Regulation of C-Met Receptor Tyrosine Kinase Signaling by Angiotensin-(1-7) in Triple Negative Breast Cancer

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REGULATION OF C-MET RECEPTOR TYROSINE KINASE SIGNALING BY ANGIOTENSIN-(1-7) IN TRIPLE NEGATIVE BREAST CANCER

GUORUI DENG

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY

GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Cancer Biology

December, 2016

Winston Salem, North Carolina

Approved by:

E. Ann Tallant, Ph.D., Co-Advisor

Patricia E. Gallagher, Ph.D., Co-Advisor

Linda Metheny-Barlow, Ph.D., Chair

Lance D. Miller, Ph.D.

Ravi N. Singh, Ph.D.

This work is dedicated to the memory of my aunt, Zhen Tse, who was crucial in helping my family and I to settle in a new environment when we first arrived in the United States, and whose life was abruptly cut short by the same disease we are striving to cure.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my mentors, Dr. Ann Tallant and

Dr. Patricia Gallagher for their guidance and support, both professionally and personally over the past five years. They are a great mentoring duo - always challenging their students with thought-provoking questions and helped mold me into the scientist I am today. They are the mentors that I strive to become. I would also like to express my gratitude for my doctoral committee members, Dr.

Linda Metheny-Barlow, Dr. Lance Miller and Dr. Ravi Singh for their valuable advice and input in regards to my studies as well as the Cancer Biology department at the Wake Forest School of Medicine for providing an excellent and supportive training program for graduate students.

In addition, I would like to thank the faculty, trainees and staffs of the

Hypertension & Vascular Research Center, who have treated me as one of their own member even though I am not a student of the Physiology and

Pharmacology graduate program. I will truly miss the annual Chilli cook-off, partaking in the NCAA tournament brackets and certainly the celebratory gatherings with members of this wonderful center. To Tenille Shields, Mark

Landrum and Rob Lanning, I am grateful for your training in the various laboratory techniques. I would like to express my gratitude and appreciation for

Dr. Alison Arter, who provided friendly guidance, insight and being patient with me as I was starting my graduate career. To the current students of the lab,

Omeed Rahimi, Pooja Patel and Marianne Collard, I have enjoyed and appreciated your enthusiasm in the lab.

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I would like to thank my family and friends for their unconditional love and support. To my childhood friend Di, thank you for the wonderful friendship and being there to play video games with me when I needed to relax. To my parents who made the difficult decision to leave behind their families in China and immigrate to the United States so that my brother and I could have the opportunity to obtain the best education in the world, words cannot express my gratitude for all the sacrifices you two have made, for that I am forever grateful. I would like to express my gratitude to my brother JR for being a great role model for me as I was growing up. To my beautiful wife Nanmeng, thank you for helping to make the past six years filled with wonderful memories and I look forward to sharing many more precious moments with you in the future. As for our dog

Sammy, your adorableness and gentle manner have given us the comfort we needed at some of the most stressing times, also - please do not eat my dissertation – it is not food.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... III

LIST OF FIGURES AND TABLES ...... VI

LIST OF ABBREVIATIONS ...... VIII

ABSTRACT ...... X

CHAPTER I: INTRODUCTION ...... 1

CHAPTER II: ANGIOTENSIN-(1-7) UP-REGULATES PTP1B TO REDUCE C- MET SIGNALING AND ATTENUATE TRIPLE NEGATIVE BREAST CANCER GROWTH ...... 63

CHAPTER III: ANGIOTENSIN-(1-7) REDUCES TRIPLE NEGATIVE BREAST CANCER BRAIN METASTATIC GROWTH IN ASSOCIATION WITH DECREASED C-MET SIGNALING ...... 113

CHAPTER IV: GENERAL DISCUSSION ...... 148

CURRICULUM VITAE ...... 197

LIST OF FIGURES AND TABLES

CHAPTER I Page

Figure 1 c-Met Receptor Tyrosine Kinase (RTK) Signaling 46

Figure 2 Synthesis of the Renin-Angiotensin System (RAS) 48 peptides

Figure 3 Chemical Structure of angiotensin-(1-7) [Ang-(1-7)] 50

CHAPTER II

Figure 1 Ang-(1-7) administration reduced 4T1 orthotopic triple 103 negative breast tumor growth

Figure 2 Ang-(1-7) decreased c-Met RTK activation in vitro and in 104 vivo

Figure 3 Ang-(1-7) inhibited c-Met RTK mediated cell proliferation 106 and invasion

Figure 4 Ang-(1-7) up-regulates the phosphatase PTP1b, a 109 negative regulator of c-Met RTK signaling

Figure 5 PTP1b knockdown prevented the Ang-(1-7)-mediated 111 reduction in c-Met RTK signaling

Figure 6 A proposed model of c-Met RTK signaling regulation by 112 Ang-(1-7)

Supplementary c-Met RTK is enriched in triple negative breast cancer 113 Figure S1

Supplementary Mas receptor in triple negative breast cancer cell lines 114 Figure S2

Supplementary Low PTP1b and Mas1 expression correlate with reduced 115 Figure S3 relapse-free survival for breast cancer patients with basal-like subtype of TNBC

CHAPTER III

Figure 1 Treatment with Ang-(1-7) reduced BR5 4T1 metastatic 141 TNBC tumor number in the brains of BALB/c mice

Figure 2 Ang-(1-7) administration decreased the size of brain 142

metastatic TN tumors

Figure 3 Ang-(1-7) treatment reduced TNBC metastatic tumor 144 proliferation

Figure 4 Ang-(1-7) mitigated c-Met RTK pathway activation in 146 BR5 4T1 brain metastatic tumors

Figure 5 c-Met RTK and PTP1b expression correlated with the 150 metastatic status of breast cancer patients

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LIST OF ABBREVIATIONS

ACE Angiotensin converting enzyme ACE2 Angiotensin converting enzyme 2 AI Aromatase inhibitor Ang II Angiotensin II Ang-(1-7) Angiotensin-(1-7) AR Androgen receptor AT1R Angiotensin type I receptor AT2R Angiotensin type II receptor BL1 Basal-like 1 subtype BL2 Basal-like 2 subtype BRCA1/2 Breast cancer 1/2, early onset CAM Chick chorioallantoic membrane cAMP Cyclic adenosine monophosphate CCNB1 Cyclin B1 CNS Central nervous system CT Computerized tomography DSS Disease-free survival ECM Extracellular matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor ER Estrogen receptor ERK1/2 Extracellular signal-regulated kinase 1/2 GBM Glioblastoma multiforme GPCR G-protein coupled receptor HER2 Human epidermal growth factor receptor 2, also known as ERBB2 HGF/SF Hepatocyte growth factor/scatter factor HIF1 Hypoxia inducible factor 1 IM Immunomodulatory subtype IR Insulin receptor LAR Luminal androgen receptor subtype M Mesenchymal subtype MAPK Mitogen activated protein kinase MMP Matrix metalloproteinases MRI Magnetic resonance imaging MSL Mesenchymal-like subtype MYBL2 V-Myb Avian Myeloblastosis Viral Oncogene Homolog-Like 2 NADPH Nicotinamide adenine dinucleotide phosphate NO Nitric oxide NSF N-ethylmaleimide sensitive fusion proteins PARP Poly ADP ribose polymerase pCR Pathologic complete response

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PET Positron emission tomography PFS Progression-free survival PI3K Phosphoinositide 3-kinase PI3KCA Phosphoinositide 3-kinase catalytic subunit PKA Protein kinase A PlGF Placental growth factor PR Progesterone receptor PTEN Phosphatase and tensin homolog PTP1b Protein tyrosine phosphatase 1b PVDF Polyvinylidene difluoride ROS Reactive oxygen species RTK Receptor tyrosine kinase SCID Severe combined immune deficiency SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SERM Selective estrogen receptor modulators SHP-1 Src homology region 2 domain-containing phosphatase-1 SRS Stereotactic radiosurgery TGF-B Transforming growth factor-beta TN Triple negative TNBC Triple negative breast cancer TP53 Tumor protein 53 VEGF Vascular endothelial growth factor VSMC Vascular smooth muscle cell WBRT Whole brain radiation therapy

ABSTRACT

Breast cancer remains a devastating disease, negatively impacting the lives of many women worldwide. In the United States, 40,450 women succumbed to breast cancer in 2015, with an estimated 247,000 new cases being diagnosed in 2016. Approximately 15% of all breast cancer cases will be classified as triple negative breast cancer (TNBC), an aggressive form of breast cancer characterized by the lack of estrogen and progesterone receptors as well as normal expression of the human epidermal growth factor receptor 2 (HER2).

These cellular properties thereby exclude patients diagnosed with TNBC from the benefits of current targeted agents. The aggressive nature of TNBC coupled with the lack of effective targeted therapies contribute to a greater patient mortality rate when compared to other breast cancer subtypes, underscoring a crucial need for novel development of actionable targets for this patient population.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven amino acid peptide hormone of the renin-angiotensin system with pleotropic anti-tumor activities.

Herein, we report that Ang-(1-7) inhibited TNBC growth in vivo as well as in vitro by mitigating the signaling of the c-Met receptor tyrosine kinase, which is implicated in TNBC development. Mechanistically, the heptapeptide hormone up- regulates the phosphatase PTP1b to mitigate c-Met activation, as depletion of

PTP1b by siRNA abrogated the Ang-(1-7)-mediated suppression of c-Met signaling. The inhibitory effects exhibited by the heptapeptide hormone were facilitated by the G protein-coupled receptor Mas, since co-treatment with the

Mas receptor antagonist D-Ala [D-alanine7-Ang-(1-7)] prevented the Ang-(1-7)- facilitated reduction in TNBC growth.

Mortality from metastatic disease accounts for approximately 90% of deaths in patients diagnosed with solid tumors. Patients diagnosed with TNBC are more likely to experience brain metastatic disease compared to patients diagnosed with other breast cancer subtypes. Using brain tropic BR5 4T1 TNBC cells, we report that Ang-(1-7) significantly reduced tumor number as well as mitigated the growth of BR5 4T1 brain metastatic tumors, demonstrated by a reduction in average tumor size in mice treated with the heptapeptide hormone as well as a reduction in the number of cells which stained positively with an antibody to the proliferation marker, Ki67. Ang-(1-7) treatment produced a significant reduction in c-Met receptor tyrosine kinase activation with a concurrent increase in protein phosphatase PTP1b, suggesting that the heptapeptide hormone may target c-Met signaling to attenuate triple negative metastatic breast tumor growth in the brain. Collectively, these results provide support for the heptapeptide hormone as a novel targeted treatment option for TNBC patients for the management of primary and metastatic tumors.

CHAPTER I: Introduction

Breast Cancer

Breast cancer is a devastating disease in the United States, claiming approximately 40,000 lives each year, with 234,000 new estimated cases in

2015. It is the most commonly diagnosed malignancy in women and the second leading cause of cancer-related mortality in women, second only to lung cancer

[1]. Globally, an estimated 450,000 individuals will succumb to the disease annually. An increased incidence of breast cancer and higher death rates are observed in non-Hispanic white (Caucasian) and non-Hispanic black (African

American) women compared to women of other ethnicities [2]. Despite a lower overall incidence of breast cancer in African American women, the breast cancer mortality rate is 42% higher for these women compared to their Caucasian peers

[2]. This difference in mortality is likely influenced by a combination of variables including genetic, environmental and socioeconomic factors [1, 2].

Breast cancer is not a single disease but rather a complex, heterogeneous disease encompassing diverse clinical and pathological presentations with dissimilar molecular characteristics. The normal breast tissue is composed of glandular and stromal tissues; within the glandular tissue, the lobules and ducts produce and transport milk for secretion, respectively. Depending on the site of origin, breast tumors arising from the lobule cells or duct cells are classified as lobular carcinoma in situ (LCIS) or ductal carcinoma in situ (DCIS). Patients diagnosed with non-invasive disease, where the carcinoma cells are still confined to the ducts or lobules, can be treated with surgical lumpectomy or mastectomy, in which the tumor tissue or entire breast is removed [3]. In contrast, tumors in patients with metastatic cancer are highly invasive and aggressive, spreading to distant tissue sites in the body that confers a poorer prognosis for the patient.

The introduction of mammography screening contributed to the marked increased in the diagnoses of DCIS and LCIS as well as other forms of breast cancer internationally. Mortality from breast cancer has dramatically decreased over the past four decades [2, 4], as a result of the availability of early screening methods such as mammography and improved treatment for advanced disease.

The tumor, node, and metastasis (TNM) staging system is the most commonly used method for pathologists to classify the extent and severity of malignancy [5]. For physicians, the TNM staging system assists in providing prognostic predictions and formulating appropriate treatment regimens for the patient. The size of the primary tumor (T), whether the tumor has infiltrated into the lymph nodes (N), and the presence of metastatic tumors (M) are parameters considered in designating the TNM stages. Each parameter is designated with a score reflective of disease progression. Collectively, these parameters assist in assigning the tumor stage, which can range from stage 0, indicative of noninvasive disease, to stage IV, indicating that the tumor has metastasized to at least one distant tissue site in the body. Additionally, tumor staging can be influenced by imaging tests (such as x-rays, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans, and positron emission tomography

(PET) scans), biopsy reports, menopausal status of the patient, estrogen receptor (ER) or progesterone receptor (PR) expression, and expression of human epidermal growth factor receptor 2 (HER2).

Breast Cancer Immunohistochemical Subtypes

Breast tumors can be divided into four immunohistochemical subtypes with unique pathological features and therapeutic implications; classification is based on the expression of the ER, PR, and HER2: 1) ER+/PR+, HER2+; 2)

ER+/PR+, HER2-; 3) ER-/PR-, HER2+, and 4) ER-/PR-, HER2- [6]. The advent of targeted molecular therapies for individuals presenting with HER2+ and ER+ breast cancer has significantly improved the overall survival of these patients [7,

8]. The incorporation of trastuzumab, a humanized monoclonal antibody targeting

HER2 to inhibit downstream growth-promoting signaling pathways, as a standard therapy for HER2+ breast cancer patients, has provided significant improvement in life expectancy for breast cancer patients with HER2+ disease, who had a poorer prognosis and shorter overall survival prior to the introduction of trastuzumab [7, 9-11]. Similarly, for ER+ breast tumors which proliferate in response to the hormone estrogen, the advent of molecules targeting aromatase

(aromatase inhibitors, AI) and selective ER modulators (SERMs) such as tamoxifen have reduced 15-year risks of ER+ breast cancer recurrence and death [12].

While substantial progress was made over the past decades in targeted therapies for ER+ and HER2+ breast cancer, little therapeutic success was achieved for patients diagnosed with ER-/PR-, HER2- (triple negative, TN) breast tumors, which accounts for up to 20% of breast cancer diagnoses [13]. Triple negative breast cancer (TNBC) is associated with an aggressive clinical behavior, often presents at a more advanced stage at the time of diagnosis, and is accompanied by poorly differentiated histology, contributing to an overall dismal prognosis. TNBC disproportionately affects women of younger age and women of African American and Hispanic descent [13, 14]. There is a significant decrease in survival for the first 3 to 5 years after diagnosis with TNBC. Indeed, the five-year relative survival for TNBC patients is 77% compared to 93% survival for women bearing non-triple negative breast tumors [13]. Strikingly, non-

Hispanic black women diagnosed with late stage TNBC have the lowest 5-year survival at 14% compared to non-Hispanic black women diagnosed with other types of breast cancer at 49% [13].

The lack of expression of hormonal receptors and HER2 overexpression precludes TNBC patients from the benefits of endocrine therapies or trastuzumab. As a consequence, cytotoxic chemotherapy remains the mainstay of systemic treatment for TNBC. Platinum compounds such as cisplatin and carboplatin exert tumoricidal activity by promoting the formation of DNA-platinum adducts, leading to single- and double-strand DNA breaks and ultimately cell death [15]. For women bearing triple negative breast tumors with mutations in the

BRCA1 gene, which encodes a protein important in repairing damaged DNA, the capacity for their cancer cells to facilitate DNA repair is reduced. For women with mutations in the BRCA1 gene, the therapeutic efficacy of platinum compounds was evaluated [16]; while modest responses were achieved in these TNBC patients, the development of nephrotoxicity remains the principal dose-limiting side effects for platinum drugs such as cisplatin [16, 17].

Other commonly administered chemotherapy for TNBC patients include taxanes (such as paclitaxel) and anthracyclines (such as doxorubicin), which are mitotic inhibitors and DNA intercalating agents, respectively. These agents, like cisplatin, exert antitumor activity by inducing cell death in rapidly-proliferating cells [18, 19]. However, taxanes and anthracyclines are non-specific, indiscriminately targeting both tumor cells and normal cells, resulting in side- effects ranging from high grade nausea and vomiting to increased susceptibility to infection, fatigue, decreased appetite, hair loss to dose-limiting cardiotoxicity and nephrotoxicity, ultimately leading to poor quality of life for these patients [17,

20, 21]. It is important to note that, while systemic chemotherapies produce undesired and adverse side-effects, some triple negative breast tumors are particularly sensitive to treatment with taxanes and anthracyclines compared to other breast tumor subtypes [14, 22, 23]. For TNBC patients that responded to chemotherapy regimens, a higher pathologic complete response (pCR) rate was observed in comparison to non-TNBC patients [22]. However, the progression- free survival (PFS) or disease-free survival (DSS) for patients diagnosed with

TNBC is significantly shorter than patients with other breast cancer subtypes, suggesting that the initial response to chemotherapeutic agents does not necessarily correlate with overall survival.

Clinical trials evaluating targeted and effective therapeutic agents for

TNBC patients are limited in number. Recent trials were initiated to examine the inhibition of poly ADP ribose polymerase (PARP), an enzyme critical for repair of

DNA single-strand breaks, in TNBC patients harboring BRCA1 mutations [24].

Preclinical studies suggested that inhibition of PARP promoted highly selective toxicity in BRCA1- and BRCA2-defective tumor cells, leading to chromosomal instability and subsequent apoptosis [25]. However, conflicting results were reported in two separate clinical trials evaluating the PARP inhibitor olaparib as a single agent in BRCA1-deficient TNBC patients. Tutt and colleagues evaluated the clinical efficacy of olaparib as a single agent therapy in advanced breast cancer harboring germline BRCA1/2 mutations. The authors reported a 41% objective response rate in these patients; however, treatment-related adverse events were seen in 81% of patients, with 24% of patients experiencing grade 3 or 4 serious adverse events [26]. In contrast, Gelmon and colleagues observed a lack of confirmed objective response in a cohort of TNBC patients, irrespective of

BRCA-mutation status [27]. The results of these two studies highlight the continuing challenges in the effort to identify a druggable target for the treatment of TNBC.

Intrinsic Molecular Subtypes of Breast Cancer

The immunohistochemical profiling of breast tumors based on the expression of the ER, PR, and HER2 stratified breast cancer into basic therapeutic subtypes, whereby targeted endocrine therapies and anti-HER2 agents can offer survival benefits for patients bearing ER+ and HER2+ breast tumors. Sequencing by The Cancer Genome Atlas (TCGA) consortium further classified breast cancer into five intrinsic molecular subtypes [28]. Insights provided by integrating genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse-phase protein arrays of primary breast tumors demonstrated the existence of five breast cancer subtypes with significant molecular heterogeneity: luminal-A, luminal-B,

HER2-enriched, normal breast-like, and basal-like.

Luminal breast tumors account for approximately 70% of all breast cancers and feature high mRNA and protein expression of a luminal gene signature, suggesting that these tumor cells were derived from the luminal epithelium of the breast. Additionally, similarities shared between luminal A and luminal B breast tumors include ER-positivity, expression of luminal cytokeratins

8/18 and genes associated with ER activation and frequent mutations in the genes encoding the phosphatidylinositol-3 kinase (PI3K) p110 catalytic subunit,

PI3KCA and genes encoding components of the mitogen-activated protein kinase (MAPK) pathways, MAP3K1 and MAP2K4, contributing to deregulated

7 tumor cell proliferation [28, 29]. Despite shared features, luminal A and luminal B tumors also exhibit dissimilar characteristics. Relative to luminal A breast tumors, luminal B tumors tend to have increased HER2 expression and are enriched in proliferative genes such as CCNB1, MKI67 and MYBL2, correlating with higher grade tumors and significantly worse clinical outcome [30]. Since luminal breast tumors depend on estrogen to support cancer cell growth, hormonal therapies such as tamoxifen and aromatase inhibitors are successfully implemented as routine clinical practice for patients bearing luminal/ER+ breast tumors, resulting in effective management of disease and favoring a better clinical prognosis [12].

HER2-enriched breast tumors represent approximately 20% of diagnosed breast cancers and are characterized by the amplification of the ErbB2 gene, encoding the HER2 oncoprotein. HER2 (human epidermal receptor 2) is a member of the epidermal growth factor (EGF) family of receptor tyrosine kinases

(RTKs). Extensive studies demonstrated that HER2 forms heterodimers with other RTKs of the EGF family to enhance and prolong signal transduction by growth factors to promote cell proliferation and survival [31-33]. HER2 overexpression is an adverse prognostic factor, associated with poorly differentiated tumors of higher grade; these tumors also tend to have a higher proliferation rate and can be resistant to certain types of chemotherapies [7, 10].

The discovery that HER2 overexpression is a marker of aggressive disease and a druggable target led to the development and introduction of trastuzumab into clinical use, improving the clinical outcome for women with HER2+ breast tumors

[34, 35]. Indeed, prior to the advent of HER2 targeted therapies, a diagnosis of

HER2+ breast cancer often conferred a poor prognosis and significantly shorter overall survival [10].

The normal-breast-like subtype of breast cancer was initially identified from hierarchical clustering of gene expression from normal and malignant human breast tissues derived from 42 individuals [29]. These tumors displayed gene expression patterns resembling normal breast tissue samples, characterized by high expression of genes associated with basal epithelial cells and adipose cells.

Basal-like breast tumors account for up to 15% of all breast cancer cases, affect younger women, are more prevalent in African American women and are significantly more aggressive than breast tumors of other molecular subtypes

[36]. The aggressive nature of basal-like breast tumors can be characterized by increased tumor size, higher tumor grade at the time of diagnosis and higher mitotic index; in addition, these patients are more likely to develop distant recurrence and have an increased risk of death due to breast cancer compared to non-basal-like breast tumors. Immunohistochemical assays and gene expression analysis demonstrated that the majority of basal-like breast tumors lack ER, PR, and do not overexpress HER2, but instead express cytokeratins 5,

6, and/or 17 and epidermal growth factor receptor (EGFR) [37]. Over half of basal-like tumors harbor mutations in the tumor suppressor gene TP53 and many

9 basal-like tumors exhibit a defunct BRCA-mediated DNA break repair pathway in part due to hypermethylation or acquisition of germline mutation of the BRCA1 gene [38]. The term triple negative breast cancer and basal-like breast cancer are used interchangeably in the literature, since the immunohistochemically defined subtype (triple negative immunotype) and the molecularly-defined basal- like subtype shared many characteristics including the lack of expression for

ER/PR and overexpression of HER2; both triple negative and basal-like breast tumors tend to be more aggressive, presenting with larger tumor sizes and higher grades at the time of diagnosis relative to breast cancer of other types and a prevalence for affecting younger, premenopausal women, and women of African

American and Hispanic ancestry. It is important to note, however, that triple- negative and basal-like breast cancers are not synonymous. Gene expression profiling suggests that approximately 71% of triple negative breast cancers cluster with the basal-like subgroup whereas only 77% of basal-like breast tumors are triple negative [28, 39].

Triple Negative Breast Cancer Molecular Subtypes

The extent of heterogeneity exhibited by TNBC tumors was shown in recent gene expression studies. Analysis of 21 gene expression data sets containing over 560 triple negative breast cancer cases demonstrated that TNBC encompasses six distinct molecular subtypes with unique biology: basal-like 1

(BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M),

10 mesenchymal stem-like (MSL), and luminal androgen receptor (LAR) subtypes

[40]. The identification of these TNBC molecular subtypes with distinctive gene expression profiles presents new opportunities to tailor treatment to more precise molecular subtypes.

Tumors belonging to the basal-like 1 (BL1) subtype exhibit a molecular signature dominated by genes associated with high proliferation (e.g. MKI67), and elevated expression of DNA damage markers (e.g. CHEK1, RAD51). Basal- like 2 (BL2) subtype tumors display genes enriched in growth factor signaling

(e.g. EGF and MET pathways) [40]. In addition, patients with the BL1 and BL2 molecular subtypes express elevated levels of TP63 and MME genes, suggestive of the basal/myoepithelial origin of these tumors. Due to the highly proliferative nature of these tumors, antimitotic agents such as taxanes were evaluated as a treatment option; analysis of TNBC patients treated with paclitaxel, an antimitotic agent, showed that tumors correlated to the basal-like subtypes achieved a significantly higher pCR when compared to tumors of mesenchymal-like or LAR subtypes [40, 41].

TNBC tumors belonging to the immunomodulatory (IM) subtype are enriched in genes involved in immune cell processes including immune cell signaling, cytokine signaling, antigen processing and presentation, and core immune signal transduction pathways (e.g. NFκβ, TNF, JAK/STAT signaling).

The genes represented within the IM subtype overlap with a gene signature

11 derived from a rare, histologically distinct form of TNBC known as medullary breast cancer (MBC). In contrast to its high-grade histology, MBC is associated with a favorable prognosis [42].

The gene expression signatures displayed by mesenchymal (M) and mesenchymal stem-like (MSL) tumor subtypes are involved in cell motility, extracellular matrix (ECM)-receptor interaction and cell differentiation pathways

(e.g. TGF-β signaling). The differentially-expressed genes in the M and MSL subtypes are similar to metaplastic breast cancer, a highly dedifferentiated and chemoresistant type of breast cancer dominated by mesenchymal/sarcomatoid or squamous characteristics [43]. In contrast to tumors of the M subtype, MSL tumors express low levels of proliferation genes accompanied by enrichment of genes associated with stem cells and mesenchymal stem cell specific markers

(e.g. BMP2, PDGFRB, VCAM1). Additionally, MSL tumors display low expression of the tight junction proteins claudin 3, 4, and 7, similar to the recently identified claudin-low subtype of triple negative breast cancer [44].

Despite being ER-, a subset of TNBC tumors are enriched in hormonally- regulated pathways (e.g. steroid synthesis, androgen/estrogen metabolism) [40].

Efforts by Lehmann and colleagues demonstrated that these tumors express higher levels of the AR gene and androgen receptor (AR) protein, along with increased expression of AR downstream targets and coactivators (e.g. FASN) than other TNBC subtypes [40]. Interestingly, the gene expression centroids of

12 the LAR subtype strongly correlated with a molecular subtype of breast cancer known as molecular apocrine breast tumors first described by Farmer and colleagues [45]. Breast tumors within the molecular apocrine subtype are characterized as ER-/AR+, further underscoring the validity of the LAR subtype gene expression signature. The identification of the luminal androgen receptor

(LAR) TNBC subtype provided a unique opportunity to develop and facilitate the use of anti-androgens as a targeted therapy for TNBC patients bearing tumors with the gene expression signature identified with LAR tumors. Several clinical trials are ongoing to examine the efficacy of anti-androgens or AR modulators in

AR+ TNBC patients (NCT02368691, NCT02353988, NCT02580448).

The positive clinical impact achieved by the identification of actionable molecular targets and the subsequent development of targeted therapies as exemplified by trastuzumab and tamoxifen for treatment of HER2+ and ER+ breast cancer, respectively, underscore the pivotal need to better characterize molecular drivers of tumor aggressiveness for TNBC patients.

c-Met Signaling in normal physiology and emerging roles in TNBC development and progression

The receptor tyrosine kinase (RTK) c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF, will hence be referred to as HGF) control a complex biological program known as invasive growth [46]. This c-Met-driven

13 process coordinates cell proliferation with cell invasion and is a physiological program that is activated during embryonic development and post-natal tissue growth and regeneration.

The c-Met RTK is synthesized as a single-chain precursor polypeptide which undergoes intracellular proteolytic cleavage to generate a disulfide-linked heterodimer [47]. The mature c-Met RTK is composed of an extracellular α subunit tethered to a β subunit by disulfide bonds. The intracellular region of the

β subunit contains the catalytic kinase domain with two tyrosine residues

(Tyr1234/1235) that are critical for receptor kinase activation [48]. Two additional tyrosine residues (Tyr1349/1356) within the C-terminal region of the β subunit form the multifunctional docking site for c-Met and are crucial for recruitment of downstream signaling mediators to facilitate intracellular signaling [49].

HGF is a large multidomain protein that shares homology with plasminogen [50]. Binding of the ligand to c-Met RTK induces dimerization and autophosphorylation of the tyrosine residues at position 1234 and 1235

(Tyr1234/1235) in the catalytic domain. Consequent phosphorylation of tyrosine

1349 and 1356 (Tyr1349/1356) in the carboxy-terminal domain generates a multifunctional docking site capable of interacting with signaling effectors and adaptor proteins. c-Met signaling activates several downstream signaling cascades shared with other growth factor receptors including signaling extracellular-signal regulated kinase (ERK) 1/2 and PI3 kinase-Akt pathways

(Figure 1). In particular, ERK1/2 and PI-3 kinases are activated by c-Met with greater intensity and duration as compared with other growth factor receptors and this hyperactivation is critical to the pro-invasive activity exerted by c-Met

[51, 52].

Activation of ERK1/2 by c-Met mediates cell proliferation and motility and facilitates cell cycle progression, whereas PI-3K/Akt signaling induces cell survival and invasion [53-55]. Gab1 is a multi-adapter protein and is a crucial substrate for c-Met RTK, as ablation of the Gab1 gene in mice results in phenotypes that resemble those of mice with deletions of c-Met and HGF/SF genes [56]. Direct interaction between Gab1 and activated c-Met is facilitated through a unique 13 amino-acid c-Met binding domain (MBD). Gab1/c-Met association allows the docking protein to be phosphorylated by c-Met RTK to generate additional binding sites for signal-relay molecules to activate downstream pathways such as PI-3K/Akt and ERK1/2 signaling. While Gab1 is phosphorylated in response to other growth factors, such as epidermal growth factor (EGF) and nerve growth factor (NGF), HGF-mediated Gab1 phosphorylation is sustained up to four times longer than EGF-induced phosphorylation [57]. The ability of activated c-Met to sustain activation of signal transduction pathways allows it to regulate differential signaling response and induce diverse downstream biological processes [58].

The HGF/c-Met signaling pathway is crucial for embryonic development and organ regeneration. Activation of this signaling cascade mediates proliferation and motility of migrating muscle progenitors by inducing epithelial-to- mesenchymal transition (EMT), where these myogenic precursors subsequently differentiate into hypaxial muscles of the limbs, tongue and diaphragm [59]. The important role that c-Met signaling plays in facilitating the formation of the hypaxial muscles was demonstrated when ablation of the HGF or c-Met gene resulted in the complete absence of hypaxial muscles without affecting the other muscle groups [60]. The proper wiring of the nervous system during early development is also dependent on c-Met signaling, as disruption of this signaling pathway resulted in reduced sensory and sympathetic neuron survival as well as compromised outgrowth and axon bundling of certain motor nerves [61-63].

Intact HGF/c-Met signaling is required for proper organ regeneration after organ damage. In response to liver damage, HGF production is significantly elevated, resulting in increased c-Met RTK activation in hepatocytes to facilitate cell proliferation and mount an anti-apoptotic response for organ repair [50, 64].

Conversely, conditional deletion of c-Met in the liver failed to facilitate organ reconstitution following toxic insult and hepatectomy [65, 66]. The loss of p-

ERK1/2 and the delay in maximal Akt activation following hepatic injury in these c-Met null mice contributed to a proliferation defect, underscoring the important role downstream ERK1/2 signaling plays in mediating the biological responses of c-Met RTK.

Additional studies also showed a protective role for c-Met RTK in the kidney. Stimulation of renal epithelial cells by HGF strongly induces cell proliferation and mediates an anti-apoptotic response by activating downstream

MAPK and PI3K-Akt signaling pathways [67]. The potent renal-protective effects exhibited by HGF prevent acute renal failure in response to tubular necrosis and quicken kidney regeneration. The HGF/c-Met signaling pathway is also activated in epithelial cells during wound healing to facilitate migration and subsequent proliferation of keratinocytes to form the so-called hyperproliferative epithelium and repopulate the wounded area. As such, keratinocytes derived from mice bearing conditional c-Met deletion in the epidermis failed to establish the hyperproliferative epithelium, preventing the wound healing process [68].

The capacity for c-Met RTK to sustain self-renewal and influence binary cell fate decisions, which is are feature of stem cells, highlights the role of HGF/c-

Met signaling in stem cell biology [46]. A recent study showed that c-Met is specifically expressed in the luminal progenitors of the mouse mammary epithelium and that HGF expression maintains these cells in stem/progenitor states to prevent differentiation towards the mature luminal phenotype [69].

Expression of c-Met RTK was also reported in neural stem cells where functional c-Met signaling is essential for self-renewal [70].

The aforementioned studies demonstrated the critical biological roles that are regulated by the HGF/c-Met signaling pathway. However, deregulation of many of these c-Met RTK mediated cellular processes may contribute to cancer formation and progression. c-Met RTK was first identified as an oncogene in the

1980s as a mutant fusion protein TRP-MET (translocated promoter region fused to c-Met) from a human osteosarcoma cell line treated with the carcinogen N- methyl-N-nitro-N-nitrosoguanidine [71]. TRP-MET exhibits constitutive dimerization and therefore persistent activation of c-Met signaling. When expressed in transgenic mice, TRP-MET promoted the development of multiple epithelial-derived tumors [72]. This fusion protein was discovered in human specimens from both the precancerous lesions of gastric cancers and the adjacent normal mucosa, suggesting that this genetic lesion can predispose to gastric carcinoma development [73]. Aberrant c-Met RTK activation can be attributed to 1) an activating point mutation in the c-Met gene, 2) c-Met gene amplification, 3) overexpression of its ligand HGF, or 4) transcriptional upregulation of c-Met signaling independent of gene amplification [74].

Further evidence implicating c-Met RTK as a potent oncogene was provided by the discovery of activating mutations within the receptor kinase domain initially in both sporadic and inherited forms of human renal papillary carcinomas and subsequently in a small number of other human malignancies

[75]. Elevated c-Met protein as a result of transcriptional upregulation in the absence of gene amplification is the common cause of constitutive c-Met

18 activation and was frequently observed in human tumors, including malignancies of the thyroid, colorectal, ovarian, pancreatic, lung and breast [76-82]. HGF, the ligand for c-Met, was reported to be frequently overexpressed in the reactive stroma of primary tumors, thereby providing a mechanism to support paracrine positive feedback loops to facilitate cancer cell dissemination to distant locals

[83]. Overexpression of c-Met and increased serum HGF, which was found in 14-

54% of breast cancer patients, correlated with short relapse-free and overall survival for breast cancer patients [84-91]. Thus, it is not surprising that deregulation of c-Met signaling is associated with cancer progression and metastasis [46, 92].

Recent studies demonstrated that deregulation of c-Met signaling is associated with TNBC progression and disease dissemination. Elevated c-Met expression is detected in human triple negative breast tumors of the basal-like and claudin-low subtypes [40, 44, 69, 93, 94]. In TNBC patients, increased c-Met expression alone is an adverse prognostic factor, conferring a reduced recurrence-free and overall survival compared to tumors of TNBC patients expressing low levels of c-Met [90].

An analysis of over 900 breast cancer specimens demonstrated a strong association between elevated c-Met and expression of basal-like markers such as CK5/6 [91]. Furthermore, Ho-Yen and colleagues found that c-Met was independently associated with basal-like features [94]. A critical role for c-Met in

19 promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland was reported by Gastaldi and colleagues [69].

In this same study, the authors demonstrated that c-Met signaling restricts terminal differentiation of luminal progenitors and favors commitment toward a more basal-like phenotype, further implicating c-Met RTK as an important mediator of TNBC development. The observations that mice harboring a mutated, constitutively-active c-Met RTK in the mammary gland promotes the formation of mammary tumors with histology, gene expression and immunohistochemical profiles similar to human basal breast cancer further validated the contribution of c-Met signaling pathway to TNBC pathology [95].

Furthermore, results of this study demonstrated that elevated c-Met and SNAIL, an EMT marker, are highly predictive of poor prognosis in lymph-node negative breast cancer patients.

The tp53 gene encodes the tumor suppressor p53 and is one of the most commonly mutated and inactivated genes in human breast cancer [96]. Knight and colleagues reported that c-Met induces mammary tumors with pathological and molecular features of the claudin-low subtype of TNBC in the presence of tp53 loss [97]. These tumors displayed spindle-like morphology and a distinct molecular signature, resembling the human claudin-low subtype of TNBC. This molecular signature encompasses diminished expression of claudins, changes in genes associated with an epithelial-to-mesenchymal transition and decreased expression of the microRNA-200 family. In these tumors, functional c-Met RTK

20 activity is required for maintenance of the claudin-low tumor phenotype as c-Met inhibition rescued claudin 1 expression and abrogated growth and dissemination of these cells in vivo.

Collectively, insights from the clinical data and experimental results suggest potential roles whereby aberrant c-Met signaling may contribute to

TNBC disease progression and provide the rationale to target c-Met RTK signaling as a therapeutic treatment for the basal-like and claudin-low subtypes of TNBC.

PTP1b is a negative regulator of c-Met RTK signaling

Regulation of c-Met RTK signaling is mediated by HGF binding and protein autophosphorylation. However, c-Met signaling can also be regulated by cellular phosphatases which dephosphorylate and thus oppose the actions of protein kinases by removing the activating phosphate groups. Dephosphorylation and subsequent inactivation of c-Met RTK at tyrosine residues 1234/1235 within the kinase domain is facilitated by the protein tyrosine phosphatase 1b (PTP1b)

[98]. PTP1b targets multiple RTKs, including c-Met, the insulin receptor (IR), insulin growth factor-1 receptor (IGF1-R), epidermal growth factor receptor

(EGFR) and platelet-derived growth factor receptor (PDGFR), by dephosphorylation in response to ligand-induced receptor activation [98-103].

PTP1b is a widely-expressed non-receptor phosphatase localized to the cytoplasmic face of the endoplasmic reticulum [104]. It is the founding member of the family of protein tyrosine phosphatases (PTPs) which play critical roles in modulating numerous cellular processes [105]. PTP1b expression is regulated in response to metabolic stresses and cellular transformation. For instance, PTP1b mRNA expression is decreased in esophageal cancer lesions whereas increased expression of the phosphatase was reported in the liver of a hamster model of insulin resistance [106, 107].

The phosphatase activity of PTP1b can also be modulated by multiple mechanisms, including oxidation and reduction reactions involving the critical cysteine residue within its catalytic domain and phosphorylation of critical residues which would either enhance or abrogate phosphatase activity.

Stimulation by insulin, for example, promotes intracellular generation of hydrogen peroxide (H2O2) which results in rapid and transient oxidation and inhibition of

PTP1b phosphatase activity [108]. In skeletal muscle and adipose tissue, insulin stimulation promotes PTP1b tyrosine phosphorylation to decrease the activity of the phosphatase [109]. Another mechanism by which insulin stimulation negatively regulates PTP1b activity is through serine phosphorylation by Akt, thereby creating a positive feedback loop for insulin signaling [110]. Additional serine residues on PTP1b can be phosphorylated by kinases such as protein kinase A (PKA) and PKC; however, the effect of these phosphorylation events on

PTP1b activity remains to be elucidated [111].

As previously noted, PTP1b displays broad substrate specificity and is a negative regulator of several kinases that influence breast tumor progression and aggressiveness including c-Met RTK, which is frequently aberrantly activated in

TNBC and is a poor prognostic marker for these patients [90]. By dephosphorylating and inactivating oncogenic kinases, PTP1b is purported to be a tumor suppressor. Indeed, mutant mice lacking both Tp53 (which encodes the tumor suppressor P53) and PTP1b displayed increased B-cell lymphomas compared to mice lacking only Tp53, demonstrating a tumor-suppressive ability of PTP1b [112]. Consistent with a role of a tumor suppressor, decreased PTP1b expression was observed in esophageal cancer, hepatocellular carcinoma, and ovarian cancer [113, 114]. A recent study by Beales and colleagues reported that an increase in PTP1b protein and activity by the adipokine adiponectin inhibited leptin-induced oncogenic signaling in esophageal cancer cells, further supporting

PTP1b as a tumor suppressor [115].

Sangwan and colleagues reported that PTP1b interacts and forms a complex with c-Met RTK upon HGF stimulation and this interaction requires phosphorylation of tyrosine 1234 and 1235 in kinase domain of c-Met [98]. The complex formation between PTP1b and activated c-Met RTK allows the phosphatase to dephosphorylate tyrosine 1234 and 1235 to decrease c-Met kinase activity. Loss of PTP1b enhances c-Met RTK phosphorylation, corresponding to increased c-Met-mediated biological activity and cellular invasion. The negative regulation of c-Met RTK by PTP1b was also reported in

23 mouse models of glioblastoma multiforme (GBM), a highly aggressive brain tumor [99]. In response to ligand-dependent activation, RTKs are removed from the plasma membrane by internalization through the endocytic pathway, resulting in either recycling of the receptor to the cell surface or targeted for lysosomal degradation [116]. PTP1b also modulates c-Met RTK internalization and attenuates signaling mediated by c-Met [117]. Dephosphorylation of the ATPase

N-ethylmaleimide sensitive fusion proteins (NSF) by PTP1b promotes its interaction with endosomal components to facilitate RTK internalization. Thus, in the absence of PTP1b catalytic activity, c-Met RTK internalization is abrogated, resulting in prolonged activation of downstream signaling mediators. Taken together, these studies identified two distinct mechanisms by which PTP1b negatively regulates c-Met RTK signaling. Lastly, the phosphatase was identified as an independent positive prognostic factor in a cohort of over 1,400 breast cancer patients, suggesting that PTP1b exerts a protective, anti-tumor role in breast cancer patients in part by negatively regulating c-Met RTK signaling [118].

Importantly, Garcia-Espinosa and colleagues reported an increase in PTP1b mRNA in the medulla of Ang-(1-7) treated rats, underscoring a potential regulatory role of the heptapeptide hormone in c-Met signaling [119].

Metastasis

Metastasis occurs when cancer cells disseminate from their original or primary location to secondary sites within the body. Cancer metastasis accounts

24 for approximately 90% of all deaths from solid tumors [120]. The metastatic cascade is a multi-step process which each disseminated cancer cell must complete in order to successfully establish metastatic lesions [121]. The first step of the metastatic process involves the invasion of the local stroma or surrounding normal tissue parenchyma by the cancer cell. Successful execution of local stroma invasion requires breaching of basement membrane, which is composed of extracellular matrix (ECM) proteins and functions as a physical barrier separating the epithelial and stromal compartments. Subsequent to local invasion, metastatic cancer cells proceed to intravasate into nearby lymph or blood vessels, allowing the escaped cancer cells to disperse through the body.

These cancer cells must survive while in the circulation by overcoming both stresses associated with matrix detachment (by overcoming a form of apoptosis called anoikis) and physical damage which occurs due to hemodynamic shear forces. Extravasation occurs when surviving cancer cells in the circulation are arrested at a distant location and gain the ability to cross from the vessel lumen into the tissue parenchyma. Formation of micrometastasis arises when the extravasated cancer cells begin to proliferate in the new tissue site. A limitation facing the newly extravasated tumor cells is the need for adequate nutrients to facilitate tumor growth. To circumvent the nutrient deprivation, new blood vessel formation or neoangiogenesis is induced by angiogenic and growth factors secreted by the tumor cells. Tumor-associated neoangiogenesis allows for the delivery of the nutrients and oxygen required by the actively proliferating tumor cells to perpetuate continued tumor growth and survival [122].

Non-triple negative breast cancers preferentially metastasize to the bone, liver and lungs but rarely to the brain. In contrast, patients with triple negative breast tumors are more likely to develop metastasis to the lungs and the brain

[36, 123, 124]. Thus, in addition to a different clinical presentation, triple negative breast tumors also display a distinct metastatic pattern. TNBC also employ a hematogenous route for metastasis rather than a route through the lymphatic system, which may account for the characteristically aggressive and highly metastatic nature of these tumors. Few treatment options are effective in the treatment of metastatic lesions, with the combination of chemotherapy, local surgery and radiation therapy as the standard of care treatment for metastatic breast cancer patients [125].

Patients bearing triple negative breast tumors experience a shorter time to relapse and metastasis when compared to patients bearing non-triple negative breast tumors. The survival outcome for metastatic TNBC is routinely 9 to 14 months after diagnosis of disease, relative to an average survival of 22 months for patients diagnosed with metastatic breast cancer from a non-TNBC subtype

[36, 126]. Furthermore, the risk of metastasis for TNBC patients is highest within the first 5 years of diagnosis, peaking between 2.5 to 4.2 years [36, 127]. In contrast, the greatest chance of developing metastasis for patients with alternative subtypes of breast cancer occurs between 5 to 6 years following initial diagnosis.

The presence of the blood-brain barrier (BBB) precludes the entry of most chemotherapy agents into the central nervous system (CNS), rendering the brain as a sanctuary site for metastatic tumor cells. The poor penetrance of chemotherapeutics largely limits the treatments for brain metastasis to whole brain radiation therapy (WBRT), stereotactic radiosurgery (SRS) and surgical resection [128]. The limited efficacy of these traditional treatments and an increased risk of developing neurocognitive dysfunction greatly reduce the quality of life for patients with metastatic brain cancer. Neurocognitive deficits remain the most undesired side effect of WBRT, whereas surgical resection has limited application for patients diagnosed with mutli-focal brain tumors. SRS delivers a single fraction, high dose radiation focally to the tumor but may put a significant patient population at risk for the development of neurological complications caused by radionecrosis [129]. Currently, there are no targeted therapies for the management of patients with breast cancer metastasis in the brain, justifying the need for druggable targets in metastatic brain tumors and the development of targeted therapies with efficacious effects in breast tumors in intracranial and extracranial sites.

In addition to contributing to primary tumor formation and growth, the

HGF/c-Met signaling cascade was also reported to facilitate cellular processes crucial to cancer cell metastasis, including induction of a pro-migratory and pro- invasive phenotype in breast cancer cells in response to HGF stimulation.

Administration of HGF significantly increased cell migration and invasion of the

27 human MDA-MB-231 TNBC cells in a wound-healing and transwell invasion assay in vitro [130]. Tumors cells exposed to HGF can undergo EMT, which confers cancer cells with enhanced invasive capability and allows these cells to overcome anoikis [131, 132]. Activation of c-Met RTK stimulates the expression and proteolytic activity of matrix metalloproteinase (MMP) 2 and MMP 9 to assist in degradation of the basement membrane, to facilitate the intravastation and extravasation steps of the metastatic process [133-135]. Furthermore, c-Met inhibition resulted in a decreased metastatic burden in mutant mice bearing claudin-low TNBC cells expressing a constitutively active c-Met RTK [97]. Lastly, c-Met RTK was reported to be overexpressed in metastatic tumors compared to primary tumors [136, 137]. Increased c-Met transcription is induced in metastatic tumor cells in the brain in response to ionizing radiation, a common treatment modality for patients presenting with metastatic brain disease [138, 139]. In turn, elevated c-Met RTK signaling in tumor cells promotes radioresistance through activation of cell survival pathways.

Glial cells such as astrocytes and microglia respond to the development of brain metastasis by adopting an activated phenotype whereby proliferative, angiogenic and inflammatory factors are secreted into the microenvironment. In response to CNS injury, HGF and c-Met expression are both up-regulated in activated GFAP+ astrocytes [140]. Expression of HGF and c-Met were also reported in microglial cells of the human CNS [141, 142]. The expression of the ligand-receptor pair in astrocytes and microglia highlights the impact and

28 potential roles for c-Met RTK signaling in facilitating brain metastatic tumor growth. HGF secretion by the glial cells can act in paracrine fashion on metastatic breast tumor cells to elicit metastatic growth and colonization of the brain [143]. Thus, the HGF/c-Met signaling cascade may represent a novel therapeutic target for treatment of metastatic TNBC tumors to the brain.

Renin-Angiotensin System (RAS)

The renin-angiotensin system (RAS) plays a critical role in modulating vascular tone, electrolyte homeostasis, and blood pressure, establishing the RAS as an important regulator of cardiovascular function and renal physiology [144].

Classically, the RAS was characterized as a peptide-based system with endocrine properties. However, this view of the RAS has changed in the past decade as “local” or “tissue” RASs were identified in cells from several organs and tissues, including the heart, kidney, the vasculature, testis, the ovaries, the brain and the breast [145-152].

The biological effects exerted by the RAS are mediated by the generation of angiotensin peptides through proteolytic cleavage of angiotensinogen and elicited by binding of angiotensin peptides to their respective receptors (Figure

2). Renin, a protease secreted by the kidney, participates in the rate limiting step by cleaving the liver-derived protein angiotensinogen to form angiotensin I (Ang

I). Subsequent cleavage of angiotensin I by angiotensin converting enzyme

(ACE) generates the octapeptide angiotensin II (Ang II), a potent vasoconstrictor and a peptide with growth-promoting properties [144]. Alternative pathways of angiotensin I catabolism were also reported; Ang I can be processed by neprilysin, thimet oligopeptidase or prolylendopeptidase to form the heptapeptide angiotensin-(1-7) [Ang-(1-7)] (Figure 3), a biologically active member of RAS that exerts opposing actions to those of Ang II, by exhibiting vasodilatory and growth inhibitory properties [153]. Additionally, the formation of Ang-(1-7) can be achieved through the actions of angiotensin converting enzyme 2 (ACE2) by cleaving Ang II [154].

Ang II exerts its biological profile by binding to the G-protein coupled receptors (GPCR) angiotensin type 1 and angiotensin type 2 (AT1R and AT2R, respectively) [155, 156]. Extensive studies showed that Ang II primarily binds to

AT1R and this binding of Ang II to the AT1R induces downstream signaling cascades to mediate cell proliferation, fibrosis, and inflammation [157-159]. In contrast, AT2R activation by Ang II was reported to inhibit cell proliferation and promote cell differentiation, indicating that Ang II activation of AT2R may serve to counterbalance the pro-proliferative activity of Ang II/AT1R axis [160, 161].

Angiotensin-(1-7) [Ang-(1-7)]

Angiotensin-(1-7) [Ang-(1-7)] is a biologically active heptapeptide hormone of the RAS. The demonstration that Ang-(1-7) is an endogenous ligand for the

30 orphan GPCR Mas further expanded the complexity of the RAS [162]. In addition to opposing the actions of Ang II by promoting vasodilatory effects, Ang-(1-7) also exhibits anti-proliferative, anti-fibrotic, and anti-inflammatory effects [163-

169]. This heptapeptide hormone functions to maintain homeostasis in various organs. In a rat model of an ischemic heart, Ang-(1-7) inhibited ischemia-induced cardiac arrhythmias and improved cardiac function [170, 171]. Furthermore, oral

Ang-(1-7) administration attenuated isoproterenol-induced impairment of heart function and cardiac remodeling in infarcted rats, highlighting the cardioprotective effects of the heptapeptide hormone [172].

The molecular mechanisms by which Ang-(1-7) counterbalances the vasoconstrictive effects of Ang II include stimulating nitric oxide (NO) release from the endothelium as well as inducing bradykinin and prostaglandin production to facilitate vasorelaxation, by binding to and activating the Mas receptor [162, 173-175]. The anti-proliferative effect of Ang-(1-7) was initially reported by Freeman and colleagues, by demonstrating that the heptapeptide hormone reduces growth factor-induced vascular smooth muscle cell (VSMC) proliferation in vitro [164]. In support of the growth inhibitory effects exerted by

Ang-(1-7), subsequent studies demonstrated that incubation with the heptapeptide hormone inhibited cardiomyocyte proliferation in vitro, an effect which was blocked by the Mas receptor antagonist, [D-alanine7]-angiotensin-(1-

7) [D-Ala] [169]. More importantly, the neointimal growth of VSMCs in response to balloon-catheter injury to rat carotid arteries was reduced by Ang-(1-7) while

31 treatment with the heptapeptide hormone had no effect on the non-proliferating medial cell layer, underscoring the selectively of Ang-(1-7) in the inhibition of actively proliferating cells [167].

The counterregulatory roles of Ang-(1-7) are not limited to cell proliferation as additional studies demonstrated that the heptapeptide hormone also opposes the fibrosis-stimulatory actions of Ang II, establishing Ang-(1-7) as a potent anti- fibrotic agent. The ability of Ang-(1-7) to inhibit fibrosis was reported in adult rat cardiac fibroblasts, where treatment with the heptapeptide hormone prevented

Ang II-stimulated synthesis of collagen, TGF-β mRNA expression, and cardiomyocyte hypertrophy [176]. Grobe and colleagues reported that infusion of

Ang-(1-7) in deoxycorticosterone acetate (DOCA)-salt rat model of hypertension prevented interstitial and perivascular collagen deposition [177]. Ang-(1-7) also mitigated the endothelin (ET-1)-stimulated proliferation and collagen synthesis of cardiac fibroblasts [165]. In a recent study, Willey and colleagues explored the potential for Ang-(1-7) as a radioprotective agent for radiation-induced fibrosis

(RIF) using a mouse model of extremity sarcoma radiation therapy [178]. The results of this study showed that Ang-(1-7) administration significantly reduced

RIF in the irradiated muscles, concurrent with decreased muscle stiffening and reduced production of profibrotic cytokines. Collectively, these studies indicate an important role for Ang-(1-7) in targeting fibrosis.

Studies aimed to elucidate the molecular mechanism of Ang-(1-7)- mediated effects demonstrated that activation of the Mas receptor by the heptapeptide induced downstream signaling through generation of cyclic adenosine monophosphate (cAMP) [168, 179]. Indeed, incubation with Rp-

CAMPS, an inhibitor of the cAMP-dependent protein kinase A (PKA), in VSMCs abrogated the Ang-(1-7)-mediated reduction in cell proliferation, indicating that activation of PKA by cAMP is necessary for the heptapeptide hormone to exert inhibitory effects on cell growth [179].

Ang-(1-7) also modulates the growth promoting mitogen-activated protein kinase (MAPK) signaling cascade to attenuate cell proliferations. Pre-treatment with the heptapeptide hormone decreased the phosphorylation and activation of extracellular regulated kinase 1/2 (ERK1/2), the final signaling effectors of MAPK signaling, in Ang II stimulated VSMCs [179, 180]. A reduction of phosphorylated-

ERK1/2 (p-ERK1/2) was observed in cardiac fibroblasts obtained from rat hearts incubated with Ang-(1-7); this reduction in the growth-promoting MAPK signal effectors was associated with decreased cardiac fibroblast proliferation, suggesting that Ang-(1-7) targets MAPK signaling to attenuate downstream cell growth effects [165]. The reduction of p-ERK1/2 by Ang-(1-7) is not limited to

VSMCs and cardiac fibroblasts; indeed multiple studies reported that treatment with heptapeptide hormone resulted in attenuation of ERK1/2 phosphorylation in proximal tubular cells and endothelial cells, suggesting that the effects mediated by Ang-(1-7) are exerted in multiple cell types [181-183].

To further delineate the molecular mechanism by which Ang-(1-7) reduces

ERK1/2 phosphorylation, experiments were carried out to test the hypothesis that the heptapeptide hormone modulates cellular phosphatase(s) to reduce MAPK signaling. MAPK phosphatases, also known as dual specificity phosphatases

(DUSPs), act as negative regulators of MAPK signaling [184]. Substrates for

DUSP1, an inducible nuclear phosphatase and a member of the DUSP family, include ERK1 and ERK2. An increase in DUSP1 expression was observed in

Ang-(1-7)-treated rat cardiac fibroblasts and rat primary astrocytes, with an associated decrease in p-ERK1/2; this reduction in ERK1/2 phosphorylation was abrogated in the presence of the phosphatase inhibitor, sodium orthovandate

[182, 185]. Collectively, these findings support the hypothesis that Ang-(1-7) modulates phosphatase expression to attenuate MAPK signaling, dampening the cellular responses mediated by this signaling cascade.

The underlying mechanism by which Ang-(1-7) upregulates DUSP1 expression is not yet well characterized. cAMP-PKA signaling was demonstrated to inhibit ERK phosphorylation in a cell-type dependent manner [186]. The promoter/enhancer regions of mkp-1, the gene encoding DUSP1, contain cAMP response element (CRE) binding sites, allowing this gene to be transcribed in response to cAMP generation [187]. Indeed, treatment with forskolin, which increases intracellular cAMP levels, inhibited growth factor-induced ERK1/2 phosphorylation in rat aortic smooth muscle cells by inducing DUSP1 expression,

34 demonstrating the positive regulatory role of cAMP on DUSP1 transcription [188].

The heptapeptide hormone Ang-(1-7) was reported to increase the generation of cAMP to inhibit cell proliferation, providing a potential mechanistic link between the regulation of DUSP1 by Ang-(1-7) [168, 179].

Renin-Angiotensin System and Cancer

As an endocrine system, the RAS peptides have the potential to influence cellular processes involve in tumor progression. The first evidence implicating

RAS involvement in malignancy came from a retrospective study encompassing over 5,000 hypertensive patients [189]. From this study, Lever and colleagues reported that the use of ACE inhibitors is associated with a reduced risk for cancer development, whereas treatment with other anti-hypertensive medications such as calcium channel blockers, beta-blockers and diuretics had no effect on the risk of developing cancer. In support of this retrospective analysis, dysregulation of components of the RAS were reported in several cancer types including breast, ovarian, prostate, pancreatic and gastric tumors and/or cell lines

[190]. Of note, AGTR1 was found to be markedly overexpressed in up to 20% of breast cancer. Intriguingly, the overexpression of the gene encoding the AT1R was enriched in ER+ breast tumors, implicating a potential influence of estrogen signaling on AGTR1 transcription [191]. Furthermore, polymorphisms of genes encoding RAS components were associated with increased risk for the development of certain types of malignancies. For example, it was reported that

35 genetic polymorphisms in the ACE gene may predispose women to breast cancer [192, 193].

Perturbation of RAS signaling was implicated in several cellular processes involved in cancer progression, including tumor cell proliferation, tumor- associated angiogenesis, and tumor cell metastasis. As noted above, Ang II exhibits pro-proliferative actions on VSMCs and endothelial cells in normal tissues. Ang II also promotes cancer cell proliferation [194-196]. Tumor- associated angiogenesis is an essential process in facilitating tumor growth and metastasis [197]. Downstream signaling mediated by the Ang II/AT1R axis induces expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and activation of the oxygen-sensing transcription factor hypoxia- inducible factor 1 (HIF1) to promote neovascularization to support tumor growth

[198, 199]. Finally, the involvement of altered RAS signaling was implicated in tumor metastasis. Analysis of a retrospective study involving stage II colorectal cancer patients showed that fewer metastatic lesions were observed in patients receiving ACE inhibitors [200]. In support of this observation, the use of ACE inhibitors and AT1 receptor blockers (ARBs) demonstrated efficacy in reducing metastasis in preclinical models whereas increased AT1 receptor expression correlated with tumor invasiveness [201-203]. Taken together, these observations provide strong evidence implicating deregulated RAS involvement in carcinogenesis.

Angiotensin-(1-7) exhibits pleotropic anti-tumor properties

The discovery that the RAS peptide Ang-(1-7) exhibits selective inhibition of actively proliferating cells, in part through mitigation of MAPK signaling, suggested that the heptapeptide hormone might serve as a novel therapeutic for the treatment of malignancy. Based on the anti-proliferative properties exerted by

Ang-(1-7), Gallagher and colleagues evaluated the effects of the heptapeptide hormone on lung cancer cell growth [204]. Ang-(1-7) reduced the proliferation of lung cancer cells by attenuating DNA synthesis and attenuated activation of

ERK1/2, a proliferative mediator of MAPK signaling [204]. In support of this finding, Ni and colleagues reported that Ang-(1-7) abrogated the serum-mediated migration and invasion of human A549 lung cancer cells in vitro through attenuation of PI3-K and MAPK signaling, two commonly deregulated signaling nodes in human malignancy [205].

The growth inhibitory effects of Ang-(1-7) were also evaluated in preclinical animal models of cancer. Tumor growth was inhibited in athymic mice bearing human lung tumor xenografts treated with Ang-(1-7), which was associated with reduced expression of the cell proliferation marker Ki67 as well as a marked decrease in the pro-inflammatory enzyme cyclooxygenase-2

(COX2), further demonstrating an anti-inflammatory role for heptapeptide hormone [166]. Pretreatment with Ang-(1-7) abolished metastatic prostate tumor formation in the bone of SCID mice, by preventing osteoclastogenesis of bone

37 marrow cells, demonstrating that Ang-(1-7) also exhibit anti-metastatic potentials

[206]. Furthermore, overexpression of ACE2, which facilitates the conversion of

Ang II to Ang-(1-7), in human A549 lung cancer cells resulted in decreased liver metastasis formation in mice [207].

The Mas receptor was identified in endothelial cells and VSMCs, suggesting that Ang-(1-7) may play a role in regulating angiogenesis, a biological process crucial for supporting tumor growth and metastasis [208]. In support of this hypothesis, Ang-(1-7) treatment facilitated a reduction in the number of blood vessels as visualized by the decrease in CD34+ endothelial cells lining the blood vessels, concurrent with downregulation in the protein and transcript levels of the pro-angiogenic factor VEGF [209]. From this same study, Soto-Pantoja and colleagues observed a reduction in in vitro endothelial tubule formation with Ang-

(1-7) treatment, indicative of the ability of the heptapeptide hormone to negatively regulate blood vessel formation. Lastly, in a chick chorioallantoic membrane

(CAM) assay, which mimics neovascularization, Ang-(1-7) treatment resulted in a

50% reduction in vessel formation when compared to an untreated control group.

The ability for Ang-(1-7) to suppress angiogenesis was also reported in preclinical models of human prostate cancer, nasopharyngeal carcinoma and hepatocellular carcinoma, further demonstrating broad tumor targeting effects of the heptapeptide hormone [210-212].

In mice bearing ER+ and HER2-amplified breast tumors treated with Ang-

(1-7), reductions in interstitial and perivascular fibrosis were observed with a concomitant decrease in tumor-associated fibroblast proliferation [163]. Ang-(1-7) administration also resulted in a decrease in the expression of the pro-fibrotic molecule tumor growth factor (TGF)-β and the extracellular matrix (ECM) protein fibronectin in tumoral fibroblasts. In support of the negative regulatory role of

Ang-(1-7) on the pro-proliferative MAPK signaling, tumor-associated fibroblasts incubated with the heptapeptide hormone exhibited reduced p-ERK1/2, with an associated increase in DUSP1. Collectively, these studies highlight the pleiotropic mechanism exhibited by Ang-(1-7) to inhibit tumor growth and progression.

The limited toxicity and benefits of Ang-(1-7) treatment of cancer patients were reported in several clinical trials. In a Phase I clinical trial completed at

Wake Forest University Comprehensive Cancer Center, treatment with Ang-(1-7) demonstrated clinical benefits with few side effects in patients with solid tumors

(NCT00471562) [213]. In this Phase I clinical trial, adult patients with advanced solid tumors refractory to standard therapy were administered Ang-(1-7) in escalating doses from 100-700 µg/kg/day by daily subcutaneous injection for 5 days in 21 day cycles. Out of fifteen evaluable patients, four exhibited clinical benefits with an associated reduction in the circulating pro-angiogenic factor, placental growth factor (PlGF). The Ang-(1-7)-associated decrease in plasma

PlGF in patients underscores the role of the heptapeptide hormone as an anti-

39 angiogenic agent and suggests that the anti-tumor effects of the patients may be mediated, in part, through a reduction in angiogenesis. Three patients experienced disease stabilization lasting longer than 3 months, while another patient diagnosed with metastatic sarcoma with progressing disease at the time of enrollment experienced an overall reduction in the sum of unidimensional measures of 19% following Ang-(1-7) treatment and continued on therapy for 10 months before developing progressive disease. Few side effects were observed in these patients, with only three serious adverse events (SAEs) possibly related to Ang-(1-7) administration reported in this study. Calf pain related to deep-vein thrombosis was experienced by one patient, while another patient developed strokes which did not subside upon removal of treatment and one patient experienced cranial neuropathy. Considering the advance stage of disease in these patients in addition to the chemotherapeutic treatment regimens received prior to the start of Ang-(1-7) treatment which may have provoked adverse side effects, these SAEs cannot be definitively attributed to Ang-(1-7) administration.

In the adjuvant setting, Ang-(1-7) reduced the frequency of high grade thrombocytopenia, anemia and lymphopenia compared to filgrastim in a breast cancer patient cohort receiving doxorubicin and cyclophosphamide [214]. Similar findings were reported in a randomized, double-blind, placebo-controlled Phase 2 study evaluating the efficacy of Ang-(1-7) as an adjuvant therapy for a group of cancer patients treated with gemcitabine and carboplatin or cisplatin [215]. Taken

40 together, these studies demonstrate minimal risk for treatment of cancer patients with the heptapeptide hormone.

While the clinical efficacy of Ang-(1-7) was evaluated in the Phase I clinical trial conducted at Wake Forest Comprehensive Cancer Center

(NCT00471562) in a patient cohort consisting of solid tumors, no breast cancer patients were enrolled in this study. Thus, the experimental findings reported in this dissertation will potentially provide the preclinical support for initiating a

Phase I clinical trial assessing the clinical activity of Ang-(1-7) in a breast cancer cohort, with emphasis on patients diagnosed with TNBC.

Hypothesis and Specific Aims

The heterogeneity of TNBC and the paucity of targeted efficacious treatment for this devastating disease have prompted intense research efforts to identify molecules which can be targeted to provide treatment of both the primary and metastatic disease. The c-Met RTK is a potent inducer of cancer cell proliferation and invasion. Recent studies identified a potential role for c-Met RTK participation in TNBC development and progression. Clinical observations and increasing evidence from experimental studies suggest aberrant c-Met RTKs signaling contributes to the development of the basal-like and claudin-low subtypes of TNBC [95, 97, 216]. Thus, c-Met RTK represents a potential therapeutic target for TNBC patients.

Increasing evidence supports the anti-cancer activities exhibited by Ang-

(1-7). The heptapeptide hormone exhibits pleotropic anti-tumor properties, including anti-proliferative, anti-metastatic, anti-fibrotic, anti-angiogenic and anti- inflammatory activities. Cardiovascular studies as well as models of non-triple negative breast cancer models demonstrated that Ang-(1-7) reduces the pro- proliferative MAPK signaling through decreasing activated ERK1/2 (p-ERK1/2)

[163, 165, 182]. These reductions were associated with a significant increase in the expression of the MAPK phosphatase, DUSP1, contributing to attenuation of cell proliferation. Ang-(1-7) treatment also increased the expression of a diverse array of regulatory phosphatases, including the tumor suppressor PTEN and

SHP-1 [217-219]. Due to the potent attenuating effect on kinase activity induced

42 by Ang-(1-7), we postulate that the heptapeptide hormone may serve as a universal regulator of phosphatases. More importantly, Garcia and colleagues reported that Ang-(1-7) administration increased PTP1b, a negative regulator of c-Met RTK signaling [119]. Thus, in Chapter 2, we hypothesize that Ang-(1-7) will inhibit the growth of triple negative breast cancer cells and tumors through upregulation of PTP1b to decrease c-Met RTK signaling.

The HGF/c-Met signaling cascade was also implicated in promoting tumor metastasis by enhancing the migratory and invasive properties of tumor cells and by protecting circulating metastatic tumors cells from anoikis. In addition, expression of HGF/c-Met was observed in glial cells, in response to the colonization of tumor cells in the brain, which is a common metastatic site for triple negative breast tumors [14]. Thus, in Chapter 3, we hypothesize that treatment with the heptapeptide hormone Ang-(1-7) will reduce metastatic breast tumor formation in the brain, in part, by reducing c-Met RTK signaling to reduce metastatic tumor growth.

The development of Ang-(1-7) as a therapy for triple negative breast cancer patients will fill a need in the currently limited available treatment options for TNBC patients due to the pleotropic anti-tumor effects exhibited by the heptapeptide hormone. Furthermore, patients diagnosed with TNBC are primarily subjected to cytotoxic chemotherapeutics that produce undesired side effects such as dose-limited cardiotoxicity, thereby contributing to poor quality of life. In

43 contrast, as shown in clinical trials of cancer patients, Ang-(1-7) produces few side effects [213-215]. Thus, Ang-(1-7) has the potential to become a first-in- class therapeutic for the treatment of primary and metastatic TNBC.

Figure 1. c-Met Receptor Tyrosine Kinase (RTK) Signaling. Following hepatocyte growth factor (HGF) binding to its cognate receptor c-Met, receptor dimerization occurs resulting in upregulation of kinase activity. The multifunctional adapter protein Gab1 is recruited to the c-Met receptor, where

Gab1 is phosphorylated at several tyrosine residues to provide docking sites for signaling effector molecules to initiate downstream PI-3K/Akt and MAPK signaling. Activation of PI-3K/Akt and MAPK signaling pathways facilitate cellular proliferation, survival, and cellular motility with increased invasive potential.

Figure 2. Synthesis of the Renin-Angiotensin System (RAS) Peptides.

Figure 3. Chemical Structure of Angiotensin-(1-7) [Ang-(1-7)]

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CHAPTER II: Angiotensin-(1-7) Up-regulates PTP1b to Reduce c-MET Signaling and Attenuate Triple Negative Breast Cancer Growth

The following manuscript is prepared for submission to Molecular Cancer Therapeutics and represents the efforts of the first author. Differences in formatting and organization reflect the requirements of the journal.

Angiotensin-(1-7) Up-regulates PTP1b to Reduce c-Met Signaling and

Attenuate Triple Negative Breast Cancer Growth

Guorui Deng,1, 2 Patricia E. Gallagher1, 2 and E. Ann Tallant,1, 2

1Hypertension and Vascular Research Center, 2Department of Cancer

Biology,

Wake Forest University School of Medicine, Winston-Salem, North Carolina

27157

Key Words: angiotensin-(1-7), triple negative breast cancer, c-Met, protein tyrosine phosphatase 1b

Running Title: Ang-(1-7) reduces triple negative breast cancer

Address for Correspondence:

E. Ann Tallant, Ph.D. Hypertension and Vascular Research Center Wake Forest University School of Medicine Medical Center Boulevard Winston Salem, NC 27157, USA Telephone: (336) 716-2108 FAX: (336) 716-2456

Abstract

Despite recent advances in targeted therapies for HER2-overexpressing and ER- positive breast cancer, no targeted therapies are available for triple negative breast cancer and patients are treated with cytotoxic chemotherapeutics that often result in cardiotoxicity and harsh side effects. Since the heptapeptide hormone angiotensin-(1-7) [Ang-(1-7)] targets a unique receptor, the G protein- coupled receptor Mas, and inhibits the growth of various types of cancer including breast cancer, the effect of Ang-(1-7) on the growth of 4T1 triple negative breast tumors and the role of the receptor tyrosine kinase c-Met in the response to Ang-(1-7) was investigated. c-Met is elevated in triple negative breast cancer relative to other breast cancer subtypes and Ang-(1-7) decreased

4T1 tumor growth with an associated reduction in phosphorylated c-Met. The decrease in 4T1 cell proliferation was also associated with a reduction in c-Met signaling, including decreased phospho-ERK1/2 as well as both tumor cell proliferation and invasion. Ang-(1-7) up-regulated protein phosphatase PTP1b which dephosphorylates phospho-c-Met and the siRNA reduction in PTP1b ablated the response to the heptapeptide hormone. Analysis of publically available gene expression datasets showed that the c-Met oncogene is enriched in triple negative breast cancer cell lines and tumors when compared to non-

TNBC samples and that decreased expression of PTP1b and Mas1 correlates with reduced relapse-free survival of TNBC patients. These results provide preclinical support for evaluation of Ang-(1-7) as a novel targeted therapeutic for patients with triple negative breast cancer.

Introduction

Triple negative breast cancer (TNBC) is an aggressive disease that is diagnosed in 15 to 20% of all breast cancer cases [14]. It is characterized by a lack of estrogen receptor (ER) and progesterone receptor (PR) expression and the absence of amplified concentrations of human epidermal growth factor receptor 2

(HER2) oncoprotein. Recent gene expression profile analysis stratified TNBC into six distinct molecular subtypes including the basal-like (basal A) and claudin- low (basal B) subtypes, indicating significant heterogeneity within triple negative breast tumors [29, 40, 44].

Unlike patients diagnosed with ER+ and HER2+ disease for whom targeted therapies are available, patients diagnosed with TNBC are treated with cytotoxic chemotherapeutics such as anthracyclines or taxanes that often result in harsh side effects including adverse quality of life symptoms and cumulative dose- dependent cardiotoxicity. As a consequence, TNBC patients have an increased risk of recurrence within the first 3 years, of cancer-related death within the first 5 years, and of visceral metastasis to the lungs and central nervous system [220].

Thus a significant need remains for the identification of actionable targets and the development of novel targeted therapies for TNBC patients.

The c-Met receptor tyrosine kinase (RTK) and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), orchestrate a complex biological program termed invasive growth. The c-Met RTK-driven process coordinates cell proliferation and invasion and is a physiological program that is activated during embryonic

66 development and post-natal tissue growth and regeneration. HGF binding induces c-Met receptor dimerization and autophosphorylation of tyrosine residues at position 1234 and 1235 (Tyr1234/1235) within its catalytic domain

[92]. Consequent phosphorylation on tyrosine residues 1349 and 1356

(Tyr1349/1356) in the carboxy-terminal domain generates a multifunctional docking site capable of interacting with signaling effectors and adaptor proteins. c-Met signaling activates several downstream signaling cascades shared with other growth factor receptors, including extracellular-signal regulated kinase

(ERK) 1/2 and the phosphatidylinositol (PI)-3 Kinase-Akt pathways. In particular,

ERK1/2 and PI-3 kinases are activated by c-Met with greater intensity and duration as compared with other growth factor receptors and this hyperactivation is critical for the pro-invasive activity [51, 52].

Deregulation of c-Met signaling is associated with tumor growth and metastasis in a variety of tumor types [46, 93]. In particular, overexpression of c-Met and increased serum HGF correlates with short relapse-free and overall survival for breast cancer patients [84-86]. c-Met overexpression was reported in 20-30% of breast cancer cases and is a strong, independent prognosticator of poor clinical outcome [82, 85, 221-223]. Emerging studies implicate increased c-Met expression and deregulated downstream signaling in TNBC progression and disease dissemination, with the highest expression of c-Met observed in the basal-like subtype of TNBC [95, 224]. A recent study suggested that c-Met activation induces mammary tumors with pathological and molecular features of the claudin-low subtype of TNBC in the absence of tp53 [95] and mice harboring

67 constitutively active c-Met mutations develop mammary tumors with a gene expression profile that clusters with human basal-like breast cancer [97].

Collectively, insights from clinical observations and experimental studies demonstrate a potential contribution of aberrant c-Met signaling to TNBC disease initiation and progression, suggesting that inhibition of the c-Met signaling pathway may serve as a target for the treatment for TNBC patients.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, seven amino acid peptide hormone of the renin-angiotensin system exerting pleotropic anti-tumor activities by activating the G protein-coupled receptor Mas [162]. The heptapeptide hormone exhibits anti-angiogenic, anti-fibrotic, anti-metastatic, anti-inflammatory and anti-proliferative properties in a spectrum of different cancer types [163, 166,

204, 206, 209]. Of note, Ang-(1-7) exerts its anti-proliferative effects in part by up-regulating the protein phosphatase DUSP1, a dual specificity phosphatase, to counteract ERK1/2 activation. Additionally, the limited toxicity and benefits demonstrated by Ang-(1-7) were reported in clinical trials of cancer patients treated with the heptapeptide hormone [213-215]. In a Phase I clinical trial completed at Wake Forest University Comprehensive Cancer Center, treatment with Ang-(1-7) demonstrated clinical benefits with few side effects in patients with solid tumors [213]. These studies underscore the minimal risk for treatment of cancer patients with Ang-(1-7). Intriguingly, lower Mas receptor expression was observed in invasive ductal carcinoma (IDC) of the breast compared to benign mammary tissues and decreased Mas receptor expression was associated with

68 tumor growth, lymph node metastasis and tumor grade, suggesting a role for

Ang-(1-7) and its receptor in IDC [225].

Protein phosphatases play critical roles in modulating protein function, activity and localization. The protein tyrosine phosphatase 1b (PTP1b), encoded by the

PTP1b gene, is a widely-expressed non-receptor phosphatase localized to the cytoplasmic face of the endoplasmic reticulum [104]. PTP1b targets multiple

RTKs, including c-Met, for dephosphorylation in response to ligand-induced receptor activation. PTP1b negatively regulates activated c-Met by dephosphorylating tyrosine residues 1234 and 1235 to decrease kinase activity

[98]. PTP1b was identified as an independent positive prognostic factor in a cohort of breast cancer patients [118]. Moreover, Garcia-Espinosa and colleagues reported an increase in PTP1b expression in the medulla oblongata of Ang-(1-7)-treated rats [119]. These studies suggest a regulatory role for the heptapeptide hormone on c-Met signaling [119].

In this study, the effects of Ang-(1-7) administration on triple negative breast tumor growth were investigated in the 4T1 syngeneic model, since i) c-Met is aberrantly activated in TNBC [95, 224], ii) Ang-(1-7) regulates phosphatase activities, and iii) Ang-(1-7) increases the protein phosphatase PTP1b, a regulator of c-Met RTK in rat brain.

Materials and Methods

Reagents and materials

Angiotensin-(1-7) [Ang-(1-7)] was purchased from Bachem while D-alanine7-Ang-

(1-7) [D-Ala] was synthesized by GenScript Corporation (Piscataway, NJ).

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, RPMI-1640, streptomycin, and fetal bovine serum (FBS) were obtained from Life

Technologies, Invitrogen. Tween-20 was purchased from Thermo Fisher

Scientific (New Jersey, USA). SMARTPOOL siRNAs against PTP1b were designed and purchased from Dharmacon (Chicago, IL) whereas non-specific

(NS) negative control siRNA was purchased from Thermo Fisher Scientific.

Recombinant human hepatocyte growth factor (HGF) was purchased from

Peprotech (Rocky Hill, NJ). Alzet osmotic pumps were obtained from Braintree

Scientific, Inc. Matrigel-coated Invasion Chambers with 5 µm PET membranes were purchased from BD Biosciences (Franklin Lakes, New Jersey).

The following antibodies were used: anti-Ki67 and anti-PTP1b (Abcam,

Cambridge, MA), anti-phosphorylated c-Met (p-c-Met) [Tyr1234/1235], anti-c-

Met, anti-phosphorylated ERK1/2 (Thr202/Tyr204) [p-ERK1/2], anti-ERK1/2 and anti-α–tubulin (Cell Signaling), anti-β-actin (Sigma-Aldrich), anti-Mas (Alomone

Lab), anti-DUSP1 (MKP-1) (Upstate Biotechnology), and horse radish peroxidase

(HRP)-conjugated antibodies (GE Healthcare Life Sciences, USA).

Cell Culture

MDA-MB-231 human adenocarcinoma cells [American Tissue Culture Collection

(ATCC) HTB-26), derived from a 51 year-old Caucasian female, were purchased from ATCC and grown in DMEM medium supplemented with 100 µg/mL penicillin, 100 units/mL streptomycin, 15 mM HEPES, L-glutamine and 10% fetal bovine serum (FBS). 4T1 mouse mammary gland carcinoma cells (ATCC CRL-

2539), derived from a spontaneously-arising mammary tumor in a female BALB/c mouse, were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 100 µg/mL penicillin, 100 units/mL streptomycin, 15 mM

HEPES, L-glutamine and 10% FBS. Subconfluent cells were incubated with serum-free media overnight to remove growth factors, prior to stimulation with

HGF (30 ng/mL) or Ang-(1-7) [100 nM]. Cells were grown at 37°C in a humidified atmosphere of 5% CO2/95% room air.

Cell proliferation assay

4T1 cells and MDA-MB-231 cells were serum-starved for 16 h prior to treatment with HGF (30 ng/mL) or Ang-(1-7) [100 nM]; cells were pretreated with Ang-(1-7) for 5 h prior to HGF stimulation (15 min). Where indicated, the Mas receptor antagonist D-Ala (1 µM) was added 15 min prior to the addition of HGF and Ang-

(1-7). The number of cells was assessed after 48 h of treatment using a hemocytometer.

Orthotopic Tumor Model

Actively growing 4T1 cells (2.5 x 105) were injected into the mammary fat pads of

6-week old female BALB/c mice (Charles River) to induce tumor growth. Tumor size was measured three times a week during the treatment period, using a caliper, and tumor volume was calculated using the formula for a semi-ellipsoid

[(4/3πr3)/2]. Mice were randomized into either a control or a treatment group when tumors reached 50 mm3 in volume. Treated mice were administered 24

µg/kg/h Ang-(1-7) delivered by subcutaneously implanted osmotic mini pumps

(Alzet). After 21 days of treatment, the mice were euthanized and the tumors were harvested for analysis. Tumor weight was determined at the time of sacrifice. All animal protocols were approved by the Wake Forest School of

Medicine Animal Care and Use Committee.

Western Blot Hybridization

Tumor tissue was homogenized and solubilized as previously described [209] and tumor cell monolayers were solubilized in lysis buffer [204]. Protein concentration was measured by a modified Lowry method. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Non- specific binding was blocked by incubating membranes in Blotto (Tris-buffered saline [pH 7.5] with 5% powdered milk and 0.1% Tween-20). The membranes were incubated overnight at 4°C with primary antibodies specific to p-c-Met

(1:1000), c-Met (1:1000), p-ERK1/2 (1:1000), ERK1/2 (1:2500), DUSP1 (MKP-1,

1:1000), PTP1b (1:1000), or Mas (1:50), followed by a 1 h incubation with polyclonal HRP-conjugated secondary antibodies (1:2500 or 1:5000) at room temperature. Immunoreactive bands were visualized by chemiluminescence

(SuperSignal Femto or Pico West, Pierce Biotechnology). Densitometric analysis of bands of interests was quantified by using MCID digital densitometry software

(Cambridge, UK).

Immunohistochemistry

Five (5) micron sections of paraformaldehyde-fixed and paraffin-embedded tumors were cut on a microtome, deparaffinized in xylene and hydrated in graded alcohols prior to antigen retrieval in citrate buffer (Target Retrieval Solution,

Dako, Carpinteria, CA). Sections were incubated with antibodies against Ki67

(1:100) to visualize actively proliferating cells, p-c-Met (Y1234/1235) [1:100] to identify tumor cells with activated c-Met RTK and PTP1b (1:100) to highlight

PTP1b positive cells. A Leica DM 4000 microscope attached to an Olympus Q

Imaging Retiga 1300R camera was used to acquire pictures of four representative regions per tumor section for each animal. The average percentage of positively-stained cells per representative region was quantified using ImageJ software (NIH).

Transfection assay

4T1 cells were transfected with PTP1b-targeted siRNA (25 nM final concentration) or control non-specific (NS) siRNA (25 nM final concentration) using DharmaFECT transfection reagents according to the manufacturer’s

73 instructions. Forty-eight h after transfection, cells were either collected for subsequent analysis or serum-starved overnight prior to HGF stimulation and

Ang-(1-7) treatment.

Quantitative RT-PCR

RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA was reverse-transcribed using a high- capacity cDNA reverse transcription kit (Life Technologies). Real-time quantitative PCR was performed using a SYBR FAST qQCR kit and PTP1b- specific primers (Hs00182260_m1, Thermo Fisher Scientific). The relative expression of PTP1B was determined relative to 18S rRNA in each reaction.

Invasion assay

MDA-MB-231 cells were seeded on top of Matrigel-coated Invasion Chambers

(BD Biosciences, Franklin Lakes, New Jersey, USA) and incubated with HGF (30 ng/mL) in the absence or presence of 100 nM Ang-(1-7) or 1 µM D-Ala. The bottom chamber contained DMEM supplemented with 1% FBS. Cells were allowed to invade for 24 h prior to fixation in 4% paraformaldehyde and staining with 0.5% crystal violet. Five fields of each transwell chamber were photographed using an inverted wide-field microscope at 200x magnification and the number of cells in each image was counted using ImageJ software (NIH).

74 c-Met expression analysis from microarray studies

Affymetrix data sets from GSE1456 were downloaded from the NCBI Gene

Expression Omnibus (www.ncbi.nlm.nih.gov/geo). The average expression of all c-

Met probes was used. c-Met expression was also evaluated in the online database, Cancer Cell Line Encyclopedia (CCLE)

(http://www.broadinstitute.org/ccle/home). The molecular subtype of each breast cancer cell line was identified according to Neve et al [226]. For c-Met expression analysis in The Cancer Genome Atlas (TCGA) Breast Cancer Nature 2012 data set, the cBioPortal (http://www.cbioportal.org/) was used [28, 227, 228].

Kaplan-Meier analysis

The online survival analysis tool KMPlot (http://kmplot.com/analysis/) was used to analyze the correlation between the expression of genes of interest (PTP1b,

Mas1, c-Met) and clinical outcome in patients diagnosed with basal-like TNBC

[229]. For genes (PTP1b, c-Met) with multiple probes, the average expression of all probes was analyzed using patients identified with the basal-like intrinsic subtype from the 2014 version dataset.

Statistical Analysis

Data are expressed as means ± SEM. Statistical significance was evaluated with the use of Student’s t-test when comparing only two data groups or with a one- way ANOVA followed by Tukey’s post hoc test to compare between multiple groups. Statistical comparisons were performed using Prism 6 (GraphPad Prism

Software Version 6.04; La Jolla, CA, USA). Differences were considered significant at p<0.05.

Results

Angiotensin-(1-7) [Ang-(1-7)] administration reduced orthotopic triple negative breast tumor growth in a syngeneic mouse model

The murine syngeneic model was used to examine the effects of Ang-(1-7) treatment on orthotopic triple negative breast tumor growth in vivo. Murine 4T1 cells, a triple negative mammary carcinoma cell line, were isolated by Fred Miller and colleagues from a spontaneously arising mammary tumor in a BALB/c mouse [230]. The 4T1 tumor model closely mimics the progression of human triple negative breast cancer when introduced orthotopically into BALB/c mice.

Actively growing 4T1 cells (2.5 x 105 cells per mouse) were injected into the mammary fat pad of female BALB/c mice. When the tumors reached a size of approximately 50 mm3, the mice were randomized for subcutaneous infusion of

24 µg/kg/h Ang-(1-7). Tumors from untreated, control mice increased in size over the course of 21 days. In contrast, Ang-(1-7) administration resulted in a marked decrease of 49% in tumor volume compared to the control mice (Figure 1A); treatment with the heptapeptide hormone produced a significant reduction in average tumor volume at the end of the treatment period (control, 1484 ± 199.2 mm3; Ang-(1-7), 763 ± 105 mm3, Figure 1B). In agreement with reduced tumor burden, Ang-(1-7) significantly decreased tumor wet weight (control, 2.1 ± 0.2 g;

Ang-(1-7), 1.5 ± 0.2 g, Figure 1C). Tumor sections were stained with an antibody

76 against the proliferation marker Ki67 and actively proliferating cells were visualized to determine whether Ang-(1-7) inhibits tumor cell proliferation to reduce tumor growth. Ang-(1-7) significantly reduced the number of Ki67-positive cells by 83%, suggesting that the heptapeptide hormone attenuates tumor growth in part by decreasing cell proliferation (Figure 1D). Ang-(1-7) administration was well-tolerated by mice. No obvious pathologic abnormalities were observed in major organs at the time of sacrifice, indicating a lack of toxic side effects at the dose administered. Collectively, these data suggest that Ang-(1-7) attenuates orthotopic triple negative tumor growth in part by regulating tumor cell proliferation.

The c-Met Proto-oncogene is overexpressed in Triple Negative Breast

Cancer (TNBC)

Breast cancers are stratified into five intrinsic molecular subtypes by gene expression profiling – luminal A, luminal B, HER2-enriched, basal, and normal breast-like subtypes [28]. TNBC, defined therapeutically by the absence of the estrogen and progesterone receptor and the lack of over-expression of the HER2 receptor, is predominantly of the basal subtype. c-Met expression was queried in a panel of breast cancer cell lines from the Cancer Cell Line Encyclopedia

(CCLE) [231], to evaluate whether the c-Met oncoprotein is enriched in a subset of breast cancer. TNBC cell lines expressed a significantly higher level of c-Met compared to non-TNBC cell lines (Supplementary Figure S1A and B). c-Met

77 expression was also examined in two public breast cancer datasets derived from patient samples - GSE1456 and The Cancer Genome Atlas (TCGA) [28, 232], to assess the clinical relevance of the CCLE expression data. Comparison of the basal-like TNBC subtype to the luminal A, luminal B and HER2-enriched breast cancer molecular subtypes indicated that c-Met is enriched in the basal-like

TNBC but not in the non-TNBC molecular subtypes (Supplementary Figures

S1C and 1D). To validate these in silico observations further, c-Met protein expression was analyzed in a panel of breast cancer cell lines representative of the HER2-enriched (BT-474, SK-BR-3), luminal (MCF-7) and TNBC subtypes

(BT-20, BT-549 and MDA-MB-231). In contrast to non-TNBC cell lines, TNBC cell lines displayed increased c-Met expression (Supplementary Figure S1E).

Taken together, these results suggest that the c-Met receptor tyrosine kinase is enriched in triple negative breast tumors and may serve as a novel target in

TNBC patients.

Ang-(1-7) attenuated HGF-induced C-MET RTK phosphorylation and activation

Having confirmed that c-Met RTK is enriched in triple negative breast tumors and therefore may serve as a novel target, the ability for the heptapeptide hormone

Ang-(1-7) to modulate c-Met RTK activation in response to its ligand HGF was assessed. The G protein-coupled receptor Mas, which selectively binds Ang-(1-

7) and elicits its downstream effects, was expressed in the 4T1 and MDA-MB-

231 cell lines (Supplementary Figure S2). Binding of HGF to the c-Met RTK induces receptor dimerization and trans-phosphorylation of tyrosine residues

Y1234 and Y1235 within the catalytic domain, resulting in receptor activation

[92]. HGF stimulation induced a pronounced c-Met RTK phosphorylation in both

4T1 and MDA-MB-231 cells (Figure 2A and B). In contrast, co-treatment with

Ang-(1-7) significantly attenuated HGF-mediated c-Met RTK activation by 30 to

49% in MDA-MB-231 and 4T1 cells, respectively. The suppressive effect of Ang-

(1-7) on c-Met phosphorylation was abrogated in the presence of the Mas receptor antagonist, D-Ala (Figure 2A). To determine whether the heptapeptide hormone displayed similarly reduced c-Met RTK phosphorylation/activation in vivo, paraffin-embedded sections from control and Ang-(1-7)-treated tumors were incubated with an antibody against phosphorylated c-Met RTK (p-C-Met

Y1234/5) to visualize tumor cells with activated c-Met. Tumors from the control mice displayed a higher percentage of tumor cells with p-c-Met Y1234/5 immunoreactivity, whereas Ang-(1-7) administration produced a marked reduction in p-c-Met+ tumor cells (control, 47.0 ± 9.5%; Ang-(1-7), 19.7 ± 3.5%, p

< 0.05; Figure 2C). Overall, these results suggest that Ang-(1-7) inhibits c-Met activation in vitro and in vivo.

HGF-induced cell proliferation and cell invasion is attenuated by Ang-(1-7) c-Met RTK signaling activates several downstream signaling cascades including the extracellular-signal regulated kinases (ERK) 1/2 to facilitate cell proliferation

79 and cell invasion [53]. The effect of Ang-(1-7) on c-Met induced cell proliferation was examined in 4T1 and MDA-MB-231 cells stimulated with HGF in the presence of the heptapeptide hormone. The growth of both 4T1 and MDA-MB-

231 cells was significantly increased by HGF (Figures 3A and B). However, co- treatment with Ang-(1-7) blunted the HGF-mediated increase in cell proliferation in both cell lines; Ang-(1-7) alone had no significant effect on cell growth.

Pretreatment with the Mas receptor antagonist D-Ala prevented c-Met-stimulated growth reduction by Ang-(1-7) in 4T1 cells, while D-ala alone had no effect

(Figure 3A).

The RAF-MEK-ERK signaling cascade is a key pathway regulating cell proliferation and its dysregulation can promote tumorigenesis [233]. Protein lysates were prepared using 4T1 tumors from untreated mice or mice treated with Ang-(1-7) and analyzed by Western blot hybridization using an antibody specific for phosphorylated ERK1/2, to determine whether Ang-(1-7) attenuates phosphorylation and activation of ERK1/2, the final mediator of RAF-MEK-ERK signaling. Treatment with the heptapeptide hormone significantly decreased both phosphorylated ERK1 and ERK2 (p-ERK1/2) (Figure 3C). The decrease in p-

ERK1/2 was associated with a pronounced increase in the phosphatase DUSP1, which dephosphorylates and inactivates p-ERK1/2 (Figure 3D). HGF also increased p-ERK1/2 in 4T1 cells which was reduced by co-administration of Ang-

(1-7). ERK1/2 is phosphorylated in 4T1 cells in the absence of HGF stimulation

(Control) and addition of Ang-(1-7) alone also reduced p-ERK1/2 in the absence of HGF (Figure 3E).

c-Met RTK signaling is also a potent inducer of tumor cell invasion [92]. MDA-

MB-231 cells were seeded on the upper chamber of transwells coated with

Matrigel, which is a mixture of extracellular matrix components (laminin, collagen, and heparan sulfate proteoglycans) and stimulated with HGF, in the presence or absence of Ang-(1-7), to determine whether Ang-(1-7) reduces HGF-mediated cell invasion. Invasion of tumor cells from the upper chamber through the membrane was then evaluated. MDA-MB-231 cells were inherently invasive and migrated through the Matrigel layer in the absence of any stimulation (Control in

Figure 3F). The addition of HGF further enhanced the number of cells invading through the Matrigel layer; in contrast, co-treatment with Ang-(1-7) blocked HGF- stimulated cell invasion while the heptapeptide hormone alone had no effect. The reduction in HGF-stimulated invasion by Ang-(1-7) was abrogated in the presence of the Mas receptor antagonist D-Ala, suggesting that the heptapeptide hormone activates Mas to inhibit HGF-induced cell invasion; D-ala alone had no effect of HGF-stimulated cell invasion. Taken together, these findings suggest that Ang-(1-7) antagonized c-Met RTK activation by its ligand HGF to attenuate downstream signaling and inhibit tumor cell proliferation and invasion.

Ang-(1-7) up-regulates phosphatase PTP1b, a negative regulator of c-Met signaling

PTP1b is a non-receptor tyrosine phosphatase that negatively regulates activated c-Met RTK by dephosphorylating tyrosine residues 1234 and 1235 in the activation loop [98]. PTP1b is induced in the medulla oblongata of rat brains in response to Ang-(1-7) administration, suggesting that up-regulation of the phosphatase may be a mechanism whereby the heptapeptide hormone could antagonize c-Met signaling [119]. 4T1 cells were incubated with Ang-(1-7) for increasing periods of time and PTP1b was measured by Western blot hybridization, to determine whether Ang-(1-7) increases PTP1b to attenuate c-

Met RTK activation in TNBC. PTP1b protein was significantly up-regulated in response to incubation with the heptapeptide hormone, with an increase of 2-3 fold by 8 h (Figure 4A). The increase in PTP1b was observed in 4T1 or MDA-

MB-231 tumor cells treated with Ang-(1-7), in the presence or absence of HGF

(Figures 4B and C). Paraffin-embedded sections of tumors from control and

Ang-(1-7)-treated mice were incubated with an antibody to PTP1b, to determine whether the heptapeptide hormone increased PTP1b expression in vivo. A significant increase in the number of PTP1b positive cells was observed in tumors from Ang-(1-7)-treated mice with a low level of expression of the enzyme in tumors from untreated mice (Figure 4D). These results suggest that Ang-(1-7) up-regulates PTP1b in triple negative breast cancer cells and tumors.

PTP1b depletion reverses the suppressive effects of Ang-(1-7) on c-Met

RTK signaling

PTP1b was depleted from 4T1 cells using PTP1b-targeted SMARTpool siRNAs

(Dharmacon), to investigate whether Ang-(1-7) up-regulates PTP1b to reduce the phosphorylation and activation of c-Met RTK. A 48 h transfection of 4T1 cells with PTP1b-targeted siRNA significantly reduced the expression of PTP1b compared to cells transfected with non-specific (NS) siRNA (Figure 5A). This corresponded to a significant decrease in PTP1b protein concentration by

Western blot hybridation in 4T1 cells transfected with PTP1b siRNA relative to cells transfected with a NS siRNA, demonstrating efficient knockdown of the phosphatase (Figure 5B). Tumor cells transfected with siRNA against PTP1b and NS siRNA were stimulated with HGF, in the absence or presence of Ang-(1-

7), to assess the effects of Ang-(1-7) on c-Met RTK signaling in the context of

PTP1b depletion. Similar to our previous observations, a significant reduction in

HGF-stimulated p-c-Met was observed in 4T1 cells transfected with a NS siRNA and treated with Ang-(1-7) when compared to treatment with HGF alone (Figure

5C). In contrast, transfection with the PTP1b-targeted siRNA impaired the suppressive effects of Ang-(1-7) on HGF-induced c-Met phosphorylation.

The ability of Ang-(1-7) to attenuate HGF-stimulated tumor cell proliferation in the context of PTP1b loss was also examined. A pronounced increase in tumor cell number was observed in NS siRNA transfected 4T1 cells stimulated with HGF

(Figure 5D); in contrast, co-treatment with Ang-(1-7) inhibited HGF-stimulated tumor cell proliferation in NS siRNA transfected cells. However, PTP1b loss in

83 cells treated with PTP1b siRNA prevented the inhibitory effects of Ang-(1-7) on

HGF-facilitated tumor cell proliferation. Collectively, these results suggest that

Ang-(1-7) exerts its suppressive effects on HGF-mediated c-Met RTK phosphorylation and tumor cell proliferation in part by increasing PTP1b protein expression.

Low PTP1b and Mas1 expression correlates with reduced relapsed-free survival for breast cancer patients with basal-like TNBC

The relationship between the expression of PTP1b, Mas1 and c-Met was investigated in a cohort of triple negative breast cancer patients with basal-like disease, to determine whether the expression of these genes correlates with disease severity [234]. Kaplan-Meier analysis demonstrated that the relapse-free survival of patients whose tumors express low levels of PTP1b, the gene encoding the phosphatase PTP1b, was significantly reduced compared to patients with tumors that express high levels of PTP1b (Supplementary Figure

S3A). A decrease in relapse-free survival benefits was also observed in the same patient cohort whose tumors express low levels of Mas1, the gene encoding the Ang-(1-7) receptor Mas (Supplementary Figure S3B). Conversely, patients whose tumors express high c-Met expression correlated with a significant decrease in relapse-free survival compared to patients with low c-Met expression (Supplementary Figure S3C), in support of previous studies implicating elevated c-Met RTK expression as a poor predictor of clinical

84 outcome [84]. These data suggest that expression of PTP1b and Mas1 are inversely correlated with relapse-free survival for breast cancer patients diagnosed with the basal-like subtype of TNBC, whereas high c-Met RTK expression correlates with an increased probability of distant metastasis for this cohort of patients.

Discussion

TNBC is an aggressive form of breast cancer that lacks effective targeted therapies. Previous studies from our group demonstrated that the heptapeptide hormone Ang-(1-7) reduces tumor growth in a number of different types of cancer including breast cancer [163, 166, 204, 206, 209, 212] and is well- tolerated in clinical studies [213-215]. We now report for the first time that Ang-(1-

7) reduces the growth of triple negative breast tumors with an associated decrease in c-Met RTK signaling and an increase in the protein phosphatase

PTP1b, a negative regulator of c-Met. Ang-(1-7) inhibited HGF-mediated c-Met

RTK phosphorylation and activation, tumor cell proliferation and invasion in vitro.

Reduction of PTP1b using selective siRNAs abrogated the suppressive effects of the heptapeptide hormone on c-Met RTK signaling, demonstrating the role of

PTP1b in dephosphorylating and inactivating c-Met kinase. These results suggest that Ang-(1-7) may be an effective targeted therapy for TNBC by positively modulating the phosphatase PTP1b to antagonize c-Met RTK signaling and attenuate tumor growth, as summarized in Figure 6.

The injection of 4T1 cells into the mammary glands of syngeneic BALB/c mice produces breast tumors that are characteristic of human TNBC [235]. We observed a significant decrease in tumor volume and wet weight in mice with 4T1 mammary tumors which were treated with Ang-(1-7) but not in the untreated mice. The reduction in tumor volume by the heptapeptide hormone was associated with a diminished number of Ki67+ proliferative tumor cells and decreased phosphorylation and activation of the pro-proliferative kinases ERK1/2 in the heptapeptide hormone-treated tumors. These results are consistent with the anti-proliferative properties of Ang-(1-7) in animal models of human lung adenocarcinoma and prostate cancer [209, 212].

Using an in silico approach, we analyzed publically available gene expression datasets and showed that the c-Met oncogene is enriched in TNBC cell lines as well as TN breast tumors when compared to non-TNBC samples. The increase in c-Met in TNBC patients is in agreement with clinical evidence showing that the highest c-Met RTK expression is observed in patients with TNBC compared to other types of breast cancer and pre-clinical studies demonstrating that mice with deregulated c-Met activation develop breast cancer that resembles human TNBC

[95, 97], implicating aberrant c-Met RTK signaling in the progression of TNBC.

We showed that select TNBC cell lines (BT-20, BT-549 and MDA-MB-231) contain elevated c-Met immunoreactive protein compared to ER+ or HER-2 overexpressing cell lines, including BT-474, SK-BR-3 and MCF-7 cells. These results suggest that c-Met RTK may be a promising therapeutic target for TNBC patients who are currently treated with cytotoxic therapies including doxorubicin and

86 taxanes which produce cardiotoxicity as well as severe side effects which reduce quality-of-life.

Treatment with Ang-(1-7) produced a significant reduction in c-Met RTK phosphorylation and activation in response to HGF in the human MDA-MB-231 and murine 4T1 TNBC cells. Similarly, decreased phosphorylation of c-Met (p-c-

Met), phosphorylated at tyrosine 1234 and 1235 within the catalytic domain, was observed in Ang-(1-7)-treated 4T1 orthotopic tumors but not in the untreated counterparts. Consistent with c-Met RTK inhibition, Ang-(1-7) administration also blocked HGF-mediated tumor cell proliferation with a concurrent reduction in the phosphorylation and activation of the pro-proliferative kinases ERK1/2 in 4T1

TNBC cells as well as diminished tumor cell invasion through Matrigel in response to HGF stimulation. The inhibitory effects of Ang-(1-7) on HGF-induced c-Met phosphorylation and activation, tumor cell proliferation and tumor cell invasion were blocked by the Mas receptor antagonist, D-Ala, demonstrating that

Ang-(1-7) regulation on c-Met RTK signaling is a receptor-mediated process.

We further defined the regulatory mechanism by which Ang-(1-7) reduces c-Met

RTK signaling in TNBC by demonstrating that the heptapeptide hormone up- regulates PTP1b. PTP1b depletion by siRNA abolished the suppressive effects exhibited by Ang-(1-7) on c-Met phosphorylation and cell growth, to counteract c-

Met RTK activation. Further supporting these findings, we report that decreased expression of PTP1b and Mas1, the genes encoding PTP1b and Mas, respectively, correlates with reduced relapse-free survival of TNBC patients, as shown in an analysis of a cohort of TNBC patients. These observations are

87 consistent with previously published results reporting that PTP1b is an independent positive predictor of improved survival for breast cancer patients

[118].

Several c-Met RTK inhibitors were investigated or are currently being evaluated in pre-clinical models or in phase I/II clinical trials [236, 237]. While data from preclinical studies showed promising results for c-Met inhibitors, the outcome of early phase I/II clinical trials were less encouraging [236, 238]. In a Phase I clinical trial, JNJ-38877605, a selective c-Met inhibitor, caused extensive renal toxicity in patients, ultimately precluding the small molecule inhibitor from further clinical development [238]. Furthermore, in a clinical trial examining the efficacy of cabozantinib in metastatic medullary thyroid cancer, severe adverse events led to dose reductions in 79% of patients and treatment discontinuation in 16% of patients administered with cabozantinib; cabozantinib is an inhibitor of c-Met, the

VEGF receptor and the RET proto-oncogene RTK of the neurotrophic factor family of RTKs [239]. The development of resistance mechanisms to small tyrosine kinase inhibitors such as cabozantinib is also a concern, as resistance to epidermal growth factor receptor (EGFR) inhibitors invariably developed in

NSCLC patients who initially displayed clinical efficacy [240]. The lack of adverse side effects to administration of Ang-(1-7) may be attributed to the observations that treatment with Ang-(1-7) does not completely inhibit critical signaling pathways required for normal cellular processes; Ang-(1-7) administration reduces the growth factors and activated mitogen signaling to basal levels, allowing the normal cellular processes requiring these molecules to still occur.

In contrast to standard anti-angiogenic and anti-proliferative therapeutics which target only a single molecule or a single cellular process implicated in tumor progression, Ang-(1-7) exerts activity against multiple growth factors and downstream effector molecules. The ability of the heptapeptide hormone to target numerous signaling pathways which are implicated in tumor progression may reduce the evasive efforts by tumors to utilize alternative signaling mechanisms to overcome the inhibitory effects of chemotherapeutic drugs. Most importantly,

Ang-(1-7) displayed clinical efficacy with limited toxicity in a Phase 1 clinical trial completed at Wake Forest University Comprehensive Cancer Center [213]. In the

Phase 1 clinical trial, patients with advanced solid tumors refractory to standard therapy were administered Ang-(1-7) in escalating doses from 100 – 700

µg/kg/day through subcutaneous injection for 5 days in 21 day cycles. Four out of

15 evaluable patients exhibited clinical benefits with an associated reduction in the pro-angiogenic factor PlGF; three patients experienced stabilize disease lasting longer than 3 months. In the adjuvant setting, Ang-(1-7) treatment decreased the frequency of high grade thrombocytopenia, anemia and lymphopenia in a breast cancer patient cohort medicated with doxorubicin and cyclophosphadmide, further highlighting the benefits of Ang-(1-7) in the clinical setting [214]. The results of our study, which demonstrate that Ang-(1-7) antagonizes c-Met RTK, an oncogenic molecule enriched in TNBC, provides the preclinical support for evaluation of the heptapeptide hormone in a clinical trial for

TNBC patients to determine whether Ang-(1-7) may serve as a novel targeted therapy for patients diagnosed with TNBC disease.

Acknowledgements

Funding was provided by Komen for the Cure (EAT, PEG), the Randi B. Weiss

Cancer Fund and the Farley-Hudson Foundation (Jacksonville, NC). The Cell &

Viral Vector Laboratory Shared Resource services were supported by the Wake

Forest Baptist Comprehensive Cancer Center’s NCI Cancer Center Support

Grant P30CA012197. The authors thank M. Landrum, T. Shields and R. Lanning for technical assistance.

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International journal of cancer. Journal international du cancer, 2005. 113(4): p. 678-82. 17. Graveel, C.R., et al., Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer. Proceedings of the National Academy of Sciences of the United States of America, 2009. 106(31): p. 12909-14. 18. Ponzo, M.G., et al., Met induces mammary tumors with diverse histologies and is associated with poor outcome and human basal breast cancer. Proceedings of the National Academy of Sciences of the United States of America, 2009. 106(31): p. 12903-8. 19. Knight, J.F., et al., Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(14): p. E1301-10. 20. Santos, R.A., et al., Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(14): p. 8258-63. 21. Gallagher, P.E. and E.A. Tallant, Inhibition of human lung cancer cell growth by angiotensin-(1-7). Carcinogenesis, 2004. 25(11): p. 2045-52. 22. Menon, J., et al., Angiotensin-(1-7) inhibits growth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase-2. Cancer research, 2007. 67(6): p. 2809-15. 23. Cook, K.L., et al., Angiotensin-(1-7) reduces fibrosis in orthotopic breast tumors. Cancer research, 2010. 70(21): p. 8319-28. 24. Soto-Pantoja, D.R., et al., Angiotensin-(1-7) inhibits tumor angiogenesis in human lung cancer xenografts with a reduction in vascular endothelial growth factor. Molecular cancer therapeutics, 2009. 8(6): p. 1676-83. 25. Krishnan, B., et al., Angiotensin-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis. The Prostate, 2013. 73(1): p. 71- 82. 26. Petty, W.J., et al., Phase I and pharmacokinetic study of angiotensin-(1-7), an endogenous antiangiogenic hormone. Clinical cancer research : an official journal of the American Association for Cancer Research, 2009. 15(23): p. 7398-404. 27. Pham, H., et al., Pharmacodynamic stimulation of thrombogenesis by angiotensin (1-7) in recurrent ovarian cancer patients receiving gemcitabine and platinum-based chemotherapy. Cancer chemotherapy and pharmacology, 2013. 71(4): p. 965-72. 28. Rodgers, K.E., J. Oliver, and G.S. diZerega, Phase I/II dose escalation study of angiotensin 1-7 [A(1-7)] administered before and after chemotherapy in patients with newly diagnosed breast cancer. Cancer chemotherapy and pharmacology, 2006. 57(5): p. 559-68. 29. Luo, Y., et al., Expression of MAS1 in breast cancer. Cancer science, 2015. 106(9): p. 1240-1248.

30. Frangioni, J.V., et al., The nontransmembrane tyrosine phosphatase PTP- 1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell, 1992. 68(3): p. 545-60. 31. Sangwan, V., et al., Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase. The Journal of biological chemistry, 2008. 283(49): p. 34374-83. 32. Soysal, S., et al., PTP1B expression is an independent positive prognostic factor in human breast cancer. Breast cancer research and treatment, 2013. 137(2): p. 637-44. 33. Garcia-Espinosa, M.A., et al., In vivo expression of angiotensin-(1-7) lowers blood pressure and improves baroreflex function in transgenic (mRen2)27 rats. Journal of cardiovascular pharmacology, 2012. 60(2): p. 150-7. 34. Neve, R.M., et al., A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer cell, 2006. 10(6): p. 515-27. 35. Cerami, E., et al., The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov, 2012. 2(5): p. 401-4. 36. Gao, J., et al., Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal, 2013. 6(269): p. pl1. 37. Comprehensive molecular portraits of human breast tumours. Nature, 2012. 490(7418): p. 61-70. 38. Gyorffy, B., et al., An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast cancer research and treatment, 2010. 123(3): p. 725-31. 39. Miller, F.R., B.E. Miller, and G.H. Heppner, Characterization of metastatic heterogeneity among subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability. Invasion & metastasis, 1983. 3(1): p. 22-31. 40. Barretina, J., et al., The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature, 2012. 483(7391): p. 603- 7. 41. Pawitan, Y., et al., Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population- based cohorts. Breast cancer research : BCR, 2005. 7(6): p. R953-64. 42. Paumelle, R., et al., Hepatocyte growth factor/scatter factor activates the ETS1 transcription factor by a RAS-RAF-MEK-ERK signaling pathway. Oncogene, 2002. 21(15): p. 2309-19. 43. Gyorffy, B., et al., Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PloS one, 2013. 8(12): p. e82241. 44. Krishnan, B., et al., Angiotensin-(1-7) reduces proliferation and angiogenesis of human prostate cancer xenografts with a decrease in angiogenic factors and an increase in sFlt-1. The Prostate, 2013. 73(1): p. 60-70.

45. Kaur, P., et al., A mouse model for triple-negative breast cancer tumor- initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease. BMC cancer, 2012. 12: p. 120. 46. Goyal, L., M.D. Muzumdar, and A.X. Zhu, Targeting the HGF/c-MET pathway in hepatocellular carcinoma. Clin Cancer Res, 2013. 19(9): p. 2310-8. 47. Ghiso, E. and S. Giordano, Targeting MET: why, where and how? Curr Opin Pharmacol, 2013. 13(4): p. 511-8. 48. Lolkema, M., et al., The c-Met tyrosine kinase inhibitor JNJ-38877605 causes renal toxicity through species specific insoluble metabolite formation. Clinical cancer research : an official journal of the American Association for Cancer Research, 2015. 49. Elisei, R., et al., Cabozantinib in progressive medullary thyroid cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 2013. 31(29): p. 3639-46. 50. Niederst, M.J. and J.A. Engelman, Bypass mechanisms of resistance to receptor tyrosine kinase inhibition in lung cancer. Sci Signal, 2013. 6(294): p. re6.

Figure Legends

Figure 1. Ang-(1-7) administration reduced 4T1 orthotopic triple negative breast tumor growth. (A) Female BALB/c mice with 4T1 triple negative breast tumors in their mammary fat pads were treated for 21 days with Ang-(1-7) [24

µg/kg/h]. Tumor volume (mm3) was measured every 3 to 4 days using a caliper. * denotes p < 0.05. (B) The average tumor volume at the end of treatment period

(day 21) is shown graphically and representative pictures of control and Ang-(1-

7)-treated tumors are shown. ** denotes p < 0.01. (C) Tumors were weighed at the time of euthanasia and wet weights were measured. * denotes p < 0.05.

Values are mean ± SEM for the average volume (A, B) or weight (C); n = 10 - 12.

(D) Ki67 immunoreactivity in tumors from untreated mice or mice treated with

Ang-(1-7) was assessed by immunohistochemistry. The data represent the average number of Ki67+ cells per field, n = 10 - 12. Representative pictures of saline-treated and Ang-(1-7)-treated tumors are shown. Representative micrographs are taken at 200X magnification.

Figure 2. Ang-(1-7) decreased c-Met RTK activation in vitro and in vivo. (A)

4T1 cells and (B) MDA-MB-231 cells were pretreated with Ang-(1-7) [100 nM] for five h prior to stimulation with HGF (30 ng/mL) for 15 min. In experiments where

D-Ala was used, the Mas receptor antagonist was added 15 min prior to the Ang-

(1-7) pretreatment. Lysates were incubated with antibodies specific for phosphorylated-c-Met(Tyr1234/1235) [p-c-Met] or c-Met; α-tubulin was used as a loading control. Representative immunoblots are shown. Total c-Met expression

(represented by the numerical values beneath the c-Met band) was not altered in

95 the experimental conditions assayed. Data are represented as mean ± SEM, n =

3 – 4 independent experiments. (C) Sections from 4T1 tumors from control mice or mice treated with Ang-(1-7) were incubated with an antibody specific to p-MET

(brown stain). Data are represented as the average percentage of p-c-Met+ cells

± SEM, n = 10 - 12. Representative micrographs of a tumor section from a control or Ang-(1-7)-treated mouse are shown along with a section that was incubated with no primary antibody. The bar represents 50 µm. * denotes p <

0.05.

Figure 3. Ang-(1-7) Inhibited c-Met RTK mediated cell proliferation and invasion. (A) 4T1 and (B) MDA-MB-231 cells were seeded overnight and serum- starved for 16 h. Cells were either treated with PBS, HGF (30 ng/mL), HGF with

Ang-(1-7) [100 nM] or Ang-(1-7) alone. In experiments where D-Ala was used, the Mas receptor antagonist was added 15 min prior to HGF and Ang-(1-7). The number of cells was assessed after 48 h. Data are presented as mean ± SEM, n

= 3 independent experiments. (C and D) Lysates from 4T1 tumors from untreated mice or mice treated with Ang-(1-7) were incubated with antibodies to phosphorylated-ERK1/2(Thr202/Tyr204) [p-ERK1/2(Thr202/Tyr204)] and

DUSP1; α–tubulin was used as loading control. (E) Lysates from 4T1 cells treated with PBS, HGF (30 ng/mL), HGF with Ang-(1-7) [100 nM] or Ang-(1-7) alone for 15 min were analyzed by Western blot hybridization using an antibody specific to p-ERK1/2(Thr202/Tyr204), ERK1/2 and α-tubulin. Representative immunoblots are shown. (F) MDA-MB-231 cells were stimulated to invade through Matrigel-coated trans-wells in response to HGF, Ang-(1-7), or HGF co-

96 treated with Ang-(1-7) in the presence or absence of D-Ala. Data are presented as the average number of cells per field ± SEM, n = 3 - 4 independent experiments (2 - 4 replicates per condition). Representative micrographs are shown. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.005, **** denotes p < 0.0001.

Figure 4. Ang-(1-7) up-regulates the phosphatase PTP1b, a negative regulator of c-Met RTK signaling. (A) 4T1 cells were incubated with Ang-(1-7)

[100 nM] for increasing periods of time and PTP1b was measured by Western blot hybridization. Representative immunoblots are shown. The data are expressed as the mean ± SEM, n = 4. (B and C) PTP1b was measured by

Western blot hybridization in lysates from 4T1 or MDA-MB-231 cells treated with

100 nM Ang-(1-7) and stimulated with HGF (30 ng/mL) for 15 min.

Representative immunoblots are shown. The data is expressed as the mean ±

SEM, n = 2 – 3. (D) PTP1b expression was evaluated by immunohistochemistry using a PTP1b-specific antibody in sections from 4T1 tumors from untreated mice or mice treated with Ang-(1-7). The bar represents 40 µm. Data are expressed as percentage of PTP1b+ cells ± SEM, n = 10 – 12. * denotes p <

0.05, ** denotes p < 0.01, *** denotes p < 0.005, **** denotes p < 0.0001.

Figure 5. PTP1b knockdown prevents the Ang-(1-7)-mediated reduction in c-Met RTK Signaling. (A and B) PTP1b mRNA and protein were measured by quantitative RT-PCR of PTPN1 mRNA (A) and Western blot hybridization of

PTP1b immunoreactive protein (B) of 4T1 cells transfected with either nonspecific (NS) siRNA or PTP1b siRNA for 48 h. The data are expressed as

97 mean ± SEM, n = 3. (C) 4T1 cells were transfected with NS siRNA or PTP1b siRNA, followed by treatment as indicated. Cell lysates were collected and analyzed by Western blot hybridization using a specific p-c-Met antibody. The data are expressed as the mean ± SEM, n = 4. (D) 4T1 proliferation was measured in cells 48 h after transfection with NS siRNA or PTP1b siRNA followed by treatment as indicated. The data are expressed as the mean ± SEM, n = 3.

Figure 6. A proposed model of c-Met RTK signaling regulation by Ang-(1-

7). Activation of the Mas receptor by Ang-(1-7) up-regulates DUSP1 and PTP1b to oppose c-Met RTK signaling, to inhibit cancer cell proliferation and invasion.

Supplementary Figure S1. The c-Met Receptor Tyrosine Kinase is enriched in Triple Negative Breast Cancer (TNBC). (A) c-Met expression was assessed in a panel of breast cancer cell lines from the Cancer Cell Line Encyclopedia

(CCLE). [231]. The intrinsic subtype of each breast cancer cell line was derived from Neve et al [226]. The TNBC group encompasses cell lines designated as basal A and basal B (claudin-low) subtype, whereas the non-TNBC group consists of cell lines characterized as luminal subtype. The dashed line represents the average c-Met expression across all breast cancer cell lines in the panel, n = 32. (B) The average c-Met expression between TNBC cell lines (n =

17) and non-TNBC cell lines (n = 16) is shown. (C and D) Met expression was analyzed in two additional datasets derived from clinical specimen, The Cancer

Genome Atlas (TCGA) and GSE1456. The data (B, C and D) are displayed as box and whiskers plots. ** denotes p < 0.01, **** denotes p < 0.0001. (E) Lysates

98 from a panel of TNBC and non-TNBC cell lines were analyzed by Western blot hybridization using an antibody specific for c-Met; β-actin was used as a loading control.

Supplementary Figure S2. Mas receptor expression in TNBC cell lines.

Lysates from the 4T1 murine TNBC cell line and the MDA-MB-231 human claudin-low TNBC cell line were analyzed by Western blot hybridization using an antibody specific for the Mas receptor; β-actin was used as a loading control. A human kidney cortex homogenate was used as positive control for Mas receptor expression.

Supplementary Figure S3. Low PTPN1 and Mas1 expression correlate with reduced relapse-free survival for breast cancer patients with basal-like subtype of TNBC. (A) Kaplan-Meier plots of percent relapse-free survival in patients diagnosed with basal-like subtype of TNBC stratified by median c-Met,

PTPN1 (B), Mas1 (C) expression in the KM Plotter breast cancer meta-analysis database [229].

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112

CHAPTER III: Angiotensin-(1-7) Reduces Triple Negative Breast Cancer Brain Metastatic Growth in Association with Decreased c-Met Signaling

The following manuscript is prepared for submission to Journal of Neuro- Oncology and represents the efforts of the first author. Differences in formatting and organization reflect the requirements of the journal.

113

Angiotensin-(1-7) Reduces Triple Negative Breast Cancer Brain

Metastatic Growth in Association with Decreased c-Met Signaling

Guorui Deng,1, 2 Wenhong Chen,3 Linda Metheny-Barlow,2, 3 E. Ann Tallant,1, 2 and

Patricia E. Gallagher1, 2

1Hypertension and Vascular Research Center, 2Department of Cancer Biology,

3Department of Radiation Oncology

Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Key Words: angiotensin-(1-7), metastatic breast cancer, triple negative breast cancer, c-Met, protein tyrosine phosphatase 1b

Running Title: Ang-(1-7) reduces metastatic breast cancer

Address for Correspondence:

Patricia E. Gallagher, Ph.D. Hypertension and Vascular Research Center Wake Forest University School of Medicine Medical Center Boulevard Winston Salem, NC 27157, USA Telephone: (336) 716-4455 FAX: (336) 716-2456 E-mail: [email protected]

114

Abstract

Patients with triple negative breast cancer often develop metastatic brain disease and have limited treatment options due to the lack of targeted therapeutics that are capable of efficiently crossing the blood brain barrier. In the current study, we investigated the effect of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone with anti-proliferative and anti-metastatic activities, on the growth of murine brain tumors using brain trophic BR5 4T1 cells in a model of metastatic triple negative breast tumor growth. Ang-(1-7) treatment significantly reduced metastatic tumor number and size in the brain, which was associated with decreased Ki67+ proliferating cells and a significant reduction in phosphorylated c-Met, a receptor tyrosine kinase which promotes cancer cell proliferation and facilitates cell invasion. Protein phosphatase 1b (PTB1b), which dephosphorylates and negatively regulates phospho-c-Met, was increased in the tumor cells of mice treated with Ang-(1-7) compared to tumor cells from untreated animals. In support of a role for c-Met signaling in the regulation of metastatic brain disease in breast cancer patients, analysis of publically available breast cancer cohort databases demonstrated a significant correlation between low c-Met/high PTP1b expression and improved survival outcome, including an increase in brain metastasis-free survival and distant-metastasis free survival. This is the first report to demonstrate that Ang-(1-7) reduces metastatic triple negative breast cancer localized to the brain in association with increased protein phosphatase

PTP1b and decreased phospho-c-Met. The results of this study suggest that the heptapeptide hormone may serve as a novel targeted therapy to reduce metastatic brain tumor burden in triple negative breast cancer patients.

115

Introduction

The development of brain metastatic disease can occur in up to 30% of breast cancer patients, resulting in increased morbidity and mortality [241].

Patients diagnosed with triple negative breast cancer (TNBC), an aggressive form of breast cancer, are more likely to develop metastatic disease in the brain than patients diagnosed with non-TNBC [220]. For TNBC patients diagnosed with metastatic brain disease, the survival outcome is dismal, with a median survival of less than 6 months after diagnosis [124]. Targeted therapies for treatment of primary triple negative breast tumors are lacking. In addition, less than 2% of drugs which target metastatic disease in the central nervous system (CNS) obtain approval for clinical use due to suboptimal penetration through the blood- brain barrier (BBB) [242, 243]. The poor penetrance of chemotherapeutics largely limits the treatment options for brain metastases to whole brain radiation therapy, stereotactic radiosurgery and surgical resection [128]. The limited efficacy of these traditional treatments and an increased risk of developing neurocognitive dysfunction greatly decrease the quality of life for patients undergoing these treatments. Stereotactic radiosurgery delivers a single fraction, high dose radiation focally to the tumor but may put a significant patient population at risk for development of neurological complications caused by radionecrosis [129].

Neurocognitive deficits remain the most undesired side effect of whole brain radiation therapy, whereas surgical resection has limited application for patients presented with multi-focal brain tumors. The aggressive nature of metastatic brain disease coupled with the increased prevalence of metastatic brain tumors

116 in TNBC patients necessitate the development of novel effective targeted therapies which target triple negative cancer tumors in the brain.

The initial step in the metastatic cascade is the migration of tumor cells from the primary tumor followed by extravasation into adjacent blood vessels, transport to distant sites through the blood stream, intravasation into tissues and implantation into receptive niches [121]. The c-Met receptor tyrosine kinase

(RTK) and its ligand, hepatocyte growth factor (HGF), orchestrate a complex biological program known as invasive growth by coordinating cell proliferation and cell invasion [46]. While c-Met-driven invasive growth is a physiological program activated during embryonic development and crucial in post-natal tissue growth and regeneration, aberrant activation of the c-Met signaling cascade is implicated in the development of various human malignancies. c-Met signaling promotes disease dissemination through activation of signaling cascades crucial to tumor cell proliferation, invasion and survival [46, 92]. For TNBC patients, elevated c-Met is associated with poor recurrence-free survival and overall survival [90]. Similarly, high levels of c-Met are associated with death resulting from metastatic disease in a cohort of auxiliary lymph node negative breast cancer patients [221]. Moreover, increased lung metastases were observed in a

TNBC mouse model harboring a constitutively active c-Met mutation and knockdown of c-Met resulted in decreased brain metastatic lesions and significantly prolonged survival time in a breast cancer model [97, 136]. These studies underscore a potential role for aberrantly activated c-Met signaling in the development of metastatic tumors for TNBC patients.

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Angiotensin-(1-7) [Ang-(1-7)] is an endogenous heptapeptide hormone of the renin-angiotensin system (RAS) exhibiting pleotropic anti-tumor properties through activation of the unique G protein-coupled receptor Mas [162, 163, 166,

204, 209, 210, 212, 244]. Ang-(1-7) administration attenuated the formation of metastatic prostate cancer cells in the bone in vivo and inhibited human lung adenocarcinoma cell migration and invasion in vitro, demonstrating the potential anti-metastatic properties of the heptapeptide hormone [205, 206]. Furthermore, expression of an Ang-(1-7) fusion protein in the brains of rats resulted in increased expression of the protein phosphatase PTP1b, a negative regulator of c-Met RTK signaling, suggesting that the heptapeptide hormone may negatively regulate c-Met signaling [98, 119]. In the present study, we examined the effects of Ang-(1-7) on metastatic tumor formation in the brain by employing an in vivo model of a brain tropic variant of the murine 4T1 triple negative breast cancer cell line and evaluated the role of PTB1b/c-Met signaling in the response to the heptapeptide hormone.

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Materials and Methods

Reagents and materials

Angiotensin-(1-7) [Ang-(1-7)] was purchased from Bachem (Torrance, CA, USA).

Alzet osmotic mini pumps were obtained from Braintree Scientific, Inc. (Braintree,

MA, USA). Anti-Ki67 and anti-PTP1b antibodies were from Abcam (Cambridge,

MA, USA) and an anti-phosphorylated c-Met (p-c-Met) (Tyr1234/1235) antibody was acquired from Cell Signaling Technology (Danvers, MA, USA). Horse radish peroxidase (HRP)-conjugated secondary antibodies were purchased from GE

Healthcare Life Sciences (Pittsburg, PA, USA).

Derivation of the BR5 4T1 Subline

A brain trophic variant of 4T1 mouse breast cancer cells, BR5 4T1 cells, was generated through intracarotid injection of the parental 4T1 cells into female

BALB/c mice followed by ex vivo expansion of tumor cells capable of metastasizing to the brain. After expansion, these cells were re-injected into

BALB/c mice to further select for cells with an enhanced capacity to establish tumors in the brain. This procedure was repeated five times to generate the BR5

4T1 subline which was subsequently used for this study.

Generation of Metastatic Triple Negative Breast Tumor in the Brain

To generate metastatic triple negative breast tumors in the brain, 1.25 x 104 BR5

4T1 cells were injected into the carotid arteries of twenty BALB/c mice in 25 µL of

PBS. Two days post-surgery, mice were randomly assigned to an untreated

119 control group or a treatment group. Mice in the treatment group received a constant infusion of 24 µg/kg/h Ang-(1-7) in saline delivered by osmotic mini- pumps subcutaneously implanted in the subscapular space. Mice were euthanized after 20 days of treatment and the brains were collected for analysis.

All animal protocols were approved by the Wake Forest School of Medicine

Animal Care and Use Committee.

Morphological Analysis

Brains were fixed in 4% paraformaldehyde for 24 h and incubated in 70% ethanol for an additional 48 h prior to paraffin embedding. The embedded tissues were sectioned into 10 µm thick serial coronal sections through the entire length of the brain. For morphological analysis, every tenth section was stained with hematoxylin and eosin (H & E). Each section was evaluated for the presence of tumors and tumor area was measured using the ImageJ software (NIH).

Immunohistochemistry

Paraffin-embedded brain sections were deparaffinized in xylene and rehydrated in graded alcohols prior to antigen retrieval in citrate buffer (Target Retrieval

Solution, Dako, Carpinteria, CA) for 40 min. Sections were incubated with an antibody against Ki67 (1:100), to visualize actively proliferating cells. Additional sections were incubated with antibodies against p-c-Met (Y1234/1235) (1:100) to identify tumor cells with activated c-Met RTK and with anti-PTP1b (1:100) to highlight PTP1b positive cells. Brain sections from each animal were stained with each antibody and four representative regions per tumor section were acquired

120 with a Leica DM 4000 microscope. The average percentage of positively-stained cells was quantified using the ImageJ software (NIH).

Kaplan-Meier Analysis

The Affymetrix datasets from GSE2034 were downloaded from the NCBI Gene

Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) to evaluate the correlation between the expression of genes of interest (c-Met and PTP1b) and brain metastases-free survival. The expression values for all c-Met and PTP1b probes were used to obtain an average expression value for the respective gene for each patient. The patient population was stratified into high and low levels of expression based on whether the value for the gene is above or below the average expression of the gene for the cohort.

The correlation between distant metastasis-free survival and the expression of genes of interest (c-Met and PTP1b) was assessed using the online survival analysis tool KMPlot (http://kmplot.com/analysis/). For genes with multiple probes

(c-Met, PTP1b), the average expression of all probes was used. Patients presenting with the basal-like intrinsic subtype of breast cancer from the 2014 version dataset were used for the survival analysis.

Statistical Analysis

All data are presented as the mean ± SEM. Statistical significance was evaluated by Student’s t test when comparing two groups of data. For the tumor area comparison between untreated and Ang-(1-7)-treated cohort, a Chi-Square test was performed. Statistical comparisons were performed using Prism 6

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(GraphPad Prism Software Version 6.04; La Jolla, CA, USA). Differences were considered significant at p < 0.05.

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Results

Ang-(1-7) administration reduced the growth of 4T1 triple negative breast cancer brain metastases

Brain-tropic BR5 4T1 cells were injected into the internal carotid artery of female BALB/c mice, to investigate the effects of Ang-(1-7) treatment on TNBC brain metastatic disease. BR5 4T1 cells are a metastatic brain tropic variant of the murine 4T1 triple negative breast cancer cell line developed by five consecutive rounds of selection of parental 4T1 cells capable of establishing metastatic tumors in the brain after intracarotid injections. An average loss of

10% of body weight was observed during the first two days following intracarotid injection and the mice were supplied with wet chow during this time period. No further pronounced weight loss was observed. Two days after the tumor cells were injected, the mice were randomly assigned to the untreated control group or a treatment group; osmotic mini-pumps to administer Ang-(1-7) were implanted subcutaneously in the midscapular space. The mice in either treatment group developed no neurological symptoms and the animals remained alert and active throughout the treatment period. After 20 days, the mice were euthanized and their brains were harvested for subsequent analysis.

The brain of each mouse was cut into 10 µm serial sections and every 10th section was stained with H & E to identify brain tumors. An approximate average of 20 metastatic lesions per mouse was observed in the brains of the untreated control cohort; in stark contrast, an average of 3 metastatic tumors per mouse was observed in the Ang-(1-7)-treated cohort. Metastatic lesions were stratified

123 into rostal (sections 1-70), mid-rostal (sections 71-140), mid-caudal (sections

141-210) and caudal (sections 211-280) regions. A significant difference in the number of metastatic tumors between the untreated control mice and the Ang-(1-

7)-treated mice was observed across the four regions; administration of the heptapeptide hormone produced a marked reduction in tumor number compared to the control cohort (p <0.0001, Chi Square test; Figure 1). Furthermore, infusion with the heptapeptide hormone resulted in a marked decrease in tumor size as shown in Figure 2A; a greater than 17-fold reduction in tumor area was observed in the Ang-(1-7)-treated tumors compared to the untreated control tumors (220,254 ± 103,029 µm2 for control tumors, 12,430 ± 2,536 µm2 for Ang-

(1-7)-treated tumors) [Figure 2B].

Sections of tumors from untreated control mice or Ang-(1-7) were stained with Ki67, a marker of cell proliferation. Tumors from the untreated mice displayed enhanced Ki67 positivity relative to tumors from the Ang-(1-7)-treated mice (Figure 3A). The number of cells per representative region that had positive

Ki67 immunostain in tumors from control mice was 29.1 ± 6.7% compared to only

6.4 ± 2.2% in tumors from mice treated with Ang-(1-7), as shown in Figure 3B.

This reduction in Ki67 immunoreactivity is in agreement with the decreased size of the tumors from Ang-(1-7)-treated mice but not the tumors in the untreated cohort. Taken together, these results suggest that Ang-(1-7) attenuated metastatic triple negative breast carcinoma cell proliferation to decrease metastatic tumor burden in the brain.

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Treatment with Ang-(1-7) attenuated c-Met pathway activation in 4T1 brain metastases

Aberrant activation of the c-Met RTK signaling pathway facilitates tumor cell invasion and metastasis. Enhanced c-Met RTK activation is associated with the development of brain metastasis in patients with non-small cell lung carcinoma [27]. Depletion of c-Met RTK using shRNA significantly reduced the size of brain metastasis and prolonged the survival of mice bearing metastatic brain tumors compared to the control cohort in a model of human breast carcinoma [14]; a comparison of the expression of c-Met in primary MDA-MB-435 tumors in mice and brain metastatic breast tumors from this model demonstrated increased c-Met expression in the metastatic tumors relative to the primary tumors [14]. These studies underscore the potential role of c-Met RTK signaling in promoting brain metastatic disease.

Paraffin-embedded brain sections were incubated with an antibody against the activated and phosphorylated c-Met RTK (at tyrosine residues

1234/1235, p-c-Met), to examine the effects of Ang-(1-7) administration on the c-

Met RTK pathway. Enhanced p-c-Met immunoreactivity was observed in tumors from untreated mice relative to tumors from Ang-(1-7)-treated mice (Figure 4A).

As shown in Figure 4B, treatment with the heptapeptide hormone significantly reduced the percentage of metastatic tumor cells exhibiting p-c-Met immunostain as compared to the untreated cohort, from 20.8 ± 3.4% in tumors from untreated mice to 6.7 ± 2.6% in mice treated with Ang-(1-7).

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The protein tyrosine phosphatase PTP1b is a negative regulator of c-Met

RTK signaling by dephosphorylating tyrosine residues 1234/1235 to inactivate the oncogenic kinase. Previous studies demonstrated that Ang-(1-7) administration increased the expression of PTP1b in rat brain [98, 119]. Brain sections were incubated with an antibody to PTP1b to determine whether PTP1b expression is increased in response to Ang-(1-7) treatment in the 4T1 brain metastatic model. Increased PTP1b+ cells were observed in Ang-(1-7)-treated, but not the untreated mice (Figure 4C). As shown in Figure 4D, the percentage of cells expressing PTP1b was significantly higher in the brain tumors of mice receiving treatment with the heptapeptide hormone when compared to the untreated cohort, from 4.1 ± 1.4% in sections of tumors from control mice compared to 8.8 ± 0.9% in sections of tumors from mice treated with Ang-(1-7).

These results suggest that Ang-(1-7) increases PTP1b to antagonize c-Met RTK signaling and decrease triple negative breast cancer brain metastasis.

Expression of c-Met RTK and PTP1b correlates with breast cancer brain metastasis

Various databases with annotated clinical information on breast cancer patients are available to assess the clinical relevance between expression of specific genes and metastatic status. The breast cancer patient cohort GSE 2034 contains 180 lymph-node negative relapse-free patients and 106 lymph-node negative patients that developed a distant metastasis, with information on the time until relapse and ER gene expression [245]. Analysis of this breast cancer

126 patient cohort indicated that elevated c-Met RTK expression in the primary tumor is significantly correlated with decreased brain metastasis-free survival when compared to patients with low c-Met RTK expression (Figure 5A). In addition, patients whose primary tumors express low levels of PTP1b correlated with reduced brain metastasis-free survival, as shown in Figure 5B.

The correlation between c-Met RTK expression and distant metastasis- free survival in breast cancer patients was also carried out using the KM-Plotter database [229]. This database contains and integrates gene expression data with relapse free and overall survival information from the European Genome- phenome Archive and The Cancer Genome Atlas. Analysis of this breast cancer cohort indicated that high c-Met RTK expression was significantly associated with reduced distant metastasis-free survival in patients with breast tumors of the basal intrinsic subtype (Figure 5C). Consistent with the negative regulatory role of PTP1b on c-Met RTK signaling, low PTP1b expression was a predictor of poor distant metastasis-free survival for these breast cancer patients (Figure 5D).

Collectively, the results of these in silico analysis suggest that c-Met RTK and

PTP1b expression may be potential biomarkers for a risk of metastatic disease in patients with triple negative breast tumors.

Discussion

We report for the first time that treatment of brain metastatic TNBC with

Ang-(1-7) significantly reduces tumor growth. Ang-(1-7) is an endogenous

127 heptapeptide hormone that demonstrates pleotropic anti-cancer activities in different types of cancer, including breast cancer [246]. Our laboratory previously reported that the heptapeptide hormone inhibits the metastatic growth of prostate cancer cells in the bone as well as reduces the growth and formation of metastatic triple negative breast tumor growth in the lungs of experimental mouse models [206]. In the current study, we evaluated the effects of the heptapeptide hormone on metastatic brain tumor formation and growth using brain tropic BR5 4T1 cells, which were derived from the murine TNBC cell line

4T1 by five cycles of injection/reinjection into the brain parenchyma.

Administration of the heptapeptide hormone significantly reduced the size as well as the number of the metastatic tumors in the brain compared to tumors in the untreated control mice. This reduction in tumor size was associated with a pronounced decrease in actively proliferating tumor cells, visualized using an antibody against Ki67. These results suggest that Ang-(1-7) reduces the growth of metastatic TNBC tumors in the brain by reducing cell proliferation. The reduction in tumor number in mice medicated with Ang-(1-7) compared to untreated mice is in agreement with previous studies showing that pretreatment with Ang-(1-7) prevented the development of metastatic lesions in the bones of mice after injection of prostate cancer PC3 cells [206]. Collectively, these results suggest that the heptapeptide hormone attenuates breast cancer cell proliferation to reduce metastatic tumor burden.

The reduction in the size and number of metastatic TNBC tumors in the brains of mice treated with Ang-(1-7) was associated with an increase in the

128 protein phosphatase PTP1B and a reduction in c-Met signaling. We observed a pronounced reduction in the number of cells which stained positively when incubated with antibody against p-c-Met using tumors from mice medicated with

Ang-(1-7) compared to tumors from untreated control mice. This decrease in p-c-

Met positive tumor cells in mice administered the heptapeptide hormone was associated with an increase in the number of cells which stained positively with an antibody to PTP1b, a negative modulator of c-Met RTK signaling. The reduction in c-Met RTK and the increase in PTP1b that we observed in mice with

TNBC brain metastases treated with Ang-(1-7) is consistent with our previous studies demonstrating a regulatory role of the heptapeptide hormone in c-Met

RTK signaling in primary 4T1 tumors; we previously demonstrated that Ang-(1-7) administration antagonized c-Met RTK signaling in two different TNBC cell lines—the MDA-MB-231 human TNCB cell line and in murine 4T1 TNBC cells, as well as in in vivo tumor xenografts [247]. c-Met RTK activation facilitates several key cellular processes crucial for cancer cells metastasis including cell proliferation, migration and invasion [92]. In addition, elevated c-Met protein expression was associated with death due to metastatic disease in a cohort of breast cancer patients [221]. Further support for the pro-metastatic role by c-Met

RTK signaling was provided by gene silencing experiments where shRNA- mediated knockdown of c-Met markedly decreased the size of metastatic brain tumors size as well as enhanced cumulative survival of tumor bearing mice [136].

Lastly, in silico analysis of patient cohort databases demonstrated that the expression of c-Met RTK and PTP1b correlate with survival outcome in breast

129 cancer patients with metastatic brain tumors. These results indicate a role for the c-Met/PTP1b signaling pathway in brain metastatic disease.

The anti-cancer properties exhibited by Ang-(1-7) are elicited through binding to and activating the unique G-coupled protein receptor, Mas [162, 209].

The Mas receptor is present in the brains of rats and mice [248, 249] and activation of the Ang-(1-7)/Mas receptor signaling axis in the brain has beneficial roles in lowering blood pressure and attenuating cardiac hypertrophy in hypertensive rats [250]. In our studies, the heptapeptide hormone was effective in reducing the size and number of metastatic TNBC brain tumors when administered by a peripherally-implanted osmotic mini-pump. Although the BBB is a physical barrier that precludes the entry of most drugs into the brain, the integrity of the BBB appears to be compromised during pathological states [251,

252]. Indeed, Yonemori and colleagues reported that metastatic brain tumors arising from patients diagnosed with triple negative breast disease often compromise the integrity of the BBB when compared to patients bearing HER2+ breast tumors [253]. Thus, Ang-(1-7), a seven amino acid peptide, may gain entry into the brain parenchyma through a leaky BBB to elicit the anti-tumor effects. Alternatively, the heptapeptide hormone may enter the brain through circumventricular organs, which are highly vascular structures in the brain that lack a BBB [254, 255].

The efficacy of c-Met RTK inhibitors in various malignancies was evaluated in several phase I/II clinical trials and additional trials are currently in progress [256, 257]. However, the outcome of these clinical trials was

130 discouraging. Extensive renal toxicity was reported in patients treated with the c-

Met RTK inhibitor JNJ-38877605 [238]. Patients with metastatic medullary thyroid cancer treated with the c-Met RTK inhibitor cabozantinib experienced elevated frequency of severe adverse events, leading to treatment discontinuation in 16% of patients [239]. In addition, acquired resistance to small tyrosine kinase inhibitors such as the c-Met inhibitor cabozantinib is also a major concern, as non-small cell lung cancer patients who initially displayed clinical efficacy to epidermal growth factor receptor (EGFR) inhibitors eventually became non- responsive to treatment [258].

Ang-(1-7) administration to patients with advanced solid tumors refractory to standard therapy resulted in clinical efficacy with limited toxicity in a phase I clinical trial [213]. Four of 15 evaluable patients exhibited clinical benefits with an associated reduction in the pro-angiogenic factor, PlGF. Disease stabilization lasting longer than 3 months was observed in three patients. The heptapeptide hormone also exhibited clinical benefit in the adjuvant setting in a group of breast cancer patients receiving doxorubicin and cyclophosphamide as treatment [214].

In this clinical trial, Ang-(1-7) treatment decreased the frequency of high grade thrombocytopenia, anemia and lymphopenia when compared to filgrastim. Ang-

(1-7) has anti-proliferative, anti-angiogenic, anti-fibrotic, anti-inflammatory and anti-metastatic effects, through regulation of multiple signaling pathways, thereby minimizing the evasive efforts of tumors to utilize alternative signaling pathways to overcome the inhibitory effects exerted by the heptapeptide hormone.

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The survival outcome is poor for patients diagnosed with brain metastatic disease and patients with TNBC are more likely to development metastasis to the brain than patients with other types of breast cancer [220]. Treatment modalities for brain metastases are restricted to whole brain radiation, stereotactic radiosurgery and surgical resection; however, the increased risk for developing neurocognitive dysfunction and the limited efficacy of the current treatment modalities demonstrate the critical need to identify new targeted therapies for patients with brain metastatic disease. Our results indicate that Ang-(1-7) administration reduced metastatic triple negative breast tumor burden in the brain, with an associated decrease in c-Met RTK activation, suggesting that the heptapeptide hormone may serve as a novel targeted therapy for patients with disseminated triple negative breast disease.

Acknowledgements

Funding was provided by Komen for the Cure (EAT, PEG), the Randi B. Weiss

Cancer Fund and the Farley-Hudson Foundation (Jacksonville, NC). The Cell &

Viral Vector Laboratory Shared Resource services were supported by the Wake

Forest Baptist Comprehensive Cancer Center’s NCI Cancer Center Support

Grant P30CA012197. The authors thank M. Landrum, T. Shields and R. Lanning for technical assistance as well as B. Westwood for statistical analysis

132

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Figure Legends

Figure 1. Treatment with Ang-(1-7) reduced BR5 4T1 metastatic TNBC tumor number in the brains of BALB/c mice. Metastatic lesions were stratified into rostal (sections 1-70), mid-rostal (sections 71-140), mid-caudal (sections

141-210) and caudal (sections 211-280) regions. A significant difference in tumor number was observed between the untreated control cohort and the Ang-(1-7)- treated cohort. *** denotes p < 0.001, Chi square test.

Figure 2. Ang-(1-7) administration decreased the size of brain metastatic TN tumors. (A) Representative pictures of untreated control tumors and Ang-(1-7)- treated tumors. (B) Average area of tumors from female BALB/c mice bearing

BR5 4T1 metastatic brain tumors treated with Ang-(1-7) (24 µg/kg/h) delivered via osmotic mini-pumps for 20 days or untreated mice. Representative regions were acquired at 100x magnification or 200x magnification (inset). * denotes p <

0.05.

Figure 3. Ang-(1-7) treatment reduced TNBC metastatic tumor proliferation.

(A) Representative pictures of brain sections from untreated control mice or mice treated with Ang-(1-7) incubated with an antibody against Ki67. (B) The number of positive cells was expressed as a percentage of 100 cells sampled per representative field and four representative fields per brain section were analyzed. The data represent the average number of Ki67+ cells per field.

Representative pictures of tumors from untreated and Ang-(1-7)-treated tumors, acquired at 100x magnification or 200x magnification (inset), are shown and a dashed red line outlines each tumor. * denotes p < 0.05.

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Figure 4. Ang-(1-7) mitigated c-Met RTK pathway activation in BR5 4T1 brain metastatic tumors. Representative pictures of sections from the brains of untreated and Ang-(1-7)-treated mice incubated with an antibody against p-c-Met

(A) or PTP1b (C). Data are representative of the average percentage of cells which stained positively for p-c-Met (B) or PTP1b (D) by sampling from 100 cells in four representative fields per section from each mouse brain. Representative micrographs of tumors from untreated and Ang-(1-7)-treated mice were, visualized at 100x magnification or 400x magnification (inset). A dashed red line outlines each tumor. * denotes p < 0.05.

Figure 5. c-Met RTK and PTP1b expression correlated with the metastatic status of breast cancer patients. Kaplan-Meier plots of brain metastasis free survival in breast cancer patients stratified by mean gene expression of c-Met (A) and PTP1b (B) from GSE2034 [245]. The correlation between c-Met (C) and

PTPb1 (D) gene expression and distant metastasis free survival (DMFS) was evaluated in patients presented with the basal-like subtype of TNBC from the KM

Plot breast cancer meta-analysis database [229]. * denotes p < 0.05.

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CHAPTER IV: General Discussion

According to the American Cancer Society, an estimated 234,000 new cases of breast cancer were diagnosed in 2015 [259]. Triple negative breast cancer (TNBC) constitutes approximately 15-20% of all diagnosed breast cancer cases [220]. TNBC is an aggressive form of the disease characterized by the lack of expression of the estrogen receptor (ER) and the progesterone receptor

(PR) and TNBC cells do not overexpress the human epidermal growth factor receptor 2 (HER2) oncoprotein. Thus, unlike patients presenting with ER+ or

HER2 amplified breast cancer who can benefit from targeted therapies such as selective ER modulators (SERM, i.e. Tamoxifen) or the HER2-targeted monoclonal antibody Herceptin, there are currently no clinically-approved targeted therapies available for patients diagnosed with TNBC. As a consequence, cytotoxic drugs such as anthracyclines and taxanes remain the standard of care for treatment of this aggressive disease. Prolonged exposure to these cytotoxic chemicals often results in severe side effects, including quality-of- life issues and cumulative dose-dependent cardiotoxicity [260, 261]. In addition, the five-year survival outcome for TNBC patients remains at 77%, as compared to 93% for patients with non-TNBC. Furthermore, patients with triple negative

(TN) breast tumors experience a greater likelihood of disease relapse and distant metastases [220]. The increase in mortality and lack of targeted therapy underscores the critical need for identifying actionable targets for TNBC patients.

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Emerging studies established a causal link between deregulated c-Met receptor tyrosine kinase (RTK) signaling and TNBC progression and disease dissemination. RTK c-Met and its ligand - hepatocyte growth factor (HGF) - play crucial roles in embryonic development and organogenesis by regulating cell proliferation, cell migration and invasion [46, 92]. Aberrant c-Met RTK signaling is associated with tumor growth and disease relapse in a spectrum of human malignancies [46]. Knight and colleagues reported that c-Met activation induces formation of mammary tumors with pathological and molecular features of the claudin-low subtype of TNBC, a distinct subtype of TNBC, in the absence of

Tp53 [97]. Ponzo and colleagues demonstrated that mice harboring constitutively active c-Met mutations develop mammary tumors with a gene expression profile that clusters with human basal-like breast cancer, another subtype of TNBC [95].

Additional analysis of clinical specimens showed that the highest c-Met RTK expression was observed in the basal-like subtype of TNBC [95, 224]. Taken together, insight from clinical observations and experimental studies underscore the potential role that aberrant c-Met RTK signaling may play in TNBC initiation and progression and suggest the c-Met RTK signaling pathway as a potential targeted therapy for TNBC patients.

Studies demonstrated that the seven amino acid peptide hormone, angiotensin-(1-7) [Ang-(1-7)], exhibits pleotropic anti-tumor activities through regulation of multiple signaling cascades crucial for cancer growth and progression [163, 166, 205, 209, 210]. Ang-(1-7) is an endogenous peptide of the

149 renin-angiotensin system (RAS) initially identified as a biologically active peptide with vasodilatory properties, by binding to and activating the unique G-protein coupled receptor (GPCR) Mas [162, 262]. Seminal studies by Tallant and colleagues demonstrated that the heptapeptide hormone exhibits anti- proliferative actions in rapidly proliferating vascular smooth muscle cells in response to vascular injury and fetal bovine serum (FBS) stimulation [164, 167].

Subsequent studies showed that Ang-(1-7) inhibited the proliferation and growth of human lung cancer cells and lung tumor xenografts [166, 204]. The anti-tumor repertoire of the heptapeptide hormone was extended to include inhibition of angiogenesis, cancer cell metastasis, inflammation and fibrosis in a wide spectrum of cancer cell lines and corresponding tumor xenografts [246]. While a detailed signal transduction pathway mediated by the Ang-(1-7)/Mas receptor interaction has yet to be fully elucidated, evidence suggests a critical role of the heptapeptide hormone as a modulator of protein kinase activation through regulation of protein phosphatases. The anti-proliferative effect demonstrated by

Ang-(1-7) is associated with decreased phosphorylation and activation of the growth-promoting signal transducers, extracellular regulated kinases 1/2

(ERK1/2) [163, 169, 204]. The phosphatase DUSP1 (dual specificity phosphatase 1) is a negative regulator of ERK1/2 signaling, dephosphorylating phospho-ERK1/2 on both tyrosine and threonine residues. DUSP1 expression is increased in response to Ang-(1-7) and this correlated with decreased ERK1/2 phosphorylation and reduced breast tumor burden [163], implicating activation of

150 a protein phosphatase and dephosphorylation of growth-stimulated protein kinases as a mechanism of action for the heptapeptide hormone.

The phosphatase PTP1b negatively regulates c-Met RTK signaling by dephosphorylating c-Met at tyrosine residues 1234/1235 within the catalytic kinase domain [98, 99]. Introduction of an Ang-(1-7) fusion peptide increased the expression of PTP1b in the brains of transgenic rats, suggesting a potential regulatory role for the heptapeptide hormone in c-Met RTK signaling [119].

Therefore, we hypothesized that Ang-(1-7) inhibits the growth and progression of

TNBC through modulation of the c-Met RTK signaling pathway.

The effects of Ang-(1-7) regulation on c-Met RTK signaling in primary triple negative breast tumors

The growth inhibitory effect of Ang-(1-7) in ER+ and HER2-amplified breast cancer cells in vitro and corresponding orthotopic breast tumor in vivo through reductions in the ERK1/2 signaling was reported previously [163]. In this current study, we examined the effects of Ang-(1-7) treatment on proliferation and c-Met RTK signaling in TN breast tumor growth and progression.

Dysregulated c-Met RTK signaling was implicated in the initiation and progression of TNBC, establishing this oncogenic kinase as a potential target for

TNBC patients.

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The anti-tumor properties of Ang-(1-7) in TNBC were tested using the syngeneic murine 4T1 TNBC mice model in vivo. The 4T1 cell line was established from a spontaneously arising mammary tumor in BALB/c mice by

Miller and colleagues [263]. 4T1 cells express basal markers such as the epidermal growth factor receptor (EGFR) and cytokeratin (CK) 5/6 but do not express ER or PR nor do they overexpress the oncoprotein HER2, establishing this murine mammary tumor cell as a basal-like subtype of TNBC [235, 264].

We injected 4T1 cells into the mammary fat pad of BALB/c mice and treated the animals with Ang-(1-7). A significant reduction in tumor volume was observed in the cohort medicated with the heptapeptide hormone but not the untreated animals. Consistent with these findings, the wet weight of harvested tumors from mice treated with Ang-(1-7) was markedly reduced compared to mice from the control group. Excised tumors were sectioned and immuno-stained with an antibody against the cell proliferation marker, Ki67, to assess whether

Ang-(1-7) regulates tumor cell growth. Tumors from the Ang-(1-7)-treated cohort displayed a significant reduction in the number of Ki67-positive cells. This observation is consistent with previous publications reporting the anti-proliferative activity of Ang-(1-7) in animal models of human lung adenocarcinoma and prostate cancer [166, 212] .

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Elevated ERK1/2 expression and activity were observed in human primary breast tumor tissues when compared to normal adjacent tissues and increased

ERK1/2 expression was identified as a negative prognostic factor for relapse-free survival of breast cancer patients [265]. We observed a reduction in activated

ERK1/2 which was associated with increased expression of its negative modulator, DUSP1. The observation that Ang-(1-7) up-regulates DUSP1 to attenuate ERK1/2 signaling is consistent with previous studies demonstrating a regulatory role for the heptapeptide hormone on the pro-proliferative kinases

ERK1/2 in breast cancer cell lines in vitro as well as ER+ and HER2+ breast cancer tumors in vivo [163].

Using an in silico approach, we queried publically available gene expression datasets and showed that the c-Met oncogene is enriched in TNBC cell lines as well as TN breast tumors when compared to non-TNBC samples.

This observation is consistent with previous studies reporting elevated c-Met expression in TN breast tumors of the basal-like and claudin-low subtypes [40,

44, 69, 93, 94]. We further validated our in silico observations by screening a panel of breast cancer cell lines for expression of the c-Met RTK. Consistent with our in silico analysis, TNBC cell lines (BT-20, BT-549 and MDA-MB-231) expressed significantly higher level of the c-Met protein compared to non-TNBC cell lines (BT-474, SK-BR-3 and MCF-7). Based on the observations that Ang-(1-

7) positively modulates the expression of PTP1b, the phosphatase responsible for inactivating c-Met RTK signaling, we investigated whether the heptapeptide hormone exerts a regulatory role on the HGF/c-Met signaling axis.

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We demonstrated that Ang-(1-7) treatment antagonizes c-Met RTK activation by its ligand HGF in murine 4T1 TNBC cells and human MDA-MB-231

TNBC cells. Stimulation with HGF resulted in robust phosphorylation at

Tyr1234/1235 within the catalytic domain of c-Met, indicative of kinase activation

[48, 266-268]. Ang-(1-7) significantly reduced the HGF-mediated receptor activation. Total c-Met protein expression was not altered in response to treatment, suggesting that Ang-(1-7) specifically modulates c-Met RTK phosphorylation under these experimental conditions. The cellular responses exerted by Ang-(1-7) are facilitated through activation of the unique G protein- coupled receptor (GCPR) Mas [162, 169, 204]. We verified that the suppressive effect of Ang-(1-7) on HGF induced c-Met RTK phosphorylation was mediated by the Mas receptor, as co-treatment with the selective Mas receptor antagonist D-

Ala abrogated the inhibitory effects exerted by the heptapeptide hormone.

Activation of c-Met RTK by HGF is a potent inducer of cancer cell proliferation [269]. Consistent with the ability of Ang-(1-7) to mitigate c-Met RTK phosphorylation and activation in response to its ligand, HGF-mediated cancer cell proliferation was markedly decreased in the 4T1 and MDA-MB-231 cells when co-treated with the heptapeptide hormone. Phosphorylation and subsequent activation of the mitogen-activated protein kinase (MAPK) ERK1/2 by c-Met RTK is a major signaling pathway facilitating cell proliferation. 4T1 cells

154 stimulated with HGF displayed marked phosphorylation of ERK1/2 at threonine

(Thr) 202 and tyrosine (Tyr) 204, indicative of kinase activation when compared to the unstimulated cells [270] and co-treatment with Ang-(1-7) blunted the phosphorylation of ERK1/2 by HGF.

In addition to providing a strong proliferative signal to cancer cells, c-Met activation can also promote cancer cell invasion. We examined the ability of Ang-

(1-7) to attenuate HGF-induced cancer cell invasion in vitro using a Boyden chamber invasion assay. Ang-(1-7) markedly reduced MDA-MB-231 cell invasion through Matrigel-coated transwells in response to HGF stimulation, and the Ang-

(1-7)-mediated suppressive effect was blocked by the Mas receptor antagonist

D-Ala, suggesting that the heptapeptide hormone activates the Mas receptor to elicit its antagonistic effects on c-Met RTK signaling. The ability of Ang-(1-7) to reduce c-Met-mediated cancer cell invasion further underscores the heptapeptide as a negative modulator of cancer metastasis, consistent with previous studies purporting the anti-metastatic effects of the heptapeptide hormone [205, 206,

271].

The antagonistic effect of Ang-(1-7) on c-Met RTK signaling was also assessed in vivo by incubating tumor sections with an antibody against the activated and phosphorylated c-Met (p-c-Met) RTK. We observed a prominent reduction in the percentage of cells stained with the antibody against

155 phosphorylated c-Met RTK in the tumors from Ang-(1-7)-treated mice when compared to the untreated cohort. The reduction in phosphorylated c-Met in tumors medicated with the heptapeptide hormone is associated with an increase in PTP1b, suggesting that Ang-(1-7) negatively regulates c-Met RTK signaling in vitro as well as in vivo by positively modulating the expression of a tyrosine phosphatase. The regulation of c-Met RTK and PTP1b in TNBC suggests a novel mechanism by which Ang-(1-7) inhibits breast cancer cell and tumor growth, underscoring a promising therapeutic target for future clinical investigations.

Our experimental findings demonstrate that Ang-(1-7) antagonizes c-Met

RTK signaling through positive modulation of PTP1b and thus would provide survival benefits for patients with tumors expressing high levels of Mas1 and

PTP1b, the genes encoding for the Mas receptor and PTP1b, respectively. We confirmed that elevated Mas1 expression is correlated with a better relapse-free survival for breast cancer patients presented with the basal-like subtype of

TNBC. In this same patient cohort, those with tumors expressing elevated levels of PTP1b also had an increase in relapse-free survival when compared to low expressers. Based on our findings, we conclude that PTP1b expression is a good prognostic marker for breast cancer patients with basal-like TNBC. Our observations that PTP1b is a beneficial prognostic factor are consistent with previously published reports. The correlation between PTP1b expression and clinical outcome was evaluated in more than 1,300 Swedish breast cancer patients. In this study, Soysal and colleagues reported that PTP1b is an

156 independent predictor of improved survival in breast cancer patients [118].

Genetic models demonstrated that loss of PTP1b in p53 deficient mice led to lymphomagenesis and decreased survival rate, providing evidence that the phosphatase may exert tumor suppressive functions in multiple malignancies

[112].

The molecular mechanism by which Ang-(1-7) modulates PTP1b expression is not clearly defined. PTP1b is a critical regulator of leptin signaling and the expression of the phosphatase can be positively modulated by the adipokine, adiponectin, to oppose leptin-mediated cellular response [272].

Several studies reported the anti-tumor actions of adiponectin in models of breast and esophageal cancer. Incubation with adiponectin stimulated PTP1b expression in human breast cancer cells to antagonize leptin-mediated cancer cell proliferation, migration and invasion, whereas the inhibition of PTP1b abrogated the suppressive effects of adiponectin on the leptin-induced oncogenic effects [273]. Similarly, Beales and colleagues reported that adiponectin antagonizes leptin-facilitated Janus Kinase 2 (JAK2) signaling to inhibit esophageal cancer cell proliferation and migration by elevating PTP1b expression [274]. In contrast, the addition of TCS 401, a specific PTP1b inhibitor, reversed the adiponectin-mediated suppression, leading the authors to conclude that PTP1b is a central mediator of the anti-cancer effects exhibited by adiponectin. Ang-(1-7) also confers a beneficial role by protecting against oxidative stress and dysregulated glucose metabolism under pathological

157 disease states such as obesity and diabetes in animal models of high-fat diet and glucose intolerance. Introduction of an Ang-(1-7) fusion protein augmented the expression of adiponectin in adipose tissues to improve lipid and glucose metabolism [275]. Treatment of primary adipocytes from C57 mice with Ang-(1-7) resulted in increased adiponectin expression, which is associated with decreased oxidative stress and increased glucose uptake [276]. The authors of both studies used the Mas receptor antagonist A779 (D-Ala) to successfully disrupt the Ang-

(1-7)-induced adiponectin expression, demonstrating that the observed effects of

Ang-(1-7) were mediated by the Mas receptor. The observation that Ang-(1-7) positively modulates adiponectin expression and decreases oxidative stress may provide molecular mechanisms by which the heptapeptide hormone up-regulates

PTP1b to antagonize c-Met RTK signaling.

Previous studies demonstrated that the heptapeptide hormone up- regulates the phosphatase DUSP1 to inactivate ERK1/2 signaling, resulting in decreased proliferation of target cells [163, 165, 179]. Increased cAMP production with a concomitant decrease in angiotensin II (Ang II)-facilitated

ERK1/2 phosphorylation and vascular smooth muscle cell (VSMC) proliferation was observed with Ang-(1-7) treatement [168]. The anti-proliferative actions of

Ang-(1-7) was dependent upon cyclic adenosine monophosphate (cAMP) and activation of the cAMP-dependent protein kinase A (PKA) as incubation with Rp- cAMPS, an inhibitor PKA, abolished the heptapeptide hormone-mediated inhibition of cell growth and blocked the deactivation of ERK1/2 signaling [179].

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Furthermore, cAMP responsive element (CRE) binding sites were identified in the promoter/enhancer region of the DUSP1 gene and are involved in its regulation [277-279]. Thus, one mechanism by which Ang-(1-7) exerts its anti- proliferative action is through modulation of phosphatases such as DUSP1, mediated by cAMP signaling. The phosphatase activity of PTP1b can also be modulated by cAMP signaling. Formation of cAMP is catalyzed by the enzyme adenylate cyclase. Treatment with forskolin, an activator of adenylate cyclase, is associated with increased PTP1b activity [109, 280]. Since Ang-(1-7) stimulates cAMP formation in VSMC, increased cAMP and subsequent activation of PKA may provide a mechanism for the increase in PTP1b in breast cancer cells and tumors.

Redox signaling is also involved in regulating the activity of PTP1b. The catalytic cysteine 215 (Cys 215) within the active site of PTP1b is vulnerable to oxidation by reactive oxygen species (ROS) such as H2O2 to generate sulphenic acid, resulting in inactivation of the phosphatase [281]. While the transition to sulphenic acid is reversible, further oxidation to the sulphinic acid and sulphonic acid is irreversible, resulting in increased cellular tyrosine phosphorylation and profound effects on phosphotyrosine-dependent signaling cascades [282].

Elevated basal ROS levels are common in cancer cells when compared to their normal counterparts [283]. Studies by Lou and colleagues demonstrated that a substantial fraction of endogenous PTP1b is constitutively oxidized in HepG2 and

A431 human cancer cells [284]. Furthermore, activation of the insulin receptor

159

(IR), which is elevated in human breast cancer, promotes the production of H2O2 to inhibit PTP1b and sustain downstream insulin-mediated signaling [285, 286].

Members of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family produce ROS in an enzymatic process requiring both NADPH and one- or two-electron reductions of oxygen to generate superoxide radical or H2O2 [287].

The expression and activity of the NADPH oxidase Nox4 is decreased in kidneys of diabetic hypertensive rats treated with Ang-(1-7), resulting in a reduction of intracellular ROS and inhibition of the development of abnormal vascular reactivity to constrictor stimuli in hypertensive animals [288]. Nox4 overexpression was reported in many human malignancies including breast cancer and ovarian cancer [289, 290]. Overexpression of Nox4 in normal breast epithelial cells confers the ability to resist apoptosis and anchorage-independent growth along with increased cell invasion, which are considered tumorigenic phenotypes [289]. Treatment with catalase, which converts H2O2 into water and oxygen, abolished the tumorigenic effects facilitated by Nox4, suggesting that the transformative properties of Nox4 is mediated, in part, by Nox4-generated H2O2.

The ability for Ang-(1-7) to down-regulate Nox4 expression and reduce H2O2 production underscores an alternative regulatory mechanism for the heptapeptide hormone to preserve PTP1b activity and thus antagonize c-Met

RTK signaling in TNBC.

Collectively, our studies elucidate a novel regulatory mechanism for the heptapeptide hormone Ang-(1-7) in TNBC progression by antagonizing c-Met

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RTK signaling. The suppressive effect of Ang-(1-7) on c-Met is mediated through increased expression of the phosphatase PTP1b, which is a negative regulator of c-Met. Furthermore, the heptapeptide hormone may potentially regulate the activity of PTP1b, either through augmenting the phosphatase activity by facilitating cAMP/PKA signaling or by preventing the ROS-induced oxidation and subsequent inactivation of PTP1b to negatively regulate c-Met RTK signaling.

The Effects of Ang-(1-7) on Metastatic Triple Negative Breast Tumor Growth in the Brain

The development of metastatic brain tumors is most common in human malignancies of the lung, skin and breast [291]. Up to 30% of breast cancer patients will develop metastatic lesions in the brain [241]. Patients with TNBC also display a distinct metastatic pattern; unlike non-TN breast tumors, which preferentially metastasize to the bone, liver and lungs and rarely to the brain, patients bearing triple negative breast tumors are more likely to develop metastases in the lungs and the brain [36, 123, 124]. A retrospective analysis of over 1,400 breast cancer patients diagnosed with stage I/II invasive breast cancer demonstrated that the overall 5-year cumulative incidence of brain metastases was the highest at 7.4% for TNBC, compared to the next highest occurrence rate at 3.4% for HER2+ breast cancer [292]. From the same study, the authors reported that the median overall survival among all breast cancer patients after diagnosis with brain metastases was 7.2 months. However, when

161 the analysis was restricted to include just patients with TNBC, the media overall survival dropped sharply to 3.7 months, highlighting the urgency in developing effective targeted therapies for these patients.

The blood-brain barrier (BBB) presents a major obstacle in the development of therapeutics for treating metastatic brain disease. The BBB is a selective barrier which acts as an interface between the brain microenvironment and the peripheral circulation critical for the normal function of the central nervous system [293]. The BBB is composed of endothelial cells that line cerebral microvessels, functionally forming a “physical barrier” between adjacent endothelial cells through tight junction complex formations that restricts molecular traffic into and out of the CNS by forming a seal. Multiple transport mechanisms span the BBB to allow the entry and exit of selective molecules. The availability of nutrients such as glucose and amino acids to supply the brain are mediated by transport proteins such as GLUT1 and LAT1, respectively. Receptor-mediated endocytosis allows the uptake of molecules such as insulin and transferrin across the BBB to be utilized by resident brain cells to maintain metabolic homeostasis.

Efflux transporters such as P-glycoprotein and the multidrug resistance-related proteins (MRPs) lining the luminal side of endothelial cells of the BBB participate in the clearance of chemotherapeutic agents such as taxol [294, 295]. Thus, with the exception of small gaseous molecules such as O2 and CO2 and small lipophilic agents, the entry of large hydrophilic molecules such as peptides and proteins are normally excluded from crossing the BBB unless they can be

162 transported by specific receptor-mediated transcytosis. As a consequence, the presence of the BBB hinders the penetrance of chemotherapeutics, resulting in poor and limited efficacy for treatment of CNS malignancies.

The high mortality and morbidity associated with patients diagnosed with brain metastases can be attributed to the limited treatment options for CNS malignancies. The standard of care for patients presenting with intracranial disease is comprised of chemotherapy, whole brain radiation therapy (WBRT), stereotactic radiosurgery (SRS), and surgical resection [128]. In cases where a solitary metastatic lesion is present in the brain, a combination of surgery and radiation is often the preferred treatment of choice. Presentation of multifocal tumors in the brain often requires radiation therapy and/or chemotherapy as surgical resection is not plausible. However, the limited efficacy and increased likelihood of developing neurocognitive dysfunction from these treatments greatly reduce the quality of life for patients. While neurocognitive deficits remained the most distressing side effect of WBRT, patients also experience manageable but nonetheless undesirable side effects such as hair loss, fatigue, headache, and skin erythema. Surgical resection is a highly invasive procedure that has limited application for patients presenting with multi-focal brain tumors. SRS employs a high dose of radiation delivered focally to a tumor, usually in a single dose. This treatment, however, may put a significant patient population at the risk of development of neurological complications due to radionecrosis [129]. The aggressive nature of malignant tumors in the brain coupled with the lack of

163 effective targeted therapies warrants the identification of druggable targets in metastatic brain tumors.

Treatment with Ang-(1-7) reduced the metastatic tumor formation of prostate cancer cells in the bone in vivo and inhibited the migration and invasion of human lung adenocarcinoma cells in vitro, demonstrating the anti-metastatic ability exhibited by the heptapeptide hormone [205, 206]. Furthermore, Yu and colleagues recently reported that the downregulation of the Ang-(1-7)/Mas receptor signaling axis in breast cancer promotes the metastatic behavior of human breast carcinoma cells in vitro and in vivo [296]. Moreover, ACE2, the enzyme primarily responsible for the physiological generation of Ang-(1-7) from

Ang II, is markedly reduced in clinical samples obtained from invasive breast carcinoma when compared to normal adjacent breast tissues, and depletion of

ACE2 in breast cancer cells enhanced the migratory and invasive capability of these carcinoma cells in transwell migration and Boyden chamber invasion assays, respectively [296]. Supplementation with exogenous Ang-(1-7) mitigated the pro-metastatic phenotypes conferred by the loss of ACE2, suggesting that restoration of the heptapeptide hormone is sufficient to attenuate the metastatic behavior of breast cancer cells. Collectively, these studies support a regulatory role for Ang-(1-7) in reducing cancer metastases.

The effects of Ang-(1-7) administration on metastatic TN breast tumor growth in the brain were investigated by employing the brain tropic subline BR5

164

4T1. This subline was derived, in the laboratory of Dr. Linda Metheny-Barlow, from the murine TNBC parental cell line 4T1 by five continuous rounds of ex vivo expansion following the selection for metastatic cells capable of colonization and tumor formation in the brain after intracarotid injection of 4T1 cells in female

BALB/c mice. Metastatic brain tumors were generated by injecting BR5 4T1 cells into the internal carotid artery of female BALB/c mice to allow a direct path for the cells to reach the brain. The mice were allowed to recover for two days after surgery prior to being randomly assigned either to an untreated group or an Ang-

(1-7) treatment group. Osmotic mini-pumps were subcutaneously implanted in tumor-bearing mice for continuous release of Ang-(1-7) at a rate of 24 µg/kg/h.

The brains were excised for subsequent analysis after 20 days of treatment.

Ang-(1-7) administration had a profound effect on metastatic brain tumor size. Infusion of the heptapeptide hormone produced a significant reduction in the size of metastatic lesions by greater than 17-fold (220,254 ± 103,029 µm2 in control mice; compared to 12,430 ± 2,536 µm2 in Ang-(1-7)-treated mice).

Furthermore, treatment with the heptapeptide hormone markedly decreased the number of metastatic lesions, as visualized in H&E-stained brain sections (an average of 20 metastatic lesions per mice in the untreated cohort compared to 3 metastatic lesions in Ang-(1-7)-treated mice), suggesting that Ang-(1-7) may prevent the seeding of circulating tumor cells to form metastasis as well as attenuating the growth of metastatic tumors. Tumors in the brains harvested from

Ang-(1-7)-treated mice had a marked reduction in the number of Ki67-positive

165 cells, similar to primary orthotopic TN breast tumors from mice medicated with the heptapeptide hormone, suggesting that Ang-(1-7) regulates cell proliferation as a mechanism for inhibiting tumor growth in both primary and metastatic TN breast tumors.

Aberrant c-Met RTK signaling is implicated the development and progression of TNBC [69, 97]. Interestingly, elevated c-Met protein expression was associated with increased mortality from metastatic disease in a cohort of breast cancer patients [221]. Moreover, it was reported that c-Met RTK is overexpressed in metastatic tumors compared to primary tumors [136, 137, 297].

Most importantly, elevated c-Met expression is significantly correlated with a lower recurrence-free survival and overall survival for TNBC patients [90]. Tumor cells stimulated with HGF, the activating ligand for c-Met, can undergo epithelial- to-mesenchymal transition (EMT), allowing the carcinoma cells to overcome anoikis [132, 298]. In addition, c-Met RTK activation induces the expression and enhances the proteolytic activity of matrix metalloproteinase (MMP) 2 and MMP 9 to facilitate in the degradation of the basement membrane, to aid in the intravasation and extravasation steps of the metastatic cascade [133, 135, 299].

Additional support for a pro-metastatic role for c-Met RTK signaling was demonstrated in gene silencing experiments where shRNA-mediated depletion of c-Met resulted in a marked reduction in breast cancer brain metastatic tumor size as well as an increase in the cumulative survival of mice lacking aberrant c-Met expression [136].

166

We observed a marked decrease in p-c-Met+ cells in tumors from mice treated with Ang-(1-7) when compared to tumors from untreated mice.

Furthermore, comparison between the tumors from control mice and Ang-(1-7)- treated mice demonstrated that an increase in PTP1b+ cells was only observed in tumors from mice mediated with the heptapeptide hormone. Thus, the decrease in p-c-Met+ cells is associated with an increase in PTP1b+ cells, which is a negative regulator of c-Met RTK signaling. The observed antagonism of c-

Met RTK signaling by Ang-(1-7) is consistent with our previous studies investigating the regulatory role of the heptapeptide hormone in c-Met RTK signaling in 4T1 primary triple negative breast tumors, suggesting that Ang-(1-7) antagonizes c-Met RTK signaling in primary TNBC tumor as well as metastatic brain tumors.

We also investigated whether the expression of c-Met RTK and PTP1b would serve as predictors of disease progression. Kaplan-Meier survival analysis of two independent breast cancer patient cohorts demonstrated that high expression of c-Met RTK in the primary tumor is highly correlated with poor brain metastasis-free survival and distant metastasis-free survival when compared with patients with low c-Met RTK expression. Conversely, high PTP1b expression is associated with a significantly better brain-metastasis-free and distant metastasis-free survival outcome those lower expressers. These in silico analysis suggest that the expressions of c-Met RTK and PTP1b may be potential biomarkers for the risk of developing metastatic disease in TNBC patients.

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Binding and activation of the Mas receptor by Ang-(1-7) is crucial for the heptapeptide hormone to exert its anti-tumor profile [162, 246]. Expression of the

Mas receptor was reported in the CNS of the mouse and other rodent animal models [248, 249]. Moreover, studies demonstrated that the Ang-(1-7)/Mas receptor signaling axis in the CNS facilitates a reduction in blood pressure and mitigates cardiac hypertrophy in hypertensive rats [250]. One limitation of our studies is that we do not know whether Ang-(1-7) directly activates the Mas receptor on the BR5 4T1 cells to decrease tumor burden by effectively crossing the BBB. While the BBB is a formidable barrier that precludes the entry of most chemotherapeutic drugs into the brain, it is important to note that the integrity of the BBB was reported to be altered and compromised under pathological conditions [300]. Studies by Yonemori and colleagues demonstrated that the

BBB surrounding brain metastatic tumors arising from patients presenting with triple negative breast cancer are often structurally compromised and thus leakier than the BBB observed in patients with HER2+ brain metastatic breast cancer

[253]. Experiments using fluorescently labeled Ang II, an octapeptide and the immediate precursor of Ang-(1-7), demonstrated that the labeled molecule crossed a disrupted BBB in hypertensive rats [252]. Therefore, it is plausible that

Ang-(1-7), a heptapeptide, may gain entry into the CNS through a compromised

BBB. An alternative route by which Ang-(1-7) may enter the CNS is through circumventricular organs (CVOs), which are highly vascular structures of the brain that lacks a BBB [301, 302]. Additional experiments employing the use of

168 labeled Ang-(1-7) peptide will assist in clarifying the mechanism by which circulating Ang-(1-7) targets and inhibits metastatic triple negative brain tumor growth.

Glial cells such as astrocytes and microglia respond to the development of brain metastasis by transitioning to an activated phenotype whereby proliferative, angiogenic, and inflammatory factors are secreted into the microenvironment

[303]. These deposited factors can serve as substrates for metastatic breast tumor cells to utilize to fuel metastatic growth. Collectively, these cells mount a coordinated response termed reactive gliosis to resolve the CNS scar tissues formed by the homing of fibroblast-like cells into the site of CNS injury to promote deposition of extracellular matrix proteins. Intriguingly, studies reported that tumor cells can recruit activated glial cells to promote metastatic growth in the brain [303].

In our studies, we demonstrated that Ang-(1-7) treatment resulted in a significant reduction in the growth of metastatic triple negative breast tumor in the mouse brain with an associated decrease in c-Met RTK signaling. Emerging evidence suggests that Ang-(1-7) may have a direct impact on glial cells under pathological states. Liu and colleagues recently reported that incubation of Ang-

(1-7) with microglial cells produced a significant reduction in the expression of pro-inflammatory cytokines and increased expression of the anti-inflammatory

169 cytokine interleukin-10 [304]. Activated astrocytes up-regulate the intermediate filament protein GFAP whereas activated microglias overexpress the lysosomal protein CD68 and the calcium binding protein Iba1. In response to CNS injury,

HGF and c-Met RTK are up-regulated in activated GFAP+ astrocytes [140].

Expression of HGF and c-Met were also reported in microglial cells of the human

CNS [141, 142]. The expression of the ligand-receptor pair in astrocytes and microglia suggest potential roles for c-Met RTK signaling in reactive gliosis and its impact on brain metastatic tumor cells. HGF secretion by glial cells can act in a paracrine fashion on metastatic breast tumor cells to elicit metastatic growth and colonization of the brain. Transcription of the c-Met gene is induced in response to ionization radiation, a common treatment modality for brain metastases, and supports a radioresistance phenotype in tumor cells [138].

Thus, further experiments are needed to evaluate whether the heptapeptide hormone inhibits the generation of tumor-supportive activated astrocytes and microglial, to reduce HGF generation and thus mitigate c-Met RTK activation in tumor cells to decrease metastatic tumor growth in the brain.

Clinical Applications of Ang-(1-7) for the Treatment of Cancer

The development of peptide-derived molecules as therapeutic agents is attractive for pharmaceutical interventions due to several advantages possessed by naturally occurring peptides. In general, peptide-based molecules have high

170 specificity and selectivity for their cellular targets, thus reducing adverse toxic effects when compared to chemically synthesized molecules. Furthermore, efficient degradation of natural peptides by circulating proteases results in decreased tissue accumulation and an increased safety profile.

The safety profile and clinical benefits of Ang-(1-7) were evaluated in several clinical trials involving cancer patients. In a Phase I clinical trial conducted at Wake Forest University Comprehensive Cancer Center, adult patients bearing non-resectable solid tumors refractory to standard therapy were administered escalating doses of Ang-(1-7) subcutaneously for five days in a 21- day cycle [213]. Clinical assessment of treated patients found no hypertensive- related events or bleeding complications. Four out of fifteen evaluable patients medicated with the heptapeptide hormone exhibited clinical benefits associated with a reduction in the circulating pro-angiogenic factor, placental growth factor

(PlGF). This Ang-(1-7)-associated response to decrease plasma PlGF in treated patients further underscores the regulatory role of the heptapeptide hormone as an anti-angiogenic agent, whereby the anti-tumor effects benefited by these patients may be mediated, in part, through the modulatory effects of Ang-(1-7) on reduced blood vessel formation. Moreover, it is important to note that three patients experienced disease stabilization lasting more than 3 months, while another patient presenting with metastatic sarcoma with progressing disease at the time of enrollment experienced an overall reduction in tumor volume following

Ang-(1-7) treatment. Few adverse effects were observed in patients medicated

171 with Ang-(1-7), with only three serious adverse events (SAEs) possibly related to treatment with the heptapeptide hormone reported. It is important to note that, in addition to the advanced stage of disease in this cohort of patients, these individuals had also received chemotherapeutic treatments prior to the start of this clinical trial, which may be related to the adverse side effects. Thus, the reported SAEs cannot be definitively attributed to Ang-(1-7) treatment.

The benefits of Ang-(1-7) used in the adjuvant setting were explored in a separate phase I/II dose escalation study in a group of breast cancer patients receiving doxorubicin and cyclophosphamide as treatment [214]. In this clinical trial, treatment with Ang-(1-7) decreased the frequency of high grade thrombocytopenia, anemia and lymphopenia compared to the neutrophil- stimulating agent filgrastim in this cohort. Comparable findings were reported in a randomized, double-blind, placebo-controlled phase 2 study evaluating the efficacy of Ang-(1-7) as an adjuvant therapy for cancer patients treated with gemcitabine and carboplatin or cisplatin [215].

While Ang-(1-7) administration demonstrated clinical benefit in the clinical trials referenced above, no information on the effect of the heptapeptide hormone in the clinical treatment of triple negative breast cancer was available, since no patients presenting with TNBC were enrolled in these studies. The aggressive nature of TNBC coupled with the lack of an effective targeted therapy

172 underscores the critical need for identification of druggable cellular targets.

Based on the effectiveness demonstrated by the heptapeptide hormone in the clinical trials involving cancer patients along with the preclinical rationale provided by the current studies, it is reasonable to propose the initiation of a phase I clinical trial for in the treatment of triple negative breast cancer patients with Ang-(1-7).

Ang-(1-7) provides several advantages over current forms of treatment for

TNBC patients. The heptapeptide hormone exhibits limited harmful, cytotoxic effects on undesired tissues associated with standard chemotherapies by targeting actively proliferating cells, whereas cytotoxic therapeutics often damage both malignant and normal cells. Moreover, while hemodynamic complications are often observed in cancer patients, Ang-(1-7) treatment does not increase blood pressure or heart rate [213]. Finally, the activity and/or synthesis of molecules targeted by Ang-(1-7) are not entirely abolished with treatment; the heptapeptide hormone decreases the signaling output to basal levels which allows normal cellular processes that are dependent on these signaling cascades to occur. This is supported by the observations that patients treated with Ang-(1-

7) experience no complications in wound-healing [213].

The anti-fibrotic properties of Ang-(1-7) may mitigate fibrosis-induced vessel compression to improve drug delivery. As cancer and cancer-associated

173 stromal cells proliferate in confined microenvironments, reduced vascular perfusion in the tumors develops due to collapsed blood vessels from the pressure of solid tissue components. As a consequence, the distorted blood vessels promote a hypoxic environment by diminishing efficient oxygen transport and more important, impair drug delivery. Indeed, it has been noted that patients with low tumor perfusion demonstrate poorer chemotherapy responses and decreased survival compared to patients with high perfusion [305, 306]. Ang-(1-

7) reduces the proliferation of cancer-associated fibroblasts (CAFs) isolated from breast tumors and decreases the expression of the fibrotic mediators TGF-β,

CTGF and tenascin C in isolated CAFs. In further support of a role for Ang-(1-7) in augmenting drug delivery, Chauhan and colleagues reported that inhibition of

Ang-II/AT1R signaling through ACE inhibitors or angiotensin receptor blockers

(ARBs), which results in increased circulating Ang-(1-7) level, enhances drug delivery and potentiates chemotherapy by decompressing tumor blood vessels

[307]. Additionally, Ang-(1-7) exhibits prominent anti-hypertensive properties through vasodilatory actions on the vasculature [308]. Therefore, there is potential to use the heptapeptide hormone to augment the delivery of therapeutics to the tumors for enhanced efficacy.

The development of drug-induced cardiotoxicity in cancer patients treated with chemotherapy has become an emerging clinical issue. While anthracyclines such as doxorubicin are effective anti-cancer agents that are widely prescribed in the clinic, patients treated with regiments of these cytotoxic drugs often develop cardiotoxic effects, contributing to a poor quality of life [309, 310]. Doxorubicin-

174 induced cardiotoxicity can occur as early as 2-3 days post administration and can result in irreversible cardiomyopathy, ultimately leading to congestive heart failure and death in as many as 50% of treated patients [311, 312].

Mechanistically, aberrant activation of p38 MAPK along with increased generation of reactive oxygen species induced by doxorubicin promotes cardiomyocyte apoptosis and impairment of cardiac function [313, 314]. In addition to the anti-cancer profile exhibited by Ang-(1-7), the heptapeptide hormone also elicits cardioprotective effects through ROS reduction as well as mitigates ROS-mediated p38 activation [315, 316].

Thus, an additional goal is to administer Ang-(1-7) as a combinatorial treatment with a standard chemotherapeutic such as doxorubicin, paclitaxel or cyclophosphamide for TNBC patients. Indeed, a recent study by Jung and colleagues reported that c-Met inhibition resensitizes breast carcinoma and colon carcinoma cells to doxorubicin [317]. Furthermore, a strategy with Ang-(1-7) combined with a cytotoxic chemotherapeutic may allow for use of decreased chemotherapy dosages to alleviate undesired side effects such as cardiotoxicity.

Lastly, co-administration of Ang-(1-7) with a chemotherapeutic may enhance the growth inhibitory effects on tumors due to complementary drug-mediated targeting of a wide range of tumorigenic and metastatic processes.

175

Similar to many endogenous peptides, Ang-(1-7) is rapidly metabolized in the plasma and is not biologically available after oral administration. Infusion of

Ang-(1-7) through a subcutaneous route increases plasma levels of the heptapeptide hormone 2-3 fold both in animal models and cancer patients, similar to changes observed in studies using ACE inhibitors [163, 213, 318].

However, the plasma half-life of Ang-(1-7) is approximately 30 minutes in humans, thus highlighting the critical need to develop an orally-active Ang-(1-7) mimetic with an increased half-life and bioavailability [213, 214].

The first small molecule mimetic of Ang-(1-7) developed and studied was

AVE-0991, a non-peptide orally-active agent that activates the Mas receptor

[319]. This mimetic competes with Ang-(1-7) for binding to the Mas receptor, to facilitate nitric oxide (NO) generation in both endothelial cells and Chinese hamster ovary (CHO) cells [320]. While AVE 0991 treatment elicits cellular responses similar to those observed with Ang-(1-7), the mimetic also binds to the

AT1 and AT2 Ang II receptors resulting in blockade of NO production, suggesting that side effects may result in patients if AVE 0991 is not administered in a specific manner [321].

CGEN 856S is another mimetic of Ang-(1-7) that demonstrated high binding for the Mas receptor without AT1 or AT2 receptor activation [322]. This mimetic induced NO-dependent vasorelaxation in isolated aortic rings from rats,

176 the effects of which were blocked by the mas receptor antagonist D-Ala [322].

Exhibiting similar cardioprotective and anti-fibrotic properties to Ang-(1-7), CGEN

856S administration attenuated isoproterenol-induced cardiac hypertrophy in rats with associated reductions in collagen and fibronectin deposition in the rat hearts

[323].

Ang-(1-7)-CyD, an orally active formulation of Ang-(1-7), was developed based on the incorporation of cyclodextrins to enhance drug stability and increase absorption across cellular membranes [324]. This novel Ang-(1-7) formulation demonstrated improved efficacy over Ang-(1-7) in animal models of diabetes, myocardial infarctions and fibrosis [172, 325]. While these newly- developed Ang-(1-7) mimetics demonstrated apparent effectiveness similar to that of the parent heptapeptide hormone, extensive experimental investigations including additional in vivo studies as well as Phase I clinical trials are necessary in order to demonstrate the specificity of these potential therapeutics as well as any side effects. Lastly, these agents have not been tested in models of malignancy, motivating researchers to consider additional modifications for the

Ang-(1-7) peptide in order to further enhance its therapeutic benefits in cancer patients.

Taken together, the findings reported in this dissertation along with previous preclinical and clinical studies demonstrate a role for Ang-(1-7) in

177 regulating multiple signaling pathways critical for cancer cell growth and disease progression. The heptapeptide hormone offers a wider range of clinical benefits than current standard chemotherapies. Therefore, Ang-(1-7) represents an attractive candidate as a first-in-class targeted therapeutic for the treatment of

TNBC, which continues to remain an devastating disease for women due to lack of effective therapies.

178

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Curriculum Vitae

Guorui (Gary) Deng

Department of Cancer Biology, Wake Forest School of Medicine

Medical Center Boulevard, Winston Salem, NC 27157

EDUCATION

2016 Ph.D. Department of Cancer Biology, Wake Forest School of Medicine Graduate School of Arts and Sciences (Expected)

2010 B.S. Biochemistry & Cell Biology, Bucknell University

RESEARCH EXPERIENCE

2011-2016 Graduate Research Student Laboratories of Ann Tallant, Ph.D. and Patricia Gallagher, Ph.D. Hypertension and Vascular Research Center Department of Integrative Physiology & Pharmacology Wake Forest School of Medicine, Winston Salem, NC

2010-2011 Post-baccalaureate Research Student Laboratory of Isabelle Berquin, Ph.D. Department of Cancer Biology Wake Forest School of Medicine, Winston Salem, NC

2009 Summer Research Student Laboratory of Amy Yee, Ph.D., Department of Developmental, Molecular & Chemical Biology, Sackler School of Graduate Biomedical Sciences Tufts University, Boston, MA

2008-2010 Undergraduate Research Student Laboratory of Marie Pizzorno-Simpson Department of Biology Bucknell University, Lewisburg, PA

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HONORS AND AWARDS

2015 Silver Award – Poster Competition, Basic Science Section 23rd Annual Wake Forest University Division of Surgical Sciences Research Day, Winston Salem, NC

2015 Wake Forest University Alumni Student Travel Award 106th AACR Annual Conference, Philadelphia, PA

2013-2015 Recipient, National Cancer Institute Institutional Research Training Grant (T32), Department of Cancer Biology, Wake Forest School of Medicine, Winston Salem, NC

2006-2010 Bucknell University-Posse Foundation Scholarship for Academic Excellence

PROFRESSIONAL MEMBERSHIP

American Association of Cancer Research (AACR) 2010-2015

TEACHING EXPERIENCE

Guest lectures on cancer metastasis 2013 Department of Cancer Biology Wake Forest School of Medicine

BIBLIOGRAPHY

Publications:

Patricia E. Gallagher, Alison L. Arter, Guorui Deng, E. Ann Tallant. Angiotensin- (1-7): A Peptide Hormone with Anti-Cancer Activity. Current Medicinal Chemistry 2014; 21(21): 2417-23. PMID: 24524765

Guorui Deng, Guangchao Sui. Noncoding RNA in Oncogenesis: A New Era of Identifying Key Players. International Journal of Molecular Sciences 2013; 14(9):18319-49. PMID: 24013378

Manuscripts:

Deng G, Gallagher PE, Tallant EA. Angiotensin-(1-7) Targets c-Met Signaling by Up-regulating PTP1b to Attenuate Triple Negative Breast Cancer Progression. (to be submitted to Molecular Cancer Therapeutics).

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Deng G, Metheny-Barlow L, Tallant EA, Gallagher PE. Angiotensin-(1-7) Reduces Triple Negative Breast Cancer Brain Metastasis in Association with Decreased c-Met Signaling. (to be submitted to Journal of Neuro-Oncology).

Arter AL, Deng G, Tallant EA and Gallagher PE. Angiotensin-(1-7) inhibits metastatic triple negative breast cancer growth in the lungs of mice associated with reduction in TGF-beta(β)-mediated extracellular matrix (ECM) protein production (in preparation)

Arter AL, Soto-Pantoja DR, Deng G, Cook KL, Gallagher PE and Tallant EA. Angiotensin-(1-7) attenuates triple negative breast cancer growth associated with a reduction in Akt (in preparation)

Abstracts & Poster Presentations:

Guorui Deng, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) Targets c- Met Signaling by Up-regulating the Phosphatase PTP1b to Attenuate Triple Negative Breast Cancer Progression. (2015) Wake Forest School of Medicine Division of Surgical Sciences Research Day. *Received 2nd Place Award.

Guorui Deng, Linda Metheny-Barlow, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) Targets c-Met Signaling by Up-regulating the Phosphatase PTP1b to Reduce Triple Negative Breast Cancer. (2015) AACR 106th Annual Meeting, Philadelphia, PA.

Guorui Deng, E. Ann Tallant, Patricia E. Gallagher. Angiotensin-(1-7) Attenuates Triple Negative Breast Cancer Progression by Targeting c-Met and Downstream ERK1/2 Signaling. (2014) AACR Special Conference on RAS Oncogenes: From Biology to Therapy, Lake Buena Vista, FL.

Guorui Deng, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) Attenuates Triple Negative Breast Cancer Progression by Targeting c-Met Activation and Downstream ERK1/2 Signaling. (2014) Wake Forest School of Medicine Division of Surgical Sciences Research Day.

Guorui Deng, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) Attenuates Triple Negative Breast Cancer Progression by Targeting c-Met Signaling. (2013) Wake Forest School of Medicine Division of Surgical Sciences Research Day.

Guorui Deng, Jing Wang, Isabelle Berquin. Elucidating the role of YB-1 in EGFR Regulation and Breast Cancer (2011) Wake Forest School of Medicine PREP Symposium, Winston Salem, NC

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Jing Wang, Chunlan Zhang, Guorui Deng, Yong Chen, Isabelle Berquin. Omega-3 fatty acids delay the growth of mammary tumors in MMTV-Erbb2 mice. (2011) AACR 102nd Annual Meeting, Orlando, FL.

Talks:

Guorui Deng. (May 2015) Angiotensin-(1-7)-mediated Reduction of c-Met Receptor Tyrosine Kinase Signaling in Triple Negative Breast Cancer. Research in Progress Seminar, Hypertension & Vascular Research Center, Wake Forest School of Medicine, Winston Salem, NC.

Guorui Deng. (January 2014) Angiotensin-(1-7) Attenuates Triple Negative Breast Cancer Progression by Targeting c-Met Signaling. Research in Progress Seminar, Hypertension & Vascular Research Center, Wake Forest School of Medicine, Winston Salem, NC.

Guorui Deng. (March 2013) Angiotensin-(1-7)-mediated Reduction of c-Met Signaling in Breast Cancer. Research in Progress Seminar, Hypertension & Vascular Research Center, Wake Forest School of Medicine, Winston Salem, NC.

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