 0.00264 1421917_at Pdgfra 0.89 1.12 0.00264 1437230_at Kcna1 1.02 1.80 0.00257 1442082_at C3ar1 0.91 1.35 0.00245 1453396_at 1700123D08Rik 1.03 0.88 0.00238 1460236_at Klk10 0.98 1.17 0.00231

3-50 Meyer et al., Developmental Biology, 2009.

1415806_at Plat 1.06 1.49 0.00227 1448269_a_at Klhl13 1.00 1.33 0.00223 1417268_at Cd14 0.99 1.54 0.00222 1443264_at Ms4a2 1.00 1.17 0.00214 1454979_at Diap1 0.99 1.09 0.00209 1416308_at Ugdh 0.97 1.24 0.00206 1451527_at Pcolce2 0.92 1.52 0.00196 1452858_at Elavl1 1.00 0.93 0.00194 1436024_at A930025D01Rik 0.99 0.94 0.00191 1452244_at 6330406I15Rik 1.02 1.59 0.00185 1423243_at Mpp1 0.96 1.13 0.00175 1457308_at Usp45 0.99 0.83 0.00175 1433278_at 5830440H09Rik 0.90 1.01 0.00168 1457672_at Chd9 1.03 1.23 0.00167 1433069_at 5730433N10Rik 1.02 0.92 0.00167 1422738_at Ddr2 0.95 1.03 0.00165 1451005_at Sumo1 1.07 0.93 0.00163 1426507_at Il1f5 0.99 1.09 0.00163 1417153_at Btbd14a 0.88 1.06 0.00158 1427980_at 4933407C03Rik 0.96 1.18 0.00157 1451551_at Krt2-16 0.99 0.85 0.00149 1430135_at Dnase2a 1.60 1.28 0.00146 1431004_at Loxl2 0.97 1.22 0.00143 1435534_a_at Tomm20 1.06 0.98 0.00142 1445878_at C920006O11Rik 0.86 1.31 0.00140 1435785_at Ehd2 0.99 1.20 0.00138 1420310_at --- 0.93 1.87 0.00137 1423140_at Lip1 0.98 1.24 0.00117 1438068_at --- 1.02 1.95 0.00102 1445882_at Cd300lb 0.88 1.61 0.00100 1449249_at Pcdh7 0.90 1.31 0.00097 1447481_at 2900045N06Rik 0.99 1.10 0.00084 1436918_at 1810038L18Rik 1.01 0.93 0.00072 1417563_at Eif4ebp1 0.93 1.37 0.00047 1442285_at Syne2 0.97 1.22 0.00044 1426937_at 6330406I15Rik 1.05 1.70 0.00043 1417524_at Cnih2 1.05 0.95 0.00041 1422704_at Gyk 0.99 1.98 0.00040 1418836_at Qprt 0.99 0.88 0.00034 1425040_at Cybrd1 1.02 0.86 0.00018 1447818_x_at Rhebl1 1.00 0.91 0.00015 1445370_at --- 1.00 0.81 0.00011 1444059_at 6330415G19Rik 1.00 0.91 0.00003

3-51 Meyer et al., Developmental Biology, 2009.

Supplemental Table S5. Genes differentially expressed at more than one age in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week

5, 6, and 7 weeks 1448340_at Tmem30a 0.99 1.01 1.13 1.74 1.72 1.73 1429963_at 2610021I23Rik 1.01 0.99 0.98 1.94 2.32 1.66 1447271_at Nck1 1.02 1.01 0.90 0.44 0.42 0.37 1442484_at D9Ertd306e 1.00 0.97 0.97 0.27 0.35 0.24 1429390_at Acpl2 0.95 0.97 1.31 0.33 0.33 0.42 1435704_at C920006O11Rik 1.16 0.95 0.70 3.41 4.75 5.08 1450089_a_at Srprb 0.91 0.98 1.10 0.38 0.43 0.43 1456735_x_at Acpl2 0.95 0.97 1.22 0.31 0.29 0.36 1456518_at 4930422I07Rik 1.11 1.01 1.02 0.22 0.23 0.24 1458491_at 4930422I07Rik 1.01 0.93 1.11 0.19 0.20 0.28

5 and 7 weeks 1431006_at Mrpl3 0.95 0.97 0.72 0.75 1441092_at 9330159M07Rik 0.94 1.06 2.10 1.98 1438239_at Mid1 0.95 1.20 0.38 0.43 1442733_at BC028799 0.87 1.47 0.32 0.37 1459314_at Cdkal1 1.06 0.94 1.57 1.50

5 and 6 weeks 1451347_at AI225782 1.03 0.88 0.83 1.12

6 and 7 weeks 1436920_at Pcdh17 1.21 0.82 3.11 1.83

3-52 Meyer et al., Developmental Biology, 2009.

Supplemental Table S6. 77 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant to the biological process of development.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1415890_at Papss1 0.86 1.00 1.07 0.90 1.03 1.06 0.16400 1416006_at Mdk 0.92 1.04 1.41 0.81 0.89 1.37 0.09590 1416293_at Nfib 1.00 1.04 0.98 1.09 0.91 0.91 0.10800 1438245_at --- 0.92 1.07 1.54 0.94 1.11 1.36 0.12600 1416330_at Cd81 1.02 0.99 1.00 0.98 0.99 1.21 0.16200 1416370_at Zipro1 0.98 0.98 1.18 0.93 0.97 1.10 0.19900 1416891_at Numb 1.00 0.94 1.08 0.94 1.11 1.10 0.15400 1416895_at Efna1 0.91 1.03 1.24 0.97 0.94 1.46 0.16000 1417086_at Pafah1b1 0.96 1.00 1.30 0.99 1.15 1.08 0.03110 1417301_at Fzd6 0.89 0.86 1.33 0.90 0.87 1.19 0.14100 1417311_at Crip2 0.90 0.88 1.27 0.96 0.91 1.28 0.17900 1417859_at Gas7 1.23 0.98 0.97 0.97 0.91 1.47 0.13600 1419057_at Slc5a1 0.83 1.00 1.61 0.98 1.03 1.46 0.18200 1419149_at Serpine1 1.25 0.77 1.02 1.19 1.03 3.56 0.16100 1419286_s_at Ift81 0.95 0.98 1.15 0.86 0.88 1.03 0.15400 1421344_a_at Jub 0.87 1.01 1.35 0.94 1.10 1.25 0.16500 1421579_at Hoxa9 0.93 1.10 0.98 1.00 1.08 0.85 0.11100 1422602_a_at Wnt5b 0.88 0.85 1.80 0.89 0.96 1.59 0.19700 1422629_s_at Shroom3 0.83 0.90 1.51 0.93 0.99 1.53 0.18500 1423174_a_at Pard6b 0.92 0.85 2.38 0.85 1.02 1.29 0.20000 1423349_at Socs5 0.98 0.99 1.25 0.94 1.06 1.20 0.14200 1423350_at Socs5 0.99 0.98 1.22 1.02 1.12 1.55 0.09530 1424762_at C1qtnf5 0.89 1.03 1.06 0.96 0.94 1.31 0.15900 1424893_at Ndel1 1.17 0.98 0.96 0.99 0.84 0.80 0.14200 1424950_at Sox9 0.73 1.01 1.96 0.96 1.01 1.50 0.19100 1425558_at Klc3 0.89 0.95 1.44 0.89 0.96 1.35 0.15400 1425779_a_at Tbx1 0.96 1.10 0.83 1.01 1.22 0.66 0.16600 1426241_a_at Scmh1 1.00 1.01 1.04 1.00 0.94 1.34 0.16200 1426411_a_at Strbp 0.93 0.89 1.24 0.94 1.03 0.98 0.19400 1426869_at Boc 0.81 0.84 1.46 0.82 0.91 1.33 0.16200 1427516_a_at Boc 0.89 1.00 1.32 0.94 0.97 1.27 0.18100 1427512_a_at Lama3 0.87 0.96 1.70 0.90 1.04 1.52 0.10800 1428938_at Gnaq 0.90 1.01 1.12 0.95 1.11 1.06 0.11300 1429559_at Gnaq 0.94 1.03 1.05 0.88 1.03 1.07 0.19600 1428940_at Gnaq 0.92 1.01 1.14 0.93 1.12 1.10 0.16200 1429250_at Dync2h1 0.94 0.94 1.16 0.87 0.97 1.00 0.16500 1429693_at Dab2 0.91 0.99 0.99 0.86 1.01 1.30 0.17500 1430135_at Dnase2a 1.07 0.94 1.60 1.05 1.18 1.28 0.12200 1430779_at Ntng1 0.94 1.02 1.27 0.89 0.86 1.21 0.17800 1431415_a_at Tbpl1 1.08 0.96 1.00 1.01 1.05 1.03 0.19800 1433857_at Fath 0.86 0.99 1.35 0.91 1.07 1.23 0.15600 1434460_at Bbs4 0.98 0.96 1.03 0.93 0.88 1.28 0.12400 1434765_at Ep300 1.11 1.06 0.87 1.00 1.27 0.86 0.12300 1435547_at --- 1.01 0.96 1.03 0.95 0.89 1.12 0.16200 1436205_at --- 0.99 1.09 0.88 1.05 1.02 0.92 0.17400 1436551_at Fgfr1 0.97 1.03 0.99 1.01 1.04 1.45 0.19100

3-53 Meyer et al., Developmental Biology, 2009.

1436911_at Ss18l1 0.99 0.97 1.19 0.96 1.14 1.09 0.16200 1438412_at --- 0.97 1.03 1.26 1.05 1.14 1.28 0.17000 1441175_at Arx 1.02 1.01 0.96 1.05 0.94 0.95 0.17900 1441923_s_at Edn3 0.98 1.11 0.98 1.05 1.00 1.00 0.14600 1442107_at Flnb 0.92 1.00 1.21 0.95 0.92 1.25 0.03780 1443785_x_at Pdlim7 1.00 0.97 1.01 1.02 0.89 1.06 0.11400 1443969_at Irs2 1.18 0.91 1.05 1.05 0.81 1.45 0.18500 1444344_at --- 1.26 1.04 0.53 0.99 0.86 0.53 0.16200 1444620_at Tcf12 1.09 1.07 0.90 0.89 0.69 0.50 0.20000 1444705_at App 0.94 0.90 1.24 0.87 0.97 1.30 0.10900 1446839_at Odz4 0.94 1.00 1.13 1.04 1.08 1.29 0.14600 1458045_at Odz4 0.86 1.41 0.88 1.41 1.25 1.90 0.15400 1447271_at Nck1 1.02 1.01 0.90 0.44 0.42 0.37 0.00004 1447964_at Ttl 0.81 1.00 1.11 0.92 0.99 0.94 0.15800 1448669_at Dkk3 0.99 0.98 1.30 0.94 1.10 1.61 0.19400 1449031_at Cited1 0.76 0.70 1.25 0.66 0.69 1.66 0.18500 1449141_at Fblim1 0.95 0.86 1.20 0.85 0.83 1.21 0.17800 1449351_s_at Pdgfc 0.98 0.96 1.44 0.84 0.96 1.15 0.14600 1449845_a_at Ephb4 0.98 0.85 1.05 0.93 0.88 1.08 0.16700 1450209_at Hoxd4 0.87 1.02 1.06 0.97 0.87 1.13 0.19900 1450388_s_at Twsg1 0.98 0.94 1.25 0.89 0.94 1.17 0.18100 1450414_at Pdgfb 0.96 0.98 1.18 1.02 0.95 1.38 0.16200 1450799_at Adcyap1r1 1.05 1.05 0.97 0.96 0.85 0.95 0.16500 1450918_s_at Src 1.00 0.94 1.11 0.95 0.97 1.21 0.20000 1451899_a_at Gtf2ird1 0.92 0.94 1.03 0.91 0.74 1.21 0.17500 1460364_at Gtf2ird1 0.97 1.00 1.08 1.03 0.93 1.31 0.15600 1452514_a_at Kit 0.81 1.03 1.56 0.95 0.99 1.28 0.17800 1452589_at Ptk7 0.96 0.92 1.32 0.98 0.97 1.12 0.18500 1452911_at Spred1 0.89 0.97 1.07 0.87 1.00 1.32 0.11800 1454037_a_at Flt1 1.08 1.02 0.77 1.09 0.95 0.96 0.18500 1455188_at Ephb1 0.92 0.98 1.11 0.92 0.89 1.22 0.12300 1456189_x_at Ltbp3 0.84 0.96 1.38 1.00 1.01 1.64 0.19700 1458361_at Dclre1c 1.26 1.08 0.74 0.93 0.92 0.62 0.15400 1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.16100 1460571_at Dicer1 0.97 0.97 1.14 0.94 1.11 1.11 0.12900 1460642_at Traf4 0.92 0.95 1.30 1.12 1.06 1.29 0.17800 1460700_at Stat3 0.99 0.95 1.08 0.96 1.14 1.37 0.14200

3-54 Meyer et al., Developmental Biology, 2009.

Supplemental Table S7. 74 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant to the biological process of transcription.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1441324_at Zfp395 0.96 1.07 1.00 0.91 1.04 1.28 0.14600 1422286_a_at Tgif 0.94 0.87 1.23 0.88 0.98 1.22 0.09870 1425779_a_at Tbx1 0.96 1.10 0.83 1.01 1.22 0.66 0.16600 1426241_a_at Scmh1 1.00 1.01 1.04 1.00 0.94 1.34 0.16200 1437892_at Zfp306 1.09 0.93 0.99 1.48 1.53 1.43 0.04800 1442080_at Creb3l2 0.95 1.03 1.07 0.93 1.00 1.29 0.12500 1456154_at Zfp444 0.98 1.08 0.97 1.06 0.92 0.96 0.11700 1452258_at Phf20 1.01 1.02 0.95 1.07 0.97 0.89 0.16500 1449370_at Sox4 0.96 0.99 1.16 0.97 0.88 0.98 0.16500 1450191_a_at Sox13 0.90 0.97 1.11 0.96 0.99 1.17 0.13200 1456037_x_at Preb 0.93 0.99 1.27 0.97 1.30 1.40 0.15300 1424950_at Sox9 0.73 1.01 1.96 0.96 1.01 1.50 0.19100 1418284_at Vps72 0.96 0.97 1.14 0.94 0.94 1.29 0.16700 1454815_at Sertad2 0.98 0.99 1.11 0.98 1.08 1.16 0.15000 1416899_at Utf1 0.99 1.08 0.97 0.97 0.86 0.90 0.14600 1449031_at Cited1 0.76 0.70 1.25 0.66 0.69 1.66 0.18500 1435994_at Kcnh1 0.71 0.85 1.70 0.73 0.90 1.87 0.16000 1428616_at Zfp131 0.99 0.98 0.95 1.04 1.19 0.91 0.11600 1427322_at Brwd1 1.03 0.91 1.09 0.97 1.07 1.03 0.19900 1451005_at Sumo1 0.98 1.01 1.07 0.94 0.99 0.93 0.10800 1425094_a_at Lhx6 0.98 0.96 1.05 0.93 0.87 1.39 0.09590 1438079_at BC050078 0.95 1.02 1.24 0.93 0.93 1.21 0.12300 1447325_at Ches1 1.18 1.00 0.93 0.99 0.97 0.95 0.16200 1423340_at Tcfap2b 0.83 0.89 2.07 0.86 0.82 1.43 0.12300 1422771_at LOC670044 1.05 1.02 0.93 1.01 0.96 1.82 0.10300 1426048_s_at Hrc 0.85 0.94 1.69 0.88 0.95 1.34 0.18100 1451983_at Irx1 0.86 0.81 1.56 0.85 0.89 1.47 0.18100 1429086_at Grhl2 0.75 0.92 1.92 0.82 0.85 1.75 0.18100 1440187_at Taf3 0.96 0.86 1.08 0.93 1.02 1.15 0.12600 1436911_at Ss18l1 0.99 0.97 1.19 0.96 1.14 1.09 0.16200 1418301_at Irf6 0.86 0.84 1.74 0.85 1.03 1.32 0.20000 1426910_at --- 1.00 0.96 1.56 0.99 1.13 1.12 0.16600 1426437_s_at Hdac3 0.95 1.02 1.08 1.00 0.99 1.13 0.19100 1432177_a_at Mnat1 0.97 0.96 1.09 0.92 0.92 1.10 0.20000 1449915_at Zfp202 0.91 0.98 1.08 0.94 0.91 1.19 0.11500 1448585_at Gtf2h4 0.95 0.98 1.16 0.97 1.00 1.30 0.19600 1434765_at Ep300 1.11 1.06 0.87 1.00 1.27 0.86 0.12300 1416370_at Zipro1 0.98 0.98 1.18 0.93 0.97 1.10 0.19900 1428594_at Garnl1 0.98 0.97 1.06 0.98 1.00 1.14 0.17000 1428985_at Ints12 0.98 0.98 1.12 0.96 1.12 1.22 0.14600 1439945_at Zfp449 0.92 0.94 0.98 0.92 0.83 1.36 0.07120 1427512_a_at Lama3 0.87 0.96 1.70 0.90 1.04 1.52 0.10800 1435500_at Rab26 1.02 1.05 0.87 1.05 0.99 0.86 0.18500 1416293_at Nfib 1.00 1.04 0.98 1.09 0.91 0.91 0.10800 1438245_at --- 0.92 1.07 1.54 0.94 1.11 1.36 0.12600 1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.16100

3-55 Meyer et al., Developmental Biology, 2009.

1436684_a_at Riok2 0.91 1.00 1.08 1.12 1.00 1.17 0.17800 1456518_at 4930422I07Rik 1.11 1.01 1.02 0.22 0.23 0.24 0.00012 1458491_at 4930422I07Rik 1.01 0.93 1.11 0.19 0.20 0.28 0.00002 1440068_at Rfxdc2 1.14 1.00 0.99 0.66 0.53 0.22 0.18500 1437023_at Zfp12 1.05 0.95 1.07 0.92 1.12 1.05 0.16200 1430435_at Aff3 1.06 0.98 0.92 1.06 0.97 0.81 0.19000 1460367_at Hbp1 1.05 0.96 1.04 0.92 0.99 1.12 0.17600 1418322_at Crem 1.00 0.93 1.09 0.95 1.15 1.20 0.17900 1454971_x_at Tsc22d1 0.99 0.98 1.09 0.95 1.01 1.10 0.19700 1460700_at Stat3 0.99 0.95 1.08 0.96 1.14 1.37 0.14200 1431415_a_at Tbpl1 1.08 0.96 1.00 1.01 1.05 1.03 0.19800 1429217_at Zfp655 1.09 1.01 0.84 1.08 1.01 1.00 0.06640 1438412_at --- 0.97 1.03 1.26 1.05 1.14 1.28 0.17000 1444620_at Tcf12 1.09 1.07 0.90 0.89 0.69 0.50 0.20000 1451899_a_at Gtf2ird1 0.92 0.94 1.03 0.91 0.74 1.21 0.17500 1460364_at Gtf2ird1 0.97 1.00 1.08 1.03 0.93 1.31 0.15600 1455173_at Gspt1 0.79 1.02 1.07 1.04 1.12 0.92 0.11500 1424297_at Zfp282 1.01 0.92 1.16 0.87 0.94 1.16 0.17300 1444344_at --- 1.26 1.04 0.53 0.99 0.86 0.53 0.16200 1450209_at Hoxd4 0.87 1.02 1.06 0.97 0.87 1.13 0.19900 1419313_at Ccnt1 0.99 1.00 1.04 1.01 1.20 0.99 0.18500 1417549_at Zfp68 1.01 0.94 1.13 0.95 1.02 1.16 0.12400 1457571_at Zfp68 0.92 0.98 1.11 0.87 1.00 1.18 0.17900 1457445_at Trps1 0.77 0.92 1.82 0.91 1.00 1.44 0.19500 1441175_at Arx 1.02 1.01 0.96 1.05 0.94 0.95 0.17900 1422752_at Polr3k 0.89 1.00 1.23 0.98 1.07 1.08 0.17900 1416303_at Litaf 0.99 0.95 1.06 0.81 1.10 1.38 0.15600 1417406_at Sertad1 1.04 0.83 1.01 0.93 1.15 1.04 0.18100 1421579_at Hoxa9 0.93 1.10 0.98 1.00 1.08 0.85 0.11100 1448860_at Rem2 0.91 0.88 1.31 0.91 0.87 1.19 0.15400 1428449_at Gtf3c2 1.00 0.99 1.11 0.96 1.08 1.13 0.18100 1435547_at --- 1.01 0.96 1.03 0.95 0.89 1.12 0.16200

3-56 Meyer et al., Developmental Biology, 2009.

Supplemental Table S8. 44 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant to the biological process of morphogenesis.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1425779_a_at Tbx1 0.96 1.10 0.83 1.01 1.22 0.66 0.16600 1447964_at Ttl 0.81 1.00 1.11 0.92 0.99 0.94 0.15800 1452589_at Ptk7 0.96 0.92 1.32 0.98 0.97 1.12 0.18500 1430779_at Ntng1 0.94 1.02 1.27 0.89 0.86 1.21 0.17800 1424950_at Sox9 0.73 1.01 1.96 0.96 1.01 1.50 0.19100 1449031_at Cited1 0.76 0.70 1.25 0.66 0.69 1.66 0.18500 1449141_at Fblim1 0.95 0.86 1.20 0.85 0.83 1.21 0.17800 1460700_at Stat3 0.99 0.95 1.08 0.96 1.14 1.37 0.14200 1429693_at Dab2 0.91 0.99 0.99 0.86 1.01 1.30 0.17500 1438412_at --- 0.97 1.03 1.26 1.05 1.14 1.28 0.17000 1425558_at Klc3 0.89 0.95 1.44 0.89 0.96 1.35 0.15400 1455188_at Ephb1 0.92 0.98 1.11 0.92 0.89 1.22 0.12300 1449351_s_at Pdgfc 0.98 0.96 1.44 0.84 0.96 1.15 0.14600 1450414_at Pdgfb 0.96 0.98 1.18 1.02 0.95 1.38 0.16200 1449845_a_at Ephb4 0.98 0.85 1.05 0.93 0.88 1.08 0.16700 1434460_at Bbs4 0.98 0.96 1.03 0.93 0.88 1.28 0.12400 1416330_at Cd81 1.02 0.99 1.00 0.98 0.99 1.21 0.16200 1450209_at Hoxd4 0.87 1.02 1.06 0.97 0.87 1.13 0.19900 1444344_at --- 1.26 1.04 0.53 0.99 0.86 0.53 0.16200 1429250_at Dync2h1 0.94 0.94 1.16 0.87 0.97 1.00 0.16500 1417859_at Gas7 1.23 0.98 0.97 0.97 0.91 1.47 0.13600 1444705_at App 0.94 0.90 1.24 0.87 0.97 1.30 0.10900 1416891_at Numb 1.00 0.94 1.08 0.94 1.11 1.10 0.15400 1436911_at Ss18l1 0.99 0.97 1.19 0.96 1.14 1.09 0.16200 1460571_at Dicer1 0.97 0.97 1.14 0.94 1.11 1.11 0.12900 1421344_a_at Jub 0.87 1.01 1.35 0.94 1.10 1.25 0.16500 1436551_at Fgfr1 0.97 1.03 0.99 1.01 1.04 1.45 0.19100 1460642_at Traf4 0.92 0.95 1.30 1.12 1.06 1.29 0.17800 1422602_a_at Wnt5b 0.88 0.85 1.80 0.89 0.96 1.59 0.19700 1447271_at Nck1 1.02 1.01 0.90 0.44 0.42 0.37 0.00004 1423349_at Socs5 0.98 0.99 1.25 0.94 1.06 1.20 0.14200 1423350_at Socs5 0.99 0.98 1.22 1.02 1.12 1.55 0.09530 1434765_at Ep300 1.11 1.06 0.87 1.00 1.27 0.86 0.12300 1428938_at Gnaq 0.90 1.01 1.12 0.95 1.11 1.06 0.11300 1429559_at Gnaq 0.94 1.03 1.05 0.88 1.03 1.07 0.19600 1428940_at Gnaq 0.92 1.01 1.14 0.93 1.12 1.10 0.16200 1454037_a_at Flt1 1.08 1.02 0.77 1.09 0.95 0.96 0.18500 1421579_at Hoxa9 0.93 1.10 0.98 1.00 1.08 0.85 0.11100 1419149_at Serpine1 1.25 0.77 1.02 1.19 1.03 3.56 0.16100 1450388_s_at Twsg1 0.98 0.94 1.25 0.89 0.94 1.17 0.18100 1416895_at Efna1 0.91 1.03 1.24 0.97 0.94 1.46 0.16000 1436205_at --- 0.99 1.09 0.88 1.05 1.02 0.92 0.17400 1416293_at Nfib 1.00 1.04 0.98 1.09 0.91 0.91 0.10800 1438245_at --- 0.92 1.07 1.54 0.94 1.11 1.36 0.12600 1423174_a_at Pard6b 0.92 0.85 2.38 0.85 1.02 1.29 0.20000 1435547_at --- 1.01 0.96 1.03 0.95 0.89 1.12 0.16200

3-57 Meyer et al., Developmental Biology, 2009.

1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.16100 1433857_at Fath 0.86 0.99 1.35 0.91 1.07 1.23 0.15600

3-58 Meyer et al., Developmental Biology, 2009.

Supplemental Table S9. 38 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant to the biological process of cell differentiation.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1425779_a_at Tbx1 0.96 1.10 0.83 1.01 1.22 0.66 0.166 1447964_at Ttl 0.81 1.00 1.11 0.92 0.99 0.94 0.158 1452589_at Ptk7 0.96 0.92 1.32 0.98 0.97 1.12 0.185 1424950_at Sox9 0.73 1.01 1.96 0.96 1.01 1.50 0.191 1430779_at Ntng1 0.94 1.02 1.27 0.89 0.86 1.21 0.178 1449031_at Cited1 0.76 0.70 1.25 0.66 0.69 1.66 0.185 1416006_at Mdk 0.92 1.04 1.41 0.81 0.89 1.37 0.096 1417086_at Pafah1b1 0.96 1.00 1.30 0.99 1.15 1.08 0.031 1460700_at Stat3 0.99 0.95 1.08 0.96 1.14 1.37 0.142 1431415_a_at Tbpl1 1.08 0.96 1.00 1.01 1.05 1.03 0.198 1426411_a_at Strbp 0.93 0.89 1.24 0.94 1.03 0.98 0.194 1441923_s_at Edn3 0.98 1.11 0.98 1.05 1.00 1.00 0.146 1429693_at Dab2 0.91 0.99 0.99 0.86 1.01 1.30 0.175 1452514_a_at Kit 0.81 1.03 1.56 0.95 0.99 1.28 0.178 1455188_at Ephb1 0.92 0.98 1.11 0.92 0.89 1.22 0.123 1434460_at Bbs4 0.98 0.96 1.03 0.93 0.88 1.28 0.124 1449845_a_at Ephb4 0.98 0.85 1.05 0.93 0.88 1.08 0.167 1450799_at Adcyap1r1 1.05 1.05 0.97 0.96 0.85 0.95 0.165 1443785_x_at Pdlim7 1.00 0.97 1.01 1.02 0.89 1.06 0.114 1444344_at --- 1.26 1.04 0.53 0.99 0.86 0.53 0.162 1430135_at Dnase2a 1.07 0.94 1.60 1.05 1.18 1.28 0.122 1417859_at Gas7 1.23 0.98 0.97 0.97 0.91 1.47 0.136 1444705_at App 0.94 0.90 1.24 0.87 0.97 1.30 0.109 1416891_at Numb 1.00 0.94 1.08 0.94 1.11 1.10 0.154 1436911_at Ss18l1 0.99 0.97 1.19 0.96 1.14 1.09 0.162 1458361_at Dclre1c 1.26 1.08 0.74 0.93 0.92 0.62 0.154 1436551_at Fgfr1 0.97 1.03 0.99 1.01 1.04 1.45 0.191 1419286_s_at Ift81 0.95 0.98 1.15 0.86 0.88 1.03 0.154 1423349_at Socs5 0.98 0.99 1.25 0.94 1.06 1.20 0.142 1423350_at Socs5 0.99 0.98 1.22 1.02 1.12 1.55 0.095 1416370_at Zipro1 0.98 0.98 1.18 0.93 0.97 1.10 0.199 1428938_at Gnaq 0.90 1.01 1.12 0.95 1.11 1.06 0.113 1429559_at Gnaq 0.94 1.03 1.05 0.88 1.03 1.07 0.196 1428940_at Gnaq 0.92 1.01 1.14 0.93 1.12 1.10 0.162 1454037_a_at Flt1 1.08 1.02 0.77 1.09 0.95 0.96 0.185 1450388_s_at Twsg1 0.98 0.94 1.25 0.89 0.94 1.17 0.181 1416895_at Efna1 0.91 1.03 1.24 0.97 0.94 1.46 0.160 1436205_at --- 0.99 1.09 0.88 1.05 1.02 0.92 0.174 1423174_a_at Pard6b 0.92 0.85 2.38 0.85 1.02 1.29 0.200 1435547_at --- 1.01 0.96 1.03 0.95 0.89 1.12 0.162 1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.161

3-59 Meyer et al., Developmental Biology, 2009.

Supplemental Table S10. 35 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant with predicted biological process of kinase activity.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1429963_at Mapk6 1.01 0.99 0.98 1.94 2.32 1.66 0.00740 1436684_a_at Riok2 0.91 1.00 1.08 1.12 1.00 1.17 0.17800 1425505_at Mylk 0.92 1.03 1.30 0.95 1.20 1.23 0.15800 1452589_at Ptk7 0.96 0.92 1.32 0.98 0.97 1.12 0.18500 1444366_at Taok3 0.99 1.07 0.95 1.06 0.93 0.94 0.20000 1455511_at Sephs1 0.99 0.97 1.14 0.84 1.02 1.04 0.19600 1428373_at Ihpk2 0.99 0.96 1.15 0.86 0.91 1.05 0.02550 1454605_a_at Pi4k2a 1.01 0.99 1.00 0.98 1.05 1.26 0.17300 1445984_at Pftk1 0.98 1.00 1.11 0.80 0.92 1.18 0.16400 1435994_at Kcnh1 0.71 0.85 1.70 0.73 0.90 1.87 0.16000 1436753_at Adck5 1.03 0.87 1.09 0.88 0.90 1.20 0.18100 1433450_at Cdk5r1 1.12 1.00 0.93 0.99 1.16 0.75 0.12500 1419834_x_at Mark1 0.96 1.02 1.14 0.96 0.94 1.17 0.09530 1425510_at Mark1 0.90 0.99 1.25 0.90 1.02 1.37 0.01600 1425511_at Mark1 0.94 0.96 1.16 0.91 0.98 1.27 0.04590 1449630_s_at Mark1 0.94 0.94 1.28 0.86 0.95 1.49 0.04720 1424633_at Camk1g 0.98 1.13 0.99 1.13 0.95 0.99 0.19600 1427246_at Magi1 0.97 0.99 1.43 1.00 0.97 1.24 0.07680 1443231_at Magi1 0.88 0.98 1.35 0.88 0.98 1.17 0.13000 1446050_at Magi1 0.94 0.97 1.25 0.86 0.87 1.31 0.13600 1458240_at Magi1 0.99 0.98 1.30 0.93 0.98 1.34 0.10300 1451893_s_at Magi1 0.97 0.96 1.30 0.90 0.92 1.30 0.16200 1452514_a_at Kit 0.81 1.03 1.56 0.95 0.99 1.28 0.17800 1455188_at Ephb1 0.92 0.98 1.11 0.92 0.89 1.22 0.12300 1415890_at Papss1 0.86 1.00 1.07 0.90 1.03 1.06 0.16400 1451003_at Map3k7ip2 0.98 0.96 1.06 0.89 0.93 0.90 0.19100 1449845_a_at Ephb4 0.98 0.85 1.05 0.93 0.88 1.08 0.16700 1437404_at Mast4 0.90 0.95 1.38 0.93 0.91 1.41 0.14200 1445866_at Mast4 0.85 1.02 1.37 0.87 0.89 1.21 0.17400 1415747_s_at Riok3 1.00 1.02 0.96 1.02 1.08 1.02 0.06450 1427358_a_at Dapk1 1.04 0.91 1.02 0.72 0.80 1.24 0.17600 1434706_at Vcpip1 1.12 1.03 0.78 1.00 1.32 0.81 0.19700 1436551_at Fgfr1 0.97 1.03 0.99 1.01 1.04 1.45 0.19100 1447271_at Nck1 1.02 1.01 0.90 0.44 0.42 0.37 0.00004 1448297_a_at Tnk2 0.96 1.00 1.02 1.05 0.90 1.26 0.15800 1448298_at Tnk2 1.00 1.05 1.10 1.07 0.96 1.57 0.18600 1427639_a_at Nek4 0.93 0.97 1.14 0.94 0.98 1.10 0.14400 1441213_at --- 0.89 0.88 3.29 0.97 1.18 1.56 0.14300 1450918_s_at Src 1.00 0.94 1.11 0.95 0.97 1.21 0.20000 1416774_at Wee1 1.05 0.95 1.09 0.97 1.07 0.72 0.19500 1454037_a_at Flt1 1.08 1.02 0.77 1.09 0.95 0.96 0.18500 1425279_at Pdik1l 1.09 0.97 1.03 1.10 0.90 0.86 0.15600 1428814_at --- 1.02 0.98 1.04 0.89 1.04 0.86 0.15800 1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.16100

3-60 Meyer et al., Developmental Biology, 2009.

Supplemental Table S11. 22 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant with predicted biological process of cell motility.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1425779_a_at Tbx1 0.96 1.10 0.83 1.01 1.22 0.66 0.16600 1444705_at App 0.94 0.90 1.24 0.87 0.97 1.30 0.10900 1447964_at Ttl 0.81 1.00 1.11 0.92 0.99 0.94 0.15800 1441175_at Arx 1.02 1.01 0.96 1.05 0.94 0.95 0.17900 1416288_at Dnaja1 0.93 0.95 1.20 0.91 1.19 1.01 0.16200 1459835_s_at Dnaja1 0.79 1.00 1.19 0.93 1.13 1.13 0.06670 1460179_at Dnaja1 0.83 0.98 1.15 0.87 1.15 1.03 0.05410 1421344_a_at Jub 0.87 1.01 1.35 0.94 1.10 1.25 0.16500 1457806_at Dock1 0.94 0.98 1.14 0.91 0.98 1.14 0.16600 1447271_at Nck1 1.02 1.01 0.90 0.44 0.42 0.37 0.00004 1417086_at Pafah1b1 0.96 1.00 1.30 0.99 1.15 1.08 0.03110 1426411_a_at Strbp 0.93 0.89 1.24 0.94 1.03 0.98 0.19400 1424893_at Ndel1 1.17 0.98 0.96 0.99 0.84 0.80 0.14200 1417693_a_at Gab1 0.87 0.89 1.33 0.88 0.88 1.11 0.11700 1417694_at Gab1 0.93 0.92 1.47 0.90 0.94 1.22 0.12100 1439786_at Gab2 1.01 0.99 1.00 0.87 1.03 0.99 0.17900 1454037_a_at Flt1 1.08 1.02 0.77 1.09 0.95 0.96 0.18500 1436205_at --- 0.99 1.09 0.88 1.05 1.02 0.92 0.17400 1427512_a_at Lama3 0.87 0.96 1.70 0.90 1.04 1.52 0.10800 1423174_a_at Pard6b 0.92 0.85 2.38 0.85 1.02 1.29 0.20000 1450414_at Pdgfb 0.96 0.98 1.18 1.02 0.95 1.38 0.16200 1459617_at Mapk14 1.03 1.01 0.89 0.94 0.91 0.87 0.16100 1455188_at Ephb1 0.92 0.98 1.11 0.92 0.89 1.22 0.12300 1449845_a_at Ephb4 0.98 0.85 1.05 0.93 0.88 1.08 0.16700 1418361_at Gas8 0.91 1.00 1.07 1.01 1.00 1.23 0.16600

3-61 Meyer et al., Developmental Biology, 2009.

Supplemental Table S12. 22 genes differentially expressed in RonTK-/- mouse mammary glands compared to RonTK+/+ mouse mammary glands relevant with predicted biological process of cell adhesion.

RonTK+/+ RonTK-/- Affymetrix ID Gene 5 week 6 week 7 week 5 week 6 week 7 week p-value

1426872_at Fcgbp 0.49 0.83 1.79 0.59 1.21 2.84 0.17 1444705_at App 0.94 0.90 1.24 0.87 0.97 1.30 0.11 1452589_at Ptk7 0.96 0.92 1.32 0.98 0.97 1.12 0.19 1420429_at Pcdhb3 0.79 0.97 1.10 0.77 1.03 1.03 0.02 1451407_at Jam4 0.86 0.86 1.63 0.90 0.88 1.61 0.12 1420798_s_at Pcdha4 0.50 0.96 1.83 0.68 1.13 2.10 0.17 1424341_s_at Pcdha4 0.57 0.94 1.75 0.61 1.13 1.84 0.18 1421344_a_at Jub 0.87 1.01 1.35 0.94 1.10 1.25 0.17 1420630_at 8430419L09Rik 0.97 0.94 1.34 0.89 1.00 1.18 0.19 1415803_at Cx3cl1 0.88 0.87 1.22 0.86 0.87 1.43 0.17 1449141_at Fblim1 0.95 0.86 1.20 0.85 0.83 1.21 0.18 1420798_s_at Pcdha4 0.50 0.96 1.83 0.68 1.13 2.10 0.17 1424341_s_at Pcdha4 0.57 0.94 1.75 0.61 1.13 1.84 0.18 1417812_a_at Lamb3 0.83 0.81 1.73 0.80 0.86 1.59 0.17 1436920_at Pcdh17 0.89 1.21 0.82 2.62 3.11 1.83 0.20 1426332_a_at Cldn3 0.88 0.79 1.52 0.82 0.83 1.33 0.10 1434651_a_at Cldn3 0.85 0.78 1.68 0.85 0.88 1.50 0.14 1460569_x_at Cldn3 0.78 0.74 1.73 0.80 0.87 1.50 0.16 1451701_x_at Cldn3 0.82 0.82 1.76 0.83 0.87 1.52 0.11 1436205_at --- 0.99 1.09 0.88 1.05 1.02 0.92 0.17 1427512_a_at Lama3 0.87 0.96 1.70 0.90 1.04 1.52 0.11 1426869_at Boc 0.81 0.84 1.46 0.82 0.91 1.33 0.16 1427516_a_at Boc 0.89 1.00 1.32 0.94 0.97 1.27 0.18 1419589_at Cd93 0.74 1.03 1.06 0.81 0.88 1.21 0.17 1419608_a_at Mia1 0.83 0.91 1.87 0.88 0.95 1.36 0.19 1438410_at Prtg 1.00 1.01 0.96 0.97 0.92 1.24 0.03 1456329_at Prtg 0.97 1.04 1.00 0.85 0.91 1.16 0.18 1433857_at Fath 0.86 0.99 1.35 0.91 1.07 1.23 0.16 1420798_s_at Pcdha4 0.50 0.96 1.83 0.68 1.13 2.10 0.17 1424341_s_at Pcdha4 0.57 0.94 1.75 0.61 1.13 1.84 0.18

3-62 Meyer et al., Developmental Biology, 2009.

Discussion

Pubertal mammary gland development is a complex and tightly controlled process.

Many positive regulators of mammary development are known, however, very few negative regulators have been identified [168]. We report for the first time evidence supporting an important role for the receptor tyrosine kinase Ron in mammary gland development. We showed that Ron is expressed during pubertal mammary development (5-8 weeks), which corresponds with Ron expression analyses published previously [32]. We also observed Ron expression in mammary glands during later stages of mammary gland development including pregnancy and lactation, which was not detected previously by Chodosh et al., 2000. This discrepancy may be due to strain-specific differences between the mice, differences in the times at which the tissues were take, or differences in methods of detection. Immunohistochemistry on mouse mammary glands and Western analysis of human and mouse mammary epithelial cells showed that Ron is expressed in the mammary epithelium. In addition,

Ron expression was undetectable in mammary fat pads by Western analysis of 3 week- old mouse mammary fat pads devoid of epithelium and in human primary adipocytes.

While this data suggests the Ron is expressed predominantly in the mammary epithelium, we cannot preclude, however, that Ron may be expressed in other cell types within the mammary gland during pubertal development.

To test the role of Ron during mammary gland development, RonTK-/- mice with a targeted deletion of the tyrosine kinase domain of Ron were analyzed for mammary growth and morphogenesis compared to wild-type RonTK+/+ mice. We found

3-63 Meyer et al., Developmental Biology, 2009. significantly accelerated ductal elongation and significantly increased branching morphogenesis during pubertal mammary development in RonTK-/- mice compared to age and weight-matched controls. This finding was unexpected given that the majority of receptor tyrosine kinases previously examined in the mammary gland play a positive role in both promoting morphogenesis and breast tumorigenesis, and that overexpression of the Ron receptor also promotes breast tumorigenesis

[30,31,168,183,239,243-245]. However, there is precedent for negative regulation of mammary gland development by a receptor tyrosine kinase, which is also overexpressed in breast cancer [168]. The well-studied receptor tyrosine kinase ligand

TGF! limits branching morphogenesis during pubertal mouse mammary development, and overexpression of TGF! is one mechanism leading to breast tumor progression

[246-248].

The classical initiator and positive regulators of mammary gland development are estrogen and progesterone. During the estrus cycle the rise in estrogen stimulates the mammary epithelium to proliferate, and progesterone can stimulate branching. The loss of either signaling pathway results in a severely stunted mammary rudiment [171].

Based on our discovery that Ron negatively regulates mammary gland development, we hypothesized that RonTK-/- mice may have altered estrogen or progesterone signaling that leads to this phenotype. However, our analysis of circulating serum levels of estrogen and progesterone showed the contrary and found no differences between

RonTK-/- and RonTK+/+ mice. Additionally, mammary glands from RonTK-/- mice ovariectomized at 3 weeks of age displayed significantly increased branching at 6

3-64 Meyer et al., Developmental Biology, 2009. weeks of age compared to ovariectomized RonTK+/+ controls, which is consistent with the phenotype observed in animals with intact ovaries. Therefore, our data suggests that Ron signaling may regulate mammary branching morphogenesis in the absence of ovarian hormone stimulation. It is possible that the RonTK-/- mammary glands are more sensitive to other factors, such as growth factors, that also contribute to mammary branching morphogenesis, and are therefore less affected by ovariectomy. It is also important to note that while removal of the ovaries severely compromises estrogen and progesterone levels, low amounts of these hormones are produced by other organs and may account for the ductal morphogenesis observed.

An alternative mechanism by which Ron could negatively regulate mammary development is by limiting cell turnover rates of the mammary epithelium, such that deletion of the tyrosine kinase domain of Ron would increase these rates and accelerate glandular development. In tumor cells, Ron overexpression has been reported to modulate cell proliferation and apoptosis, however, nothing is known about the influence of Ron on cell turnover in the normal mammary epithelium [7]. To test this, RonTK+/+ and RonTK-/- mice injected with BrdU were analyzed for BrdU incorporation, as a measure of proliferation, and TUNEL staining, as a measure of potential cell death, in mammary gland end buds and ducts. Although mammary glands from RonTK-/- mice harbor significantly more ductal structures than RonTK+/+ controls, there were no significant differences in the percentage of proliferating or apoptotic cells in the ductal and end bud epithelium between RonTK+/+ and RonTK-/- glands. In both genotypes, the end buds displayed the greater amounts of proliferation (approximately

3-65 Meyer et al., Developmental Biology, 2009.

17%) and apoptotic staining (approximately 3%) when compared to ducts as expected

[167]. Our finding is consistent with others that have shown that while some cell proliferation is essential in budding epithelium to extend the bud, it may not be necessary for bud formation or branching morphogenesis [249,250].

To determine whether modification of Ron receptor function in the mammary epithelium is responsible for the observed modulation of branching morphogenesis in vivo, ductal epithelial fragments (organoids) from RonTK+/+ and RonTK-/- mice were purified, and embedded in a three-dimensional Matrigel matrix. After 6 days in culture, the majority,

71%, of RonTK-/- organoids, and only 32% of RonTK+/+ organoids formed buds and branches. This significant increase in RonTK-/- mammary organoid branching suggests that the loss of Ron receptor expression in the mammary epithelium is sufficient to increase branching morphogenesis ex vivo. Our results, however, do not exclude the possibility that loss of Ron in other cellular compartments may contribute to the branching morphogenic phenotype in vivo. In contrast to the in vivo setting where differences in branching morphogenesis early in pubertal development resolve overtime between the RonTK+/+ and RonTK-/- glands, the differences between the RonTK+/+ and RonTK-/- organoids cultured in Matrigel do not normalize over extended periods of time (up to three weeks). This finding supports the prospect that other factors may be playing a role to orchestrate branching morphogenesis in vivo. We also investigated the possibility that treatment of RonTK+/+ organoids ex vivo with the Ron ligand, HGFL, would inhibit branching morphogenesis. Interestingly, however, we found that addition of HGFL had no additional effect on branching in culture. This finding could be due to

3-66 Meyer et al., Developmental Biology, 2009. several factors. First, it is not known whether the Matrigel or the mammary epithelium growth supplement (which is derived from pituitary extracts) in which the organoids were grown contain HGFL. In addition, it is not known whether a component of the organoid cultures produces HGFL. There is also a possibility that crosstalk between

Ron and other signaling pathways may regulate branching morphogenesis independent of HGFL. Cross-talk with Ron and other receptor tyrosine kinases has been reported by a number of independent groups. [251].

We continued our investigation of Ron signaling by assessing Akt and MAPK, two known downstream targets of Ron receptor signaling. Activation of Akt and MAPK has been shown to be essential for branching morphogenesis in numerous organs [252].

Phosphorylation of Akt and MAPK were examined in RonTK+/+ and RonTK-/- mammary glands by Western and immunohistochemical analyses. RonTK-/- mammary glands demonstrated increased phosphorylation of both Akt and MAPK, however, while

Akt phosphorylation appeared more diffuse throughout the mammary glands, MAPK phosphorylation primarily localized to the epithelium. To examine whether MAPK activity was required for modulation of branching morphogenesis, RonTK-/- mammary organoids embedded in Matrigel were treated with the MAPK inhibitor PD98059, which yielded a significant decrease in the number of organoids with buds and branches.

Treatment of RonTK+/+ organoids with the MAPK inhibitor at the same concentration had no significant impact on branching in Matrigel. Although a dose of PD98059 that blocked branching in both RonTK+/+ and RonTK-/- organoids was achieved, there were signs of toxicity associated with lack of proper sphere formation at this concentration.

3-67 Meyer et al., Developmental Biology, 2009.

Conversely, treatment of RonTK+/+ and RonTK-/- organoids with the Akt inhibitor

LY294002 resulted in a modest reduction in the percent of organoids with branches, but was not statistically significant or dependent on genotype. While Akt signaling does not appear to be an important factor in the differential branching of mammary organoids under our culture conditions, these data do not exclude the potential importance of Akt activity in branching morphogenesis in RonTK+/+ or RonTK-/- mice in vivo, in the context of the other cellular compartments where we also observed Akt phosphorylation by immunohistochemistry. Given that the Ron receptor is well established as an activator of Akt and MAPK activity [10,78], we were surprised to observe increased Akt and MAPK phosphorylation in RonTK-/- mammary glands. This result is seemingly contradictory to a previous study wherein loss of Ron tyrosine kinase in the MMTV- polyoma-middle T antigen (PyMT) breast cancer model resulted in reduced Akt and

MAPK phosphorylation [98] in lysates from Ron-deficient mammary tumors compared to

Ron expressing tumors. Our current study, however, differs greatly from the tumor biology studies previously reported. First, the studies herein are examining the normal physiologic levels of Ron during mammary gland development versus examining the loss of Ron function during tumorigenesis whereby Ron expression levels are upregulated in the mammary tumors. Second, the PyMT model itself is dependent on

Akt activation in mammary tumors directly coupled to polyoma-middle T antigen overexpression and the mechanisms by which Ron may modulate PyMT signaling and de novo mammary gland development (which is not dependent on this viral oncogene) may be different. Third, it is also not clear as to whether increased phosphorylation of

Akt and MAPK in RonTK-/- mouse mammary glands is a direct or indirect consequence

3-68 Meyer et al., Developmental Biology, 2009. of the loss of Ron. Abrogation of another the transmembrane receptor tyrosine kinase

IGF-IR in mouse prostate, also increased phosphorylation of Akt and MAPK [253].

Together, these data suggest that receptor tyrosine kinases are able to differentially regulate Akt and MAPK activation in a context specific manner.

Microarray and functional annotation analyses of genes altered in pubertal mammary glands from RonTK-/- mice compared to RonTK+/+ controls revealed that several key cellular processes were significantly altered by deletion of the Ron tyrosine kinase domain including development, transcription, morphogenesis, differentiation, kinase activity, and cell adhesion. It is evident that all these processes are essential during pubertal mammary gland development [167,168,171]. Gene families differentially regulated in RonTK-/- mammary glands within these functional categories include Wnt, sprouty, laminin, protocadherin, and ephrin. We have validated several targets identified by the microarray, which have known functions with broad implications and may potentially play a role in our model, but have not yet been studied with respect to mammary development including the glycoprotein Ceacam10 and acid phosphatase

Acpl2. Two genes found by DAVID analysis in the cell adhesion and motility cellular processes important for branching morphogenesis are the tyrosine kinase adapter Nck1

(data not shown) and protocadherin Pcdh17. The overall changes observed in our studies are consistent with other microarray analyses implementing these gene families in mammary gland development [254]. Together, these microarray data support the conclusion that Ron likely impacts mammary development through a process that is multifactoral.

3-69 Meyer et al., Developmental Biology, 2009.

In summary, we have shown that ablation of Ron receptor tyrosine kinase accelerates pubertal mammary gland development. Specifically, the loss of Ron impacts mammary gland branching morphogenesis independently of ovarian estrogen and progesterone stimulation. Moreover, based on three-dimensional ex vivo analyses, the absence of

Ron in the epithelium is sufficient to produce branching. Ron receptor ablation alters

Akt and MAPK phosphorylation, which is known to be essential for proper mammary epithelial branching morphogenesis. Finally, deletion of Ron tyrosine kinase profoundly alters the genetic program in the pubertal mammary gland. Taken together, our data demonstrate that Ron is an important regulator of pubertal mammary gland development.

3-70 Meyer et al., Developmental Biology, 2009.

Materials and Methods

Animals: A germline deletion of the tyrosine kinase domain of Ron (RonTK-/-) has been previously described and was backcrossed 8 generations onto the FVB/N background for the studies herein [17,98]. FVB/N mice containing wild-type Ron

(RonTK+/+) were used as controls for all experiments. For ovariectomization experiments, 3 week-old RonTK+/+ and RonTK-/- female mice (n=3 per gentoype) were ovariectomized and mammary glands were allowed to develop for an additional 3 weeks in the absence of ovarian hormones. At 6 weeks of age, inguinal mammary glands were harvested and prepared for whole mount analyses. All experiments involving animals were performed under protocols approved by the Institutional Animals and Use

Committee of the University of Cincinnati.

Whole Mount and Histological Analyses: Mammary glands from 5, 6, 7, 8, and 10 week-old virgin female RonTK+/+ and RonTK-/- mice (n=10 per genotype) were harvested. Thoracic glands were frozen for protein and RNA analysis, one inguinal gland was formalin fixed for histology and immunohistochemistry, while the other was while mounted for morphological assessment. For whole mount preparation, glands were spread on glass slides and fixed overnight in Carnoy’s Fixative, rinsed in 70% ethanol, and transferred into Carmine Alum stain overnight. Glands were rinsed in a graded series of ethanol and cleared in Xylene before mounting with Permount. Images of whole mounts were taken using a Nikon D1x digital camera with a Nikon AF MICRO

NIKKOR 105mm 1:2:8D lens. For histological analysis, glands were fixed overnight in

10% neutral buffered formalin then changed to 70% ethanol, processed, and paraffin

3-71 Meyer et al., Developmental Biology, 2009. embedded. 4µm-thick sections were stained using Harris Hematoxylin and Eosin.

Images of histological sections were taken with a Nikon FX-35DX camera affixed to the

Nikon Microphot microscope and Spotcam Advanced software (Nikon).

Ductal Elongation, TEB, and Branch Point Analyses: Axiovision Release 4.5 software was used to measure ductal elongation, terminal end bud number, and number of secondary and tertiary branch points from images of 5, 6, 7, 8, and 10 week-old

RonTK+/+ and RonTK-/- inguinal mammary whole mounts (n=10 per group), and from images of 6 week-old ovariectomized RonTK+/+ and RonTK-/- inguinal mammary whole mounts (n=3 per genotype). Ductal elongation was measured as the distance from the center of the lymph node to the furthest terminal end bud at the leading edge of the mammary epithelium. All terminal end buds of greater than or equivalent to 0.03mm2 were quantified. The longest primary duct directly above the lymph node was used for branch quantification from one inguinal gland per mouse (n=10 per genotype). A secondary branch was defined as any branch that bifurcates from the primary duct. A tertiary branch was defined as any branch that bifurcates off a secondary branch.

BrdU and TUNEL Analyses: Five, 6, and 7 week-old RonTK+/+ and RonTK-/- mice

(n=4 per genotype) were injected intraperitoneally with 10µl per gram body weight of a

10mM BrdU solution (Amersham Biosciences, Piscataway, NJ) 2 hours prior to sacrifice. The left side thoracic and inguinal glands were snap frozen in liquid nitrogen for use in the microarray analysis procedure. The right side thoracic and inguinal glands were fixed in 10% neutral buffered formalin overnight then changed to 70%

3-72 Meyer et al., Developmental Biology, 2009. ethanol. Mammary glands were processed and embedded in paraffin. 4µm-thick sections of RonTK+/+ and RonTK-/- inguinal mammary glands were stained using a

BrdU Staining Kit (Zymed Laboratories, Inc., San Francisco, CA) and In Situ Cell Death

Detection Kit, POD (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions. The percentage of BrdU-positive cells was determined by quantifying the number of BrdU-positive cells out of the total number of cells from 4 end buds and 4 ducts per mouse. The same quantification procedure was also used for

TUNEL analysis.

Primary Mammary Cell Purification and Stromal Fat Pad Isolation: Mammary epithelial cells were purified from RonTK+/+ and RonTK-/- mice. Two 6-8 week-old female mice per genotype were sacrificed and thoracic and inguinal mammary glands were removed, diced using razor blades, and placed into 25ml digestion media containing DMEM/F12, 1mg/ml collagenase (Worthington Biochemical, Lakewood, NJ),

Penicillin/Streptomycin, and 2mg/ml bovine serum albumin (Sigma, St. Louis, MO) for approximately 2 hours at 37°C with shaking at 200rpm. Cells were then centrifuged for

5 minutes at 1000 rpm and supernatant removed. To further dissociate epithelial cells, the pellet was resuspended in DMEM/F12 containing 2U/ml DNase for 5 minutes with vigorous shaking. DNase was inactivated with equal volume of DMEM/F12 containing

5% FBS. Organoids were centrifuged for 5 minutes at 800rpm, supernatant was removed and pellet was resuspended in 1X PBS plus 5% FBS. To remove fibroblasts, organoids were shaken vigorously then pulse spun for 10 seconds to a maximum of

1000 rpm, supernatant was removed and repeated 6 additional times. Epithelial

3-73 Meyer et al., Developmental Biology, 2009. organoids were rinsed twice in 1X PBS to remove traces of FBS. For characterization of the mammary fat pad without epithelium, fat pads were dissected, excluding nipple and lymph node, from 3 week-old RonTK+/+ mouse inguinal mammary glands. Fat pads from at least 2 mice were pooled, per sample.

Primary Mammary Epithelial Organoid Cultures: Mammary epithelial organoids purified from RonTK+/+ and RonTK-/- female mice were resuspended in Growth Factor

Reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) and plated on Matrigel pre- coated 24-well plates. 171 Medium (Cascade Biologics, Portland, OR) plus Mammary

Epithelial Growth Supplement (Cascade Biologics, Portland, OR) was added on top of the matrix/organoid mixture once gelled. Organoids were treated with 2µM of the MAPK inhibitor PD98059 (Calbiochem, San Diego, CA), or equivalent volume DMSO vehicle control, and cultured for 6 days with media plus inhibitor refreshed every 2 days. On day 6, the number of organoids with buds and/or branches was quantified and the percentage of budding/branching organoids out of total organoids plated was determined. Approximately equal numbers of organoids were plated per genotype and experiments were repeated three times with similar results.

Western Analyses: For Ron protein expression, mammary glands excised were from

5, 6, 8, and 10 week-old RonTK+/+ and RonTK-/- mice (a minimum of 2 mice per group) and snap frozen in liquid nitrogen. Glands from pregnant mice were isolated at day 16 following timed matings, lactating glands were isolated at day 10 after birth, and involuting glands were isolated 4 and 10 days following pup withdraw. Briefly, frozen

3-74 Meyer et al., Developmental Biology, 2009. glands were homogenized in 1.5X Laemmli Buffer, sonicated, and centrifuged at

12,000rpm for 15 minutes. Telomerase immortalized normal human mammary epithelial cells (HMEC) were donated by Dr. Robert Weinberg (MIT, Cambridge, MA) grown in Medium 171 (Cascade Biologics, Portland, OR) plus Mammary Epithelial

Growth Supplement (Cascade Biologics, Portland, OR) and antibiotics, then lysed in

1.5X Laemmli Buffer. Human primary adipocytes cells (HPAC) were grown in preadipocyte media (Zen-Bio, Research Triangle Park, NC), and lysed in Laemmli

Buffer. Eph4 immortalized normal mouse mammary epithelial cells were grown in

DMEM supplemented with 5% FBS and antibiotics, then lysed in Laemmli Buffer.

Stromal fat pads were isolated as indicated and lysed in Laemmli Buffer. Protein concentrations were determined using the MicroBCA Kit (Pierce Biotechnology,

Rockford, IL) according to manufacturer’s instructions. Anti-Ron ! (C-20) rabbit polyclonal antibody was used at a concentration of 0.2µg/ml (Santa Cruz Biotechnology,

Santa Cruz, CA). For Akt and Erk Western analyses, proteins lysates were generated from 6 week-old RonTK+/+ and RonTK-/- mice (n=4 per genotype). Rabbit monoclonal anti-phospho-Erk1/2, rabbit polyclonal anti-Erk1,2,3, rabbit polyclonal anti-phospho-Akt, and rabbit monoclonal anti-Akt(pan) were purchased from Cell Signaling Technology

(Danvers, MA) and used according to manufacturer’s instructions. All primary antibodies were detected using peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Antibody detection was performed according to manufacturer’s instructions with ECL Plus

Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) and

3-75 Meyer et al., Developmental Biology, 2009. developed on film. All Western anlyses were repeated thrice using different sets of mice.

Immunohistochemical Analyses: Phosphorylated MAPK (n=4) (phospho-Erk1/2, Cell

Signaling Technology, Danvers, MA) and Ron expression (n=3) (Santa Cruz

Biotechnology, Santa Cruz, CA) were detected on tissue sections from 6 week-old virgin female RonTK+/+ and/or RonTK-/- mice as indicated. Primary antibodies were detected using 0.75µg/ml goat anti-rabbit biotinylated secondary antibodies. Antibody immunoreactivity was amplified using the VECTASTAIN ABC kit (Vector Laboratories,

Burlingame, CA), and visualized using DAB substrate (Vector Laboratories, Burlington,

CA). The sections were counterstained in hematoxylin, dehydrated in a graded series of alcohols ending with Xylene, and mounted. Experiments were repeated twice. All images were captured using a Nikon FX-35DX camera attached to the Nikon Microphot microscope and Spotcam Advanced software.

Serum Steroid Hormone Assays: Whole blood was collected from 5, 6, and 7 week- old virgin female RonTK+/+ and RonTK-/- mice (n=6 per group) via cardiac puncture and placed into serum separator tubes. Tubes were spun and serum was placed in a

0.5ml tube and stored at -80°C until assays were performed. The serum from these mice was analyzed for circulating estradiol and progesterone levels using 17!-Estradiol

ELISA Kit (Cayman Chemical, Ann Arbor, MI) and Progesterone ELISA Kit (Cayman

Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.

3-76 Meyer et al., Developmental Biology, 2009.

Statistical Analyses: Statistical significance for all analyses, except for microarray analysis, was determined by a Student’s t-test using Sigma Stat 3.5 software (Cranes

Software International, Karnataka, India).

Microarray Analysis: RNA was harvested from whole mammary glands of 5, 6, and 7 week-old RonTK+/+ and RonTK-/- virgin female mice using TriZol (Invitrogen, Carlsbad,

CA) according to the manufacturers instructions with one modification. To remove fat from the TriZol preparation, centrifugation at 12,000g for 10 minutes at 4°C was preformed prior to the addition of chloroform. RNA samples were submitted to the

Cincinnati Children’s Hospital Medical Center Affymetrix Core, Cincinnati, Ohio. The

Agilent Bioanalyzer 2100 (Hewlett Packard, Palo Alto, CA) using the RNA 6000 Nano

Assay was applied to the RNA for quality assessment. Next, 400-500ng of total RNA per sample was used in the TargetAmp 1-Round Aminoallyl-aRNA Amplification Kit

(Epicentre Biotechnologies, Madison, WI) to generate cRNA following the manufacturer’s instructions. Biotin-X-X-NHS (Epicentre Biotechnologies, Madison, WI) was used to label the aminoallyl-aRNA with biotin. The biotin-labeled cRNA target was then chemically fragmented, and a hybridization cocktail was prepared and hybridized to the Affymetrix Mouse Genechip 430 2.0 array. The probe arrays were scanned using the Affymetrix GeneChip Scanner 3000 and Genechip Operating Software 1v4

(Affymetrix, Santa Clara, CA).

Gene Spring Analysis: Changes in gene expression were then analyzed using Gene

Spring GX Mouse-6v.1.1 software. 45,000 probe sets were first filtered on expression

3-77 Meyer et al., Developmental Biology, 2009. using a minimum raw intensity value greater than or equal to 120. The resulting 32,181 probe sets were then normalized to the median intensity value of all wild-types. Next, all probes were assessed by genotype (RonTK+/+ and RonTK-/-) by a parametric

ANOVA assuming equal variance with multiple testing correction Benjamini and

Hochberg false discovery rate of p=0.2. This resulted in 686 probe sets that differed from the wild-type average raw intensity value, which were then subjected to another parametric ANOVA p=0.005 with no multiple testing correction to find differences between genotypes by each age time point (5, 6, and 7 weeks), yielding 114 probes at 5 weeks, 200 probes at 6 weeks, and 106 probes at 7 weeks. A Venn diagram was generated by pooling the genes from individual time points (393 probe sets). Complete raw gene expression data and analyses can be obtained through the Gene Expression

Omnibus (GEO) website (http://www.ncbi.nlm.nih.gov/geo) accession number

GSE16629.

Functional Annotation Analysis: The 393 gene list generated from the Gene Spring analysis was uploaded onto the DAVID Bioinformatics Resources 2008 National

Institute of Allergy and Infections Diseases, NIH website (http://david.abcc.ncifcrf.gov/) and pathway and functional annotation analysis was performed.

Quantitative Real-Time PCR: RNA was isolated from whole glands, purified epithelial cells, or from glandular areas devoid of epithelial cells (consisting of adipocytes, fibroblasts and associated stromal cells). The RNA was used to generated cDNA using the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA) according to

3-78 Meyer et al., Developmental Biology, 2009. manufacturer’s instructions. To measure Ron transcript expression in wild-type, or

RonTK+/+ mice, whole gland and purified gland components were analyzed using the following mouse Ron primers; Forward: 5’-GTC CCA TTG CAG GTC TGT GTA GA-3’ and Reverse: 5’-CGG AAG CTG TAT CGT TGA TGT C-3’. These primers encompass part of the kinase domain deleted in the RonTK-/- mice. An additional set of primers were utilized for Ron that would detect a possible truncated product produced upstream of the TK deletion in the RonTK-/- glands. For these experiments, the following primers were utilized: Forward: 5’-TGG AGC CAG TGC TGA CAT C-3’ and Reverse: 5’-GAT

AGC GTG AAG TGC CAT G-3’. Human Ron expression in immortalized Human

Mammary Epithelial Cells (HMEC) and immortalized Human Primary Adipocyte Cells

(HPAC) the following human Ron primers were used; Forward: 5’-GAC CAG GCC CAG

AAT CGA AT-3’, Reverse: 5’-CAG GTC ACC GTG GCA CAT ATA G-3’, and Taqman probe: 5’-TGT GCC ATC AAG TCA CTA AGT CGC ATC A -3’. To confirm microarray results the following genes and corresponding sequences were chosen: Acpl2

(Forward: 5’-CCT TAA ATT CCC TGC CTC TC-3’; Reverse: 5’-GTT GGG CAG AAG

TTT GTG T-3’), Ceacam10 (Forward: 5’-ACT CCG ATT TCT GTG CGA-3’; Reverse:

5’-AAG AAC GTT TTC CCC TTC G-3’), and Pcdh17 (Forward: 5’-TCG GAT GTC CAT

AAT TCA GAC AGA-3’ and Reverse: 5’-CTG CCT GCT GCC CAT GTA AT-3’). Gene expression values were normalized to 18S (Forward: 5’-AGT CCC TGC CCT TTG TAC

ACA-3’; Reverse: 5’-GAT CCG AGG GCC TCA CTA AAC-3’) as internal control.

Relative gene expression results are reported. Real-time analyses were repeated twice with similar results using samples from 3 individual mice per genotype.

3-79 Meyer et al., Developmental Biology, 2009.

Acknowledgments

The authors would like to acknowledge the excellent technical assistance provided by

Sarah Kader, Kenya Toney, and Jerilyn Gray as well as Dr. Nelson Horseman for the helpful discussions and MCF10A protein lysates. This work was supported by the

Public Health Services Grants CA-100002 (S.E.W.), T32-CA59268 (S.E.M.), and the

Digestive Diseases Research Development Center grant DK-064403 (S.E.W.) from the

National Institutes of Health, as well as by grant project #8950 (S.E.W.) from Shriner’s

Hospital for Children.

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Chapter 4

Discussion

The Ron receptor regulates tissue morphogenesis

Morphogenesis collectively describes the processes of cell proliferation, cell death, fate determination, differentiation, cell adhesion, and motility, which lead to the organization of cell populations into complex three-dimensional structures, and together these structures build organs like the intestine and mammary gland [255].

These morphogenic processes are tightly regulated during development and give rise to a well-organized tissue, but are deregulated in tumorigenesis. Interestingly, tissues that go through postnatal development and homeostasis, like the mammary gland, intestine, and lung, for example, may be at greater risk of acquiring somatic gene mutations, or other alterations that may account for, at least in part, why these organs have the highest incidences of cancer in men and women in the US (ACS, 2009).

Correspondingly, many of the same signaling molecules that regulate development are often deregulated in tumorigenesis. The results from my thesis projects presented herein have identified novel roles of the Ron receptor tyrosine kinase in the regulation of intestinal crypt homeostasis, intestinal tumor formation, and pubertal mammary gland development. These in vivo findings are summarized in the Tables 1 and 2 below to illustrate the contributions of my thesis research toward our understanding of development and tumorigenesis. By comparing the effects of Ron signaling on intestinal and mammary gland morphogenesis with the molecules presented in Tables 1 and 2, some of which have either been shown to participate in Ron signaling or exhibit similar morphogenic effects as Ron, one can begin to postulate the mechanisms by which Ron may regulate intestinal and mammary gland morophogenesis.

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Table 1. Molecules important for intestinal homeostasis and tumorigenesis. Intestine Homeostasis Colon Cancer Model Phenotype Model Phenotype Receptor tyrosine kinases: EphB2 EphB2-/- Mixed proliferation zone DNEphB2; Increased colon tumors EphB3-/- and differentiated cells ApcMin/+ [257] [256]

Egfr EGF/TGF! Stimulate crypt EgfrWa2;ApcMin/+ Decreased adenomas proliferation [258] [160]

Ron RonTK-/-; Increased crypt RonTK-/-; Increased adenomas ApcMin/+ proliferation [28] ApcMin/+ [28]

Orthotopic Decreased metastasis HCT116 siRON [102]

Other kinases: JNK JNK1-/- Increased crypt JNK1-/- Few spontaneous proliferation, decreased intestinal adenomas differentiation [259] [259]

Transcription factors: "-catenin Villin-Cre-"- Loss of crypts [122] "-cateninAODN; Decreased adenoma catenin-/lox ApcMin/+ formation [260]

Notch1 Villin-Cre-Nic Loss of goblet cells [261] Villlin- Increased crypt CreERT2/Nic; proliferation, earlier and Apc1638N/+ increased adenomas [261]

Math1 Math1#intestine Loss of paneth, goblet, Math1#intestine; Increased tumor enteroendocrine cells ApcMin/+ number and size [263] [262]

EphB2, Ephrin receptor B2; DN, dominant negative; Egfr, epidermal growth factor receptor; TGF, transforming growth factor; Wa2, wave 2 mutation; Apc, adenomatous polyposis coli; Min, multiple intestinal neoplasia; TK, tyrosine kinase domain; HCT116, human colorectal carcinoma cell line; siRON, siRNA-mediated RON knockdown; JNK, c-Jun NH2-terminal kinase; AODN, antisense oligodeoxynucleotides; Nic, constitutively active Notch1; CreERT2, tamoxifen inducible Cre recombinase; Math1, also known as ATOH1, atonal homolog 1.

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Table 2. Molecules important for mammary gland development and tumorigenesis. Mammary Development Breast Cancer Model Phenotype Model Phenotype Receptor tyrosine kinases: ErbB2/Neu MMTV-Cre Decreased ductal MMTV-Neu Adenocarcinomas, lung fl/flErbB2 elongation, branching, metastasis [265] can lactate [264]

Egfr Egfr-/- Decreased ductal MMTV-Egfr Hyperplasia [266] reciprocal elongation, branching in transplants Egfr-/- stroma [183]

EphA2 EphA2-/- Decreased ductal MMTV- Decreased tumor elongation, proliferation, Neu,EphA2-/- formation, metastasis branching [185] [267] c-Met Eph4 Matrigel Induced branching/tube pMIG-tTA/TRE- Neoplasia [268] + HGF formation [245] Met

Ron RonTK-/- Increased ductal MMTV-Ron Adenocarcinomas, lung elongation, branching and liver metastasis [36] [30]

Other kinases: TGF!IIR Metallothionein Increased branching T!IIR-4T1 Tumor formation, lung -DNIIR [269] metastasis [270]

Nuclear hormone receptors: ER MMTV-ERAKO No pubertal development p53-/- E and P enhance [176] tumorigenesis [271]

PR PRAKO Normal branching, CMV-PRA Increased branching, lobuloalveolar hyperplasia [273] development [272] PRBKO Decreased branching, CMV-PRB Decreased branching, lobuloalveolar alveolar development development [274] [275]

ErbB2, also known as Her2/Neu, human epidermal growth factor receptor 2; fl/fl, floxed or flanked by loxP sites; MMTV, mouse mammary tumor virus promoter; Cre, Cre recombinase; Egfr, epidermal growth factor receptor; EphA2, Ephrin receptor A2; Eph4, immortalized non-transformed mouse mammary epithelial cells; HGF, hepatocyte growth factor; pMIG, retroviral vector with GFP, green fluorescent protein, tag; tTA, tetracycline-repressible transactivator protein; TRE, tetracycline response element; TK, tyrosine kinase domain; TGF!IIR, transforming growth factor ! type-2 receptor; DN, dominant negative; 4T1, mouse mammary tumor cell line; ER, estrogen receptor; ERAKO, estrogen receptor " knockout; PR progesterone receptor; PRAKO, progesterone receptor A isoform knockout; PRBKO, progesterone receptor B isoform knockout; CMV, cytomegalovirus promoter.

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Expression of the Ron tyrosine kinase domain influences crypt proliferation but is not required for adenoma formation in Apc-mutant mice

Previously, it was demonstrated that exogenous Ron overexpression in intestinal epithelial cells leads to anchorage independent cell growth in vitro [8]. Knockdown of

Ron expression in human colorectal cancer cell lines, HT29, SW480, and HCT116 using targeted siRNA resulted in decreased cell proliferation, increased cell death, and decreased anchorage independent cell growth [24]. Stable knockdown of Ron in

SW620 colon cancer cells reduced metastasis formation in an orthotopic model of colon cancer [102]. These studies also found that Ron may signal through the PI3K and !- catenin pathways to mediate these tumorigenic effects [24,102]. Blocking HGFL binding to Ron by neutralizing antibody or small molecule inhibition of Ron kinase activity both reduced tumor growth and metastasis in colon cancer cell xenografts

[1,14]. Taken together, these data suggest that Ron expression or overexpression in human colon cancer cells is important for their tumorigenicity, and that Ron inhibition may be useful as a therapy for the treatment of colon cancer.

Mutational inactivation of the tumor suppressor APC or aberrant stabilization of

!-catenin is found in the majority of colorectal cancers [22,127]. It is not known whether

Ron expression is required for intestinal tumor initiation, or whether Ron expression cooperates with aberrant APC loss and/or !-catenin activity to mediate colon tumorigenesis. In addition, the targets of Ron signaling and mechanisms of Ron activation in colon cancer cells have not been thoroughly investigated. Lastly, nothing is known about the normal role for Ron receptor signaling in the intestine. Therefore, we

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sought to test the contribution of Ron tyrosine kinase to intestinal homeostasis and tumorigenesis in vivo using the well-established ApcMin/+ mouse model [10,74,201].

By comparing ApcMin/+ mice with and without the Ron tyrosine kinase domain, we found that Ron has a regulatory role in limiting the number of proliferating small intestine and colonic crypt cells, and that Ron is not required for Apc-mediated intestinal tumor formation (Chapter 2) [28]. This study is important because it showed for the first time that Ron affects crypt homeostasis, which could be a new area of investigation in the field of Ron receptor biology. This was also the first study to address the role of Ron in intestinal tumor initiation. It was unexpected to find that Ron is not required for Apc- mediated intestinal tumor formation. Our model showed that Apc mutant intestinal epithelia with de novo Ron loss are still capable of forming tumors [28]. However, other in vitro and in vivo studies have shown that colon cancer cell lines and established tumors that overexpress Ron have reduced growth upon Ron inhibition [1,14,15].

Together, these data suggest that although Ron is not required for intestinal tumor formation in the presence of an Apc mutation, colon cancer cells that gain Ron overexpression during tumor progression can become sensitive to Ron inhibition.

Future research questions derived from the study of Ron in ApcMin/+ intestinal tumorigenesis

Through the study of Ron loss in ApcMin/+ mice, novel information about Ron in the intestine was discovered, including that Ron is not required for adenoma formation, but importantly Ron loss leads to expansion of the proliferative crypt compartment in

Apc-mutant mice [28]. The working model presented in Figure 1 integrates my new

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findings into the context of well-established signaling pathways that regulate crypt homeostasis. It is important to note that it is not known whether Ron regulates crypt homeostasis in the absence of an APC mutation, thus Figure 1 is a generalization of our findings, which may or may not be recapitulated in the presence of functional APC. In addition, three potential mechanisms by which Ron may affect crypt proliferation are indicated by numbered question marks in Figure 1. The fourth question mark in Figure

1 refers to a potential explaination for the result that significantly more ApcMin/+;RonTK-/- mice developed a high tumor burden compared to ApcMin/+;RonTK+/+ controls [28].

Given that Ron overexpression in colon cancer cells promotes tumor cell proliferation, growth, and metastasis [1,14,24,102], and that Ron downstream signaling targets, such as the PI3K/Akt and Mapk pathways, are important growth and survival signals (see Figure 2 in Chapter 1, page 1-11), the finding that Ron loss increases crypt cell proliferation suggests that Ron may regulate crypt homeostasis through an indirect mechanism. In the normal intestine and colon, studies have shown that the location of an epithelial cell along the vertical axis dictates the level and type of morphogen that it is exposed to, which in turn, plays a major role in the determination of cell fate, differentiation, and proliferative status [276,277]. Therefore, as indicated below in

Figure 1, I propose three mechanisms that may account for increased proliferation in the small intestine and colonic crypts, and how this increased proliferation may lead to more tumors in ApcMin/+ mice.

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Figure 1. A working model of the role of Ron in the normal intestine. Our recent findings of Ron signaling in the intestine [28] have been placed in the context of well described extrinsic and intrinsic regulators of crypt morphogenesis (i.e. proliferation, migration, and differentiation), adapted from Humphries and Wright, 2008 and Scoville et al., 2008 [107,127]. On the left is a simplified model of some of the key factors that regulate intestinal crypt homeostasis. On the right, is a representation of our finding in

ApcMin/+;RonTK-/- mice that exhibited approximately a 2-fold increase in the number of proliferating crypt cells in the small intestine and colon compared to ApcMin/+;RonTK+/+ controls [28]. A red bar between the RonTK+/+ and RonTK-/- crypts illustrates this difference in proliferation. On the left, three numbered question marks indicate potential

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means by which Ron might affect the number of proliferating crypt cells and intestinal tumorigenesis. ?1 – Ron loss may alter the morphogenic proliferation/differentiation gradient. ?2 – Ron loss may limit vertical cell migration leading to expansion of the crypt proliferative compartment. ?3 – Ron loss may lead to expansion of the stem cell niche by regulation of stem/progenator daughter cell fate. ?4 – Increased crypt proliferation in RonTK-/- mice leads to increased loss of Apc and increased tumor initiation. PCNA, proliferating cell nuclear antigen; Ki67, proliferation marker; Apc, adenomatous polyposis coli; Wnt, wingless and INT; Lgr5, leucine-rich repeat- containing G protein-coupled receptor 5; CD44, cell surface glycoprotein; TK, tyrosine kinase domain.

Hypothesis 1: Ron loss alters the morphogenic proliferation/differentiation gradient. It is well established that the Wnt pathway is essentsial for the maintenance of the stem cell compartment [122-124], and the expression of Wnts are greatest in the crypts [278]. BMP [279] and Notch [280] differentiation factor expression increase toward the intestinal lumen, thus along with Wnts creates an antagonistic proliferation/differentiation morphogenic gradient along the vertical axis of the small intestine and colon [107]. Wnt, BMP, and Notch are major regulators of crypt homeostasis and the removal of any of these signaling pathways leads to a drastic loss of the crypt proliferative compartment (Wnt/!-catenin/Tcf), or loss of differentiated cell types (BMP, Notch) [107,281,282]. Further, since gross examinatin of H&E staining did not reveal obvious loss of a particular cell type within ApcMin/+;RonTK-/- intestines and colons, it is likely that if Ron impacts these signaling pathways it does so in a modest

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fashion that requires a more detailed immunohistochemical assessment of expression patterns of these particular factors and cell type-specific staining techniques, an idea expanded further in hypothesis 3. Whether a gradient expression pattern of Ron,

HGFL, or both exisst along the vertical crypt axis has not been tested, nor is it known whether epithelial-derived Ron expression, or Ron expression on other cell types is important for regulation of crypt homeostasis. It was also not been tested whether

RonTK-/- mice with wild-type Apc have altered crypt homeostasis.

More specifically, Ron has been shown to regulate the expression and activity of

!-catenin, a major target of Wnt signaling [24,203]; therefore, we tested whether Ron loss in ApcMin/+ mice atered !-catenin signaling. We did not find significant differences in !-catenin localization or target gene expression compared to ApcMin/+ mice with Ron

[28]. These data suggest that Ron may not affect crypt homeostasis through modulation of !-catenin signaling, but does not preclude the possibility that Ron may intersect with other intestinal extracellular morphgen or intracellular signaling pathways.

Hypothesis 2: Ron loss decreases vertical cell migration leading to expansion of the crypt proliferative compartment. Ron has been shown to regulate cell migration of a variety of cell types, including epithelium [24,49,283]. In our model, Ron loss results in expansion of the crypt proliferative zone with the proliferating cells still confined to the crypt base and not mislocalized to the differentiated villus region [28]. In another model, knockout of the receptor tyrosine kinase Ephrin receptor B4 (EphB4), also lead to the expansion of the crypt compartment in ApcMin/+ mice [284]. Unlike EphB2 and EphB3, which restrict proliferative and differentiated cell intermingling [256], EphB4 confines the height of the proliferative compartment. While the exact mechanism of EphB4-mediated

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regulation of crypt proliferation is not known, it was shown that EphB4 expression regulated intestinal cell migration and invasion in vitro [284]. Therefore, it is plausible that receptor tyrosine kinase-mediated intestinal cell migration may impact crypt proliferation. The effect of Ron on crypt cell migration can be measured in vivo using a previously described technique [277] wherein mice are injected intraperitoneally with

BrdU, then sacrificing the animals at defined intervals between 1-96 hours post- injection, with the idea that the intestine should self-renew in three-four days in normal mice. The intestines from these mice can be stained for BrdU incorporation into dividing cells and evaluating the number and location of BrdU-positive cells overtime as a measurement of cell migration distance.

Hypothesis 3: Ron loss leads to an expansion of the stem cell niche by regulation of cell fate of stem/progenator cell division. Crypt stem cell and progenitor cells are thought to be the only populations of cells that undergo proliferation in the intestine and colonic crypts [107,276]. Crypt stem cells undergo aschyronous division to give rise to a daughter cell that will go on to become a differentiated cell type of the intestine, and another stem cell [107]. While no evidence to date has demonstrated that Ron regulates cell fate determination, or differentiation of epithelial cells in normal tissues;

Ron signaling has been shown to influence the activity and expression of numerous signaling molecules (eg. PI3K/Akt, Mapk, JNK, Src, EGFR, and !-catenin) implicated in cell fate decisions within the intestine [78,88,203,276]. This hypothesis can be tested by performing immunohistochemical or flow cytometric analyses on intestinal tissues with and without Ron for cell type markers such as CD44+ or Lgr5+ for stem/progenitor cells, Alcian blue stain for Goblet cells, and Alcian blue stained tissue can be destained

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with N-cetylpyridinium chloride for Paneth cell visulaization [108,285,286]. These cell- specific staining techniques may help to visualize and quantify differences in cell types that may not be as obvious by routine histological examination of H&E staining, and will confirm whether the proliferating population are stem/progenitor cells.

Hypothesis 4: The increased crypt proliferation in ApcMin/+;RonTK-/- mice leads to increased susceptibility to loss of Apc and subsequent tumor formation. We havested adenomas from all segments of the small intestine and colon, and performed a simple

PCR reaction using primers that will detect both the wild-type and mutant Apc alleles.

Of the tumors tested, 100% showed loss of the wild-type Apc allele [28], an early initiating event in intestinal and colon tumor formation [18,20,205]. Further, since the crypt region is the origin of tumor initiation in Apc-mediated intestinal tumorigenesis

[151,152,206], and I found Ron loss increases crypt proliferation, it is possible that Ron loss may therefore lead to increased loss of wild-type Apc and subsequent tumor initiation. To test this, intestines from ApcMin/+;RonTK-/- and ApcMin/+;RonTK+/+ mice can be analyzed for presence of wild-type Apc by in situ hybridization. The number of Apc- negative crypt cells can be quantified and compared between genotypes.

Lastly, even though the tyrosine kinase domain of Ron is not required for intestinal adenoma formation, others have shown using colon cancer cell lines that Ron plays a role in colon cancer progression at later stages [1,6,24,102]. A recent publication showed that HCT116 colon cancer cells with stable shRNA-mediated Ron knockdown are still able form tumors upon cecal orthotopic injection in nude mice.

Importantly, even though Ron knockdown cells formed intestinal tumors, they developed fewer metastases than the Ron expressing control cells [102]. Together with our

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observations, these data show that Ron is not required for intestinal tumor initiation in vivo in two different models of intestinal tumorigenesis; one with mutant Apc [28] and the other with mutant constitutively active !-catenin [102] driving tumorigenesis. This conclusion is also supported by findings in other tumor models of the skin [99] and mammary gland [98], wherein deletion of the kinase domain of Ron may have delayed, but did not block tumor formation. Together, this information supports the hypothesis that Ron may be a critical mediator of advance-stage colon cancer progression, specifically with respect to metastasis. This hypothesis can be tested in a strictly in vivo setting by generating mice that overexpress Ron specifically in the intestinal epithelium under the control of the villin gene promoter. If Ron overexpression alone is not sufficient to drive intestinal tumorigenesis, these mice could be crossed with ApcMin/+ mice to study whether Ron overexpression induces progression of Min/+ adenomas.

In summary, the results obtained from these prospective future studies would enhance our understanding of Ron receptor biology and cancer progression, as well as regulation of intestinal homeostasis. These proposed future directions also have the potential to generate useful animal models in which to test Ron targeted molecular therapies for the treatment of colorectal cancer.

Ron regulates pubertal mammary gland development

In vitro and in vivo studies have identified Ron as an important oncogene in breast cancer cells. Specifically, Ron is expressed or overexpressed in greater than

50% of human breast cancers [1,29,194]. Our laboratory previously demonstrated that mammary-specific Ron overexpression is sufficient to induce breast tumorigenesis and

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metastasis in mice [30]. In addition, Ron loss in the polyoma middle T antigen model of breast cancer lead to increased tumor latency and decreased metastasis [98]. In breast epithelial and cancer cells, both HGFL–dependent and –independent Ron signaling regulates cell survival, spreading, migration, and invasion [29,49,192,194]. Despite the importance of Ron in breast cancer, our study presented in Chapter 3 of this thesis was the first to investigate the role of Ron in the normal mammary gland, specifically during puberty, in mice. Some preliminary evidence suggesting that Ron might play a role in the normal mammary gland was that mammary specific Ron overexpression altered the appearance of adult mammary glands in female mice [30], and expression studies on mouse and human tissues showed that Ron is expressed in the normal mammary gland

[29,32]. Pubertal mammary gland development in mice (5-10 weeks of age) generates the mammary ductal network, which supports lactation after pregnancy. Mammary gland development during puberty is a complex process with epithelial cell proliferation, apoptosis, differentiation, migration/invasion, and branching morphognesis occurring simultaneously at different locations within the mammary gland. Mammary gland development not only requires the participation of the epithelium in these processes, but it is well documented that the stromal cells (i.e. macrophages, fibroblasts, adipocytes) and endocrine signaling also play critical roles in generating a phenotypically normal adult mammary gland. Although Ron function has not been extensively studied in normal cell types, it has been shown to regulate proliferation, adhesion, and migration of epithelial cells, plus cell shape change, differentiation, and migration of macrophages

[69,90,283]. In a model of kidney injury Ron was shown to participate in tubulogenesis during repair, the only model in which prior to our study Ron had ever been shown to

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participate in branching morphogenesis [35]. In addition, the Ron family member, Met has been shown to regulate mammary epithelial branching morphogenesis [287,288].

By comparing wild-type (RonTK+/+) and Ron tyrosine kinase domain-deficient (RonTK-

/-) mice, the role of the Ron tyrosine kinase in mouse mammary gland development was investigated [36]. This study identified a role of Ron in limiting branching morphogenesis in the mammary gland during puberty, which was not previously known

[36]. Also, using three-dimensional in vitro branching morphogenesis assays, I was able to show that Erk, but not Akt, activity was important for this effect of Ron on branching [36]. Lastly, this was the first study to examine the gene expression prolife associated with Ron receptor signaling during pubertmal mammary development, and also provided a wild-type mammary gland gene signature associated with the changes occurring in the glands from 5-7 weeks of age [36]. Together, these data show that the breast oncogene Ron is also important for normal mammary gland development.

Future research questions derived from the study of Ron in pubertal mouse mammary gland development

It has been shown that mammary branching morphogenesis requires proliferation of the mammary epithelium, however, there was no evidence of a correlation between the location of proliferation and where a budding branch formed [170]. This data suggests that there are pathways, such as ER!, that impinge upon mammary epithelial growth and survival, and are therefore, required for branching morphogenesis [176], but there are other pathways that may be critical to regulate the number, location, or spacing of the branches independently of cell proliferation [170]. Based on the data

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presented in Chapter 3, it is plausible that Ron, either directly or indirectly, may be one such factor that regulates the number or spacing of these branches. Figure 2A presents a model representing our finding that Ron loss increases the extenion and number of branch points in 6-8 week old mammary glands and by 10 weeks results in fewer TEBs as compared to wild-type mice. At the level of a single representative duct, Figure 2B shows both negative (in red) and positive (in green) regulators of mammary gland lateral side branching and TEB bifurcation, in addition to our contributions shown in larger font (Ron and Erk1/2). While there are few negative regulators of mammary branching morphogenesis through which Ron may cooperate to regulate branching, it is possible that Ron may act to negatively regulate factors that promote branching.

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Figure 2. A working model of the role of Ron in mammary gland development. The findings of our recent analysis of mammary gland development in mice lacking the tyrosine kinase domain of Ron (RonTK-/-) compared to age-matched wild-type controls are summarized [36]. A) The 6-8 weeks of age pubertal time point illustrates our finding that RonTK-/- mammary glands exhibited significantly increased secondary and tertiary

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branching, with no significant differences in TEB numbers [36]. This time period is the time frame of reference in B. The 10-week time point reflects our finding that RonTK-/- glands had significantly decreased TEB numbers compared to controls. B) A representative duct with the two types of branching morphogenesis, lateral side branch and TEB bifurcation, that occur during pubertal mammary gland development. An overly simplified version of the epithelial compartments (ducts and terminal end buds) in purple and the stromal compartment (fat pad, fibroblasts, ECM, macrophages) in gray are shown. The molecules that inhibit branching morphogenesis are in red and those that promote branching are in green. The location of action of these molecules coincides with their placement within the illustration (epithelial vs. stromal origin). ER! and GHR are required for post-natal mammary gland development, but modulation of their expression levels also alters development [176,180,181].

Figure 2A summaries only what is known through our studies of Ron during pubertal mammary gland development, and it remains unknown whether Ron impacts later stages of mammary gland development. Therefore, the next step in continuation of our analyses would be to test the hypothesis that Ron is important for proper mammary gland development during pregnancy, lactation, and involution. We found that Ron was most highly expressed in normal mouse mammary glands during pregnancy and lactation [36], thus one potential future direction would be to ascertain whether Ron has a role in other stages of mammary gland development. One preliminary observation is that RonTK-/- mice are fertile, able to nurse pups to weaning through multiple rounds of breeding, and the pups appear to be within a normal weight

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range both at the time of birth and at weaning [36], suggesting that any defects that may

be present in RonTK-/- mammary glands do not to impair basic mammary gland

function. However, to understand the actual role of Ron in the mammary gland during

pregnancy, lactation, and involution further studies will be necessary. For instance,

analysis of the glandular architecture during the first pregnancy, lactation, and involution

and then again after several pregnancies to see if over longer periods of successive

rounds of development, there are differences in mammary glands without Ron. Milk

protein production can be analyzed and also susceptibility to carcinogenesis during

these developmental stages.

During puberty, branches occur by two distinct mechanisms, TEB bifurcation and

lateral budding from an existing duct [168]. We found that the number of secondary and

tertiary branches in RonTK-/- pubertal mammary glands (6-8 weeks of age) was

significantly greater than the number of branches in RonTK+/+ controls, and this

phenotype was also found in ovariectomized (OVX) 6 week-old mice [36]. While

RonTK-/- mammary glands increased numbers of terminal end buds, this difference was

not statistically significant. Nor was there a difference in TEB numbers in

ovariectomized RonTK-/- compared to wild-type mice [36]. Together these data lead to the hypothesis that Ron limits lateral branching during postnatal mammary gland development, as illustrated in Figure 2B by the placement of Ron (in red) on the lateral

branch. Since estrogen is the main driver of TEB growth and mammary gland

development during puberty [171], I more closely examined the ovariectomized wild-

type and RonTK-/- mammary gland whole mounts, which have smaller and sparser

ductal trees than mice with intact ovaries, making it easier to observe branching

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morphogenesis while at the same time perhaps limiting the extent of TEB growth and bifurcation. Preliminarily, it appeared that there was an overabundance of lateral branching in OVX RonTK-/- mammary glands; however, additional studies are needed to more closely examine the the type of branching in mice with intact ovaries. Examples of well-studied molecules that regulate lateral branching morphogenesis during pubertal development include the matrix metalloproteinase’s MMP2 and MMP3 (Figure 2B).

MMP2-/- mouse mammary glands have greater numbers of secondary branches,

MMP3-/- glands have fewer secondary and tertiary branches, and interestingly these phenotypes, like our RonTK-/- model [36], normalize by the end of pubertal development at 10-12 weeks of age [188]. There were also no differences in TEB numbers in the MMP knockout mice compared to wild-type, thus the authors concluded that these MMPs specifically regulate lateral branching [188]. Whether Ron regulates lateral branching only, or both lateral branching and TEB bifurcation is not yet clear.

One caveot to our study was that the mice were not tested for stage in estrus cycle.

TEB growth is driven by the estrus cycle, so segregating animals by stage in estrus may help to resolve whether Ron impacts TEB bifurcation.

There are several potential mechanisms by which Ron regulates mammary branching morphogenesis, including cell adhesion, migration, and invasion. Cell-cell and cell-matrix adhesion differences between RonTK-/- and wild-type primary mammary epithelium can be quantified in vitro using microscopy. Differences in cell migration between RonTK-/- and wild-type mammary epithelial cells can be tested using wound healing or scratch assays. Ron has been shown previously to regulate claudin-1 expression [75] and crosstalk with integrins [86,289], which have been shown to play

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roles in cell adhesion, motility, and branching morphogenesis [290,291]. In addition, purified mammary epithelium from RonTK-/- mice branched significantly more than epithelium from RonTK+/+ mice when embedded in Matrigel suggesting that Ron may regulate mammary epithelial cell invasion [36]. Erk phosphorylation was increased in

RonTK-/- mammary glands compared to wild-type, and the increased branching of

RonTK-/- organoids in vitro was significantly reduced upon Erk inhibition [36]. However, the signaling mechanisms that lead to Ron modulation of Erk phosphorylation in our model are not known. It is possible that the Matrigel or mammary epithelial growth medium may contain signal(s) that stimulate Ron regulated branching morphogenesis in vitro, as no difference in branching between RonTK-/- and RonTK+/+ epithelium were observed in preliminary experiments using a purified collagen matrix. Growth factor reduced Matrigel consists mostly of the ECM component laminin, in addition to some collagen, and limited quantities of several growth factors including IGF, FGF, EFG, and

TGF!. Since Ron has been shown to crosstalk with other growth factor signaling pathways like EGF/EGFR, TGF", TGB! and with cell adhesion pathways like integrins, claudins, and E-cadherin it is possible that these molecules known to be important for mammary development and branching morphogenesis, may play a role in our model

[75,77,84,86,88,289,292-294]. Furthermore, by microarray analyses we found approximately 90 genes altered in pubertal (5-7 weeks of age) RonTK-/- mouse mammary glands compared to age-matched wild-type controls that are known to have roles in cell adhesion, cell motility, differentiation, and morphogenesis including genes in such families as protocadherins, claudins, laminins, ephrins, TGF!s, and FGFs [36].

One possibility to test the importance of these factors on Ron-mediated branch

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regulation would be to compare RonTK+/+ and RonTK-/- organoid branching behavior in purified type I collagen matrix, wherein growth factors or ECM components can be added individually or in combination.

Given that RonTK-/- mammary glands exhibit increased branching morphogenesis [36], and that MMTV-Ron mice have fewer mammary gland branches than wild-type controls (12 weeks of age), but develop tumors with 100% penetrance

[30], its possible that during puberty Ron may influence breast tumorigenesis. During puberty, the tips of ducts/branches consist of proliferating end buds consequently,

RonTK-/- glands have a greater number of proliferating epithelial structures, even though the rate of cell turnover within these structures is similar to that of wild-type controls [36]. It is possible that females are at increased risk of environmentally- induced breast carcinogenesis during puberty because of the greater abundance of dividing cells to acquire mutations [295,296]. Thus, it is important to test whether Ron loss renders mammary glands more susceptible to chemically-induced carcinogenesis.

Since Ron is a breast oncogene that also restricts pubertal mammary development,

RonTK-/- mice would provide a useful model in which to test whether accelerated pubertal development and environmental or chemical carcinogen exposure, such as to dimethylbenz(alpha)anthracene (DMBA), collaborate to increase susceptibility the to mammary tumorigenesis. If upon carcinogen treatment RonTK-/- mice developed more tumors than RonTK+/+, then this outcome would suggest that precocious pubertal development is a risk factor for breast cancer. The converse result would also be important because if RonTK-/- mice developed fewer tumors than wild-type mice treated

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with the same carcinogen, then this would suggest that Ron loss is protective against multiple mechanisms of breast cancer, oncogene- and carcingoen-induced.

Lastly, it is not known whether the ligand from Ron, HGFL, contributes to Ron- mediated regulation of mammary gland development. HGFL-/- mice, which are not known to have been analyzed for mammary gland development previously [92], can be used to test the requirement for HGFL in mammary gland development and branching morphogenesis in vivo by comparing pubertal mammary branching morphogenesis between wild-type, HGFL-/-, RonTK-/-, and HGFL-/-;RonTK-/- mice.

To summarize, these future directions are important toward our understanding of

Ron receptor biology. The mechanisms of Ron signaling unraveled by these future studies may contribute to the underlying mechanisms of Ron signaling not only in mammary gland development, but in other models of tissue injury, wound healing, kidney disease, liver disease, and cancer and thus have broad implications. Our results may also lead to future investigations to determine whether Ron is an important regulator of branching morphogenesis in other organs such as the lung and billiary tree.

Clinical implications of Ron receptor loss in the intestine and breast

In general, receptor tyrosine kinases are known to be deregulated in a variety of tumor types and have proven to be successful cancer targets by molecular therapeutics including such well known examples as Herceptin (Trastuzumab) which binds Her2 triggering immune system-mediated cancer cell death, Gleevac (Imatinib) which binds the kinase domains of Bcr-Abl and c-Kit blocking downstream signaling, and Tarceva

(Erlotinib) which competes for the ATP binding pocket in Egfr preventing receptor

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autophosphorylation, and Met small molecule inhibitors are currently being developed

[14,297,298]. Over the last decade many efforts have contributed toward validating the

Ron receptor tyrosine kinase as a therapeutic target for cancer intervention [15].

Studies have shown that antibody and small molecule inhibition of Ron signaling was able to reduce or completely block the growth of human pancreatic, colon, lung, and mammary tumor cell lines that overexpress Ron in xenografts in nude mice [1,14].

These are amongst the first studies to show a benefit to blocking Ron function in tumors in vivo. My studies suggest that the de novo loss of the Ron tyrosine kinase domain could increase crypt proliferation, an adverse effect, in the presence of other favorable mutations [28]. However, this does not preclude the utility of Ron inhibitors to block the progression and metastasis of established tumors. In addition, our laboratory has demonstrated that Ron overexpression is a causal factor in breast cancer, and many studies have shown that Ron is overexpressed in a large fraction of breast cancers and correlates with poor prognosis [29,30]. Together, these studies illustrate the somplexities of colon and breast cancers and that Ron is important for motile/invasive phenotypes, but not required for formation of some tumor types. Therefore, it is imperative to continue to better understand the consequences of Ron inactivation, including long-term and short-term Ron inhibition, in normal tissues as well as during tumorigenesis in vivo.

Summary

To summarize, studying Ron kinase loss in the intestine and mammary gland revealed several new and unexpected roles of Ron receptor signaling. The data

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described herein demonstrate the first evidence that, when combined with an Apc mutation, Ron regulates intestinal crypt homeostasis, but that Ron expression is not required for initiation of intestinal tumors in ApcMin/+ mice. Future studies are aimed at addressing whether Ron regulates crypt homeostasis in a non-mutant background, and investigating the mechanism by which Ron loss expands the crypt compartment. Also described is the first evidence showing that Ron is an important regulator of mammary gland branching morphogenesis during pubertal development. With this new information about Ron receptor signaling, future studies are aimed at determining whether Ron also plays a role during other stages of mammary gland development, or even other organs. Together, these data represent the first in vivo evidence of a new outlook on the Ron receptor as an important regulator of normal tissue morphogenesis.

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Abbreviations

Akt also known as protein kinase B (PKB)

AODN antisense oligodeoxynucleotides

APC adenomatous polyposis coli

ATP adenosine triphosphate

Bcr-Abl gene fusion of Bcr and Abl genes, Philadelphia chromosome c-Kit stem cell factor receptor

CD44 type 1 transmembrane glycoprotein, intestinal stem cell marker

CMV cytomegalovirus promoter

Cox2 cyclooxygenase 2

Cre Cre recombinase

DMBA dimethylbenz(alpha)anthracene

DN dominant negative

DSS dextran sodium sulfate

ECM extracellular matrix

EGF epidermal growth factor

EGFR epidermal growth factor receptor

Eph Ephrin

Eph4 immortalized mouse mammary epithelial cell line

ER estrogen receptor

ErbB2 also known as Her2/neu, human epidermal growth factor receptor 2

Erk extracellular signal-regulated kinase

ERT2 tamoxifen inducible estrogen receptor mutant

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FGF fibroblast growth factor

GI gastrointestinal

H&E hematoxylin and eosin

HCT116 human colorectal adenocarcinoma cell line

Her2/Neu human epidermal growth factor receptor 2

HGF hepatocyte growth factor

HGFL hepatocyte growth factor-like protein, same as MSP

IBD inflammatory bowel disease

IGF insulin growth factor

JNK c-Jun N-terminal kinase

KO knockout

Lgr5 Leucine-rich repeat-containing G protein-coupled receptor 5, intestinal

stem cell marker

LOH loss of heterozygosity

LY294002 small molecule PI3K inhibitor

Mapk mitogen activated protein kinase, same as Erk

Math1 also known as ATOH, atonal homolog 1

MEK also known as MAPKK, mitogen activated protein kinase kinase

Min multiple intestinal neoplasia

MMP matrix metalloproteinase

MMTV mouse mammary tumor virus

Nic constitutively active Notch1

NO nitric oxide

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OVX ovariectomized

PD98059 small molecule MEK inhibitor

PI3K phosphoinositide 3-kinase pMIG retroviral vector with GFP, green fluorescent protein

PR progesterone receptor

PyMT polyoma virus middle T-antigen

RON Recepteur d’Origine Nantaise shRNA short hairpin RNA siRON small interfering RNA targeted against Ron

TEB terminal end bud

TGF transforming growth factor

TK tyrosine kinase domain

TRE tetracycline response element tTA tetracycline-repressible transactivator protein

US United States

Wa2 wave 2 mutation

4T1 cell line derived from spontaneous mouse mammary tumor

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Chapter 5

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Chapter 6

Declaration of Original Research

Publications resulting from thesis research:

1. Meyer SE, Waltz, SE, Goss, KH. 2009. The Ron Receptor Tyrosine Kinase is Not Required for Adenoma Formation in ApcMin/+ Mice. Molecular Carcinogenesis.

2. Meyer SE*, Zinser, GM*, Stuart, WD, Pathrose, P, Waltz, SE. 2009. The Ron Receptor Tyrosine Kinase Negatively Regulates Mammary Gland Branching Morphogenesis. Developmental Biology. 333(1):173-185.

*Both authors contributed equally to this work.

Specific contributions of the authors to these manuscripts:

Manuscript 1 (Chapter 2), S.E.M performed all published experimental analyses and wrote the manuscript.

Manuscript 2 (Chapter 3), S.E.M. wrote the manuscript and the authors’ specific contributions to each figure is described below:

Figure 1: G.M.Z performed quantitative real-time PCR analyses for Ron expression; W.D.S. performed Western analyses for Ron; P.P. performed immunohistochemical analyses of Ron expression on RonTK+/+ mammary glands; S.E.M. and G.M.Z harvested primary cells and tissues for the analyses.

Figure 2: G.M.Z. performed developmental whole mount analyses including terminal end bud, ductal elongation, 5-6 week branching quantification, and H&E staining; S.E.M performed 7-10 week branching quantification.

Figure 3: S.E.M and G.M.Z. performed hormone ELISAs; S.E.M. performed ovariectimizations and associated whole mount analyses.

Figures 4, 5, 6, and 7: S.E.M performed all analyses including BrdU analyses, TUNEL analyses, Western blots for phosphorylated and total Akt and Mapk, actin and immunohistochemical analyses for phosphorylated Mapk, in vitro branching morphogenesis assays with and without inhibitors, microarray analysis, and quantitative real-time PCR for microarray validation.

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