The Role of Bone-Derived Osteopontin in the Migration and Stem- Like Behavior of Breast Cancer Cells

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6-22-2015 12:00 AM

Graciella Pio The University of Western Ontario

Supervisor Dr. Alison Allan The University of Western Ontario

Graduate Program in Anatomy and Cell Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Graciella Pio 2015

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This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. THE ROLE OF BONE-DERIVED OSTEOPONTIN IN THE MIGRATION AND STEM- LIKE BEHAVIOR OF BREAST CANCER CELLS

(Thesis format: Monograph)

Graciella Pio

Graduate Program in Anatomy and Cell Biology

A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science

The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada

ABSTRACT

The majority of breast cancer deaths occur due to metastasis, with the most common site of distant breast cancer metastasis being bone. Previous work in our laboratory has established that stem-like breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and expression of a CD44+CD24- phenotype have enhanced metastatic capacity. This study tested the hypothesis that bone-derived osteopontin (OPN) promotes the migration and stem-like behavior of breast cancer cells.

We demonstrate that bone-derived OPN promotes migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDHhiCD44+CD24- breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment). We observed that cell surface receptors CD44 and RGD-dependent integrins facilitate these behaviors via OPN, potentially through activation of WNK-1,

PRAS40 and HSP60 signaling in breast cancer cells. Our analysis of the interactions between bone-derived OPN and breast cancer cells provides valuable insight regarding breast cancer cell metastasis to bone.

Keywords: Breast cancer, Metastasis, Bone microenvironment, Osteopontin, Cancer stem cell, CD44, Integrins

CO-AUTHORSHIP STATEMENT

All sphere-limiting dilution assays (SLDA) and colony-forming assays were performed by Matt Piaseczny (MSc Candidate, Allan Lab, Anatomy and Cell Biology, Western University).

Fluorescence-activated cell sorting (FACS) analysis was performed by Kristin Chadwick (London Regional Flow Cytometry Facility, Western University).

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ACKNOWLEDGMENTS

Firstly, I would to like to thank my supervisor, Dr. Alison Allan, for allowing me the opportunity to pursue an interesting and meaningful MSc degree in a helpful, inclusive, and comfortable work environment. I would like to thank her for constantly supporting and challenging me throughout, for accepting my mistakes and for always giving me the opportunity to improve and showcase my work both in London and abroad! Alison, you have played a major role in developing my skills as a scientist and a professional and in allowing me to grow intellectually over the past three years – I am sincerely grateful and will forever be changed by this experience.

I would also like to thank all of the members of the Allan Lab for being such fantastic peers to work with; Ying Xia, David Goodale, Lori Lowes, Matt Piasecnzy,

Mauricio Rodriguez and Maria Vieito Villar. Special thanks to Ying, for your constant, incredible patience and for teaching me many of the lab techniques I know today – I am a competent MSc student because of you. To David for your constant help, support and expertise in all things laboratory. To Lori, for being a great friend and professional role model and to Matt, for always making me laugh and for generously donating your time to do experiments for my project.

I would also like to thank my committee members, Dr. Moshmi Bhattacharya, Dr.

Stephen Sims and Dr. Paul Walton. Thank you for donating your time to support my work and for you constructive feedback, encouraging words and invaluable scientific input.

Finally, I would like to thank my family for always believing in me and pushing me to be the best I can be – Pop, Mom and Mark, I am so very lucky to have you as my family!

And to Brendan, for encouraging me to pursue my MSc and for always reminding me that I am on the right track. This thesis is dedicated to you.

TABLE OF CONTENTS

CERTIFICATE OF EXAMINATION ...... Error! Bookmark not defined.

ABSTRACT ...... ii

CO-AUTHORSHIP STATEMENT ...... iii

ACKNOWLEDGMENTS ...... iv

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 3

2.1 Cancer ...... 3

2.2 Breast Cancer ...... 4

2.3 Metastasis ...... 5

2.4 Cancer Stem Cells ...... 5

2.5 Metastatic Organ Tropism ...... 10

2.6 Breast Cancer Metastasis to Bone ...... 10

2.7 Osteopontin ...... 16

2.8 Project Rationale ...... 21

3 HYPOTHESIS and OBJECTIVES ...... 22

3.1 Hypothesis ...... 22

3.2 Objectives ...... 22

4 MATERIALS and METHODS ...... 23

4.1 Cell Culture and Reagents ...... 23

4.2 Bone Marrow-Conditioned Media (BMCM) ...... 23

4.3 RayBio® Biotin Label-Based Mouse Antibody Arrays ...... 24

4.4 Immunodepletion of OPN ...... 24

4.5 Migration Assays ...... 25

4.6 Sphere-Limiting Dilution Assays (SLDA) and Colony-Forming Assays .... 25

4.7 Flow Cytometry ...... 26

4.8 Fluorescence-Activated Cell Sorting (FACS) ...... 26

4.9 Human Phospho-Kinase Array ...... 27

4.10 Data Analysis ...... 30

5 RESULTS ...... 31

5.1 Bone marrow-conditioned media contains multiple soluble factors that

could contribute to breast cancer’s affinity for bone ...... 31

5.2 Bone-derived OPN promotes human breast cancer cell migration ...... 31

5.3 Bone-derived OPN promotes stem-like behavior of breast cancer cells ..... 34

5.4 Breast cancer cell migration promoted by bone-derived OPN is mediated by

CD44 and RGD-dependent cell surface integrins ...... 39

5.5 Promotion of breast cancer stem-like behavior by bone-derived OPN is

mediated through CD44 and RGD-dependent cell surface integrins ...... 43

5.6 Bone-derived OPN causes phosphorylation changes in migratory and stem-

like related pathways in breast cancer cells ...... 44

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6 DISCUSSION ...... 48

6.1 Summary of Experimental Findings ...... 49

6.2 Implications of Experimental Findings ...... 49

6.2.1 Bone-Derived OPN Promotes the Migration of Breast Cancer Cells 52

6.2.2 Bone-Derived OPN Promotes the Stem-Like Phenotype of Breast

Cancer in Bone ...... 54

6.2.3 The Role of CD44 and RGD-Dependent Cell Surface Integrins in the

Bone-Derived OPN Mediated Migration and Stem-Like Behavior of

Breast Cancer Cells ...... 57

6.2.4 Bone-Derived OPN Causes Phosphorylation and Expression Changes

in Migratory and Stem Cell-Related Pathways in Breast Cancer Cells

...... 60

6.3 Limitations to the Study ...... 64

6.4 Future Directions ...... 65

7 CONCLUSIONS ...... 68

8 REFERENCES ...... 69

9 APPENDICES ...... 88

Curriculum Vitae - Graciella Pio ...... 94

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LIST OF TABLES

Table 1: Metastasis-associated proteins identified in bone marrow-conditioned media with the RayBio® Biotin label-based mouse antibody array ...... 33

Table 2: Malignancy-associated kinases and protein of interest identified by the Human Phospho-Kinase Array in response to bone-derived OPN…………………………. 47

LIST OF FIGURES

Figure 1. Stem-like ALDHhiCD44+CD24- Breast Cancer Cells ...... 9

Figure 2. Breast cancer cells and the bone microenvironment interact in a vicious cycle...... 14

Figure 3. Osteopontin Protein Structure ...... 18

Figure 4. Mechanism of the ALDEFLUORTM assay (StemCell Technologies) ...... 28

Figure 5: Strategy for isolation of MDA-MB-231 ALDHhiCD44+CD24- and ALDHloCD44-CD24+ breast cancer cells...... 29

Figure 6. Bone marrow-conditioned media contains potential mediators of metastasis .. 32

Figure 7. Bone-derived OPN promotes the migration of MDA-MB-231 and SUM-159 human breast cancer cells ...... 35

Figure 8. Bone-derived OPN promotes the stem-like behavior of MDA-MB-231 breast cancer cells ...... 38

Figure 9. MDA-MB-231 cells express OPN receptors including CD44 and multiple different cell surface integrins...... 40

Figure 10. SUM-159 cells express OPN receptors including CD44 and multiple different cell surface integrins...... 41

Figure 11. Promotion of breast cancer cell migration by bone-derived OPN is mediated by the cell surface receptor CD44 and various RGD-dependent cell surface integrins .... 42

Figure 12: Promotion of breast cancer cell tumorsphere-forming capacity by bone- derived OPN is mediated by the cell surface receptor CD44 and various RGD-dependent cell surface cell surface integrins ...... 45

Figure 13. Bone-derived OPN causes phosphorylation changes in migration- and stem cell-related signaling pathways in breast cancer cells ...... 46

Figure 14 Summary of Experimental Findings ...... 51

LIST OF APPENDICES

Appendix A: Animal Protocol Approval ...... 88

Appendix B: RayBio® Biotin Label-Based Mouse Antibody Array Map and Target List ...... 89

Appendix C: R&D Human Phospho-Kinase Array Target List ...... 92

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LIST OF ABBREVIATIONS, SYMBOLS, NOMENCLATURE

7-AAD 7-Aminoactinomycin

AF Alexafluor

AIDS Acquired Immune Deficiency Syndrome

ALDH Aldehyde Dehydrogenase

AML Acute Myeloid Leukemia

ANOVA Analysis of Variance

APC Allophycocyanin

ATP Adenosine Triphosphate

BAA- BODIPYTM-aminoacetate

BAAA BODIPY-aminoacetaldehyde bFGF Basic Fibroblast Growth Factor

BM Basal Media

BMCM Bone Marrow-Conditioned Media

BMP Bone-Morphogenetic Protein

BrdU Bromodeoxyuridine

CAF Cancer-Associated Fibroblast

CCAC Canadian Council of Animal Care

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COX-2 Cyclooxygenase-2

CSC Cancer Stem Cell

CTGF Connective Tissue Growth Factor

DCIS Ductal Carcinoma In Situ

DEAB Diethylaminobenzaldehyde

DMEM Dulbecco's Modified Eagle Medium

DNA Deoxyribonucleic Acid

ECM Extracellular Matrix

EGF Epidermal Growth Factor

EGFR Epidermal Growth Factor Receptor

ELISA Enzyme-linked Immunosorbant Assay

EMT Epithelial-to-Mesenchymal Transition

ER Estrogen Receptor

ERK Extracellular-Signal Related Kinases

FACS Fluorescence-Activated Cell Sorting

FAK Focal Adhesion Kinase

FBS Fetal Bovine Serum

FITC Fluorescein Isothiocyanate

GTP Guanosine-5'-Triphosphate

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HA Hyaluronan

HCC Hepatocellular Carcinoma

HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid

HER2 Human Epidermal Growth Receptor 2

HGF Hepatocyte Growth Factor

HIF-1α Hypoxia-Inducible Factor-1α

HRP Horseradish Peroxidase

HSC Hematopoietic Stem Cell

HSP60 Heat Shock Protein 60

ICAM-1 Intracellular-Adhesion Molecule-1

Id1 Inhibitor of DNA Binding-1

IGF-1 Insulin-Like Growth Factor-1

IGF-2 Insulin-Like Growth Factor-2

IL Interleukin kDa Kilodalton

LCIS Lobular Carcinoma In Situ

LPA Lysophosphatidic Acid

MAPK Mitogen-Activated Protein Kinase

MMP Matrix Metalloprotease

MSC Mesenchymal Stem Cell mTOR Mammalian Target of Rapamycin

NC Negative Control

OPN Osteopontin

PDGF Platelet-Derived Growth Factor

PE Phycoerytherin

Pen/Strep Pencillin/Streptomycin

PI3K Phosphoinositide 3-Kinase

Ppi Inorganic Pyrophosphate

PR Progesterone Receptor

PRAS40 Proline-Rich Substrate of Akt

PTHrP Parathyroid Hormone-Related Peptide

RANK Receptor/Activator for NF-κB

RANKL Receptor/Activator for NF-κB Ligand

SDF-1 Stromal Cell-Derived Factor-1

SLDA Sphere-Limiting Dilution Assay

Sox2 Sex Determining Region Y-Box 2

SP Side Population

SRE Skeletal Related Events

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TAM Tumor Associated Macrophage

TGF-β Transforming Growth Factor β

TNF-α Tumor-Necrosis Factor-α uPA Urokinase-Type Plasminogen Activator

VCAM-1 Vascular Adhesion Molecule-1

VEGF Vascular Endothelial Growth Factor

WNK-1 WNK Lysine Deficient Protein Kinase 1

xvii 1

1 INTRODUCTION

Cancer is responsible for 1 in 7 deaths worldwide; it causes more deaths than

AIDS, malaria and tuberculosis combined [1]. According to the International Agency for

Research on Cancer, there were approximately 14 million new cancer cases in 2012 - by

2030, this number is expected to surge to 21.7 million new cases [1]. This projected increase in cancer prevalence is due to the expanding and aging global population and the adoption of lifestyles known to increase cancer risk, including poor diet, physical inactivity and exposure to carcinogens [1].

Breast cancer is an example of a type of malignancy that is and will remain a major global health challenge [1, 2]. It is currently the most frequently diagnosed cancer amongst women and is the second leading cause of cancer-related deaths in North

America. It is estimated that breast cancer cases will also increase in the coming years due to less frequent and delayed pregnancies in women, as well as reduced breast-feeding

[1, 2]. Even though we have seen a drastic improvement in the treatment of breast cancer patients over the last 25 years, there remains a deplorable gap in our ability to manage metastatic breast cancer patients. In order to significantly reduce the impact of breast cancer on women worldwide, it is imperative that we focus on researching the biology, prevention and treatment of metastatic disease.

This thesis focuses on investigating the potential mechanisms of breast cancer metastasis to bone, since bone is the most common site of distant metastases in breast cancer patients [3, 4]. Specifically, it aims at elucidating the role of the soluble bone-

2 derived factor osteopontin (OPN) and its interactions with breast cancer cells that may contribute to the establishment of bone tumor burden in breast cancer patients.

2 LITERATURE REVIEW

2.1 Cancer

Cancer is a group of diseases characterized by dynamic changes in the genome that cause normal cells to evolve progressively to highly malignant derivatives. The multistep nature of tumorigenesis includes the acquisition of sustained proliferative signaling, replicative immortality, the evasion of growth suppressors and cell death, induced angiogenesis and the ability to invade and metastasize to other tissues [5]. These neoplastic characteristics are further amplified by tumor-promoting inflammation and the deregulation of cellular energetics, as well as the tumor cell’s ability to evade the immune response and recruit ostensibly healthy cells to contribute to the progression of the disease [6].

Cancer is currently a major health challenge worldwide and in Canada [7].

Approximately 2 in 5 Canadians are expected to develop cancer in their lifetime and a quarter of Canadians will die from their disease [7]. Cancer is responsible for 30% of all deaths in Canada, making it the leading cause of death and premature death (measured in potential years of life lost) in the country [7]. In addition to the social ramifications this disease entails, cancer has major economic consequences for our country [7]. In 2000, it was the fourth most costly disease in Canada, amounting to $17.4 billion in physician and hospital expenditure and loss of productivity due to premature death [7]. The social and economic toll that cancer has on Canadians is expected to increase as the aging population continues to grow.

2.2 Breast Cancer

Breast cancer is the most frequently diagnosed cancer among Canadian women, accounting for 26.1% of all newly diagnosed cancer cases in 2015 [2]. Discovering and implementing effective treatments for this disease is dependent on elucidating the underlying mechanisms in its formation and progression. Research advances in the past half century have shown that breast cancer is a genetically and clinically heterogeneous disease, arising from the malignant transformation of epithelial cells lining the milk ducts or the milk-producing lobules in the breast [8]. It can be broadly classified into in situ carcinoma and invasive (infiltrating) carcinoma; further sub-classifications are determined based on the cell type of origin, histological representation and molecular phenotypes [9]. Breast cancers classified as in situ (still confined within the duct or lobule of origin) have an excellent prognosis if treated appropriately; when caught at this stage, patients with ductal carcinoma in situ (DCIS) have a 99.2% overall 5-year survival rate [10].

Breast cancer patients diagnosed with invasive carcinoma do not fare as well, since their disease has invaded the surrounding breast tissue and has possibly spread to lymph nodes and other organs. Molecular classification of breast cancers has proven to be particularly useful in stratifying patients based on relative risk of recurrence and progression; these classifications are based on estrogen and progesterone receptor status, human epidermal growth receptor 2 (HER2/neu) status, and proliferative index as measured by Ki67 [9, 11]. One major benefit of these molecular classifications is their use in the determination of which patients are likely to respond to targeted therapies (e.g. tamoxifen or aromatase inhibitors for ER+/PR+ patients and trastuzumab, pertuzumab or

5 lapatinib for HER2/neu patients) [11]. Despite these recent advances in targeted therapy, breast cancer still ranks second in mortality amongst all other cancers, since patients with metastatic breast cancer have a mere 22% chance of surviving longer than ten years. This is primarily due to the failure of conventional therapy to mitigate and eliminate metastatic disease [2, 7].

2.3 Metastasis

Understanding the underlying molecular mechanisms of the metastatic process is vital to the development of novel therapeutics that can effectively treat metastatic cancer.

Past research has helped elucidate the multistep nature of cancer metastasis. Tumor cells first escape the primary tumor and intravasate into the circulatory or lymphatic system.

The blood and lymphatic systems are used as transportation routes to the secondary site, where cancer cells can exit the vasculature into the tissue site, at which point they can grow into micrometastases and subsequently to clinically detectable macrometastases

[12]. The metastatic process is surprisingly inefficient; Luzzi et al (1998) determined that only 0.02% of a melanoma cell population formed macrometastases in the mouse liver after being injected intraportally. It is the ability to initiate growth after extravasation and to continue to grow into clinically detectable macrometastases that proves most difficult for cancer cells during the metastatic cascade [13].

2.4 Cancer Stem Cells

The cancer cells that do successfully complete the metastatic process are not random. It has been shown that breast cancer tumors exhibit distinct molecular subtypes

(luminal A, luminal B, basal-like, HER2-overexpressing, normal breast-like, and claudin-

6 low) and consist of a hierarchy of phenotypically and functionally different cells [9, 14].

A subpopulation of aggressive cells is believed to exist at the apex of this hierarchy, and these cells have the propensity to initiate the primary tumour and successfully metastasize to adjacent tissues and distant organs [14-17]. These cells have been termed “cancer stem cells” (CSCs) because of their unique ability to self-renew and differentiate into a heterogeneous tumor, contrasting with their non-CSC counterparts that are incapable of tumor propagation [14, 15, 18-20]. Although the cell-of-origin of the CSC has not yet been definitively identified, CSCs are hypothesized to arise via one of three different mechanisms: a normal stem cell may acquire tumorigenic properties through aberrant mutations; a more differentiated progenitor may dedifferentiate into a stem-like state; or mature differentiated cancer cells acquire mutations that result in de-differentiation and co-opting of primitive stem cell signaling pathways, thus forming a CSC. Recent work by multiple groups support the progenitor hypothesis, as it has been shown that subpopulations within breast cancer cells can interconvert between stem-like, basal or luminal phenotypes and CSCs and non-CSCs can be generated after oncogenic reprogramming of differentiated fibroblasts [21, 22].

Stem-like breast cancer cells can be isolated by selecting for specific cell surface markers. Using cells isolated from pleural effusions or primary tumors of breast cancer patients, Al-Hajj et al (2003) identified and isolated tumor-initiating cells by fluorescence-activated cell sorting based on a CD44+CD24-/low phenotype [23]. As few as

100 cells of this phenotype were able to form tumors in immune-compromised mice while tens of thousands of cells with alternate phenotypes failed to form tumors [23].

They also showed that CD44+CD24-/low cells could be serially passaged; these cells where

7 able to recapitulate the heterogeneous nature of the original tumor, containing

CD44+CD24-/low cells in addition to a myriad of other cellular phenotypes present within the primary tumor [23]. Sheridan et al (2006) later showed that breast cancer cell lines containing >30% of CD44+CD24- cells (MDA-MB-231, MDA-MB-436, Hs578T,

SUM1315, and HBL-100) express higher levels of pro-invasive genes, including IL-1α,

IL-6, IL-8, and urokinase plasminogen activator (UPA) and have highly invasive properties [24].

The value of CD44 as a CSC marker is of little surprise considering that it is capable of promoting multiple tumorigenic properties. CD44 participates in a variety of signaling networks, including the activation of Rho GTPases (integral in cytoskeletal remodeling and invasion) and the PI3K/Akt and MAPK-Ras pathways (promotion of growth, survival and invasion) [25]. CD44 facilitates cell migration by serving as a docking site for matrix metalloproteases (MMPs) and matrix-modifying enzymes that degrade components of the extracellular matrix, such as collagen [25].

Ginestier et al (2007) added an additional component to the breast CSC phenotype by demonstrating that cells with high aldehyde dehydrogenase activity (ALDHhi) isolated from healthy human breast tissue have phenotypic and functional characteristics of mammary stem cells and that ALDHhi human breast carcinoma cells contain a CSC subpopulation [26]. This study also showed that ALDHhi cells are capable of generating tumors when as little as 500 cells are implanted into the mammary fat pad of NOD/SCID mice. Investigation of the relative hierarchy of the ALDHhi and CD44+CD24- phenotypes demonstrated that the combined phenotype of ALDHhiCD44+CD24- resulted in the greatest tumor-initiating capacity, while cells displaying an ALDHloCD44+CD24-

8 phenotype were not tumorigenic even when implanting 50,000 cells/fat pad [26], suggesting that ALDH activity is essential for identifying the tumor-initiating subpopulation in breast cancer cells (Figure 1).

Prior to the elucidation of ALDH’s function as a biomarker for cancer stem cells,

ALDH was known to play a self-protective role in normal stem cells. The ALDH enzyme superfamily catalyzes the oxidation of endogenous and exogenous aldehydes into their corresponding carboxylic acids and thus protects organisms from potentially harmful aldehydes [27]. ALDH also offers cellular protection against cytotoxic drugs in both normal mammary stem cells and CSCs; Tanei et al (2009) showed that ALDH+ tumor cells and ALDH expression significantly increased in breast cancer patients following neoadjuvant chemotherapy consisting of paclitaxel and epirubicin and that ALDHhigh breast tumors are correlated with resistance to neoadjuvant chemotherapy [28]. ALDH also plays a role in the expansion and differentiation of normal and cancer stem cells; when ALDH activity is inhibited, hematopoietic and cancer stem cells shift into a more differentiated state [29-31]. This shift in CSCs increases their sensitivity to chemotherapy and renders them less aggressive [31-33].

The Allan lab has recently pioneered functional characterization of

ALDHhiCD44+CD24- breast cancer cells in relation to metastatic behavior. Croker et al

(2012) demonstrated for the first time that ALDHhiCD44+CD24- stem-like breast cancer cells have enhanced capacity for cell growth, colony formation, migration, invasion through Matrigel and adhesion to select extracellular matrix components in vitro. Croker et al (2012) also used in vivo studies employing standard experimental metastasis assays,

Figure 1. Stem-like ALDHhiCD44+CD24- Breast Cancer Cells

The primary breast tumor is made up of cancer cells with multiple different cell phenotypes. Stem-like breast cancer cells can be isolated by their high aldehyde dehydrogenase activity and CD44+/CD24- phenotype (ALDHhiCD44+CD24-); these cells have the ability to self-renew and differentiate into a secondary tumor [16, 34].

10 which involved injection into the tail vein, or spontaneous metastasis assays involving orthotopic injection into the mammary fat pad and showed that the ALDHhiCD44+CD24- phenotype selects for breast cancer cells with enhanced tumorigenic and metastatic capacity relative to non stem-like ALDHlowCD44-CD24+ breast cancer cells [34].

2.5 Metastatic Organ Tropism

In addition to the findings that only a subset of aggressive cancer cells can successfully metastasize, clinical observations demonstrate that metastatic cells have a specific affinity for certain tissues and organs, a quality referred to as organ tropism [35,

36]. Breast cancer, for example, commonly metastasizes to the lymph nodes, lung, liver, bone and brain [37]. It was first proposed in 1889 by Stephen Paget that certain tissues are predisposed to developing secondary metastases. Paget’s ‘seed and soil’ hypothesis states that metastatic dissemination depends on crosstalk between a subset of cancer cells

(the ‘seeds’) and specific organ microenvironments (the ‘soil’) [37]. A cancer cell’s altered genetic or molecular signature and unique cell surface receptors result in a predilection for certain microenvironments, and in turn a favorable niche will provide conditions to promote tumor growth [38].

2.6 Breast Cancer Metastasis to Bone

Of particular interest is breast cancer’s preference for bone, as this is the most common site of metastasis in breast cancer patients. Coleman and Rubens (1987) found that the incidence of bone metastases was significantly higher (69%) than lung (27%) and liver (27%) metastases and that bone was the most common site for first distant relapse

[39]. These observations are not unique to breast cancer, as bone is also a common site of

11 metastasis in myeloma, prostate, thyroid, bladder and lung cancer [40]. Bone metastases pose significant challenges for a patient’s quality of life and treatment of their disease as skeletal metastases are associated with significant morbidity and poor prognosis; breast cancer patients that develop bone metastases survive approximately 22 months post- diagnosis. Breast cancer bone lesions are generally osteolytic in nature; bone homeostasis is disrupted by the upregulation of osteoclast activity, bone resorption dominates and there is little new bone formation [40]. These physiological changes compromise skeletal integrity and result in bone pain, pathological fractures, spinal cord compression and reduced patient mobility [39, 40]. Hypercalcaemia (serum calcium > 3.0 mmol/L) is also a significant metabolic complication in patients and if left untreated, can result in dysfunction of the gastrointestinal tract, renal failure and central nervous system complications such as cardiac arrhythmias [40].

Breast cancer mainly forms bone metastases in the metaphyseal bone of the axial skeleton (ex. vertebrae, ribs, sternum) and the ends of long bones [40, 41]. The metaphysis is largely composed of metabolically active trabecular bone and is surrounded by hematopoietic marrow, fatty marrow and blood vessels [42, 43]. In agreement with

Paget’s seed and soil hypothesis, bone metastases of all cancer types occur almost exclusively in active red marrow, an attractive site for metastatic involvement because of its characteristic sinusoidal vascular spaces and relatively easy barrier for tumor cell penetration [3, 40]. Physical properties of the circulation within the bone marrow cavity, such as capillary structure and slow blood flow, likely also contribute to this observed pattern of metastases [40].

In conjunction with the physical properties and blood flow patterns characteristic of metaphyseal bone, bone marrow offers circulating tumor cells an optimal pre- metastatic niche via a wide variety of chemokines, such as stromal cell-derived factor-1

(SDF-1), C-X-C motif chemokine 10 (CXCL10) and other proteins, including osteopontin (OPN) and angiopoietin [44, 45]. Once breast cancer cells arrest in the bone, they influence the activity of osteoclasts and osteoblasts in such a way that results in constitutive osteoclast activation and a down-regulation of osteoblast production of bone matrix, causing sustained bone degradation [3, 46]. In healthy bone, osteoblasts externally express receptor/activator for NF-κB ligand (RANKL), which binds to RANK on the surface of monocytes to promote fusion of several of these cells into a multinucleated osteoclast [42]. Activated osteoclasts adhere to the bone matrix through

αvβ3, αvβ5 and α2β1 cell surface integrins and secrete acidic and lysosomal enzymes that cause bone resorption and increased bone turnover [42]. Breast cancer cells have the ability to hijack this mechanism by secreting a variety of cytokines, chemokines and other factors including parathyroid hormone-related peptide (PTHrP), interleukin 1 (IL-

1), IL-6, IL-8, IL-11, cyclooxegenase 2 (COX-2) and prostaglandin E2 that have all been shown to stimulate osteoclastogenesis by up-regulating RANKL expression [3, 46].

These secreted factors can influence osteoclast physiology in a RANKL independent manner by binding directly to receptors on the surface of osteoclasts; for example, IL-8 can bind directly to CXCR1 to effect osteoclast differentiation and activity [47].

During breast cancer cell-induced bone resorption, growth factors usually locked in the bone matrix such as transforming growth factor β (TGF-β), insulin-like growth factor (IGF) 1 and 2, fibroblast growth factor (FGF) 1 and 2, platelet-derived growth

13 factor (PDGF), and bone-morphogenetic proteins (BMPs) are released into the bone microenvironment, creating a niche in which cancer cells can grow [44, 45, 48, 49].

These growth factors also stimulate the secretion of metastasis-promoting factors from breast cancer cells such as connective tissue growth factor (CTGF), IL-11 and PTHrP, thereby creating a vicious cycle of bone resorption and tumor growth [50, 51] (Figure 2).

The bone metastatic niche is also influenced by bone marrow stromal cells and transient cells (erythrocytes, T-cells and platelets) [3, 46]. Mesenchymal stem cells in the bone marrow give rise to stromal cells that can differentiate into osteoblasts, chondrocytes, adipocytes or fibroblasts. These stromal cells are known to support the proliferation, migration, and survival of breast cancer cells, specifically through vascular cell adhesion molecule-1 (VCAM-1). When VCAM-1 is blocked via a neutralizing antibody, bone osteolysis decreases [52]. Additionally, adipocytes are known to secrete IL-6, tumor- necrosis factor-α (TNF-α) and leptin, all responsible for stimulating bone resorption and inhibiting osteoblast proliferation [53, 54], while fibroblasts can secrete inactive MMP-2 that is activated by breast cancer cells to increase invasiveness and migration [55].

Platelets and T-cells may also assist in the establishment of metastases by expressing

RANKL secreting factors such as lysophosphatidic-acid (LPA) and tumour necrosis factor α (TNF-α) to further increase osteoclast activity [56, 57]. Interestingly, as bone resorption is up-regulated, TGF-β is released and functions to suppress the activity of T- cells; this suppresses the immune response and may allow breast cancer cells to escape immune surveillance [58]. Notably, the bone marrow represents a rich supportive niche for hematopoietic stem cells [59] and it has been shown that most early-disseminated breast cancer cells in bone have a stem-like phenotype, observations that complement the

Figure 2. Breast cancer cells and the bone microenvironment interact in a vicious cycle.

The bone microenvironment contains multiple cell types, including osteoblasts, osteoclasts and stromal cells that secrete a myriad of growth factors and cytokines to offer a niche conducive to the establishment and progression of breast cancer metastases. Once breast cancer cells arrest in bone, they cause bone degradation via the upregulation of osteoclastic activity. Bone resorption causes more soluble factors to be released into the microenvironment, which further fuel the development of metastases in the bone, creating a “vicious cycle” of bone resorption and skeletal metastases development. Adapted from Sterling et al [3, 45, 46, 60].

CSC hypothesis of cancer metastasis [61]. Specifically, the CD44+ stem cell-like phenotype has shown increased adherence to human bone marrow endothelial cells in breast cancer [62].

Despite the extensive research conducted in the area of breast cancer metastasis to bone, this condition remains incurable. However, bone-targeted therapies do exist that attempt to reduce and delay skeletal related events (SREs) such as fractures, spinal cord compression and hypercalcaemia. Bisphosphonates were the first agents used clinically to reduce SREs and were introduced over three decades ago [63]. There are multiple different types of bisphosphonates, including clodronate, ibandronate, pamidronate and zoledronic acid; all are structural analogs of inorganic pyrophosphates (PPi) [63, 64]. PPi inhibit calcification and bone resorption by incorporating into sites of active bone remodeling, binding to hydroxyapatite crystals and inhibiting their breakdown [63].

Bisphosphonates also promote osteoclast apoptosis either by becoming incorporated into molecules of newly formed adenosine triphosphate (ATP; this nonhydrolyzable analogue of ATP is cytotoxic to osteoclasts because it inhibits multiple ATP-dependent processes) or by binding to and inhibiting farnesyl pyrophosphate, resulting in the inhibition of the posttranslational modification of multiple different proteins essential for osteoclastic function [63]. Although bisphosphonates substantially relieve skeletal pain and complications in breast cancer patients, absorption in the gastrointestinal tract and subsequent localization is poor and use of the drug is with multiple adverse effects including osteonecrosis of the jaw, atrial fibrillation, hypocalcaemia and acute inflammatory response [63]. More recently, denosomab, a monoclonal antibody that binds to RANKL and inhibits its ability to bind to RANK, has been introduced as a

16 therapeutic to reduce SREs. Efficacy and side effects of denosumab are similar to those of bisphosphonates and although both drugs are widely used, clinical practice guidelines do not specifically state which therapeutic to use over the other [65]. Unfortunately, neither bisphosphonates nor denosumab have any effect on progression-free or overall survival and are purely palliative in nature [65]. An agent that shows recent promise is everolimus, an inhibitor of mammalian target of rapamycin (mTOR); an exploratory analysis of the effect of everolimus on bone metastases found that patients using everolimus had a significant reduction in bone turnover markers and rates of progressive disease [65]. Even if everolimus proves to be useful in clinical applications, breast cancer treatment is in dire need of therapeutics that prevent and reduce bone metastases in breast cancer patients, rather than just slowing the rate of progression. Elucidation of other players within the bone microenvironment that promote the recruitment of breast cancer cells to the bone and the establishment of bone metastases is therefore essential to the discovery of such therapeutics.

2.7 Osteopontin

Osteopontin (OPN) is one of the most abundant non-collagenous extracellular matrix proteins in bone and as such, is a protein of interest when considering the bone as a favorable niche for breast cancer metastases [66]. OPN is a secreted, acidic glycophosphoprotein that was discovered independently multiple times. It was first discovered as a 60-kDa malignant transformation-specific phosphoprotein secreted by many different tumorigenic cell types, subsequently rediscovered as bone sialoprotein I, a major non-collagenous component of the bone extracellular matrix, and then identified as a protein associated with T-cell dependent genetic resistance to bacterial infections [67-

69]. The name “osteopontin” was finally introduced to reflect its inherent ability to form bridges between hydroxyapatite (HA) and multiple cell types in bone [70]. OPN is expressed by multiple tissues in the body other than bone including the kidney, brain, gastrointestinal tract, hypertrophic cartilage, dentin and cementum. However the highest levels of the protein in a healthy human body are associated with mineralized tissues and bodily fluids that have high concentrations of calcium, such as breast milk, urine and seminal fluid [71, 72]. In the bone, OPN is produced by osteoblasts, osteoclasts, osteocytes and macrophages [66, 73]. Human OPN can range in size from 41- to 75-kDa due to the myriad of post-translational modifications it undergoes, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination [73]. Its structure consists of various highly conserved regions that are indicative and essential to its various biological functions [72, 73] (Figure 3). It contains a calcium binding motif and many electronegative glutamic and aspartic acid residues that allow it to tightly bind to HA in bone. Binding of OPN to HA renders it a potent inhibitor of mineralization, as it inhibits the growth of HA crystals. It also has RGD

(arginine-glycine-aspartate) and SVVYGLR (serine-valine-valine-tyrosine-glycine- leucine-arginine) domains for integrin binding and a heparin binding site that mediates

CD44 receptor binding [73, 74]. The ability of OPN to bind to cells that express cell surface receptors such as integrins and CD44 allows it to mediate cell attachment, cell migration, chemotaxis and intracellular signaling in various cell types, including monocytic cells/macrophages, smooth muscle cells, endothelial cells and epithelial cells

[75].

Figure 3. Osteopontin Protein Structure

OPN contains a number of highly conserved structural features that are indicative of its biological functions. It contains a thrombin cleavage domain, which results in proteolytic cleavage and of OPN into two similarly-sized fragments. The N-terminal fragment contains as aspartate rich domain which allows OPN to bind hydroxyapatite in bone. The RGD and SVVYGLR domains allow for binding to ligands expressing RGD-dependent integrins (αvβ1, αvβ3, αvβ5, α5β1) as well as SVVYGLR-dependent integrins (α4β1, α9β1). The C-terminal fragment contains two heparin binding domains which assist in the binding of OPN to CD44 and a calcium binding domain. Adapted from Tuck et. al (2003) [76].

OPN expression is observed in many pathophysiological responses and pathological conditions, including cardiovascular disease (e.g. atherosclerosis, and ventricular hypertrophy), kidney diseases (e.g. kidney stone formation), wound healing, fibrosis, autoimmune diseases and multiple different cancers [72, 73, 75]. Its presence in traumatized, diseased and inflamed tissues can be accounted for by heavy secretion from activated macrophages and T-lymphocytes. In these settings, OPN promotes adhesion of

T-cells and contributes to type-1 cell mediated immunity by modulating macrophage interleukin expression.

OPN’s role in malignancy is well documented; it is secreted by several types of cancer cells including breast, lung, gastric, ovarian and myeloma cancer cells [77-81]. In these cancers, it has been shown to mediate cell-matrix and cell-cell communication through interactions with a variety of cell surface receptors (i.e. CD44, α9β1, αvβ3 and

αvβ5) to activate pathways that contribute to multiple steps in the metastatic cascade [82-

87]. Firstly, OPN can protect cells against apoptosis and induce survival and proliferation via in multiple ways, including through NK-κB downstream of the αvβ3 cell surface integrin, the PI3K-Akt axis downstream of CD44 or via epidermal growth factor (EGF)- dependent mechanisms [75]. It has been shown that breast carcinoma cells are protected against apoptosis when they adhere to OPN via αvβ3 [88]. Studies have also shown that

OPN can induce angiogenesis by up-regulating vascular endothelial growth factor

(VEGF) expression through the activation of PI3K/Akt and ERK-mediated pathways.

OPN also has a direct effect on cancer cell migration via CD44 and RGD-dependent cell surface integrins; it has been shown to induce migration via growth factor pathways involving hepatocyte growth factor (HGF), its receptor Met and epidermal growth factor

(EGF), as well as interact with focal adhesion complexes to impact cell motility [83].

OPN signaling via αvβ3 also induces uPA and multiple types of MMP secretion by breast cancer cells that allows them to invade through the basement membrane.

In the establishment of bone metastases specifically, OPN assists in the attachment of breast cancer cells to human bone marrow endothelial cells and thus plays a vital role in the early steps of bone colonization [83]. It also contributes to the upregulation of bone resorption during breast cancer metastasis to bone as neutralization of

OPN can suppress osteoclastogenesis, whereas addition of OPN enhances osteoclastogenesis by up-regulating RANKL and decreasing osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor. OPN’s role as a metastasis-enhancing protein is supported by the observation that OPN-deficient mice have a reduced number of metastases to bone and soft tissue [89]. OPN can also generate a favorable growth environment for breast cancer cells in bone by activating a variety of growth factors, including HGF, TGF-β and bFGF via the upregulation of µPA and IL-11 [83].

OPN has been identified as a clinical prognostic marker; clinical studies have revealed a correlation between elevated plasma OPN levels, increased tumor burden and poor prognosis in cancer patients [83, 84]. The amount of plasma OPN in healthy women ranges from 22 to 122 µg/L, with a median level of 47 µg/L. A higher level of OPN, upwards of 300 µg/L, exists in the plasma of breast cancer patients as well as colon, lung and prostate cancer patients [83, 84, 90]. In breast cancer specifically, high levels of OPN in carcinoma cells are associated with increased metastases to the bone [91]. Although extensive research has demonstrated that tumor-derived OPN can contribute to several

21 steps of the metastatic cascade, the role of bone-derived OPN in the metastatic progression of breast cancer has yet to be fully investigated.

2.8 Project Rationale

Extensive work has focused on characterizing the role of the bone microenvironment in breast cancer metastasis in hopes that this knowledge would lead to the discovery of clinically applicable agents that could prevent, reduce and slow the growth of bone metastases in breast cancer patients. However, there remain unanswered questions regarding the mechanism in which the bone niche promotes the establishment of breast cancer bone lesions remains unclear. Further work must be done in order to characterize what makes the bone a suitable “soil” for breast cancer cells, and identifying soluble factors in bone marrow that contribute to breast cancer cell recruitment and establishment of metastatic lesions is necessary in this regard. Previous work by Jenny

Chu in the Allan lab lead to the establishment of a comprehensive ex vivo model system for investigating the influence of organ-specific soluble factors on the metastatic behavior of breast cancer cells (described in further detail below) [92]. Preliminary work indicated that both whole population and stem-like ALDHhiCD44+CD44- breast cancer cells show enhanced migration towards bone marrow conditioned-media relative to control [92]. The goal of the current project is to investigate the role of bone-derived OPN in the migration and stem-like behavior of whole population and ALDHhiCD44+CD24- breast cancer cells in bone.

3 HYPOTHESIS and OBJECTIVES

3.1 Hypothesis

Bone-derived OPN promotes the migration and stem-like behavior of breast cancer cells.

3.2 Objectives

1. To determine the role of bone-derived OPN in promoting breast cancer migration and

stem-like behavior of breast cancer cells.

2. To investigate the receptor-ligand interactions between breast cancer cells and bone-

derived OPN and the functional consequences on migration and stem-like behavior.

3. To assess the OPN-associated downstream signaling responses within breast cancer

cells in response to bone-conditioned media.

4 MATERIALS and METHODS

4.1 Cell Culture and Reagents

MDA-MB-231 cells [93] were obtained from American Type Culture Collection

(Manassas, VA) and maintained in DMEM/F12+10% fetal bovine serum (FBS).

SUM159 cells [94] were obtained from Asterand Inc. (Detroit, MI) and maintained in

HAMS:F12 + 5% FBS + 5 µg/mL insulin + 1 µg/mL hydrocortisone + 10mM HEPES.

Media/supplements were from Invitrogen (Carlsbad, CA); FBS was from Sigma-Aldrich

(St. Louis, MO).

4.2 Bone Marrow-Conditioned Media (BMCM)

Bone marrow-conditioned media (BMCM) was generated as previously described

[92]. Briefly, healthy female nude mice (Hsd: Athymic Nude-Foxn1nu; Harlan Sprague-

Dawley, Indianapolis, IN) were maintained as per the Canadian Council of Animal Care

(CCAC) under a protocol approved by the Western University Animal Use

Subcommittee (AUS; #2009-064). Mice (6-12 weeks old) were euthanized and bone marrow was collected by flushing femur cavities as previously described [95]. Aspirates were dissociated by pipetting and cells were washed and plated in DMEM + 10% FBS + pen/strep. Resulting adherent bone marrow stromal cells (BMSCs) were passaged 2-3 times, washed, and exposed to DMEM/F12 + Mito+™ + pen/strep. Conditioned media was collected after 72 hr and stored at -20oC. To account for mouse-to-mouse variability,

BMCM from multiple mice was pooled prior to use.

4.3 RayBio® Biotin Label-Based Mouse Antibody Arrays

The Raybio® Biotin label-based mouse antibody array (AAM, BLM-1,

RayBiotech Inc, Norcross, GA) were used to assess the expression of 308 murine soluble factors within BMCM. Basal media samples (DMEM/F12 + Mito+™ + pen/strep) or

BMCM protein samples (0.12 mg each) were dialyzed and labeled with a biotin-labeling reagent. Free biotin was removed using spin columns, membranes were blocked and samples were added to the membranes and incubated at 4oC overnight. Streptavidin- horseradish peroxidase (HRP) and chemiluminescent detection reagents were used to produce a signal at each capture spot corresponding to the amount of protein bound.

Membranes were visualized using ChemiDoc Detection Software (n=3 for each media condition) (BioRad, Hercules, CA). The concentration of resulting proteins of interest present in BMCM was confirmed by Quantkine ELISA (R&D Systems, Minneapolis,

MN).

4.4 Immunodepletion of OPN

Osteopontin was immunodepleted from BMCM using a rat anti-mouse osteopontin-specific antibody (R&D Systems). Antibodies were incubated for 20 minutes at RT with Dynabeads Protein G (8 µg OPN-specific antibody per mg of beads; Life

Technologies, Burlington, ON). Bead-antibody complexes were then incubated with

BMCM for 30 minutes at RT. Resulting bead-antibody-antigen complexes were removed from BMCM using a DynaMag-2 magnet (Life Technologies). The concentration of OPN in depleted BMCM was assessed by Quantikine ELISA kits specific for OPN (R&D

Systems). Negative controls included BMCM exposed to beads only (no antibody). GST- tagged human OPN (GST-hOPN), a kind gift from Dr. Ann Chambers, London Regional

Cancer Program, London, Ontario, is a functional representative of native OPN [85]. For rescue experiments, GST-hOPN was added back to BMCM depleted of OPN at the same concentration that was originally depleted from BMCM.

4.5 Migration Assays

Transwells® (6.5 mm, 8 µm pore size; Falcon, Corning, NY) were coated with 6

µg of gelatin per well. Osteopontin-depleted or non-depleted BMCM or basal media

(DMEM/F12 + Mito+™) were placed in 24-well dishes (n = 3 per condition). MDA-MB-

231 or SUM-159 cells (5 x 104 cells per well) were plated on top of the gelatin-coated

Transwells®, then inserted into 24-well dishes. In experiments involving functional blocking of CD44 or RGD (Arginin-Glycine-Aspartic acid)-sequences, cells were incubated for 30 minutes at RT with a rat anti-human CD44 antibody (10 µg/5 x 105 cells,

Calbiochem, Mississauga, ON) or an RGD-sequence specific peptide (50 µg/5 x 105 cells for MDA-MB-231 breast cancer cells, 100 µg/5 x 105 cells for SUM-159 breast cancer cells; Sigma-Aldrich). After 18 hours incubation at 37°C and 5% CO2, Transwells® were removed, fixed and non-migrated cells were removed from the inner surface of the

Transwell®. Membranes were then carefully cut from the rest of the Transwell® and migrated cells were stained with DAPI. Five high-powered fields (HPFs) of view were counted for each membrane using ImageJ [National Institutes of Health (NIH), Bethesda,

MD] software. Results are expressed as a fold-increase from negative control (n = 3).

4.6 Sphere-Limiting Dilution Assays (SLDA) and Colony- Forming Assays

MDA-MB-231 cells were seeded in 96-well plates for colony-forming assays

(Corning, Lowell, Massachusetts) or 96-well ultra-low attachment plates (Corning) for

26 the sphere-limiting dilution assay (SLDA) in a serial-diluted fashion ranging from 1000 cells/well to 0.001 cells/well. In experiments involving functional blocking, and anti-

CD44 antibody (10 µg/5 x 105 cells; Calbiochem) or an RGD-sequence specific peptide

(50 µg/5 x 105 cells; Sigma-Aldrich) were used in a similar fashion as in the TranswellTM migration assays. Osteopontin-depleted or non-depleted BMCM or basal media

(DMEM/F12 + MITO+™) was added to the wells and cells were incubated with control media or conditioned media for 5 days at 37°C and 5% CO2. After incubation, each well was scored for the presence or absence of colonies (n = 3) or tumorspheres (n = 3) using

L-Calc™ Software (Stem Cell Technologies, Vancouver, British Columbia).

4.7 Flow Cytometry

Cells were grown to 80% confluence in normal growth media, harvested and resuspended at 1 x 106 cells/ml. Cells were then incubated with phycoerytherin (PE)- conjugated CD44 (BD Biosciences, San José, CA), fluorescein isothiocyanate (FITC)- conjugated αvβ3 (R&D Systems), Alexafluor (AF)-488-conjugated αvβ5 (R&D

Systems), AF-488-conjugated β1 (R&D Systems) or AF-488-conjugated α9β1 (R&D

Systems) antibodies for 1 hour at 4°C. Negative controls included cells only (no antibody) and cells incubated with an isotype-matched IgG-control. Samples were run on a Beckman-Coulter EPICS XL-MCL flow cytometer.

4.8 Fluorescence-Activated Cell Sorting (FACS)

ALDHhiCD44+CD24- and ALDHloCD44-CD24+ cell subpopulations were isolated from the MDA-MB-231 breast cancer cell line as previously described [33, 34]. Briefly, cells were concurrently labeled with 7-amino-actinomycin D (7-AAD), ALDEFLUORTM

27 assay kit (StemCell Technologies; Vancouver, BC) (Figure 4) and fluorescently- conjugated antibodies including anti-CD44 (clone IM7) conjugated to allophycocyanin

(APC) and anti-CD24 (clone ML5) conjugated to phycoerytherin (PE) (BD Biosciences).

ALDH activity was used as the primary sort criteria (top ~20%=ALDHhi; bottom

~20%=ALDHlow) and the CD44+CD24- phenotype as the secondary sort criteria (top

~10% gated on ALDHhi; bottom ~10% gated on ALDHlow) (Figure 5). Cell viability was assessed by 7-AAD staining during cell sorting, and confirmed by trypan blue exclusion post-sorting. FACS-isolated cells were used immediately for in vitro assays.

4.9 Human Phospho-Kinase Array

MDA-MB-231 cells were incubated in basal media, BMCM and BMCM depleted of OPN and cell lysates were harvested after 2 hr. Protein concentrations were determined with a BioRad DC protein assay (BioRad) and 400 µg of cell lysates were incubated with the Human Phospho-Kinase Array membranes (ARY003B, R&D

Systems) overnight at 4°C. According to the manufacturer’s instructions and using kit contents, arrays were washed to remove unbound proteins and a cocktail of biotinylated detection antibodies was applied. Streptavidin-HRP and chemiluminescent detection reagents were used to produce a signal at each capture spot corresponding to the amount of phosphorylated protein bound. Membranes were visualized using ChemiDoc Detection

Software (BioRad). Densitometry analysis was performed using the Protein Array

Analyzer for ImageJ (n = 3 for each media condition) [96].

Figure 4. Mechanism of the ALDEFLUORTM assay (StemCell Technologies)

The ALDEFLUORTM assay is used to identify cells that demonstrate activity of the enzyme aldehyde dehydrogenase. The assay uses a fluorescent, non-toxic reagent, BODIPYTM-aminoacetylaldehyde (BAAA) that can diffuse freely into viable cells. BAAA is a substrate for ALDH and is converted into BODIPYTM-aminoacetate (BAA-), which is retained in the cell due to its negative charge. The active removal of BAA- is inhibited with the use of cold ALDEFLUORTM assay buffer. The resulting fluorescence of the cell is assessed by flow cytometry. Diethylaminobenzaldehyde (DEAB) is used as a negative control because of its ability to inhibit the activity of ALDH; when DEAB is used, the intrinsically neutral BAAA is free to passively diffuse back out of the cell. The fluorescence of the DEAB-treated cells are used to create the gate for the ALDHhi cells. Adapted from Chu and Allan (2011) [18].

FACSDiva Version 6.1.3

FACSDiva Version 6.1.3 FACSDiva Version 6.1.3

A B FACSDiva Version 6.1.3

FACSDiva Version 6.1.3

C D

MDA MB 231 Printed on: Fri May 23, 2014 04:20:01 PDT

Figure 5:MDA Strategy MB 231 for isolation of MDA-MB-231 ALDHhiCD44+CD24- and Printed on: Fri May 23, 2014 04:20:01 PDT ALDHloCD44-CD24+ breast cancer cells.

MDA-MB-231 cells are labeled with 7-AAD, CD44-APC, CD24-PE and the ALDEFLUORTM assay kit. Cell subsets are isolated using a four-colour protocol on a MDA MB 231 Printed on: Fri May 23, 2014 04:20:01 PDT FACS ARIA III.MDA (A) MB 231Cells are first isolated based on expected light scatter and then (B) Printed on: Fri May 23, 2014 04:19:50 PDT MDA MB 231 viability based on 7-AAD exclusion. Cells are then analyzed for (C) PrintedALDH on: activity;Fri May 23, 2014 the 04:20:01 PDT top 20% most positive for ALDH activity are deemed ALDHhi and the bottom 20% for ALDH activity are deemed ALDHlo. Then, (D) ALDHhi cells are selected for the CD44+CD24- phenotype (top 50%) and (E) ALDHlo cells are selected for the CD44- /lowCD24+ phenotype (bottom 50%).

4.10 Data Analysis

In vitro experiments were performed a minimum of three times with three technical replicates within each experiment. Unless otherwise noted, all data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA) using one- way analysis of variance (ANOVA) with Tukey’s or Bonferroni’s post-hoc tests. Values of p<0.05 were classified as being statistically significant.

5 RESULTS

5.1 Bone marrow-conditioned media contains multiple soluble factors that could contribute to breast cancer’s affinity for bone

Assessment of soluble factors present in bone marrow-conditioned media

(BMCM) was carried out using the RayBio® Biotin Label-Based Mouse Antibody Array

(AAM, BLM-1, RayBiotech Inc), a protein array that tests for the presence of 308 soluble factors of interest. Of the proteins identified in BMCM using the protein array (Figure 6), five were found to be associated with steps of the metastatic cascade (Table 1). Of these, three soluble factors of interest, osteopontin (OPN), matrix metalloproteinase-14 (MMP-

14) and insulin-like growth factor-2 (IGF-2) were chosen for further functional validation based on evidence in the literature of their possible involvement in cancer migration and growth.

5.2 Bone-derived OPN promotes human breast cancer cell migration

OPN, MMP-14 and IGF-2 concentrations were analyzed in BMCM samples with

Quantikine® ELISA. IGF-2 concentrations were observed to be minimal (below 30 pg/mL) in BMCM samples tested (data not shown). However, OPN and MMP-14 were observed to be present in BMCM samples tested. To assess the effect of bone-derived

OPN and MMP-14 on human breast cancer cell migration, OPN was depleted from

BMCM using Dynabeads® Protein G (Novex by Life Technologies) and depletion efficiency was confirmed with a Quantikine ELISA (R&D Systems)

B 39

176 158

326 333

Figure 6. Bone marrow-conditioned media contains potential mediators of metastasis

The Raybio® Biotin label-based mouse antibody array (AAM, BLM-1, RayBiotech Inc) was used to identify soluble factors present in BMCM. Membranes were incubated with biotinylated samples of (A) basal media or (B) BMCM at 4oC overnight and visualized using chemiluminescence. Dotted boxes indicate internal positive controls, lined boxes indicate internal negative controls and solid boxes indicate proteins of interest that are potentially involved in the metastatic cascade.

Table 1: Metastasis-associated proteins identified in bone marrow-conditioned media with the RayBio® Biotin label-based mouse antibody array Location on Array Protein Name Function/Involvement in References (Figure 6) Malignancy 39 Basic Fibroblast • Overexpressed in breast cancer Growth Factor patients (bFGF) • Implicated in osteoblastic bone [90, 97] metastases • Regulates neoplastic cell growth

158 Intracellular • Elevated in metastatic breast Adhesion cancer patients Molecule-1 • Downregulation leads to a strong (ICAM-1) suppression of breast cancer cell [98] invasion

176 Insulin-Like • Up-regulated in triple-negative Growth Factor-2 breast cancer (IGF-2) • Down-regulation of bone- derived IGF-2 suppresses the [99, 100] growth of bone metastases in prostate cancer

326 Matrix- • Modulates migratory ability of Metalloproteinase- breast cancer cells 14 (MMP-14) • High levels of MMP-14 correlate [101, 102] with bone metastases

333 Osteopontin • Most abundant extracellular (OPN) matrix protein in the bone Associated with multiple steps of • [83, 84, 90] the metastatic cascade, including proliferation, migration, adhesion and invasion

(Figure 7A). However, MMP-14 could not be successfully depleted from BMCM using

Dynabeads® Protein G and available MMP-14 antibodies (data not shown), thus MMP-14 was no longer pursued. The TranswellTM migration assay was then used to assess the migration of MDA-MB-231 and SUM-159 cells to basal media, BMCM and BMCM depleted of OPN. These two breast cancer cell lines were chosen since the Allan lab has previously shown that they both display increased migration toward BMCM [92]. Both cell lines exhibited increased migration toward BMCM compared to basal media (P<

0.05, Figure 7B,C) and exhibited significantly decreased migration to BMCM depleted of

OPN (P< 0.05, Figure 7). In fact, the depletion of OPN from BMCM decreased the migration of both cells lines to a level not significantly different than basal media

(P>0.05; Figure 7B,C). To validate that bone-derived OPN was specifically responsible for the observed effects on breast cancer cell migration, recombinant human OPN (GST- hOPN) was added back into BMCM depleted of OPN at the same concentration originally depleted from the BMCM. The addition of GST-hOPN to BMCM rescued the migratory effect of both MDA-MB-231 and SUM-159 cells, causing cells to migrate to similar levels as to non-depleted BMCM (Figure 7B,C). These results demonstrate that bone-derived OPN mediates breast cancer cell migration towards BMCM.

5.3 Bone-derived OPN promotes stem-like behavior of breast cancer cells We next wanted to investigate if bone-derived OPN mediates the stem-like behavior of breast cancer cells, since the stem-like subpopulation of breast cancer cells has been shown to play a role in the metastatic process. We used a sphere limiting dilution assay (SLDA) to assess the effect of bone-derived OPN on the tumorsphere-

35 forming capacity of breast cancer cells. The SLDA tests the ability of cells to form tumorspheres in vitro, a marker for cell “stemness”. Whole population MDA-MB-231 cells show increased tumorsphere-forming capacity in BMCM compared to basal media

(P<0.05, Figure 8A). This tumorsphere-

A 15000

* 10000 *

5000

Concentration of OPN (pg/mL) 0

only ® OPN BMCM Δ

Basal Media BMCM B DynaBeads C MDA-MB-231 SUM-159

5 4 * δ δ 4 * * 3 *

3 φ φ 2

2 Migration Migration (Fold Change) (Fold Change) 1 1

0 0

OPN OPN BMCM Δ BMCM Δ

BMCM BMCM Negative Control Negative Control OPN + GST hOPN OPN + GST hOPN Δ Δ

BMCM BMCM

Figure 7. Bone-derived OPN promotes the migration of MDA-MB-231 and SUM- 159 human breast cancer cells (A) Goat anti-mouse OPN primary antibody was incubated for 20 min at RT with DynaBeads® Protein G (EMD Millipore) prior to addition and incubation with BMCM for 30 min at RT. The beads-antibody-antigen complex was removed with a DynaMag™-2 magnet. Concentration of OPN in BMCM, BMCM with Dynabeads® only (no antibody) and BMCM depleted of OPN (ΔOPN) was tested with Quantikine® ELISA (R&D Systems). Significance determined by one-way ANOVA and Tukey’s post-hoc test. (B) MDA-MB-231 and (C) SUM-159 human breast cancer cells were plated on gelatin

36 coated TranswellsTM (5 x 104 cells/well; 8µm pore size) before placement into either basal media (DMEM/F12 + Mito+), BMCM, BMCM ΔOPN or BMCM ΔOPN with GST- o TM hOPN added. Plates were incubated at 37 C at 5% CO2 for 18 hr. Transwells were then fixed with 1% gluteraldehyde and stained with DAPI. Five high-powered fields of view (HPFs) were captured/TranswellTM and migrated cells were enumerated through the use of ImageJ software (NIH). Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). Significance determined with one-way ANOVA and Tukey’s post-hoc test;* = significantly different from basal media; ϕ = significantly different from BMCM; δ = significantly different from BMCM ΔOPN: P< 0.05.

37 forming capacity decreases when BMCM is depleted of OPN (P<0.05, Figure 8A), suggesting that bone-derived OPN mediates this function in MDA-MB-231 cells. We also investigated the role of bone-derived OPN in the limiting dilution colony-forming ability of MDA-MB-231 cells. Whole population MDA-MB-231 cells show increased colony-forming abilities in BMCM compared to basal media (P<0.05, Figure 8B). When cells are exposed to BMCM depleted of OPN, their colony-forming capacity decreases significantly (P≤0.05, Figure 8B), suggesting that bone-derived OPN also plays a role in the colony-forming ability of breast cancer cells in a limiting dilution fashion.

As previously discussed, stem-like ALDHhiCD44+CD24- breast cancer cells are important mediators of metastasis to multiple different organs, including bone [17, 34].

We therefore also investigated if bone-derived OPN mediates the stem-like phenotype of these breast cancer cells. We have previously shown that this subpopulation of MDA-

MB-231 cells is responsible for the increased migration to BMCM, compared to their non-stem like ALDHloCD44-CD24+ cells [92]. Thus, we first investigated if bone-derived

OPN influenced the migration of this stem-like population. We observed that FACS- isolated ALDHhiCD44+CD24- cells showed significantly increased migration toward

BMCM compared to basal media (P<0.05, Figure 8C). When OPN is depleted from

BMCM, the ALDHhiCD44+CD24- subpopulation shows decreased migration, suggesting that bone-derived OPN influences the migration of these stem-like cells. The non stem- like ALDHloCD44-CD24+ cells did not display significantly increased migration to

BMCM or BMCM depleted of OPN compared to basal media (P>0.05, Figure 8C), as expected from our previous work [34, 92]. We also observed that the tumorsphere- forming capacity of ALDHhiCD44+CD24- breast cancer cells decreases in BMCM

A C Basal Media 300 0.4 * BMCM * BMCM Δ OPN

0.3 200 φ

0.2 Migration

(Cell Count) 100 0.1 φ Tumorsphere Frequency Frequency Tumorsphere (proportion of population) 0.0 0 ALDHhiCD44+CD24- ALDHloCD44-CD24+ OPN BMCM Δ

Basal Media B BMCM D

0.15 0.10 Basal Media * BMCM BMCM Δ OPN 0.10 *

0.05 0.05

Colony Frequency φ Tumorsphere Frequency Frequency Tumorsphere (proportion of population) (proportion of population) φ 0.00 0.00 ALDHhiCD44+CD24- ALDHlowCD44-CD24+ OPN BMCM Δ

Basal Media BMCM

Figure 8. Bone-derived OPN promotes the stem-like behavior of MDA-MB-231 breast cancer cells

Whole population MDA-MB-231 cells were plated in a limiting dilution fashion for 7 days in basal media (DMEM/F12 + Mito+), BMCM or BMCM ΔOPN on (A) ultra-low adhesion 96-well plates for sphere-limiting dilution assays or (B) on a normal 96-well plates for limiting dilution colony-forming assays. (C) FACS-isolated ALDHhiCD44+CD24- or ALDHloCD44-CD24+ cells from the MDA-MB-231 cell line 4 were plated on gelatin-coated Transwells® (5 x 10 cells/well; 8µm pore size) before placement into either basal media (DMEM/F12 + Mito+), BMCM or BMCM ΔOPN. Plates were incubated at 37oC at 5% CO2 for 18 hr. TranswellsTM were then fixed with 1% gluteraldehyde and stained with DAPI. Five high-powered fields of view (HPFs) were captured/Transwell® and migrated cells were enumerated with ImageJ software (NIH) (D) FACS-isolated ALDHhiCD44+CD24- and ALDHloCD44-CD24+ cells were also used in the SLDA as in (A). Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). Significance (P< 0.05) was determined with one-way ANOVA and a Bonferroni’s post-hoc test (SLDA and colony formation) or a Tukey’s post-hoc test (migration); * = significantly different from basal media; ϕ = significantly different from BMCM; δ = significantly different from BMCM ΔOPN.

39 depleted of OPN compared to BMCM (P<0.05, Figure 8D), suggesting that these stem- like cells are responsible for the tumorsphere-forming capacity of MDA-MB-231 cells in

BMCM and that bone-derived OPN may mediate this.

5.4 Breast cancer cell migration promoted by bone-derived OPN is mediated by CD44 and RGD-dependent cell surface integrins In order to investigate the mechanism by which bone-derived OPN promotes the migration of breast cancer cells in BMCM, we chose to explore the functional role that select cell surface receptors play in this interaction. OPN is known to influence multiple steps in the metastatic cascade through the cell surface receptor CD44, as well as multiple different cell surface integrins, including αvβ1, α9β1 αvβ3 and αvβ5 [83]. Thus, we first evaluated the expression of CD44, β1, α9β1 αvβ3 and αvβ5 on the MDA-MB-231 and

SUM-159 cell lines. Flow cytometry indicated that both cell lines are positive for the expression of CD44 and these four cell surface integrins (Figures 9 and 10).

Next, we investigated the role of these cell surface receptors on the migration of breast cancer cells using an anti-CD44 functional blocking antibody (Calbiochem) or the

Arg-Gly-Asp (RGD) peptide (Sigma-Aldrich). OPN contains an RGD recognition sequence that is recognized by a number of cell surface integrins, including the ones examined; therefore incubating the cells with the RGD-sequence specific peptide prior to use in functional assays should block the recognition of OPN via the RGD sequence [85,

103]. We observed that blocking CD44 on the MDA-MB-231 and SUM-159 cells significantly decreases migration to BMCM compared to migration of untreated cells (P<

0.05, Figure 11A, 11C). The migration of CD44-blocked MDA-MB-231 to BMCM was reduced to a level similar of untreated cells to BMCM depleted of OPN, suggesting that

A B Cell Count Cell Cell Count Cell

CD44-PE Log Fluorescence β1-AF-488 Log Fluorescence C D Cell Count Cell Cell Count Cell Cell Count Cell

αVβ3-FITC Log Fluorescence αVβ5-AF-488 Log Fluorescence

E IgG Control CD44/Cell Surface Integrins

α9β1-AF-488 Log Fluorescence

Figure 9. MDA-MB-231 cells express OPN receptors including CD44 and multiple different cell surface integrins.

Representative histograms are shown from flow cytometry characterization of MDA- MB-231 cells incubated with (A) phycoerytherin-conjugated anti-CD44, (B) AlexaFluor- 488-conjugated anti-integrin β1, (C) fluorescein isothiocyanate-conjugated anti-integrin αvβ3, (D) AlexaFluor-488 anti-integrin αvβ5 or (E) AlexaFluor-488 conjugated anti- integrin α9β1 antibodies (black) for 1hr at 4°C compared to cells incubated with an isotype-matched IgG-control (white). Samples were analyzed on a Beckman-Coulter EPICS XL-MCL flow cytometer.

A B Cell Count Cell Cell Count Cell

CD44-PE Log Fluorescence β1-AF-488 Log Fluorescence C D Cell Count Cell Cell Count Cell

αVβ3-FITC Log Fluorescence αVβ5-AF-488 Log Fluorescence

E IgG Control CD44/Cell Surface Integrins Cell Count Cell

α9β1-AF-488 Log Fluorescence

Figure 10. SUM-159 cells express OPN receptors including CD44 and multiple different cell surface integrins.

Representative histograms are shown from flow cytometry characterization of SUM-159 cells cells incubated with (A) phycoerytherin-conjugated anti-CD44, (B) AlexaFluor- 488-conjugated anti-integrin β1, (C) fluorescein isothiocyanate-conjugated anti-integrin αvβ3, (D) AlexaFluor-488 anit-integrin αvβ5 or (E) AlexaFluor-488 conjugated anti- integrin α9β1 antibodies (black) for 1hr at 4°C compared to cells incubated with an isotype-matched IgG-control (white). Samples were analyzed on a Beckman-Coulter EPICS XL-MCL flow cytometer.

MDA-MB-231 A MDA-MB-231 C Basal Media Basal Media BMCM BMCM 6 4 BMCM Δ OPN * BMCM Δ OPN

3 * φ φ 4 φ

2 φ Migration Migration 2 φδ (Fold Change) (Fold Change) φ δ 1

0 0

CD44 Blocked RGD Blocked

SUM-159 B SUM-159 D

Basal Media Basal Media BMCM 3 BMCM 3 * BMCM Δ OPN * BMCM Δ OPN φ 2 φ 2 φ φ

φ Migration Migration δ 1 φ δ 1 (Fold Change) (Fold Change)

0 0

CD44 Blocked RGD Blocked

Figure 11. Promotion of breast cancer cell migration by bone-derived OPN is mediated by the cell surface receptor CD44 and various RGD-dependent cell surface integrins

(A) MDA-MB-231 and (B) SUM-159 cells were blocked with an anti-CD44 antibody for 30 min and plated on gelatin coated TranswellsTM (5 x 104 cells/well; 8µm pore size) before placement into basal media (DMEM/F12 + Mito+), BMCM or BMCM ΔOPN. Plates were incubated at 37oC at 5% CO2 for 18 hr. TranswellsTM were then fixed with 1% gluteraldehyde and stained with DAPI. Five high-powered fields of view (HPFs) were captured/TranswellTM and the migrated cells were enumerated through the use of ImageJ software (NIH). (C) MDA-MB-231 and (D) SUM-159 cells were blocked with an RGD sequence-specific peptide for 30 min and used in the TranswellTM migration assay as described above. Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). Significance was determined with one-way ANOVA and Tukey’s post-hoc test;.* = significantly different from basal media; ϕ = significantly different from BMCM; δ = significantly different from BMCM ΔOPN: P< 0.05.

CD44 is responsible for the effect of bone-derived OPN of breast cancer cell migration

(P>0.05, Figure 11A). In contrast, CD44-blocked SUM-159 cell migration to BMCM was significantly lower than untreated SUM-159 cell migration to BMCM depleted of

OPN (P< 0.05, Figure 11C). This suggests that CD44 may mediate the interaction of

SUM-159 with bone-derived OPN as well as other, as yet uninvestigated soluble CD44- ligands within the BMCM.

Our results also indicate that blocking the RGD-recognition sequence on MDA-

MB-231 and SUM-159 cells significantly reduces the migration to BMCM compared to migration of untreated cells (P<0.05, Figure 11B, 11D). RGD-blocked MDA-MB-231 cells showed significantly decreased migration to BMCM compared to untreated cells to

BMCM depleted of OPN (P<0.05, Figure 11B). These results suggest that RGD- dependent integrins may mediate the interaction of MDA-MB-231 with bone-derived

OPN and other soluble factors that interact with integrins within the BMCM. In contrast,

RGD-blocked SUM-159 cells showed similar migratory levels to BMCM as untreated cells to BMCM depleted of OPN, suggesting that RGD-dependent cell surface integrins mediate the interaction of SUM-159 cells with bone-derived OPN (P>0.05, Figure 8D).

5.5 Promotion of breast cancer stem-like behavior by bone- derived OPN is mediated through CD44 and RGD-dependent cell surface integrins Given that CD44 and RGD-dependent integrins mediate the interaction between breast cancer cells and bone-derived OPN to influence migration to BMCM, we wanted to explore if these cell surface receptors also influence the stem-like behaviour of breast cancer cells in response to bone-derived OPN. An anti-CD44 blocking antibody

(CaliBiochem) and RGD-sequence specific peptide (Sigma Aldrich) were used to block

MDA-MB-231 in the SLDA. Both CD44-blocked and RGD-blocked MDA-MB-231 cells showed decreased tumorsphere-forming capacity when exposed to BMCM and BMCM depleted of OPN compared to untreated cells exposed to BMCM depleted of OPN

(P<0.05, Figure 12A, 12B), suggesting that both CD44 and RGD-dependent integrins can mediate the tumorsphere-forming capacity of breast cancer cells.

5.6 Bone-derived OPN causes phosphorylation changes in migratory and stem-like related pathways in breast cancer cells

We have shown that bone-derived OPN influences the migratory ability and stem- like behavior of breast cancer cells via CD44 and RGD-dependent cell surface integrins.

Considering this, we wanted to begin to investigate the effect of bone-derived OPN on downstream pathways within breast cancer cells. For this, we used the Human Phospho-

Kinase Array (R&D Systems), which simultaneously detects the relative site-specific phosphorylation of 43 kinases and upregulation of 2 related total proteins. We exposed

MDA-MB-231 cells to basal media ((DMEM/F12 + MITO+), BMCM and BMCM depleted of OPN for 2 hr prior to harvesting cell lysates for use with the array. Figure 13 shows 15 different kinases and 1 protein that were shown to be significantly different between treatments. Results show that BMCM causes the up-regulation of WNK-1,

PRAS40 and HSP60, compared to basal media (P≤0.05; Figure 13) and BMCM depleted of OPN (P≤0.05; Figure 13). Table 2 includes further details about these proteins of interest. We are currently in the process of validating these findings by immunoblotting.

A B MDA-MB-231 Basal Media BMCM MDA-MB-231 Basal Media * * BMCM Δ OPN * * BMCM 0.25 0.25 BMCM Δ OPN

0.20 0.20

0.15 0.15

0.10 0.10

0.05 0.05 Tumorsphere Frequency Frequency Tumorsphere Tumorsphere Frequency Frequency Tumorsphere (proportion of population) (proportion of population) 0.00 0.00

CD44 Blocked RGD Blocked

MDA-MB-231 human breast cancer cells were blocked with (A) anti-CD44 antibodies or (B) an RGD sequence-specific peptide for 30 minutes prior to plating in a limiting dilution fashion on ultra-low adhesion 96-well plates for 7 days in basal media (DMEM/F12 + Mito+), BMCM or BMCM Δ OPN in the sphere-limiting dilution assay. Significance determined with a two-way ANOVA and a Bonferroni’s post-hoc test; * = significantly different from basal media; ϕ = significantly different from BMCM; δ = significantly different from BMCM ΔOPN: P< 0.05.

Negative Control BMCM BMCMΔΟPΝ

50000 * 45000 30000 φ 25000 20000 15000 φ * 10000 φ φ

Mean Pixel Density φ φ * * 5000 φ φ φ φ φ φ φ φ * * 0

α 2 γ1 β α Lck p38 TOR FAK CREB c-Jun PYK2 EGF-R PLC- WNK1 HSP60 ERK1/2 PRAS40 AMPK PDGF R RSK1/2/3

Figure 13. Bone-derived OPN causes phosphorylation changes in migration- and stem cell-related signaling pathways in breast cancer cells

MDA-MB-231 human breast cancer cells were incubated in basal media, BMCM or BMCM ΔOPN and cell lysates were harvested after 2 hr. Cell lysates were incubated with the Human Phospho-Kinase Array membranes (ARY003B, R&D Systems) overnight at 4°C. Biotinylated detection antibodies were applied and membranes were visualized using chemiluminescence. Densitometry analysis was performed using the Protein Array Analyzer for ImageJ and significance (P<0.05) was determined with a Tukey’s test; * = significantly different from basal media; ϕ = significantly different from BMCM; δ = significantly different from BMCM ΔOPN. Only kinases and proteins with significantly different phosphorylation or expression between at least two treatments are shown.

Table 2: Malignancy-associated kinases and protein of interest identified by the Human Phospho-Kinase Array in response to bone-derived OPN Protein Phosphorylation Function/Involvement in Malignancy References Name Site • Serine/threonine protein kinase WNK-1 T60 • Participates in cell proliferation and migration [104] • Involved in differentiation of neural progenitor cells

• Acts at the intersection of the Akt PRAS40 T246 and mammalian-target of rapamycin (mTOR)-mediated signaling pathways [83, 105] • Acts downstream of OPN to influence cell migration

• Increased levels of HSP60 observed HSP60 - in multiple different cancers, plays an essential role in malignant cell survival [106] • Interaction between HSP60 and β- catenin promotes metastasis

6 DISCUSSION

One in nine Canadian females are expected to develop breast cancer in their lifetime and one in 32 of all Canadian females are expected to die of breast cancer [7].

About 85% of all breast cancer patients that die from their disease are expected to harbor bone metastases [107]. Bone metastases pose an extreme burden for breast cancer patients, resulting in pain, fractures and hypercalcaemia. The current clinical course of management lacks treatments that cure, reduce or prevent malignant skeletal lesions

[107].

Elucidation of the steps involved in the metastatic cascade is vital for the development of therapeutics that can increase progression-free and overall survival of breast cancer patients. So far, scientific research efforts have shown that the process of metastasis is highly inefficient and that subsets of malignant cells with a stem-like phenotype are most likely to form metastases in a secondary site [13, 23, 34, 39].

Moreover, breast cancer repeatedly metastasizes to specific organs in the human body.

This observed organ tropism implies a role for the microenvironment in the establishment and progression of metastases in distant organs [12, 37].

Bone is the most common site of secondary growth in metastatic breast cancer patients, suggesting an environment particularly conducive to the development of metastases [60]. It is now clear that both soluble and insoluble factors within the bone microenvironment contribute to the progression of malignant disease. This thesis focuses on the soluble factors within the bone microenvironment that promote the formation of breast cancer lesions in the bone. We used an ex vivo representation of the bone microenvironment to study soluble factors in bone and hypothesized that one specific

49 soluble factor, OPN, promotes the migration and stem-like behaviour of breast cancer cells in BMCM and that the interaction of bone-derived OPN with breast cancer cells activates downstream intracellular signaling responses.

6.1 Summary of Experimental Findings

1. Bone marrow-conditioned media (BMCM) contains multiple soluble factors that

could assist in the establishment of breast cancer metastases in bone, such as

osteopontin (OPN), basic fibroblast growth factor (bFGF), intracellular adhesion

molecule-1 (ICAM-1), insulin-like growth factor-2 (IGF-2) and matrix-

metalloproteinase-14 (MMP-14).

2. Bone-derived OPN promotes the tumorsphere and colony-forming abilities in

MDA-MB-231 breast cancer cells.

3. Bone-derived OPN promotes the migration and tumorsphere-forming abilities of

stem-like ALDHhiCD44+CD24- MDA-MB-231 breast cancer cells to BMCM.

4. Promotion of MDA-MB-231 and SUM-159 breast cancer cell migration and

MDA-MB-231 breast cancer cell tumorsphere-forming capacity by bone-derived

OPN is mediated by the cell surface receptor CD44 and various RGD-dependent

cell surface cell surface integrins.

5. Bone-derived OPN causes alterations in migration- and stem cell-related signaling

pathways in MDA-MB-231 breast cancer cells, including activation of HSP60 and

phosphorylation of PRAS40 and WNK-1.

6.2 Implications of Experimental Findings

This study supports the hypothesis that bone-derived OPN promotes the migration and stem-like behaviour of breast cancer cells via CD44 and RGD-dependent cell surface integrins and affects the phosphorylation and activation of downstream signaling pathways in breast cancer cells (Figure 14). There exists in the literature a wide breadth of knowledge on the effect of tumor-derived OPN in multiple steps of the metastatic cascade, however much less research has been conducted on the effect of host-derived

OPN in the tumor microenvironment.

It is thought that host-derived OPN differs from tumor-derived OPN both structurally and functionally. Current literature suggests that tumor-derived OPN is less phosphorylated than host-derived OPN [83]. One study showed that transformation of normal rat kidney cells changes OPN from highly phosphorylated to less phosphorylated and OPN produced by Ras-transformed fibroblasts is less phosphorylated than OPN produced by osteoblasts [108]. Kazanecki et al (2007) proposed that the differing phosphorylation profiles of host-derived and tumor-derived OPN affects its function and that the less phosphorylated, tumor-derived OPN is more effective at promoting cancer progression by protecting cells from the immune response, inhibiting apoptosis and increasing anchorage independence and metastasis while host-derived OPN serves as a chemoattractant for multiple cells of the immune system, such as macrophages, that inhibit tumor growth when recruited to the lesion site [109, 110]. It is also suggested that tumor-derived OPN better supports invasiveness while host-derived OPN promotes adhesion to components of the extracellular matrix such as fibronectin and vitronectin.

Figure 14 Summary of Experimental Findings

Bone-derived osteopontin promotes the migration of breast cancer cells toward bone as well as the stem-like behavior of breast cancer cells in bone by interacting with cells via the CD44 cell surface receptor and the RGD-dependent cell surface integrins.

Our work helps elucidate the contribution of host-derived OPN – specifically bone- derived OPN – to the metastatic progression of breast cancer within the bone.

6.2.1 Bone-Derived OPN Promotes the Migration of Breast Cancer Cells

Firstly, we showed that host-derived OPN, specifically soluble bone-derived

OPN, can in fact contribute to the metastatic potential of breast cancer cells by increasing their migration towards bone-conditioned media. This finding is supported by the work of others; Nemoto et al (2001) found that OPN-deficient mice displayed less colonization and growth of injected melanoma cells to bone than wild-type mice [89]. Chakraborty et al (2008) showed similar results, demonstrating that OPN-null mice have a slower growth rate of tumors compared to wild type mice [89, 111]. These studies, taken together with our results, suggest that OPN increases breast cancer cell migration to and colonization in bone, thus promoting breast cancer bone metastases.

Both the MDA-MB-231 and SUM-159 breast cancer cells displayed reduced migration towards BMCM depleted of OPN compared to non-depleted BMCM.

However, levels of migration toward BMCM depleted of OPN were still significantly higher than migration toward basal media, suggesting that other factors within the

BMCM may be contributing to the migration of breast cancer cells. The RayBio® Biotin label-based mouse antibody array identified multiple soluble factors that could be contributing to this observed migratory effect. One such protein is matrix metalloproteinase-14 (MMP-14; MTI-MMP). MMP-14 can be membrane bound or found in the soluble form; the type 1 transmembrane MMP is expressed by both stromal cells and breast cancer cells and undergoes shedding to produce multiple soluble active and inactive fragments [112]. Multiple different types of MMPs are known to contribute to

53 carcinogenesis through extracellular matrix remodeling and cancer cell migration.

Elevated levels of soluble MMP-14 specifically have been detected in the plasma of breast cancer patients and it is known to be a critical protein in cancer progression as it is responsible for invasion through collagen networks and collagenolysis [113]. Tobar et al

(2014) showed that MMP-14 produced by bone marrow stromal cells sheds epithelial endoglin, a membrane bound glycoprotein that has tumor suppressor effects by attenuating the expression of TGF-β1. By silencing endoglin, MMP-14 enhances the migratory properties of human breast cancer cells [101]. Zarrabi et al (2011) have shown that MMP-14 can crosstalk with CD44, activating major players in migratory signaling pathways downstream of CD44 such as MAPK and PI3K [114].

Intercellular adhesion molecule (ICAM)-1 is also of interest when considering breast cancer migration toward BMCM. ICAM-1 is expressed on the surface of endothelial cells, leukocytes and cancer cells and can also be shed into the circulation in soluble form. It is an immunoglobulin involved in facilitating adhesion of immune cells to endothelia in the healthy human body [115]. However, extensive research shows that

ICAM-1 also plays a role in malignancy. Elevated levels of soluble ICAM-1 are present in breast cancer patients and recently, Schröder et al (2011) showed that ICAM-1 expression is significantly associated with poor prognosis in breast cancer patients, specifically a poorly differentiated phenotype, negative ER status and positive lymph node involvement [116]. Binding of soluble ICAM-1 to target cells has been shown to modulate migration and angiogenesis via the activation of signaling cascades involving

MAPK and Src, thus it is possible that ICAM-1 plays a role in the migration of breast cancer cells to BMCM [117].

6.2.2 Bone-Derived OPN Promotes the Stem-Like Phenotype of Breast Cancer Cells in Bone

In this study, we also assessed the ability of bone-derived OPN to influence the stem-like behaviours of limiting dilution tumorsphere-forming ability and colony- forming ability of breast cancer cells in BMCM. To assess these properties, we used the sphere-limiting dilution assay (SLDA) and the colony-forming assay. The SLDA uses a limiting-dilution analysis to quantify the number of tumorsphere initiating cells present in populations grown in nonadherent conditions [118] and the colony-forming assay measures the ability of cells to differentiate and proliferate into colonies at very low numbers. To our knowledge, our study is the first to show that bone-derived OPN promotes the tumorsphere-forming capacity and colony-forming ability of breast cancer cells.

In normal tissues, stem cells reside in a specialized niche that influences their stem-like properties and differentiation fates via cell-to-cell interactions, various secreted soluble factors and extracellular matrix components [59, 119]. This niche helps maintain homeostasis, influencing a stem cell’s ability remain quiescent, self-renew, or differentiate; thus carefully regulating the size and activity of the stem cell pool [59, 119].

The bone marrow is known to provide an optimal niche for the maintenance of hematopoietic stem cells (HSCs). HSCs migrate toward and reside within the endosteal region of bone that lies between the bone and bone marrow interface [59]. It has been shown that contact between osteoblasts and HSCs at the endosteal surface and within trabecular bone is particularly important in modulating HSC proliferation and hematopoiesis [59, 120]. It has recently become clear that OPN is integral in mediating the effects of osteoblasts on HSCs within healthy bone. Nilsson et al (2005) have shown

55 that osteoblast-secreted OPN promotes migration and lodging of HSCs within the endosteal region; when transplanting HSCs into a normal hematopoietic environment, they settle almost exclusively into the endosteal region of the bone, however when transplanting HSCs into an OPN-/- environment, there is a random distribution of the cells throughout the bone marrow [121]. Studies by this group also suggest that OPN maintains hematopoietic progenitor cell (HPCs) quiescence within the bone marrow

[121]. HPCs were isolated from wild type and OPN-/- mice fed bromodeoxyuridine

(BrdU) continuously for four weeks; HPCs harvested from wild type animals did not show any incorporation of BrdU, suggesting that HPCs were maintained in the quiescent state, whereas almost all HPCs isolated from OPN-/- mice displayed incorporation of

BrdU [121].

Similar to healthy stem cells within the normal bone marrow niche, stem-like cancer cells are also influenced and maintained by the microenvironment in which they reside. Stromal cells play a major role in maintaining a microenvironment suitable for cancer stem cells; these cell types include osteoblasts, osteoclasts, cancer-associated fibroblasts (CAFs), infiltrating leukocytes, mesenchymal stem cells (MSCs), and tumor associated macrophages (TAMs) which interact with CSCs via direct contact or by secreting various growth factors and cytokines [122].

Recent work has implicated OPN in the maintenance of “stemness” in different types of cancers. Pietras et al (2014) showed that OPN was able to promote stemness in proneural glioblastoma [123]. This study used two functional assays to measure stemness; (1) the drug efflux-based side population (SP) assay, which was previously described to enrich for stem-like tumor initiating cells in human and murine glioma by

56 their ability to exclude Hoechst dye via ABC transporter activity [124] and (2) the colony-forming assay following irradiation, as well as the expression of a panel of stemcell markers, including Nanog, Sox2, Oct4 and Id1. This study found that OPN had the ability to increase the amount of cells displaying the SP phenotype within glioma primary cultures, suggesting that OPN was necessary to induce the stem-like phenotype. They also found that glioma primary cultures treated with OPN displayed up-regulated Nanog,

Sox2, Oct4 and Id1 stem cell markers. Finally, glioma primary cultures treated with OPN formed more colonies in a colony-forming assay following irradiation relative to control cells.

Cao et al (2015) showed that OPN promotes a cancer stem cell-like phenotype in hepatocellular carcinoma cells. They found that OPN expression is correlated with hepatocellular carcinoma (HCC) SP fractions that display high self-renewal and tumorigenic properties and was highly expressed in dormant cells, spheroids and chemo- resistant cancer cells [125]. They also found that HCC cells exhibited a dramatically decreased ability to form colonies and spheroids in colony-forming assays and tumorsphere-forming assays when OPN is knocked down with an shRNA construct in

HCC cells [125]. These cells also displayed a reduction in stemness-related genes, including HIF-1α, OCT4, Nanog, CK19, BMI-1 and Notch1 and decreased tumorgenicity in immunodeficient mice [125].

The aforementioned studies by Pietras et al (2014) and Cao et al (2015) focused on OPN derived from carcinoma cells however, given the role of the microenvironment in maintaining CSC phenotype and activity and our data showing that bone-derived OPN promotes the tumorsphere and colony-forming capabilities of breast cancer cells in vitro,

57 it is possible that OPN within the bone microenvironment also plays a crucial role in metastatic tumor progression by maintaining the stemness of breast cancer cells that have metastasized to bone. Our work proposes a novel function for bone-derived OPN in the maintenance of breast cancer cell “stemness” and thus suggests a role for bone-derived

OPN in the establishment and maintenance of heterogeneous breast cancer tumors in bone.

6.2.3 The Role of CD44 and RGD-Dependent Cell Surface Integrins in the Bone-Derived OPN Mediated Migration and Stem-Like Behavior of Breast Cancer Cells

Given the observed functional role of bone-derived OPN in promoting the migration and stem-like behaviour of breast cancer cells, we next explored the mechanism by which bone-derived OPN influences these functions of breast cancer cells.

Our results demonstrate that bone-derived OPN influences the migration and tumorsphere-forming ability of breast cancer cells via the cell surface receptor CD44 and

RGD-dependent cell surface integrins.

CD44 is encoded by 20 exons, 7 of which make the extracellular domain of the standard form of CD44 (CD44s). Variants of CD44 arise through the alternative splicing of at least 12 of the 20 exons [126]. Cell surface expression of CD44 on macrophages and osteoclasts has been shown to effect normal cell motility through interaction with OPN

[127, 128]. Receptor-ligand interactions between CD44 and OPN have also been implicated in malignancy, specifically related to migration as well as other steps in the metastatic cascade. Weber et al first showed in 1996 that OPN interacts with CD44 and then went on to show that the interaction between CD44 and cancer cell-secreted OPN promotes the migration of tumor cells to specific sites of metastasis formation [129, 130].

Katagiri et al (1999) later showed that OPN is able to bind to several CD44 variants in an

RGD-dependent and independent manner on both the N-terminal and C-terminal fragments of OPN to promote cell spreading, motility and chemotactic behavior [131].

Khan et al (2005) then showed that incubating T1NT tumorigenic human breast cancer cells with monoclonal antibodies recognizing all isoforms of CD44 or the specific variants of CD44 v6 or v9 resulted in a significant decrease in OPN-mediated cell migration and that OPN enhances CD44 expression at both the mRNA and protein level and increases the localization of CD44 to the cell membrane surface [132]. Our lab also recently showed that breast cancer cells preferentially migrate toward lung-derived OPN in a CD44-dependent manner [92].

The CD44-OPN interaction is also thought to facilitate OPN’s effect on cancer cell stemness. The ability of OPN to maintain glioma stemness phenotypes in the perivascular niche is shown to occur in a CD44-dependent manner; mouse models of glioma that were CD44-/- or CD44+/- showed better survival compared to controls, an effect shown to be mediated through downstream enhancement of HIF-2α activity [123].

The current study adds to this breadth of knowledge, showing that bone-derived OPN influences breast cancer cell migration and the tumorsphere-forming ability of breast cancer cells via CD44.

The RGD-dependent cell surface integrins include α5β1, α8β1 and all αv- containing integrins. These integrins recognize ligands that contain the RGD motif (Arg-

Gly-Asp), mainly found in components of the extracellular matrix such as OPN, fibronectin, vitronectin and nephronectin [133]. The interaction between OPN and these cell surface integrins is used in multiple different settings in the healthy body, including

59 facilitating the migration and adhesion of cell types such as osteoclasts, multiple different types of epithelial cells and smooth muscle cells via clustering and functional activation of the focal adhesion kinase complex [134]. Cell surface integrins also prove particularly important in malignancy, as increased expression of these integrins on tumor cells enhances their tumorigenic properties, specifically migration and adhesion. OPN- mediated migration in breast cancer has been shown to involve many of these integrins, including αvβ1, αvβ5 [135, 136] and α8β1 [137]. The cell surface integrin that seems to be most commonly associated with OPN’s effect in malignancy is the αvβ3 heterodimer.

Increased expression of αvβ3 during breast cancer tumorigenesis and progression makes tumor cells more responsive to OPN and thus enhances tumorigenic properties such as migration and adhesion [86]. Interestingly, it has been shown that breast cancer cell lines can express variable amounts of different cell surface integrins and thus depend on certain integrins over others in their migratory and adhesive functions. Wong et al (1998) showed that the highly metastatic MDA-MB-435 breast cancer cell line expresses high levels of αvβ3 while the non-metastatic MCF-7 breast cancer cell line expressed significantly lower levels of αvβ3 [138]. Similarly, van der Pluijm et al (1997) found higher levels of expression of α2β1, α3β1, α5β1 and αvβ3 in the highly metastatic MDA-

MB-231 breast cancer cell line compared to the less malignant MCF-7, T47D and ZR75-

1 breast cancer cell lines [139] and found that use of the GRGDS peptide blocked the adhesion of MDA-MB-231 to bone matrix molecules. Tuck et al (2000) then showed that the highly metastatic MDA-MB-435 breast cancer cell line depends more on the αvβ3 heterodimer for OPN-mediated migration than αvβ5 or β1 integrins, whereas the non-

60 metastatic breast cancer cell lines, 21PT and 21NT, migrated in an αvβ5 or β1-dependent and an αvβ3-independent manner [140].

In keeping with these studies, our results show cell line-specific migratory responses when the OPN-CD44 or the OPN-RGD-dependent cell surface integrin interactions are abrogated. The MDA-MB-231 cells display significantly decreased migration to BMCM depleted and non-depleted of OPN when the RGD motif is blocked compared to when the CD44 cell surface receptor is blocked. In contract, the SUM-159 cells display the opposite effect; they show significantly reduced migration when the

CD44 cell surface receptor is blocked compared to when the RGD motif is blocked. Flow cytometry analysis revealed that both cell lines express significant amounts of CD44 and similarly moderate amounts of RGD-dependent β1, αvβ3 and αvβ5 and RGD- independent α9β1, suggesting that the differences in migratory function observed could be dependent on the expression of other cell surface integrins on the MDA-MB-231 such as α5β1 and α8β1.

We also showed that blocking RGD-dependent cell surface integrins on MDA-

MB-231 cells attenuates their tumorsphere-forming capacity. These results are supported by those of Cao et al (2015), who showed that OPN promotes the stem cell-like phenotype of HCC cells via the αvβ3 cell surface integrin, which in turn activates the downstream transcription factor NF-κB and hypoxia-related protein HIF-1α [125].

6.2.4 Bone-Derived OPN Causes Phosphorylation and Expression Changes in Migratory and Stem Cell-Related Pathways in Breast Cancer Cells

OPN influences a number of signaling pathways via communication with CD44 and RGD-dependent cell surface integrins to mediate steps of metastatic cascade,

61 including migration, cell proliferation and survival, adhesion and angiogenesis. Soluble

OPN can promote the migration, proliferation and survival of tumor cells via αvβ1, αvβ3,

αvβ5 and CD44 while adhesion and angiogenesis are promoted via αvβ3 [83]. The interaction of OPN with αvβ1 and αvβ5 typically result in the upregulation of paxillin,

Crk and Ras while signaling via αvβ3 activates Src and the focal adhesion kinase complexes and their downstream effectors such as Erk1/2, uPA, NF- κB, OPG and the

FAK and PI3K/Akt axis [83]. Interaction of OPN with CD44 activates the CD44-ERM complex, as well as promoting the phosphorylation of Akt and the activation of MAPK

[83]. The migratory response of cells to OPN also includes the upregulation of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) and induces EGF receptor

(EGFR) receptor expression and tyrosine kinase activity as well as an increase in the kinase activity of the HGF receptor Met [140, 141].

Given the effect of OPN on multiple downstream signaling pathways within tumor cells, we wanted to explore the effect that bone-derived OPN has on the phosphorylation of common kinases and the activation of 2 related proteins in MDA-MB-

231 human breast cancer cells using the Human Phospho-Kinase Array (R&D Systems).

This array contained proteins that are commonly activated and phosphorylated in response to soluble OPN, including Akt, EGFR, Erk1/2 and FAK. With use of this array, we identified three novel factors that appear to be influenced by bone-derived OPN. Our results indicate that the presence of bone-derived OPN causes significant increases in the phosphorylation of PRAS40 and Wnk-1 and an upregulation in the expression of HSP60.

We have yet to validate the results of the array with immunoblotting, however these three

62 proteins are interesting candidates for the downstream signaling that may be activated in breast cancer cells in response to bone-derived OPN.

6.2.4.1 PRAS40

PRAS40 is a proline-rich substrate of Akt and mTORC1. It acts at the intersection of the Akt and mammalian target of rapamycin (mTOR) signaling pathways and has been used in studies as a marker for PI3K pathway activation. The PI3K-Akt signaling pathway regulates many normal cellular processes including cell motility, proliferation, cell growth and cell survival. It has been extensively studied in relation to carcinogenesis and the aberrant expression of many components within this pathway has been implicated in numerous human cancers [142]. Andersen et al (2010) found that expression of phospho-PRAS40 was up-regulated in 40% of primary breast cancer samples and correlates with PI3K-Akt signaling and thus predicts Akt inhibitor sensitivity in triple- negative breast cancer tissues [143]. As previously mentioned, OPN activates the

PI3K/Akt/mTOR pathway via interaction with αVβ3 to promote migration, invasion and cell survival and proliferation thus it is likely that bone-derived OPN may also indirectly cause increased phosphorylation of PRAS40 [144].

6.2.4.2 WNK1

WNK1 is a seronine-threonine protein kinase that is widely expressed in many human tissues. Most research on the WNK protein kinase family has focused on their role in the regulation of ion transporters in the kidney and extrarenal tissues, however growing evidence suggests that they are involved in various signaling cascades related to human cancer. WNK1 is involved in the MAPK cascade and has been shown to be involved in

EGF-dependent stimulation of ERK1/2 and ERK5 in neural progenitor cells and HeLa

63 cells [104, 145]. Knockdown of WNK1 in these cell types lead to the suppression of

ERK1/2 by EGF and resulted in greatly reduced cell growth and migration [104]. WNK1 is also known to phosphorylate Smad2 and negatively affects Smad-mediated gene expression, which results in suppression of TGF-β signaling. TGF-β is integral in inducing the epithelial-to-mesenchymal transition (EMT) thus loss or inactivation of

WNK1 activity could promote EMT of epithelial tumor cells [146]. WNK1 is also linked to Rho GTPases, which control the dynamics of the cytoskeleton and are integral in cell migration and invasiveness [147, 148]. Notably, upregulation of WNK1 in neural tumor cells has been correlated with increased invasiveness. WNK1 is also a substrate of Akt, suggesting that it may play a role in the PI3K/Akt pathway [145]. Its role in this pathway could cause WNK1 to be indirectly phosphorylated by the interaction of bone-derived

OPN with αvβ3 on breast cancer cells, and thus could contribute to breast cancer migration, invasiveness and cell growth downstream of OPN.

6.2.4.3 HSP60

Finally, heat shock protein 60 (HSP60) is a chaperone protein that is essential in assisting many different newly synthesized proteins fold into their native forms. Research focused on HSP60’s role in disease has mostly shown its role in innate immunity and cardiac ischemia, however increased HSP60 expression is also observed in a wide variety of cancers, including prostate, ovarian, pancreatic and large bowel carcinoma [149].

Cellular distribution of HSP60 changes during carcinogenesis; while it is normally found in the mitochondria, instead it accumulates in extramitrochondrial sites, such as the cytosol, plasma membrane, and secretory vesicles [150, 151]. HSP60 has also been shown to accumulate in lymph node-invading tumor cells, possibly suggested a role for

HSP60 in metastasis, and to accumulate in tumor cells as a result of treatment with the systemic agent 5-azacytidine, conferring tumor cell resistance [149]. Tsai et al (2009) recently demonstrated that overexpression of HSP60 induces metastasis in head and neck cancer cell lines by interacting with β-catenin and activating its downstream targets, such as MMP-14 [106]. Our results from the Human Proteome Profiler array suggest bone- derived OPN may also have an effect on the expression of HSP60 in breast cancer cells.

6.3 Limitations to the Study

This study’s methods rely heavily on the use of an ex vivo model system as a surrogate for the bone microenvironment. While this system provided an excellent platform in which to study the effect of bone-derived OPN on the migration and stem-like behaviour of breast cancer cells, this approach is not without limitations.

Firstly, this model only allows the examination of soluble factors within the bone marrow microenvironment and excludes insoluble components such as the extracellular matrix and the various cell types that are present in bone, including osteoblasts, osteoclasts, osteocytes, hematopoietic and mesenchymal stem cells, T cells, erythrocytes and platelets. These insoluble factors are known to influence the metastasis of breast cancer cells within bone marrow and would be valuable factors to include when examining the effect of bone-derived OPN on breast cancer metastasis to bone [3].

Secondly, the BMCM is harvested from nude mice and thus consists of murine soluble factors. When using this model, it is assumed that these murine soluble factors can interact with human breast cancer cells. The mouse genome and human genome are highly homologous; of the 4,000 genes that have been studied, less than ten exist in one species but not in the other [152]. Many murine factors are capable of interacting with

65 and stimulating human cells however, it is known that some murine factors are not capable of being recognized by human cells and are not compatible with human signaling pathways [153]. Thus, this model may not be ideal for studying the effect of all soluble factors within the BMCM on breast cancer cell malignant cell behavior. Additionally, the use of nude mice excludes a significant proportion of the immune system contribution to breast cancer cell metastasis in the bone. The immune system is particularly important when studying the effects of OPN on breast cancer cells, as OPN is known to recruit macrophages that can inhibit tumor growth at the site of metastasis [83]. However, the use of the nude murine platform is beneficial in that the results obtained can be closely related to previous and future in vivo studies in the same mouse strain.

Finally, the results of this study rely on the use of immortalized breast cancer cell lines as opposed to primary human breast cancer cells. Although our cells are routinely authenticated, cell lines have the ability to mutate in vitro as a result of being maintained outside of their native environment (tissue plastic vs. the extracellular matrix) and due to their inherent lack of fully functioning DNA repair mechanisms. Therefore although immortalized cell lines are well-characterized, commercially available, and easy to grow in culture, they still may not accurately represent the true behavior that might be seen if primary patient-derived breast cancer cells were used.

6.4 Future Directions

While this study contributes to the breadth of knowledge implicating OPN in the metastasis of breast cancer to bone, some unanswered questions remain that should be addressed to help form a more complete picture of breast tumorigenesis in the bone microenvironment.

Firstly, rescue experiments should be performed with respect to OPN’s effect on the tumorsphere-forming and colony-forming capacity of breast cancer cells in BMCM.

OPN should be added to BMCM that has been depleted of OPN and used in the SLDA and colony-forming assay to show that OPN is solely responsible for the reduction in the tumorsphere- and colony-forming capabilities of breast cancer cells in BMCM.

Secondly, it would be beneficial to show the localization of OPN in the bone and its cell surface receptors and integrins on in vivo breast cancer bone metastases to support the conclusion that bone-derived OPN is interacting with CD44 and RGD-dependent cell surface integrins to facilitate the metastatic process.

Additionally, in order to appropriately conclude that bone-derived OPN results in the phosphorylation of PRAS40 and WNK1 and the upregulation of HSP60, immunoblotting needs be performed with cell lysates exposed to basal media, BMCM and BMCM depleted of OPN. Immunoblotting would support the findings of the Human

Proteome Profiler array, as the array’s results alone are not robust enough to draw definite conclusions. It would also be interesting to repeat the array with other breast cancer cell lines (i.e. SUM-159) to identify any differences in cellular signaling in response to bone-derived OPN. As shown in the results of this study, MDA-MB-231 and

SUM-159 cells both respond to the presence of bone-derived OPN but their response seems to depend more heavily on one cell surface receptor/integrin over another in a cell line-specific manner. It is possible that the different cell lines have differing cell signaling responses to OPN as well.

It would also be beneficial to our conclusions to show the effect of bone-derived

OPN on breast cancer metastases in vivo. Some studies have used OPN knockout mice to

67 explore the metastatic behavior of breast cancer in relation to OPN, however these models have all excluded the effect on the immune system as they have used nude mice.

Repeating these studies in an OPN knockout mice strain using a syngeneic injection model would be interesting, as it has been hypothesized that the recruitment of macrophages to the tumor site by OPN can slow tumor growth and thus may also play a role in initially inhibiting the development of bone metastases [66, 154].

Finally, it is clear that other soluble factors within the BMCM can influence breast cancer cell migration to bone; although migration was significantly reduced in response to BMCM depleted of OPN compared to non-depleted BMCM, migratory levels were still higher than migration toward basal media. Other factors to explore include

MMP-14 and ICAM-1.

7 CONCLUSIONS

This thesis investigated the role of bone-derived OPN in the metastatic behavior of breast cancer cells and their penchant for bone as a secondary growth site. Our work has shown that bone-derived OPN is integral to the metastatic cascade culminating in the establishment of skeletal breast cancer metastases. Bone-derived OPN promotes the migration of breast cancer cells to bone marrow and thus, assists in attracting breast cancer cells to bone. It also contributes to maintaining the stem-like behavior of breast cancer cells in bone marrow and thus, may promote the establishment of metastases in bone. This thesis also suggests that bone-derived OPN activates a downstream signaling response in breast cancer cells involving the phosphorylation of PRAS40 and WNK-1, as well as the activation of HSP60.

Broadly, this thesis focused on the contribution of the bone microenvironment to metastatic breast cancer. While the cancer research community has known about specific metastatic patterns and organ preferences that breast cancers exhibit for over a century, only recently have efforts focused on the role of the secondary site or “soil” in promoting and supporting metastatic growth at these sites. It is becoming increasingly clear that the microenvironment, specifically the bone, offers an optimal niche for the establishment and progression of metastases; our study shows that bone-derived soluble factors – specifically OPN – contribute to this process, possibly by interacting with stem-like breast cancer cells. This knowledge is valuable to our efforts in attempting to treat metastatic disease in breast cancer patients, as targeting the OPN-breast cancer stem cell interaction in addition to using chemotherapies and radiotherapies could help mitigate breast cancer bone metastases.

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88 88

APPENDICES 9 APPENDICES

Appendix 1. Animal Protocol Approval

Appendix A: Animal Protocol Approval

Appendix B: RayBio® Biotin Label-Based Mouse Antibody Array Map and Target List

60 60 180 180 210 210 240 240 270 270 300 300 330 330 360 360 390 390 P-1b P-1b 292959 30 59 30 8989 90 90 119119 120 120 179 179 209 209 239 239 269 269 299 299 329 329 359 359 389 389 P-2b P-2b 28 28 58 58 88 88 118 118 178 178 208 208 238 238 268 268 298 298 328 328 358 358 388 388 P-3b P-3b 57 57 177 177 207 207 237 237 267 267 297 297 327 327 357 357 387 387 262656 27 56 27 8686 87 87 116116 117 117 176 176 206 206 236 236 266 266 296 296 326 326 356 356 386 386 p – Larger 25 25 55 55 85 85 115 115 175 175 205 205 235 235 265 265 295 295 325 325 355 355 385 385 24 24 54 54 174 174 204 204 234 234 264 264 294 294 324 324 354 354 384 384 al al support at 770- 8383 84 84 113113 114 114 173 173 293 293 323 323 353 353 383 383 BlankBlank BlankBlank Blank BlankBlank NEG Blank Blank NEG Blank Blank NEG Blank NEG Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank 82 82 112 112 172 172 292 292 322 322 352 352 382 382 412 412 442 442

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7777 78 78 107107 108 108 167 167 287 287 317 317 347 347 377 377 407 407 437 437 76 76 106 106 136136 137166 137166 138 138 139 139 140 140 141286 141 142286 142316 143316 143346 144346 144376 145376 145 146406 146406 147436 147436 148 148 149 149 150 150

BlankBlank BlankBlank Blank BlankBlank Blank Blank BlankBlank Blank Blank Blank BlankBlank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank NEGBlank Blank Blank Blank NEGBlank Blank NEG Blank BlankBlank Blank NEG Blank Blank BlankBlank Blank Blank Blank Blank Blank Blank Blank Blank BlankBlank Blank BlankBlank Blank Blank BlankBlank Blank Blank Blank BlankBlank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank Blank Blank BlankBlank Blank Blank Blank Blank Blank NEG Blank Blank Blank Blank Blank NEG Blank Blank Blank NEG Blank BlankBlank Blank NEG Blank Blank BlankBlank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank 13 13 43 43 73 73 103 103 163 163 193 193 223 223 253 253 283 283 313 313 343 343 373 373 403 403 433 433 42 42 162 162 192 192 222 222 252 252 282 282 312 312 342 342 372 372 402 402 432 432 111141 12 41 12 7171 72 72 101101 102 102 161 161 191 191 221 221 251 251 281 281 311 311 341 341 371 371 401 401 431 431 10 10 40 40 70 70 100 100 160 160 190 190 220 220 250 250 280 280 310 310 340 340 370 370 400 400 430 430 9 9 39 39 159 159 189 189 219 219 249 249 279 279 309 309 339 339 369 369 399 399 429 429 686898 69 98 69 99 99 158 158 278 278 308 308 338 338 368 368 67 67 97 97 157 157 277 277 307 307 337 337 367 367

156 156 276 276 306 306 336 336 366 366 656595 66 95 66 96 96 155 155 365 365 64 64 94 94 154 154 364 364 BlankBlank Blank Blank NEG NEG NEG NEG Blank Blank 153 153 363 363 62629292 63 63 93 93 152 152 362 362 12 3456789101112131415161718192021222324252627282930 61 61 91 91 121121151 122151 122 123 123 124 124 125271 125271 126301 126 272 127301 272 127331 302 128331 273 302 128361 273 332 129361 303 332 129 274 303 130 274 333 130 275 304 131 333 275 304 131 305 334 132 305 334 132 335 133 335 133 P-1aP-1a P-2a P-2a P-3a P-3a BlankBlank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank BlankBlank BlankBlank Blank BlankBlank Blank BlankBlank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank NEG Blank Blank Blank NEG Blank NEG Blank Blank NEG Blank Blank Blank Blank Blank Blank Blank Blank Blank BlankBlank BlankBlank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank NEG Blank NEG Blank NEG NEG Blank Blank RayBio® Biotin AntibodyLabel-based Mouse Array I Ma technic contacting by obtained can this of versions 729-2992.

RayBio® L-Series Mouse Antibody Array 308 Protocol

RayBio® L-Series Mouse Antibody Array 308 (L-308) List 90

Number Name Number Name Number Name Number Name 1P-1a 61CCL8 / MCP-2 121Fas Ligand 181Blank 2P-2a 62CCR10 122 FCrRIIB / CD32b 182 Blank 3P-3a 63CCR3 123 FGF R3 183 Blank 4Blank 64CCR4 124 FGF R4 184 Blank 5Blank 65CCR6 125 FGF R5 beta 185 Blank 6NEG 66CCR7 126 FGF-21 186 Blank 7NEG 67CCR9 127 Fit-3 Ligand 187 Blank 8Blank 68CD11b 128 FLRG (Follistatin) 188 Blank 96Ckine 69CD14 129 Follistatin-like 1 189 IL-1 R9 10 Activin A 70 CD195 130 Fractalkine 190 IL-1 RI 11 Activin C 71 CD27 / TNFRSF7 131 Frizzled-1 191 IL-1 RII 12 Activin RIB / ALK-4 72 CD27 Ligand / TNFSF7 132 Frizzled-6 192 IL-2 13 Adiponectin / Acrp30 73 CD30 L 133 Frizzled-7 193 IL-2 R alpha 14 Blank 74 Blank 134 Blank 194 Blank 15 Blank 75 Blank 135 Blank 195 Blank 16 Blank 76 CD30 136 Galectin-3 196 Blank 17 Blank 77 CD40 137 G-CSF 197 Blank 18 Blank 78 CD40 Ligand / TNFSF5 138 GDF-1 198 Blank 19 Blank 79 Cerberus 1 139 GDF-3 199 Blank 20 Blank 80 Chordin-Like 2 140 GDF-5 200 Blank 21 NEG 81 Coagulation Factor III / Tissue Factor 141 GDF-8 201 Blank 22 NEG 82 Common gamma Chain / IL-2 R gamma 142 GDF-9 202 Blank 23 Blank 83 CRG-2 143 GFR alpha-2 / GDNF R alpha-2 203 Blank 24 AgRP 84 Cripto 144 GFR alpha-3 / GDNF R alpha-3 204 IL-2 R beta 25 ALCAM 85 Crossveinless-2 145 GFR alpha-4 / GDNF R alpha-4 205 IL-3 26 Angiopoietin-like 2 86 Cryptic 146 GITR 206 IL-3 R alpha 27 Angiopoietin-like 3 87 Csk 147 GITR Ligand / TNFSF18 207 IL-3 R beta 28 AR (Amphiregulin) 88 CTACK 148 Glut2 208 IL-4 29 Artemin 89 CTLA-4 / CD152 149 GM-CSF 209 IL-4 R 30 Axl 90 CXCL14 / BRAK 150 Granzyme B 210 IL-5 31 Blank 91 CXCL16 151 Granzyme D 211 Blank 32 Blank 92 CXCR2 / IL-8 RB 152 Granzyme G 212 Blank 33 Blank 93 CXCR3 153 Gremlin 213 Blank 34 Blank 94 CXCR4 154 Growth Hormone R 214 Blank 35 Blank 95 CXCR6 155 HGF R 215 Blank 36 Blank 96 DAN 156 HGF 216 NEG 37 Blank 97 Decorin 157 HVEM / TNFRSF14 217 NEG 38 Blank 98 DKK-1 158 ICAM-1 218 Blank 39 b FGF 99 Dkk-3 159 ICAM-2 / CD102 219 IL-5 R alpha 40 B7-1/CD80 100 Dkk-4 160 ICAM-5 220 IL-6 41 BAFF R / TNFRSF13C 101 DPPIV / CD26 161 ICK 221 IL-6 R 42 BCMA / TNFRSF17 102 DR3 / TNFRSF25 162 IFN-alpha / beta R1 222 IL-7 43 beta-Catenin 103 Dtk 163 IFN-alpha / beta R2 223 IL-7 R alpha 44 Blank 104 Blank 164 Blank 224 Blank 45 Blank 105 Blank 165 Blank 225 Blank 46 Blank 106 EDAR 166 IFN-beta 226 Blank 47 Blank 107 EGF R 167 IFN-gamma 227 Blank 48 Blank 108 EG-VEGF / PK1 168 IFN-gamma R1 228 Blank 49 Blank 109 Endocan 169 IGFBP-1 229 Blank 50 Blank 110 Endoglin / CD105 170 IGFBP-2 230 Blank 51 Blank 111 Endostatin 171 IGFBP-3 231 NEG 52 Blank 112 Eotaxin 172 IGFBP-5 232 NEG 53 Blank 113 Eotaxin-2 173 IGFBP-6 233 Blank 54 BLC 114 Epigen 174 IGFBP-rp1 / IGFBP-7 234 IL-9 55 BTC (Betacellulin) 115 Epiregulin 175 IGF-I 235 IL-9 R 56 Cardiotrophin-1 116 Erythropoietin (EPO) 176 IGF-II 236IL-10 57 CCL1 / I-309 / TCA-3 117 E-Selectin 177 IL-1 alpha 237 IL-10 R alpha 58 CCL28 118 FADD 178 IL-1 beta 238 IL-11 59 CCL4 / MIP-1 beta 119 FAM3B 179 IL-1 R4 / ST2 239 IL-12 p40/p70 60 CCL7 / MCP-3 / MARC 120 Fas / TNFRSF6 180 IL-1 R6 / IL-1 R rp2 240 IL-12 p70

RayBio® L-Series Mouse Antibody Array 308 Protocol

RayBio® L-Series Mouse Antibody Array 308 (L-308) List continued91

Number Name Number Name Number Name Number Name 241 Blank 301 LIX 361 Spinesin Ectodomain 421 Blank 242 Blank 302 LRP-6 362 TACI / TNFRSF13B 422 Blank 243 Blank 303 L-Selectin 363 TARC 423 Blank 244 Blank 304 Lungkine 364 TCA-3 424 Blank 245 Blank 305 Lymphotactin 365 TCCR / WSX-1 425 Blank 246 Blank 306 Lymphotoxin beta R / TNFRSF3 366 TECK 426 NEG 247 Blank 307 MAdCAM-1 367 TFPI 427 NEG 248 Blank 308 MCP-1 368 TGF-beta 1 428 Blank 249 IL-12 R beta 1 309 MCP-5 369 TGF-beta 2 429 Urokinase 250 IL-13 310 M-CSF 370 TGF-beta 3 430 VCAM-1 251 IL-13 R alpha 2 311 MDC 371 TGF-beta RI / ALK-5 431 VE-Cadherin 252 IL-15 312 MFG-E8 372 TGF-beta RII 432 VEGF 253 IL-15 R alpha 313 MFRP 373 Thrombospondin 433 VEGF R1 254 Blank 314 Blank 374 Blank 434 Blank 255 Blank 315 Blank 375 Blank 435 Blank 256 Blank 316 MIG 376 Thymus Chemokine-1 436 VEGF R2 257 Blank 317 MIP-1 alpha 377 Tie-2 437 VEGF R3 258 Blank 318 MIP-1 gamma 378 TIMP-1 438 VEGF-B 259 Blank 319 MIP-2 379 TIMP-2 439 VEGFC 260 Blank 320 MIP-3 alpha 380 TIMP-4 440 VEGF-D 261 Blank 321 MIP-3 beta 381 TL1A / TNFSF15 441 WIF-1 262 Blank 322 MMP-2 382 TLR1 442 WISP-1 / CCN4 263 Blank 323 MMP-3 383 TLR2 443 Blank 264 IL-16 324 MMP-9 384 TLR3 444 NEG 265 IL-17 325 MMP-12 385 TLR4 445 NEG 266 IL-17BR 326 MMP-14 / LEM-2 386 TMEFF1 / Tomoregulin-1446Blank 267 IL-17C 327 MMP-24 / MT5-MMP 387 TNF RI / TNFRSF1A 447 Blank 268 IL-17D 328 Neuregulin-3 / NRG3 388 TNF RII 448 P-3b 269 IL-17E 329 Neurturin 389 TNF-alpha 449 P-2b 270 IL-17F 330 NGF R / TNFRSF16 390 TNF-beta / TNFSF1B 450P-1b 271 IL-17R 331 NOV / CCN3 391 Blank 272 IL-17RC 332 Osteoactivin / GPNMB 392 Blank 273 IL-17RD 333 Osteopontin 393 Blank 274 IL-18 R alpha/IL-1 R5 334 Osteoporotegerin 394 Blank 275 IL-20 335 OX40 Ligand / TNFSF4 395 Blank 276 IL-20 R alpha 336 PDGF C 396 Blank 277 IL-21 337 PDGF R alpha 397 Blank 278 IL-21 R 338 PDGF R beta 398 Blank 279 IL-22 339 Pentraxin3 / TSG-14 399 TPO 280 IL-22BP 340 PF-4 400 TRAIL / TNFSF10 281 IL-23 341 PlGF-2 401 TRAIL R2 / TNFRSF10B 282 IL-23 R 342 Progranulin 402 TRANCE / TNFSF11 283 IL-24 343 Prolactin 403 TREM-1 284 Blank 344 Blank 404 Blank 285 Blank 345 Blank 405 Blank 286 IL-27 346 P-Selectin 406 TROY 287 IL-28 / IFN-lambda 347 RAGE 407 TSLP 288 IL-31 348 RANTES 408 TSLP R 289 IL-31 RA 349 RELM beta 409 TWEAK / TNFSF12 290 Insulin 350 Resistin 410 TWEAK R / TNFRSF12 291 Integrin beta 2 / CD18 351 S100A10 411 Ubiqultin 292 I-TAC 352 SCF 412 uPAR 293 KC 353 SCF R / c-kit 413 Blank 294 Kremen-1 354 SDF-1 414 Blank 295 Kremen-2 355 Serum Amyloid A1 415 Blank 296 Lefty-1 356 Shh-N 416 Blank 297 Leptin R 357 SIGIRR 417 Blank 298 LEPTIN(OB) 358 SLPI 418 Blank 299 LIF 359 Soggy-1 419 Blank 300 LIGHT / TNFSF14 360 SPARC 420 Blank

RayBio® L-Series Mouse Antibody Array 308 Protocol

Appendix C: R&D Human Phospho-Kinase Array Target List • Akt (S473) • Akt (T308) • AMPK alpha1 (T174) • AMPK alpha2 (T172) • beta-Catenin • Chk-2 (T68) • c-Jun (S63) • CREB (S133) • EGF R (Y1086) • eNOS (S1177) • ERK1/2 (T202/Y204, T185/Y187) • FAK (Y397) • Fgr (Y412) • Fyn (Y420) • GSK-3 alpha/beta (S21/S9) • Hck (Y411) • HSP27 (S78/S82) • HSP60 • JNK pan (T183/Y185, T221/Y223) • Lck (Y394) • Lyn (Y397) • MSK1/2 (S376/S360) • p27 (T198) • p38 alpha (T180/Y182) • p53 (S15) • p53 (S392) • p53 (S46) • p70 S6 Kinase (T421/S424) • PDGF R beta (Y751) • PLC gamma-1 (Y783) • PRAS40 (T246) • Pyk2 (Y402) • RSK1/2/3 (S380/S386/S377) • Src (Y419) • STAT2 (Y689) • STAT3 (S727) • STAT3 (Y705) • STAT5a (Y694) • STAT5a/b (Y694/Y699) • STAT5b (Y699)

• STAT6 (Y641) • TOR (S2448) • WNK-1 (T60) • Yes (Y426)

Curriculum Vitae - Graciella Pio

EDUCATION

Honors BSc London, ON Western University September 2008-June 2013 • Achieved an Honors Specialization in Biology with a Major in Medical Cell Biology

RESEARCH EXPERIENCE

Honors Thesis Project, Allan Lab, London, ON Department of Anatomy and Cell September 2012-April 2013 Biology, Western University • Research area: cellular and molecular biology of metastasis • Identified two soluble factors that attract whole population breast cancer cells to bone marrow

Obstetrics and Gynecology Clinic, Toronto, ON St. Michael’s Hospital May 2011-August 2011 • Research area: reproductive and infectious diseases, maternal fetal medicine • Research roles: managed various ongoing clinical studies, organized information sessions for potential study participants, recruited clinic patients as potential study participants, organized and analyzed data

Sinclair Lab, Department of Biology, London, ON Western University September 2010-April 2011 • Research area: animal physiology, biology of arthropods in cold environments • Responsible for general laboratory and fly maintenance

SCHOLARSHIPS and AWARDS

Scholarships • Ontario Graduate Scholarship, (15,000/year) 2014-2015 (accepted) • CIHR Strategic Training Program in Cancer Research and Technology Transfer Studentship ($9,050/year), 2013-2015 (accepted) • Translational Breast Cancer Unit Studentship from the London Regional Cancer Program ($9,050/year), 2014-2015 (accepted) • Western Graduate Scholarship ($4500/year), 2013-2015 (accepted)

Awards • CIHR Institute of Cancer Research Travel Award for the Canadian Cancer

Research Conference ($1,000) (accepted) • Dean’s Honor List (UWO), 2011-2013

PUBLICATIONS

Pio GM, Piasecnzy M, Xia Y, Goodale D and Allan AL. Bone-derived osteopontin mediates the migration and stem-like properties of breast cancer cells. [In preparation for International Journal of Cancer]

ORAL AND POSTER PRESENTATIONS

External Presentations • Pio GM, Piaseczny M, Chu JE, Xia Y and Allan AL. The role of bone-derived OPN in breast cancer metastasis. Poster presentation, American Association of Cancer Research Conference, Philadelphia, PA. April 2015 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications of bone derived factors in breast cancer migration. Poster presentation, Metastasis Research Society Conference, Heidelberg, Germany. June 2014 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications of bone derived factors in breast cancer migration. Poster presentation, Canadian Cancer Research Conference, Toronto, ON. November 2013 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications of bone derived factors in breast cancer migration. Oral presentation, Ontario Biology Day at McMaster University, Hamilton, ON. March 2013

Internal Presentations • Pio GM, Piaseczny M, Chu JE, Xia Y and Allan AL. The role of bone-derived OPN in breast cancer metastasis. Oral Presentation, London Regional Cancer Program Seminar Series. March 2014. • Pio GM, Piaseczny M, Chu JE, Xia Y and Allan AL. The role of bone-derived OPN in breast cancer metastasis. Poster presentation, London Health Research Day. March 2015 • Pio GM, Piaseczny M, Chu JE, Xia Y and Allan AL. The role of bone-derived OPN in breast cancer metastasis. Oral Presentation, Anatomy and Cell Biology Research Day. October 2014 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications of bone derived factors in breast cancer migration. Poster presentation, London Health Research Day, London, ON. March 2014 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications of bone derived factors in breast cancer migration. Oral presentation, London Regional Cancer Program Seminar Series. March 2014 • Pio GM, Chu JE, Xia Y and Allan AL. Identification and functional implications

of bone derived factors in breast cancer migration. Poster presentation, Anatomy and Cell Biology Day. October 2013

PROFESSIONAL EXPERIENCE

Teaching Assistant, London, ON Oral Histology, September 2014-April 2015 Western University • Course topic: histology of the oral mucosa and tissue Teaching Assistant, Medical Sciences 4900 F/G London, ON Western University September 2013-December 2014 • Course topic: laboratory techniques with focus on animal models of human disease, real time PCR, biochemical assays, histology and medical imaging

Teaching Assistant, London, ON Translational Models of Cancer 4461b, January 2014-April 2014 Western University • Course topic: histology, prognosis, biomarkers, imaging and sites of metastasis of main cancer types including historical perspectives of how findings in the basic sciences have led to better treatments for cancer