ELUCIDATING THE ROLE OF 7A IN BREAST CANCER

by

Ramon Antonio Garcia-Areas

A Dissertation Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Florida Atlantic University

Boca Raton, FL

December, 2016 Copyright 2016 by Ramon Antonio Garcia-Areas

ii ELUCIDATING THE ROLE OF SEMAPHORIN 7 A IN BREAST CANCEl.~

by

Ramon Antonio Garcia-Areas

1 This dissertation was prepared under the direction of the candidate's dissertation advisor, Dr. Vijaya lragavarapu-Charyulu, Department of Biomedical Science\ and has been approved by the members of his supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accept~d in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 1

I SUPERVISORY COMMITTEE: ~7 -tt~L Vijaya lrag~ha~h. D . ~a~~visor

Michael Lu, Ph 1 Ta Ja Godenschwege, Ph.D ~ .. ~ ...... h . }iana LOpez. Ph.D. ~Q

Dec.eMber lo, 2ol6 Date

iii ACKNOWLE DGEMENTS

I would like to acknowledge my Dissertation advisor, Dr. Vijaya

lragavarapu-Charyulu. for her constant support, dedication and generosity. would like to thank Dr. Stephania Libreros for always pushing us to bigger and

better things, but most of all, for her friendship. The completion of my dissertation would not have been achieved without the diligence and dedication of: Samantha

Amat, Camila Castro-Silva, Nathalia Gazaniga and Michael Simoes. To my

collaborators, Dr. Patricia Keating, Dr. Kathy Schilling and Lillian Onwuha­

Ekpete, I extend my deepest gratitude. I would like to thank Dr. James Hartmann for introducing me to the greatest joy, the joy of discovery. I also thank Dr.

Michael Lu for selflessly sharing with me his deep knowledge of science through

his example of integrity and discipline. I thank Dr. Tanja Godenschwege for teaching me the intricate art of scientific communication. I thank Dr. Mahyar

Nouri-Shirazi for his insightful feedback and perspectives. I thank my partner

Andy Litvack for his unwavering and loving support. I thank the Biological

Sciences Department for supporting my degree, specially to Dr. Evelyn Frazier, the late Dr. John Nambu and especially Geri Mayer for her friendship and

guidance. Finally, I thank all the great scientists, teachers, students, friends and family members who may be unmentioned in this dedication, but who's

contributions in my life have enriched the privilege ofthis undertaking.

iv ABSTRACT

Author: Ramon Antonio Garcia-Areas

Title: Elucidating the role of Semaphorin 7 A in breast cancer

Institution: Florida Atlantic University

Thesis Advisor: Dr. Vijaya lragavarapu-Charyulu

Degree: Doctor in Philosophy

Year: 2016

Solid tumors can hijack many of the same programs used in neurogenesis to enhance tumor growth and metastasis, thereby generating a plethora of

neurogenesis-related molecules including . Among them, we have

identified Semaphorin7A (SEMA7A) in breast cancer. We first used to the DA-3

mammary tumor model to determine the effect of tumor-derived SEMA7A on

immune cells. We found that tumor-derived SEMA7A can modulate the

production of proangiogenic chemokines CXCL2/MIP-2 and CXCL1 , and pro­

metastatic MMP-9 in macrophages. We next aimed to determine the expression

and function ofSEMA7A in mammary tumor cells. We found that SEMA7Ais

highly expressed in both metastatic human and murine breast cancer cells. We

show that both TGF-f) and hypoxia elicits the production of SEMA7 A in mammary

cells. SEMA7A shRNA silencing in 4T1 cells resulted in decreased mesenchymal

markers MMP-3, MMP-13, Vimentin and TGF-1). SEMA7Asilenced cells show

v increased stiffness with reduced migratory and proliferative potential. In vivo,

SEMA?A silenced 4T1 tumor bearing mice showed decreased tumor growth and

metastasis. Genetic ablation of host-derived SEMA 7 A synergized to further

decrease the growth and metastasis of 4T1 cells. Our findings suggest novel functional roles for SEMA?A in breast cancer and that SEMA?A could be a novel therapeutic target to limit tumor growth and metastasis

iv DEDICATION

I dedicate my dissertation to my parents, Gloria and Benigno Garcia, who

sacrificed much to afford me a world of possibilities with their unconditional

love and support. To my mother, who showed the value of hard work and the

importance of kindness. To my father, who taught me a love for knowledge and the value of integrity. To my siblings, Maria and Gabriel, who's presence in my

life has always fueled my desired to excel. I also dedicate this dissertation to my

godparents Francis and Brian Waite who without any obligation gave me the

opportunity to fulfill my dream of a higher education in the United States of

America. Their boundless generosity towards me is only surpassed by their love

and support. To God, for all the good that surfaces in my life through his eternal

mercy. ELUCIDATING THE ROLE OF SEMAPHORIN 7A IN BREAST CANCER

LIST OF FIGURES ...... xi

GENERAL INTRODUCTION ...... 1

Semaphorin7A ...... 1

Semaphorin7A and its receptors ...... 2

Role ofsemaphorin7A in innate immunity ...... 4

Role ofsemaphorin7A in adaptive immunity ...... 6

Semaphorin?A's role in inflammatory diseases ...... 9

Semaphorin7A and Cancer ...... 10

CHAPTER 1. SEMAPHORIN7A PROMOTES TUMOR GROWTH AND

EXERTS A PRO-ANGIOGENIC EFFECT IN MACROPHAGES OF

MAMMARY TUMOR-BEARING MICE ...... 13

1.1 lntroduction ...... 13

1 .2 Materials and methods ...... 15

1.2.1 Mice and cellli nes ...... 15

1.2.2 Cell cultures ...... 16

1.2.3 lmmunoflluorescence ...... 17

1.2.4 RNA isolation and real-time reverse transcriptase-polymerase

chain reaction ...... 17

1.2.5 Flow cytometry studies ...... 18

1.2.6 Silencing of SEMA 7 A in macrophages ...... 18

vii 1.2.7 Silencing of SEMA7 A in DA-3 murine mammary tumor cells ...... 19

1.2.8 Monocyte migration assay ...... 19

1.2.9 Protein determination ...... 20

1.2.10 lmmunohistochemistry ...... 21

1.2.11 Tumor measurements and in vivo imaging for ...... 22

1.2.12 Statistical analysis ...... 22

1.3 Results ...... 22

1.3.1 SEMA7A is expressed in DA-3 mammary tumor cells and

expression is increased in peritoneal elicited macrophages of DA-3

mammary tumor-bearing mice ...... 22

1.3.2 Expression of SEMA7 A , ~1 (CD29) is

increased in DA-3 mammary tumor cells and macrophages from

mammary tumor-bearing mice ...... 24

1.3.3 Treatment of macrophages with rmS EMA7A induces production

of angiogenic CXCL2/MIP-2 ...... 25

1.3.4 Decreased tumor-derived SEMA7 A results in reduced in vitro

macrophage migration and CXCL2/MIP-2 production ...... 27

1.3.5 Decreased tumor growth in mice bearing SEMA7A silenced

mammary tumors ...... 29

1.3.6 Peritoneal elicited macrophages from mice bearing SEMA7A KD

tumors produce decreased levels of angiogenic molecules ...... 30

1.4 Discussion ...... 31

viii CHAPTER 2. SEMAPHORIN 7A PLAYS AN IMPORTANT ROLE IN

BREAST CANCER GROWTH AND METASTASIS ...... 42

2.1 lntroduction ...... 42

2.2 Materials and Methods ...... 44

2.2.1 Mice and cell lines ...... 44

2.2.2 RNA isolation and real-time reverse transcriptase-polymerase

chain reaction ...... 45

2.2.3 Flow cytometry studies ...... 45

2.2.4 Silencing ofSEMA?A in 4T1 murine mammary tumor cells ...... 46

2.2.5 AFM Cell Stiffness Measurements ...... 46

2.2.6 Wounding assay ...... 47

2.2.7 Statistical Analysis ...... 47

2.3 Results ...... 48

2.3.1 SEMA7A expression is increased in metastatic breast cancer

cells ...... 48

2.3.2 Hypoxia induces expression of SEMA?A in murine mammary

cell lines and inhibition of HIF-1 a decreases SEMA7A ...... 49

2.3.3 SEMA7A gene knock down in 4T1 mammary tumor cells results

in morphologic and functional changes ...... ,...... 51

2.3.4 SEMA7A gene silencing decreases proliferation and migration of

4T1 cells ...... ,.53

2.3.5 SEMA?A gene silencing reduces tumor growth and metastasis

and increases survival ...... 53

ix 2.3.6 Ablation of host-derived SEMA7 A decreases rate of tumor

growth and metastasis and increases survival ...... 54

2.4 Discussion ...... 55

CONCLUSIONS ...... 70

REFERENCES ...... 75

X LIST OF FIGURES

Figure 1 DA-3 mammary tumor cells express SEMA7A ...... 36

Figure 2 ~1 integrin expression is increased in DA-3 cells and macrophages

of mammary tumor -bearing mice ...... 37

Figure 3 Increased expression of angiogenic chemokine, CXCL2/MIP-2 in

rmSEMA7A treated RAW264.7 macrophages and peritoneal elicited

macrophages from mammary tumor-bearing mice ...... 38

Figure 4 Decreased tumor-derived SEMA7A results in reduced in vitro

macrophage migration and CXCL2/M IP-2 production ...... 39

Figure 5 Decreased tumor growth and angiogenesis in mice bearing

SEMA7A silenced mammary tumors ...... 40

Figure 6 Macrophages from mice bearing SEMA7A shRNA knockdown

tumors produce decreased levels of angiogenic molecules ...... 41

Figure 7 SEMA7A is highly expressed in metastatic human and murine cell

lines and TGF-~ induces SEMA7A expression via AKT signaling ...... 59

Figure 8 Hypoxia induces SEMA7A expression in mammary cells ...... 60

Figure 9 HIF-1 a production correlates with SEMA7A expression in 4T07

cells ...... 61

Figure 10 Gene silencing of SEMA7A in 4T1-LUC cells decreases

expression of mesenchymal and pro-metastatic genes ...... 62

Figure 11 SEMA7A alters tumor cell stiffness ...... 63

xi Figure 12 Silencing of SEMA7A gene in 4T1-LUC mammary tumor

decreases cell motility and proliferation...... 64

Figure 131nhibition of tumor-derived SEMA7Adecreases tumor growth rate

and metastasis in 4T1 -LUC tumor-bearing mice ...... 65

Figure 14 Genetic ablation of host-derived SEMA7A decreases tumor growth

rate and metastasis in 4T1 -LUC tumor-bearing mice ...... 66

Figure 151nhibition of host-derived and tumor-derived SEMA7A decreases

tumor growth rate and metastasis in 4T1 tumor-bearing mice ...... 67

xii GENERAL INTRODUCTION

*This chapter contains selected portions or full text from the followi ng publication

and is reproduced here with the permission from authors and the publisher*

Garcia-Areas R, Libreros S , lragavarapu-Charyulu V. Semaphorin7A: branching

beyond axonal guidance and into immunity. lmmuno/ogic research.

2013;57(0):81-85. doi :10.1 007/s12026-013-8460-5.

Semaphorin7A

Semaphorins are a that plays important roles in many

morphological and physiological processes from to humans (1).

Semaphorins are classified based on their structure and protein domains. The

structure of the semaphorins consists of a conserved cysteine-rich (-500 amino

acids) residue called the "sema" domain, a - semaphorin-integrin (PSI)

domain, and variable protein domains (2). Variations inC-terminal motifs are the

key differentiating factors among semaphorins. There are eight classes of

semaphorins: Class 1 and 2 are invertebrates, class 3 through 7 are found in vertebrates, and class 8 are viral-encoded (3). Semaphorins in class 1 and class

4- 7 are mainly membrane-associated, whereas semaphorins in class 2, 3, and 8

are mostly secreted (1). The membrane association/secretion behavior allows

semaphorins to mediate direct cell- cell interactions or

chemoattraction/. Semaphorins were initially described for their

1 role in guidance during neurogenesis. It has now been found that

semaphorins branch beyond the to cover an ample overlap of

molecular repertoires. Semaphorins are also involved in physiological processes

such as organogenesis, immune cell regulation, and vascular growth (1).

Semaphorin7A (SEMA7A) is the only class 7 semaphorin, and to date, it is the

only semaphorin that is GPI-anchored (4). SEMA7A was first identified as

CDw1 08 on erythrocytes and includes the John Milton Hagen antigen. It is

located on chromosome 15 (15q23-24) (5). It was not until 1998 that CDw108 was found to be part of the semaphorin family, as SemaK-1 (4). In 1999, it was

renamed SEMA7A and made into a separate class of semaphorins based on its

unique GPI anchorage (3). Yamada et al. (5) were the first to clone the human

CDw1 08 eDNA sequence, which encodes for 666 amino acids. The murine

counterpart is composed of 664 amino acids and shares roughly 90% homology with human SEMA7A (4). Both human and mouse SEMA7A contain a seven­

bladed ~-propeller semaphorin N terminus domain, a plexin, semaphorin, and

integrin domain (PSI), an immunoglobulin-like domain, and the characteristic GPI

anchor in their C-terminus (6). It was shown that SEMA7 A can dimerize through the Serna and immunoglobulin domains (6).

Semaphorin7A and its receptors

Semaphorin7A was originally identified as the vertebrate homolog of virally encoded semaphorins A39R and AHV(1, 7). Like the viral semaphorins,

SEMA7A binds a -encoded semaphorin protein receptor (VESPR) that is

also known as plexin C 1. This receptor is expressed by dendritic cells,

2 monocytes, and neutrophils (8). Unlike the A39R semaphorin, the induction of

SEMA?Aon monocyte chemotaxis and activation was not found to be mediated through the receptor plexin C1 (9). Pasterkamp et al. (10) were the first to

propose that SEMA?A's RGD motif (Arg267- Giy268- Asp269) can bind to the very late antigen 1 dimer, a1 p1 , and promote axon growth. Similarly, Suzuki et

al. (1 1) showed that the modulatory effects of SEMA?Aon T cell inflammatory

responses are also mediated through the very late antigen 1 dimer, a1 p1-

integrin. In contrast, the binding of endothelial SEMA? A to macrophage avp5

integrin in a colitis model induced the production of anti -inflammatory IL-10 (1 2).

These findings depart from the traditional notion that semaphorins signal through

and . A recent report by Liu et al. (6) revisited the SEMA? A­

plexin C1 interaction and showed that the p-propeller domains of SEMA?A and

plexin C1 can bind in an edge-to-edge manner. Their crystallographic protein findings propose that SEMA?A's RGD is hidden within the protein structure, therefore making it difficult to bind . Although most SEMA?Astudies

have investigated its role as a , a novel role for SEMA?A as a receptor for

P. fa lciparum merozoite-specific TRAP homolog (MTRAP) has been proposed.

In platelets, it has been found that the 80-kDa membrane-bound SEMA?A can be

cleaved off the membrane by ADAM-17 ( 13). The extracellular soluble version of

SEMA?A has been found to have chemotactic properties in monocytes (9) and

promote axon branching (10) and melanocyte dendricity (14), among others. In vitro treatment of recombinant SEMA?A with caspase-9 resulted in 24-kDa fragments (15). They further demonstrate that mutating the conserved

3 VHQDQA YDD motif in SEMA? A impairs caspase-9 cleavage. In vivo, they

showed that Apaf-1 is required for caspase-9 cleavage ofSEMA?A (15).

However, it is still largely unknown whether the different cleaved forms of

SEMA? A serve differential functions in comparison with full-length SEMA?A , and whether the cleaved forms may lead to alternative signaling programs. Future

studies will have to elucidate whether binding of secreted or membrane-bound

SEMA? A in cis or trans associations can mediate differential effects through

integrins, plexins, or other potential co-receptors.

Role of semaphorin7A in innate immunity

Holmes et al. (9) first showed a functional role for SEMA?A as an activator

of human monocytes in vitro. In a dose-dependent manner, soluble SEMA?A was able to induce the production of proinflammatory : IL-8, TNF-a, IL-

6, and IL-1 (9). To a lesser extent, SEMA?A was also able to induce the

production of IL-8, TNF-a, IL-6, and IL-1 in human neutrophils. In both monocytes

and neutrophils, IL-8 was most significantly up-regulated by SEMA?A. SEMA?A was able to induce a phenotypic change in these human monocytes, and skewed them to become CD11b+/CD14-/CD1a-/CD83+/CD40+/CD86+dendritic-like cells

(9), consistent with the reports of SEMA?A promoting cell dendricity (10, 14).

Furthermore, it was found that SEMA? A could chemoattract monocytes at femtomolar concentrations but was less effective in promoting neutrophil

migration (9). Recently, hypoxia-elicited endothelial SEMA 7A was found to

increase neutrophil transmigration across vasculature (1 6). SEMA?A-deficient

mice showed decreased neutrophil vascular transmigration despite hypoxic

4 stimuli (16). TGF-13 also plays a role in skin inflammation as this protein was found to induce increased expression of SEMA7A on human keratinocytes (17).

Increased SEMA7A contributed to skin inflammation by activating monocytes via

131-integrin to produce IL-8, while blocking 13-integrin neutralized the effect of

SEMA7A(17). SEMA7A was also found to influence inflammatory processes in a

cornea inflammation model by mediating macrophage infiltration to the cornea

(18).

Semaphorin7A (SEMA7A) has been shown to exert either a pro- or anti­

inflammatory effect depending on the cell type and receptor subunit engaged by this protein ( 11, 12). Contrary to the pro-inflammatory effects associated with

SEMA7A , intestinal epithelial cell (IE C)-derived SEMA7A was found to negatively

regulate development of colitis (19). Intestinal macrophages maintain quiescent

immune response under constant onslaught by commensal bacteria. SEMA7A was found to play a role in the maintenance of quiescence by inducing the

production of immunoregulatory , IL-1 0, by intestinal macrophages (19).

Furthermore, these same authors showed that SEMA7A-deficient mice exhibit

severe signs of dextran sodium sulfate-induced colitis due to reduced levels of

intestinal IL-10. These studies in total indicate differential roles of SEMA7A

depending on the receptor engagement and cellular sources. SEMA7A is pro­

inflammatory through T cell-monocyte interactions vi a a1 131-receptor (12) and

anti-inflammatory through avl31-integrin expressed on intestinal macrophages

(19).

5 Macrophages and monocytes not only respond to SEMA 7 A, but can also

express SEMA 7A . THP-1 cells treated with respiratory sensitization chemicals

showed increased expression of SEMA7A (20). Human primary monocytes from

West Nile virus expressed significantly higher mRNA levels of SEMA7A ,

compared to normal healthy controls. SEMA7A is also expressed in

macrophages from the lungs of idiopathic pulmonary disease patients (21 ).

Role of semaphorin7A in adaptive immunity

SEMA7A is expressed in both myeloid and lymphoid lineages (9).

SEMA7A has been reported to be expressed on activated lymphocytes (22).

While this protein is highly expressed on activated T lymphocytes, it is expressed

at lower levels in 8 lymphocytes (23).

Investigation of SEMA7A expression in tissue during immune system

development revealed strong signals in thymus tissue with weak expression in the spleen and lymph nodes. Flow cytometric analysis of thymocytes during

development showed that 15 % of thymocytes express this molecule. Further

analysis of thymic subpopulations revealed expression in double-positive subsets

and in C04brightC08dunsub populations with the absence of expression on single­

positive CD4 or CDS T lymphocytes (22). There is no further expression of

SEMA7A in T cell precursors until single-positive T cells exit the thymus and are

activated in the periphery (5). SEMA7A is expressed in early lymphocyte

activation and has been reported to be expressed on most T and NK cells and 8

cells as early as 4 h after activation with maximal expression at 48 h post-stimuli

6 and then gradually disappears (24). Thus, this may indicate a specific function that is related to the activation and differentiation of lymphocytes.

In the periphery, SEMA?A has been found to play an important role in the

effector phase of cell-mediated immune responses and inducing the production

of pro-inflammatory cytokines by macrophages. Activated CD4 + T cell-expressed

SEMA?A has been found to accumulate at the contact zone between T cells and

macrophages. Thus, SEMA?A is recruited to lipid rafts in the immunological

synapse between T lymphocytes and macrophages, resulting in clustering of

a1 ~1-integrins and formation of focal adhesion complexes in monocytes and

macrophages (1 1). Thus, stimulation of macro phages through a1 ~1-integrins

induces the activation of the downstream mitogen-activated protein kinase

pathway, resulting in the production of proinflammatory cytokines. While

SEMA?A has an activating effect on monocytes, it has been reported to

negatively regulate T cell-mediated immune responses (25). ~ was shown that

SEMA? A-t- T cells were hyper-proliferative upon antigenic stimulation. Further,

SEMA?A + mice had more severe responses in experimental autoimmune

encephalomyelitis (EAE) conditions compared to wild-type mice. In contrast to these findings, Okuno et al. (26) reported that SEMA? A-deficient mice

immunized with a myelin oligodendrocyte (MOG) peptide in CFA generate MOG­

peptide-specific CD4 + T cells and are resistant to EAE development. Further, it was shown that SEMA?Aon antigen-primed T cells induces inflammation in EAE through interactions with a1 ~1- i ntegrin (1 1) and that antigen-specific T cells and

7 cytokine-producing effector T cells are not impaired in SEMA7_,_ mice (11 ).

These findings indicate that SEMA7A is pathologically involved in EAE.

Regulatory T lymphocytes are important in maintaining homeostasis of the

immune system in healthy and pathological conditions. Idiopathic pulmonary fibrosis is characterized by epithelial injury and inflammation wi th an abnormal wound-healing response (27). Lymphocytes are reported to be involved in the

immunopathogenesis of fibrosis(28-31). Since abnormalities in regulatory T cells

are seen in lungs of patients with fibrosis, Reilkoff et al. (21)assessed SEMA7A

expression in human and mouse models of fibrosis. Increased levels of

SEMA7A +co4+ CD25+FoxP3+ Tregs were observed in the blood of patients with

rapidly progressive IPF. It was further shown that SEMA7A-expressing Tregs in

contrast to SEMA7-CD4+CD25+FoxP3+ cells show reduced expression of

regulatory mediators, such as IL-10 (21 ). It was postulated that since SEMA?A­

Tregs were not associated with progressive IPF, SEMA7Aexpression identifies a

population of Tregs that traffic to the lung and contribute to disease progression via impaired suppressor capabilities (21 ). To date, the expression of SEMA7A by

8 cells and the effect of SEMA7A on 8 cells have not yet been extensively

explored. Holmes et al. (9) noted that exogenous SE MA7Adid not induce 8 cell

production of proinflammatory cytokines. CD19+ 8 cells from patients suffering from scleroderma-related interstitial lung disease showed high levels of SEMA7A

compared to healthy controls.

8 Semaphorin7A's role in inflammatory diseases

Inflammation induced by the interaction between SEMA 7A and its

receptors contributes to inflammation by stimulation of macrophage chemotaxis

(9), regulation of monocyte differentiation into dendritic cell type (9), chemokine

production, and modulation ofT cell function (11 ). In its pro-inflammatory role,

SEMA7A has been found to play a role in li ver fibrosis (32), neuronal injury(33),

pulmonary fibrosis](23), skin inflammation (1 7), corneal inflammation (17, 18),

and multiple sclerosis (26). Murray et al. (34) demonstrated that M2-type

macrophages promote transforming (TGF-~1 )-induced lung fibrosis

and that SEMA7A regulates TGF - ~1 - induced M2 and fibrocyte differentiation.

Additionally, they demonstrated that SEMA7A and its receptors are induced by

TGF - ~1 and that SEMA7A plays a vital role in a Pl3 K/PKB/AKT-dependent

pathway that contributes to TGF-~1-induced fibrosis and remodeling. Gan et al.

(23) determined the role of SEMA7 A in TGF-~1-induced pulmonary fibrosis.

Using TGF-~ transgene-positive mice, it was found that SEMA7A mediates fibrocyte accumulation in the TGF-~1-exposed lungs by ~1-integrin-dependent

pathway (23). In determining the signaling pathway by which SEMA7A induces fibrosis, it was observed that TGF-~ activates PIP-3 K and its downstream Akt

pathway in a SEMA7 A dependent way (2, 12). In hepatic fibrosis, the expression

of SEMA7A and its receptor ~1-integrin subunit was up-regulated in hepatic

stellate cells (HSCs) during liver injury. Overexpression of SE MA7A in HSCs

revealed increased fibrosis with inflammatory mediator expression while

9 SEMA?Agene silenced HSCs has decreased expression of inflammatory mediators (32).

A link between inflammation and hypoxia has been recently noted. In hypoxia-induced inflammation, proinflammatory cytokines are released by the immune cells. SEMA?A was also found to induce the production of cytokines in macrophages and monocytes that play a role during effector phase of inflammatory immune response (9). The role of SEMA?A in the development of hypoxia-elicited inflammatory response was determined by Morote-Garcia et al.

(16). This study showed that SEMA?A is significantly induced by hypoxia in a process regulated by the hypoxia-inducible factor. These studies suggest that

SEMA?A plays a vital role in varied inflammatory diseases.

Semaphorin7A and Cancer

The involvement of SEMA?A in cancer is still largely unknown. To date, it has been found that SEMA?A is up-regulated in glioblastoma cell line (35). A recent study has shown a novel role for SEMA 7 A in regulating epithelial to mesenchymal transition (EMT) in a mammary epithelial cell line. Inhibition of

SEMA?A in EpRas cells resulted in an impaired ability to undergo TGF - ~ - induced

EMT. Our laboratory first showed that murine mammary carcinomas, E0771 and

DA-3, have increased SEMA?A mRNA levels compared to the expression of nontumorigenic EpH4 mammary cells . Similar to its role in migratory function on monocytes, SEMA?A was found to directly induce migration of DA-3 tumor cells in a dose-dependent manner. Further, we demonstrated that gene silencing of

SEMA?A in DA-3 mammary tumor decreased tumor growth and reduced the

10 angiogenic potential of macrophages (36) Using an shRNA approach, we also

have demonstrated that silencing of SEMA7A in4T1 mammary tumor cells

decreased not only tumor growth but also metastasis. We postulate that inhibition

of SEMA7A decreases expression of mesenchymal proteins that assist in tumor

cell dissemination. In agreement with our murine mammary model findings, Black

et. (37) al has recently showed that inhibition of SEMA 7 A in human MCF1 ODCIS

breast tumor cells via shRNA decreased growth, motility and invasion of these

cells (37). Allegra et al., (38)also postulates a role of SEMA7A in mammary tumor cell EMT by showing that exogenous ofSEMA7A in ERF-overexpressing

EpRas cells enabled, these once epithelial cells back into a mesenchymal

phenotype (38). The function of SEMA7A in promoting tumor cell metastasis has

been shown in other models other than breast cancer. It has also been shown that inhibition of SEMA7 A also by shRNA in oral squamous cell carcinoma cells

decreased their proliferative and metastatic potential by decreasing cyclins 01

and E1 and downregulating the activity of MMPs (39). Systematic administration

of shRNA viral particles targeting the SEMA7A in a murine model of melanoma

also inhibited tumor growth and prolonged survival (40)lt is important to note that these studies collectively have shown that targeting the SEMA7A mRNA with

shRNA proves effective in inhibiting the pro-tumorigenic effects of SEMA7A. ~ is

still undetermined if the mRNA coding for SEMA7A could have a direct effect on

driving tumorigenesis or if the tumor cells could alternatively splice the SEMA7 A

mRNA in ways that enhance their selective advantage. Although these studies

11 have used animal models to elucidate the role of SEMA?A in tumors, there are no published clinical studies that have targeted SEMA7A in tumors.

12 CHAPTER 1. SEMAPHORIN7A PROMOTES TUMOR GROWTH AND EXERTS

A PRO-ANGIOGENIC EFFECT IN MACROPHAGES OF MAMMARY

TUMOR-BEARING MICE.

*This chapter contains selected portions or full text from the following publication

and is reproduced here with the permission from authors and the publisher*

Garcia-Areas R, Libreros S, Amat S, et al. Semaphorin7 A promotes tumor

growth and exerts a pro-angiogenic effect in macrophages of mammary tumor­

bearing mice. Frontiers in Physiology. 2014;5:1 7. doi:10.3389/fphys.2014.00017.

1.1 Introduction

Semaphorins (SEMAs) comprise a large family of transmembrane and

secreted proteins that have been described as molecules during

neuronal development ( 41-43). Semaphorins, grouped into eight classes, are

characterized by the presence of a conserved large (-500 amino

acids) at the N-terminal domain and differentiated by their C-terminus (4 1). Of the

8 classes of semaphorins, classes 1 and 2 are mostly found in invertebrates

while classes 3- 7 are found in vertebrates and the viral (V) class encoded by viruses. Emerging evi dence is revealing additional roles for semaphorins in the

immune system where they seem to exert di verse effects on leukocyte migration,

adhesion, and inflammatory responses (43, 44).

13 A growi ng body of evidence demonstrates the participation of classical

neuronal developmental molecules in either tumor growth or inhibition by their

effects on angiogenesis (45-50). Semaphorins have been found to affect tumor

progression by either modulating tumor angiogenesis, recruiting bone marrow

cells that could then influence tumor progression, or by directly affecting the

behavior of tumor cells. While some semaphorins were found to inhibit

angiogenesis, others enhanced new blood vessel growth. Proangiogenic

semaphorins include semaphorin 4A (SEMA4A), semaphorin 40 (SEMA4D), and

semaphorin SA (SE MASA) Capparuccia (1). However, some members in

semaphorin 3 (SEMA3) class have antiangiogenic effects (44, 51-54). Although

many classes of semaphorins have been studied in different cancers, the role of

sempahorin7A (SEMA7A) in cancer progression is largely unknown. SEMA7A is

a novel transmembrane GPI-anchored protein that has been described to function through plexin C1 and beta-integrins in multiple systems(2). Recently,

SEMA7A has been reported to be one of the proteins secreted by glioblastoma tumor cells that contribute to the highly invasive phenotype (35). In this study we

explore the role of SEMA7A in breast cancer progression using the DA-3

mammary tumor model. Specifically, we are investigating how SEMA7A can

affect macrophage production of angiogenic molecules.

There is scarce information in literature on how SEMA7A affects

macrophage induced angiogenesis. An angiogenic role for SEMA7A has been

recently described to mediate vascular growth by bFGF stimulated fibroblasts in

an experimental model of corneal neovascularization (55). In this manuscript

14 using peritoneal elicited macrophages, a rich source of peripheral macrophages, we describe that SEMA7A induces macrophages to produce angiogenic

molecules such as CXCL2/MIP-2 and that silencing the SEMA 7 A gene results in

decreased production of these growth promoting molecules.

1.2 Materials and methods

1.2.1 Mice and cell lines

Female BALB/c mice were used in all studies (Charles River Laboratories,

8- 12 week-olds), and were housed and used according to the National Institutes

of Health guidelines, under protocols approved by Florida Atlantic Uni versity

Institutional Animal Care and Use Committee. In these studies, we used the DA-

3 cell line which was derived from the D1-DMBA-3 mammary tumor syngeneic to

BALB/c mice and were provided by Dr. Diana M. Lopez, Uni versity of Miami

School of Medicine, Miami, FL (56). EpH4 mammary cells, a normal mammary

cell line, were provided by Dr. Jenifer Prosperi, Indiana University School of

Medicine-South Bend, IN. Both DA-3 and EpH4 cells were grown in complete

DMEM media (DMEM with 10% FBS). RAW 264.7 cells (American Type Culture

Collection, Manassas, VA, USA) were grown and maintained in RPMI 1640

containing 5% FBS as described previously (57, 58). Female BALB/c mice were

inoculated in the lower right ventral quadrant with 7.5 x 105 mammary tumor

cells of the following types: (1) DA-3 cells silenced for the SEMA7 A gene, (2) DA-

3 cells with scramble shRNA, or (3) wild-type DA-3 cells. Imaging studies and

caliper measurements of the primary tumors were performed up to 3 weeks post­ tumor cell implantation and discontinued after this time point since the tumors

15 become necrotic and fall off after 3 weeks. Tissues from 5-week tumor bearers were used in most of the studies, unless specified, based on our previous studies that production of tumor-derived factors peak at this time point (59). At 5 weeks, tumors are not observed in the lung, liver, and bone. The establishment of

metastastic colonies at distant sites occur at 1 0- 12 weeks if 500- 750 x 103 cells

are inoculated. For determination of angiogenesis by AngioSense (PerkinEimer,

Waltham, MA), mice were implanted with SEMA?A shRNA silenced mammary tumor cells or scramble shRNA control mammary tumor cells and imaged at 21

days post-tumor implantation while tissues were collected at 5 weeks post-tumor

cell implantation.

1.2.2 Cell cultures

To obtain peritoneal elicited macrophages (PEMs), mice were injected

intraperitoneally with 1.5 ml of 3% thioglycollate and 4 days post-thioglycollate

injection and the peritoneal exudate cells were collected by peritoneal lavage with ice-cold RPMI 1640 with 10% fetal bovine serum. It is well-established that the optimal time point for harvesting PEMs is 4 days post-thioglycollate injection

(60). As our previous studies have shown increased chemokine and MMP-9

expression at 4- 5 weeks post tumor cell inoculation, we chose 5 week time point to assess the role of SEMA?A in inducing proangiogenic factors by macrophages

(61, 62). PEMs from normal (N-PEM) and DA-3 tumor-bearing (DA-3 PEM) mice were then purified using CD11 b magnetic beads (Miltenyi Biotec Inc., Auburn,

CA). 2 x 106 cells/ml were preconditioned by culturing with rmS EMA?A (5

IJg/mL) (R&D Systems, Minneapolis, MN) and incubated for 24 h followed by

16 stimulation with LPS (500 ng/ml) (Sigma Aldrich, St.Louis, MO) for an additional

12 h for RNA and 18 h for protein collection. RAW 264.7 macrophages were also

conditioned as described above. For cell signaling inhibition studies, RAW 264.7

cells were pretreated with 1 !JM MAPK inhibitor, U0126 (Calbiochem, inhibitors,

EMD Millipore, Billerica, MA) for 1 h, conditioned wi th rmS EMA7A for 12 hand then stimulated with LPS (500 ng/ml) for an additional 12 h.

1.2.3 Immunofluorescence

To determine the expression of SEMA7A, DA-3 mammary tumor cells were plated onto a confocal cover slide, post-fixed in 4% paraformaldehyde,

blocked in 4% BSA and labeled wi th 0.1 j.Jg/ml rat anti-SEMA7A (R&D Systems) followed by incubation in secondary antibody using donkey anti -rat lgG

conjugated to AlexaFiuor 488 (Molecular Probes, Eugene, OR). To visualize

nuclei, DAPI (Vector Laboratories, Burlingame, CA) was added, cover-slipped with Vectashield and examined by confocal microscopy (Carl Zeiss

Microimaging, Inc., Thornwood, NY).

1.2.4 RNA isolation and real-time reverse transcriptase-polymerase ch ain

reaction

Total RNA was extracted from murine tumor cells, RAW 264.7

macrophages or peritoneal elicited macrophages using the RNeasy Protect Mini

Kit (QIAGEN) according to manufacturer's instructions. Briefly, eDNA was

synthesized using Quantitech Reverse Transcription Kit (Qiagen, Valencia, CA)

and gene expression was detected by SYBR Green real-time PCR analysis using

SYBR RT2qPCR primers (Qiagen, proprietary primers, sequence not disclosed).

17 The mRNA levels of gene of interest were normali zed to ~ - actin mRNA levels.

PCR cycles followed the sequence: 10 min at 95°C of initial denaturation; 15 sat

95°C; and 40 cycles of 1 min each at 60°C for annealing. The samples were

amplified using the Strategene MX3005P cycler.

1.2.5 Flow cytometry studies

The expression of CD11 band CD29 on macrophages was assessed by flow cytometry (FACS-Calibur, BD Biosciences, San Jose, CA). N-PEMs and DA-

3 PEMs were stained by incubating with FITC-CD29 (0.125 f.Jg/106 cells) and

APC-CD11b (0.1 IJg /106 cells) (both from Biolegend , San Diego, CA) for 20 min

at 4°C. Surface expression was assessed by counting 10,000 cells and analyzed

by FloJo software (Tree Star, Inc., Ashland, OR).

1.2.6 Silencing of SEMA7A in macrophages

SEMA7Agene silencing in DA-3 PEMs was achieved by RNA interference vi a short hairpin RNA (Origene, Rockville, MD ) as described above. Briefly, PT-

67 packaging cells were transfected with one of the following plasmids: (1)

plasmid encoding for shRNA sequence specifically for the SEMA7 A gene and (2)

scramble shRNA plasmid not specific for the SEMA7A gene, using Lipofectamine

2000 according to manufacturer's protocol. 0.45 f.Jm fi ltered PT-67 transfected

supernatants containing the retrovirus were used to si lence SEMA7 A gene in

DA-3 PEMs for 36 h. Macrophages were then stimulated with LPS (1 00 ng/ml) for 12 hand q-PCR was performed to confirm SEMA7A gene silencing.

18 1.2.7 Silencing of SEMA7A in DA-3 murine mammary tumor cells

Semaphorin 7 A gene silencing in DA-3 mammary tumor cells was

achieved using RNA interference via short hairpin RNA (Origene). A retrovirus

shRNA plasmid system was used for stable SEMA7A gene knockdown. To

generate the retrovirus infecting particles, PT-67 packaging cells were transfected with one of the following plasmids: (1) plasmid encoding for shRNA

sequence specifically for the SEMA7 A gene and (2) scramble shRNA plasmid

not specific to the SEMA7 A gene. Transfection was performed using standard

Lipofectamine 2000 according to manufacturer's protocol. The different variants

of transfected PT-67 cells were selected for 2 weeks with puromycin (2 j.Jg/ml)

and the cell-free/retrovirus-rich supernatants from the different PT-67 variants

and controls were used to infect DA-3 cells for 24- 48 h. The different DA-3 variants were then selected with puromycin (1 j.Jg/ml) for 4 weeks. To confirm

gene knockdown, real time quantitative polymerase chain reaction (q-PCR)

(Qiagen) was performed using the SEMA?A specific primers according to

manufacturer's protocol. Cells were passaged and selected until at least a 5-fold

decrease in the SEMA7Agene expression was achieved when compared to the

scramble control. The results of gene expression were then confirmed by western blotting for the SEMA7A protein.

1.2.8 Monocyte migration assay

To test migration, RAW 264.7 murine monocytes were labeled with

Calcein-AM (1 0 !JM) and used in a modified Boyden Chamber assay. Briefly, 105

RAW264.7 were placed in the transwell insert (8 !JM pores) (BD Biosciences) of

19 the upper chamber wi th lower chamber containing supernatants from: (1) DA-3

cells silenced for the SEMA7A gene, (2) DA-3 cells with scramble shRNA, and

(3) wild-type DA-3 cells and incubated at 3rC in a C02 incubator for 12 h. RAW

264.7 macrophage migration was measured using a plate reader set at an

excitation wavelength of -485 nm and an emission wavelength of -520 nm.

Absorbance values among the various groups were measured at least 2 times in triplicate and fitted to a 7-point standard curve.

1.2.9 Protein determination

DA-3 murine mammary tumor cells were cultured under optimal conditions

using DMEM culture media with 10% FBS until -80% confluency was achieved.

DA-3 tumor cells and DA-3 SEMA7A-silenced cells or intraperitoneal

macrophages from 5-week DA-3 mammary tumor-bearing mice were lysed with

sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol,

10 mM NaF and bromophenyl blue) and used to extract total protein. 20 f.Jg of total protein from DA-3 cells and PEMs were resolved on 4- 20% Mini-Protean

SDS-PAGE gradient gels (BioRad Life Sciences, Hercules, CA) and transferred to PVDF membrane (Pierce, Rockford, IL) using a semi-dry transfer transblotter

(BioRad) at 20 Volts for 40 min. The membrane was blocked overnight at 4°C in

SeaBiock (Calbiochem), and subsequently incubated at room temperature with

anti-mouse SEMA7A monoclonal antibody (1 f.Jg/ml) (R&D Systems) and anti ­

mouse beta actin polyclonal antibody (0.25 f.Jg/ml) (Li -Cor Biosciences, Lincoln,

NE). Western blots were washed for 10 min three times with 0.5% Tween-PBS followed by 1 h incubation at room temperature with corresponding fluorescent

20 antibodies (Li-Cor Biosciences). Blots were washed again for 10 min three times with O.S% Tween-PBS and then dried at 3JOC for 20 min. The membranes were then were vi sualized with Li-Cor imager. Protein concentration was normalized to

beta-actin as loading control. ELISAs of CXCL 1, CXCL2/MIP-2 and MMP-9 (R&D

Systems) were performed followi ng manufacturer suggested protocol from DA-3 tumor control mice.

1.2.10 Immunohistochemistry

Formalin-fixed tissue from controls, SEMA?A scramble controls and

SEMA?A silenced tumors was paraffin embedded and sectioned at 4-micron thickness. Pre-treatment of formalin-fixed, paraffin-embedded tissue sections with heat-induced epitope retrieval (HIER) was done using diluted EnVision™

FLEX Target Retrieval Solution, High pH (SO x) (Dako Omnis, Carpinteria, CA) following manufacturer protocol. The sections were deparaffinized and stained with hematoxylin and eosin (H&E) with automated Tissue Tek® 2000 processor

(Sakura-Finetek, Torrance, CA). Adjacent tumor sections were assessed for vascularity using CD31 antibody. Dako FLEX monoclonal mouse anti -human

CD31 antibody (diluted 1:30, DAKO) was used to highlight the vasculature of the tumors. CD31, expressed almost exclusively on endothelial cells, is a brown

antibody stain against a hematoxylin counter stain. Photographs were taken at

SO x magnification with mineral oil immersion using Olympus MDOB3 microscope

and photographed with OlympusDP21 digital camera (Center Valley, PA).

21 1.2.11 Tumor measurements and in vivo imaging for angiogenesis

Tumor size determination was performed by measuring the two longest

perpendicular axes in the x/y plane of the tumor nearest to 0.1 mm by caliper

measurement. The depth was assumed to be equivalent to the shortest of the

perpendicular axes, defined as y and tumor volume = x(y)2/2. To account for vascularization in mice injected with either wild type DA-3 tumor cells or those

silenced for SEMA7 A, near infrared blood pool agent AngioSense 680 probe (2

nmol/mouse in 150 !JL volume) (Perkin Elmer, Waltham, MA) was injected vi a tail vein 24 h before imaging. Mice were imaged using a bioluminescence optical

imager (IVIS Lumina LT E, Perkin Elmer). Maximal near infrared signals were

quantified using Living Image 2.5 (Xenogen, Perkin Elmer) image analysis

software. Infrared signals are reported as photons/s.

1.2.12 Statistical analysis

Results are expressed as means ± standard deviation. Statistical analyses were performed using GraphPad Prism 3 software (LaJolla, CA). Statistical

comparisons of paired groups were determined by Student's t-tests. Values of p

< 0.05 were considered statistically significant.

1.3 Results

1.3.1 SEMA7A is expressed in DA-3 mammary tumor cells and expression

is increased in peritoneal elicited macro phages of DA-3 mammary tumor­

bearing mice

Semaphorins have been described to be expressed by various cell types.

Although it is known that SEMA?A is expressed by monocytes, activated T cells,

22 and keratinocytes, it is not known if tumor cells express SEMA7 A . We therefore

cultured DA-3 mammary tumor cells and assessed for SEMA7A expression.

Confocal image shows that SEMA7A is expressed by the DA-3 mammary tumor

cell line (Fig. 1A). We then asked if SEMA7A is expressed by EpH4 mammary

cells, a normal mammary cell line, and how do these levels compare with those

in DA-3 tumor cells? qPCR revealed very low levels of SEMA7A expression in

EpH4 cells compared to DA-3 mammary tumor cells (Fig. 1B ).

Members of the semaphorin family have been reported to be cleaved to generate

soluble forms that have effects on immune function (63). It was not known if

SEMA7A is solubilized in our tumor model. Since there are no reliable ELISAs

available to quantify secreted SEMA7A protein, dot blot analysis was used to

determine if SEMA 7 A is solubilized. Analysis of total protein from supernatants of

3 day DA-3 mammary tumor cell cultures confirmed the soluble protein

expression of SEMA7A, with increased levels reflected by increased cell

numbers (Fig. 1C ). It is possible that circulating levels of cleaved SEMA7A could

have effects on other cells.

In the immune system, SEMA7A has been reported to be expressed in the

myeloid and the lymphoid lineage cells (64). There are no studies to date

describing the expression of SEMA7 A in macro phages of mammary tumor

bearers. Thus, thioglycollate peritoneal elicited macrophages from normal (N­

PEMs) and DA-3 mammary tumor-bearing mice (DA-3 PEMs) were therefore tested to determine SEMA 7 A expression. tt is well-established that the optimal time point for peritone·al elicited macrophages is 4 days post-thioglycollate

23 injection (60). At earlier time points (e.g., 4- 24 h post-thioglycollate) the majority

of cells in the peritoneal cavity consist of neutrophils (65, 66). SEMA?A

expression was determined at 3, 4, and 5 days post-thioglycollate injection in

normal and DA-3 mammary tumor-bearing mice. There were no significant

differences in SEMA7A expression at these days in peritoneal elicited cells from

either normal or tumor-bearing mice. We therefore opted for 4 days as our set time point for these studies. A 3-fold increase in SEMA?A expression at the

mRNA level was found in peritoneal elicited macrophages from DA-3 PEMs (Fig.

1 D) compared to the expression in N-PEMs. Similarly, increased protein

expression of SEMA7Awas found in DA-3 PEMs compared to normal PEMs

(Fig. 1 E). Quantification of the bands from western blot analysis confirmed

increased SEMA?A protein expression in DA-3 PEMs.

1.3.2 Expression of SEMA7A receptor, 131 integrin (CD29) is increased in

DA-3 mammary tumor cells and macro phages from mammary tumor­

bearing mice

The principal signaling function of SEMA?A in the nervous and immune

systems is mediated through a1~ 1 integrin (10, 11, 67). Increased ~ 1 signaling

has previously been shown to be associated with decreased survival in invasive

breast cancer (68). We first determined if there is a differential ~ 1 integrin

expression in EpH4 and DA-3 mammary tumor cells. Flow cytometric analysis

showed that even though the percentage of~ 1 integrin (CD29) positive cells

remained unchanged between the normal EpH4 cells and the DA-3 mammary tumor cells, the mean fluorescence intensity was almost doubled in the tumor

24 cells (Fig . 2A). The expression of SEMA7A's receptor, ~ 1 integrin, in peripheral

macrophages between normal and tumor bearers has not yet been well

characterized. We determined if there are altered levels of~ 1 integrin

expression in peritoneal elicited macrophages (PEMs) from normal and DA-3

mammary tumor-beaning mice. PEMs were gated based on the fluorescent

intensity ofCD11bexpression (Fig. 28). Flow cytometric analysis ofCD11blow

PEMs from normal and DA-3 tumor bearing mice revealed no significant

differences in the frequency of CD29+ cells (Fig. 2C). In contrast, expression of

CD11 bhiCD29+ in DA-3 PEMs was higher (p < 0.05) compared to the expression

in normal PEMs (Fig. 2C).

1.3.3 Treatment of macro phages with rmSEMA7 A induces production of

angiogenic CXCL2/MIP-2

Macrophages from tumor-bearing mice are known to produce angiogenic

molecules (69). Previous studies have shown that tumor-derived factors induce

macrophages to produce angiogenic and proinflammatory molecules (70).

Holmes et al. have shown that SEMA7A induces the production of

proinflammatory molecules including the IL-8 homolog of chemokine

CXCL2/M IP-2, which also has angiogenic properties (9). As shown in the

previous section, DA-3 mammary tumor cells express and shed SEMA7A. We therefore determined whether soluble SEMA7A has an effect on macrophage function. Toward these studies we used the macrophage cell line RAW 264.7 in which SEMA7A mRNA was undetectable (CT value > 37). RAW264.7

macrophages, as a model of tissue macro phages isolated from normal mice,

25 have been used frequently for in vitro studies of macrophage function. qPCR

analysis of RAW 264.7 macrophages preconditioned with rmSEMA7A revealed that expression of proangiogenic molecules CXCL2/M IP -2 was increased by 5- fold (p < 0.001 ) (Fig.3A) after LPS stimulation. We found a significant (p < 0.01 )

increase in CXCL2/M!IP-2 protein in RAW 264.7 macrophages treated with

rmSEMA7A and LPS (Fig. 38). These studies also included culturing of RAW

264.7 cells with rmSEMA7A alone, which also showed an increase in

CXCL2/MIP-2 (data not shown). SEMA7A has previously been reported to function through ~ 1 integrin activation of MAPK signaling pathway to promote

monocyte inflammatory response (11). To get insight if SEMA7A induces

CXCL2/M IP-2 via MAPK pathway, RAW 264.7 macrophages were pretreated with a MAPK inhibitor (U0126). We found that U0126 conditioned and

rmSEMA7A treated cells exhibited decreased (p < 0.01 ) production of

CXCL2/MIP-2 compared to those cultured with rmSEMA7A alone (Fig. 3C).

To determine if freshly isolated macrophages from normal and DA-3 mammary tumor bearers express CXCL2/MIP -2, peritoneal elicited macrophages from

normal and DA-3 mammary tumor bearers were obtained and assessed for

CXCL2/MIP-2 expression by qPCR. A greater than 5-fold increase (p < 0.001 ) in

CXCL2/MIP-2 expression was observed in DA-3 PEMs compared to normal

PEMs (Fig. 30). We have previ ously shown that tumor-derived factors have an

effect on profile of PEMs (71-74). Therefore, peritoneal elicited macrophages were used as we wanted to determine the effect of SEMA7A in circulation on

macrophages. Since RAW 264.7 macrophages treated with rmS EMA7A had

26 increased expression of CXCL2/M IP -2, we determined if treatment of N-PEMs with rmS EMA7A had an effect on production of angiogenic molecule,

CXCL2/M IP-2. A considerably (p < 0.05) enhanced expression of CXCL2/M IP-2 was observed in N-PEMs pretreated with rmS EMA7 A and then stimulated with

LPS (Fig. 3E). Given that SEMA7A is known to induce CXCL2/MIP-2, and PEMs from DA-3 mammary tumor bearers have increased CXCL2/MIP-2 and SEMA7A, we silenced the SEMA7 A gene in DA-3 PEMs using shRNA. Effectiveness of

SEMA7 A gene silencing as indicated in the 1st set of bars shows that SEMA7A

gene was significantly (p < 0.001) silenced compared to the scramble control

(Fig. 3F). SEMA7A gene silenced DA-3 PEMs expressed significantly less

CXCL2/M IP-2 compared to scramble control as determined by q-PCR (Fig. 3F).

~ is important to note that our previous studies show that DA-3 cells express

CXCL2/M IP-2. ~is possible that SEMA7A could function in an autocrine manner to upregulate the expression of CXCL2/M IP-2.

1.3.4 Decreased tumor-derived SEMA7A results in reduced in vitro

macrophage migration and CXCL2/MIP-2 production

Holmes et al. demonstrated that SEMA7A is a potent monocyte

chemoattractant with 1000-times greater chemotactic activity than monocyte

chemotactic protein, MCP-1 (9). We hypothesized that silencing SEMA7A gene

in DA-3 mammary tumor cells would result in decreased secretion ofSEMA7A in tumor cell cultures and treatment of macrophages with this conditioned media would therefore have a negative influence on their migration. Thus, SEMA7A

gene was silenced in DA-3 mammary tumor cells by shRNA. Western blotting

27 was performed to test the effectiveness of SEMA7A gene silencing. Lane 1

indicates DA-3 wild ty[pe, lane 2 shows DA-3 scramble shRNA and lane 3

consists of DA-3 SEMA7A shRNA knockdown (Fig. 4A, top panel). Integrated

intensity graphs show a 6-fold decrease in SEMA7A expression in DA-3

SEMA7A shRNA knockdown cells compared to either DA-3 wi ld type or DA-3

scramble shRNA cells (Fig. 4A, bottom panel). Although DA-3 cells express

lower levels of CXCL2/MIP-2 compared to macrophages, silencing the SEMA7A

gene also lead to a decrease in tumor-derived CXCL2/MIP-2. To determine if

SEMA7A plays a role in monocyte migration, a modified Boyden chamber assay was performed using RAW 264.7 murine macrophages and conditioned media from wild type DA-3 tumor, DA-3 scramble shRNA, or DA-3SEMA7A shRNA

knockdown cells as possible chemoattractants. Fewer number of RAW 264.7

monocytes migrated towards the conditioned media from SEMA7A silenced DA-3

cells compared to media from either wild type DA-3 tumor cells or DA-3 cells with

scramble shRNA (Fig. 48). Since we demonstrated that DA-3 mammary tumor

cells produce SEMA7A, and that treatment of macrophages wi th rmS EMA7A

induced the production of proangiogenic CXCL2/MIP -2, we hypothesized that

silencing SEMA7 A gene in DA-3 mammary tumor cells would have an inhibitory

effect on production of CXCL2/M IP -2 by macrophages treated with tumor cell

supernatants silenced for the SEMA7A gene. We therefore tested to see if

SEMA7A gene silencing in tumor cells has an effect on CXCL2/MIP-2 chemokine

expression. In macrophage cultures with conditioned media from SEMA7A

28 shRNA knockdown DA-3 cells, there was a significant (p < 0.01 ) reduction in

CXCL2/MIP-2 expression compared to the cultures with SEMA7A (Fig. 4C).

1.3.5 Decreased tumor growth in mice bearing SEMA7 A silenced

mammary tumors

Culturing of RAW 264.7 orthioglycollate elicited macrophages with

rmSEMA7A induced the expression of CXCL2/MIP-2, a pro-angiogenic

chemokine. We have previously shown that mice bearing either the parental 0 1-

DMBA-3 or DA-3 mammary tumors exhibit higher levels of pro-angiogenic

molecules (62). It is well-established that angiogenesis is required for invasive tumor growth and that tumors do not grow more than 1 mm3 in the absence of

angiogenesis (75). We have shown in the previ ous section that SEMA7A induces

production of angioge·nic molecules by macrophages. We therefore determined if

implantation of BALB/c mice with SEMA7A knockdown DA-3 mammary tumors

has an inhibitory effect on tumor growth. To determine the in vivo role of

SEMA7A , mice were implanted with either wi ld -type DA-3, scramble shRNA DA-

3, or SEMA7A gene knockdown DA-3 (SEMA7A KD ) mammary tumor cells. Mice

implanted with SEMA 7 A KD tumors had significantly (p < 0.01 ) decreased

primary tumor volume compared to the wild type or SEMA7A scramble control

DA-3 mammary tumors (Fig. SA). Since SEMA7A KD tumors had lower tumor volume, we tested to see if there is decreased angiogenesis in these mice by use

of AngioSense fluorescent probe and CD31 staining by immunohistochemistry.

Thus, an AngioSense fluorescent probe was used to determine the extent of

angiogenesis in the tumors by an in vivo imaging system. Shown in the upper

29 panel are mice bearing wild type DA-3 tumors; the middle panel, scramble

control for shRNA; while the bottom panel shows mice bearing SEMA7A KD tumors. Significantly (p < 0.01 ) decreased angiogenesis was observed in mice

bearing the SEMA7A KD tumors compared to the scramble controls or wild type

DA-3 mammary tumors (Fig. 58). We also show the quantification results of in vivo imaging indicating a similar trend in tumor growth. Decrease in angiogenesis

in SEMA7A KD tumor sections was also observed by immunohistochemistry.

H&E and immunohistochemical staining for CD31 highlighted angiogenesis in

control tumors but minimally in SEMA7A KD tumors (Fig . 5C).

1.3.6 Peritoneal elicited macrophages from mice bearing SEMA7A KD tumors produce decreased levels of angiogenic molecules

4- 5 weeks post-tumor cell implantation, thioglycollate elicited

macrophages from DA-3 scramble shRNA control or DA-3 SEMA 7 A shRNA

mammary tumor-beani ng mice were analyzed for the production of pro­

angiogenic chemokines CXCL2/M IP -2, CXCL 1 and matrix metalloprotease MMP-

9. LPS-stimulated macrophages from mice implanted with SEMA7A gene

silenced DA-3 mammary tumors produce significantly (p < 0.01 ) lower amounts

of pro-angiogenic molecules compared to those implanted with SEMA7A

scramble control DA-3 tumor cells. While there were no major differences in

secretion of CXCL2/M IP-2 and CXCL 1 in unstimulated macrophages from either

SEMA7A scramble control or SEMA7A silenced mammary tumor-bearing mice, there were significant (p < 0.01 ) differences in the production of both these

chemokines from LPS-stimulated (100 ng/ml) cultures (Fig . 6A-B). Thus, LPS

30 stimulated macrophages from scramble control DA-3 mammary tumors produced

-25 ng/ml of CXCL2/MIP-2 while those from SEMA7A silenced tumor bearers

produced -18 ng/ml (Fig. 6A). Similarly, LPS stimulated macrophages from

scramble controls produced -24 ng/ml and those from SEMA7A silenced DA-3 tumor-bearers' macrophages produced 16.8 ng/ml of CXCL 1 (Fig. 68).

Interestingly, implantation of SEMA7 A knockdown tumor cells decreased the

production of MMP-9 by intraperitoneal macrophages in both unstimulated and

LPS-stimulated cultures (Fig. 6C). Furthermore, we assayed a series of tumorigenesis-related genes by qPCR on peritoneal macrophages from SEMA?A

shRNA KD or shRNA scramble control DA-3 mammary tumor-bearing mice.

PEMs from SEMA?A KD tumor bearing mice showed a significant reduction in

VEGF-A expression but not VEGF-8 expression (Fig. 60). In contrast,

expression of both (EGF) and platelet growth factor

(PGF) was significantly reduced in PEMs from SEMA?A KD tumor bearing mice

(Fig. 6E). Interestingly, the levels of serpinf1 , a secreted protein that has both

anti-angiogenic and anti-tumorigenic functions, was significantly increased in

PEMs from SEMA?A KD tumor bearers (Fig. 6F).

1.4 Discussion

The biological role of SEMA?A in breast cancer progression was explored

in this study. First, we find that SEMA?A is expressed by mammary tumor cells.

Second, we show that SEMA7 A expression is up regulated in macrophages of

mammary tumor-beani ng mice. Third, we demonstrate that SEMA?A induces the

expression of proangiogenic molecule CXCL2/MIP-2 in macrophages. Fourth, we

31 find decreased tumor growth in mice implanted with SEMA?A shRNA DA-3

mammary tumor cells . Lastly, we find that there is decreased angiogenesis in

mice implanted with SEMA?A knockdown mammary tumors. These findings

suggest that SEMA7 A could have a direct effect on tumor cell growth and

macrophage function. We are the first to show that SEMA?A plays a role in

breast cancer progression.

SEMA?A was first identified in the immune system, as myeloid and

lymphoid lineage cells have been reported to express this molecule (4, 76, 77)

There are very few reports on SEMA?A expression as it relates to cancer. We

are the first to clearly demonstrate that SEMA?A is expressed by mammary tumor cells. Formolo et al. identified SEMA?A as one of the proteins in highly

invasi ve astrocytoma cell line US? while the less aggressive cells do not express this protein (35). Our results parallel with these results as DA-3 mammary tumor

cells had greater intensity in expression of this SEMA? A compared to the

nontumorigenic mammary EpH4 cells. This raises the possibility that metastatic tumors express higher levels of SEMA?A. We are actively pursuing this in our

laboratory by assessing different breast tumor cell lines with varying levels of

metastatic potential for SEMA7 A expression and correlating with aggressive

behavior. Interestingly., while SEMA?A is known to affect monocyte activation in vitro via ~ 1 integrin-mediated effects (9), the role of SEMA?A in the activation of tumor cells has not yet been studied. We found that while PEMs from normal

mice express low levels ofSEMA?A, the expression of this protein is increased in

PEMs from tumor bearers. So what induces the expression of this molecule in

32 macrophages? In a murine fibrosis model, TGF - ~ has been reported to induce the expression ofSEMA7A in the murine lung (1 2)). We are testing tumor- and/or

host-derived factors in inducing SEMA 7 A expression in PEMs.

Although the identification of SEMA7A receptors remains controversial, two potential receptors have been identified, i.e., plexin C1 and the ~ 1 subunit of

integrin receptor. The biological activities of SEMA 7 A in the immune system have

only recently been elucidated. SEMA7A induces the production of inflammatory

cytokines such as IL-6 , TNF-a and IL-8 (11 ), an effect that could be mediated through direct interaction of GPI-anchored SEMA7A protein with a1 ~ 1 integrins

on target cells. Alternatively, SEMA7Acould be cleaved by ADAM-17 and have

paracrine effects on other cells. Cell surface bound semaphorins have been found to be proteolytically cleaved in order to exert their biological function. For

example, in order to exert proangiogenic effect, SEMA40 is proteolytically

cleaved by membrane type 1-matrix metalloproteinase, and the resulting soluble form acts on endothelial cells to enhance angiogenesis (78). SEMA?A is a GPI­

anchored protein that has been found to be cleaved in platelets by ADAM-17

(13). We have previously reported increased expression of ADAM-17 in

mammary tumor-beani ng mice (61). ~ is possible that ADAM-17 in the tumor

bearers could affect cleavage of SEMA7A. Biological effects of SEMA7A have

been reported to function through both the soluble and membrane forms. Soluble

SEMA7A has been shown to be an extremely potent monocyte chemoattaractant

(9) while membrane bound SEMA7A has been reported to stimulate monocytes

and macrophages through a1 ~ 1 integrin and increase production of

33 proinflammatory cytokines including IL-6 and TN F-a (1 1). SEMA?A has been

shown to promote spreading and dendricity in human melanocytes through its

receptor, (3 1-integrin. In this study, we report that peritoneal elicited

macrophages from mammary tumor-bearing mice express higher levels of (31

integrins as well as its ligand SEMA?A compared to the control mice in tumor

bearers' macrophages, suggesting that SEMA?A could function in a paracrine

manner. In a cancerous system, it is probable that SEMA7 A could mediate its functions through both membrane and soluble forms.

We have previously shown that macrophages from mammary tumor­

bearing mice produce angiogenic molecules in response to tumor-derived factors

(79). Angiogenesis plays a crucial role in growth of tumors since solid tumors

cannot grow beyond 1- 2 mm3 without establishing an adequate blood supply

(75). Using immunohistochemistry and an AngioSense probe, an in vivo blood

pool vascular fluorescent imaging agent, we determined the in vivo role of

SEMA?A by comparing angiogenesis in mice bearing scramble shRNA DA-3

mammary tumors with those bearing SEMA?A shRNA knockdown DA-3

mammary tumors. Since these studies showed a significant reduction in tumor volume in SEMA?A shRNA knockdown DA-3 mammary tumors, we hypothesized that these mice would produce decreased levels of angiogenic molecules. It is

also possible that although we have knocked down the gene in the tumor cells,

host derived SEMA?A may contribute toward angiogenesis. Using SEMA?A

knockout mice, we are determining the effects of tumor-derived vs. host-derived

SEMA?A.

34 Axonal guidance molecule expression is dysregulated in many types of cancer,

including breast cancer, suggesting that they may be excellent targets for

effective therapeutic strategies (80). In this report we provide novel data showing that macrophages from SEMA?A shRNA knockdown mammary tumor bearers

have decreased production of angiogenic chemokines CXCL2/MIP-2 and CXCL 1

as well as matrix degrading enzyme, MMP-9. Although it is known that cytokines

such as TNF-a induce MMP-9 through MAPK pathway (81, 82), there are no

studies in literature describing induction of MMPs by SEMA?A. We are the first to

show a relationship between MMP-9 and SEMA?A. We speculate that SEMA?A-

13 1 integrin ligation may activate MAPK pathway.

Activation of MAPK pathway has been shown to play an important role in tumor invasion and metastasis via interaction of integrins with specific receptors

(83). Further, integrins have been reported to associate with receptor tyrosine

kinases (RTKs) to activate signaling pathways, including MAPK pathways that

are necessary for tumor invasion and metastasis. We have also shown that

macrophages from SEMA7 A shRNA knockdown mammary tumor bearers have

increased levels of serpinf1 , a secreted protein known to have anti -angiogenic

and anti-tumorigenic functions (84). ~ is possible that SEMA?A could act in an

autocrine manner to upregulate the expression of not only angiogenic molecules,

but also the integrins to enhance metastatic growth. We continued this study by

characterizing the effect of SEMA?A on different mammary tumor cells and their

ability to migrate and metastasize.

35 A

IU-J i\lanunllr) Tumor Cells

B c

2S ••• 5 [-.{17 T ~ 1 ~ .t E<07 ..... s ~ ~ f•07 .. I E+07 0 E-{10 ...... __._ ...... _._,,..._...... , " Ohio• 1,1o• 2,1o• Cpll ~ DA-3 - DA-3TumorCcUs / nlL -j; ,•:J D E l><>nnal DA·' I'EJ\b I'E~h

15 ~Do - SEJ\1A7A

50 kOo - - I""3•3CIIII I l +O I

NonmiPEI\Ih Oi\-3 PEMs Nonml PEMs DA-3 Pe..ls

Figure 1 DA-3 mammary tumor cells express SEMA7A. (A)Shown is the confocal images of DA-3 mammary tumor cells for SEMA7A expression; (B) SEMA?A expression in EpH4 and DA-3 mammary tumor cells as determined by qPCR; (C) DOT blot analysis of DA-3 mammary tumor cell culture showing that SEMA?A is solubilized; (D) mRNA expression of SEMA?A in peritoneal elicited macrophages (PEMs) from normal and DA-3 mammary tumor­ bearing mice; (E) Western blot analysis of total protein from PEMs from control and DA-3 mammary tumor bearers with 7 mice/group; also shown is integrated intensity graph of western blot indicating higher levels of SEMA?A expression in tumor bearers' PEMs. In-vitro experiments are representative of three independent experiments, ' "'p::; 0.001.

36 A VNSfAINEO u: 800 ., 600 ~ u z !-u: -1 00 --' 0. u0 ... , 200 0 u 0 Ep ll-.1 Or\-.3 C029-FI TC

B Normal DA-3 c PF.i\ls PEl\ Is + ss 1"• "' .c "'0 40 --~ u 2~ - ·- .. 3 .,... 0 20 ;::; .. 0 - ....)

Figure 2 J31 integrin expression is increased in DA-3 cells and macrophages of mammary tumor-bearing mice. (A) Flow cytometric analysis of DA-3 mammary tumor cells and EpH4 mammary epithelial cells for the expression of ~1 integrin (CD29) (a representative plot of 2 independent experiments); (B) scatter plot of CD11 b+ and CD29 expression in PEMs from normal and DA-3 mammary tumor-bearing mice; (C) flow cytometric analysis of PEMs from control and DA-3 mammary tumor bearers gated on either CD11 blow or CD11 bhigh and assessed for the expression of CD29, N = 8, *p ~ 0.05.

37 0 RA W2h-1 7 1\hu.wphul!cs ~ =i c.:..,. (I ... T ~ T 1 e 11, r,o ~ -1 ~0 ' :J 2 '-.)

'-.):.-: I I II I - I ti 1(1\llh llH.!dlll 11nSFMA?A NORMAl I'I~M" DA-"1 I'I'Ms

_, OCI"<''f' ug.:s E 611 Nnnnal PFMs

.j - )II ~.-Ills ~ lll j :!II ~ Ill 11 "' Gnm rh 1\lo:

c F RA W1(>-1 7 Mn~mphn~tcs DA·l PI·Ms I ~0 D\chiJ< - D l'HI.26 a;.r \1 \1 \ >hRN \ E 1111 ~ ~ (l(l :;;;: 8 )(J ~ () ~--~~~~~~~ !-.FMA7A tXli.:!/MIJ>-2

Figure 3 Increased expression of angiogenic chemokine, CXCL2/MIP-2 in rmSEMA7A treated RAW264.7 macrophages and peritoneal elicited macrophages from mammary tumor-bearing mice. The effect of treatment with rmS EMA7A in RAW264.7 macrophages is shown in panels (A- C). Expression of CXCL2/M IP -2 at mRNA level (A) and at protein level determined by ELISA (B) is increased in RAW 264.7 macrophages treated with rmSEMA7A; and (C) effect of treatment with U0126, a MAPK inhibitor (1 1-JM) on CXCL2/M IP-2 expression by RAW 264.7 macrophages treated with rmSEMA7A. CXCL2/M IP-2 expression is increased in peritoneal elicited macrophages (PEMs) from DA-3 mammary tumor bearers compared to N-PEMs: (D) increased mRNA expression of CXCL2/MIP-2 in DA-3 PEMs compared to PEMs from normal as determined by qPCR, N = 8; (E) CXCL2/MIP-2 ELISA of normal PEMs treated wi th rmSEMA7A and stimulated with LPS; and (F) the effect of SEMA7A gene knockdown in DA-3 PEMs on mRNA expression of SEMA7A and CXCL2/MIP-2 as determined by qPCR In-vitro experiments are representative of three independent experiments. ·p::; 0.05, ""p::; 0.01, ..* p::; 0.001

38 A J

;,: E 3 E-03 ~ ~ ] j 2 E-HJ 1:-r a:! 1E-m .S""' ~ 0 F+OO ._..----il...r-l.__..&.....r...__....a..., D.\-1 \\'lid- DA-3 D \-J I'1"' Smunblo St~L\7 .\ B >hkN.\ •hK..'Io\ t-.0

c

ll\-3 \l'1l

Figure 4 Decreased tumor-derived SEMA7A results in reduced in vitro macrophage migration and CXCL2/MIP-2 production. (A) Effect of SEMA7Agene silencing on SEMA?A expression in DA-3 mammary tumor cells as determined by western blot analysis; (B) migration of Calcein AM labeled RAW 264.7 macrophages toward cell-free supernatants from either wild type DA-3 tumor cells, SEMA?A scramble shRNA DA-3 tumor cells, SEMA?A shRNA knockdown DA-3 cells or serum free media alone. (C) mRNA expression of CXCL2/M IP-2 on macrophages from normal mice in vitro treated with conditioned media from either wild type, SEMA?A scramble or SEMA?A knockdown DA-3 mammary tumor cells. In-vitro experiments are representative of three independent experiments. p :s: 0.01 .

39 A c (IJJI . 0\·' \\ll

B

l}fl<

11\·J

0 ·\·J 0 ,., 0 \· \ \\ •ldot)'pe ~ .... we "l"\1~1 \ ~~R~ \ ohR.' \ ._,,

Figure 5 Decreased tumor growth and angiogenesis in mice bearing SEMA7 A silenced mammary tumors. (A) Tumor volume is decreased in mice bearing SEMA?A shRNA knockdown DA-3 mammary tumor compared with either wild type or SEMA7 A scramble shRNA DA-3 mammary tumors; (B ) ventral image of DA-wild type, SEMA?A scramble shRNA DA-3 or SEMA?A shRNA knockdown DA-3 mammary tumor bearing mice showing 1 out 20 mice/group; quantification (photons/sec) of AngioSense specific fluorescent signal indicating decreased angiogenesis at 21 days post-tumor implantation in the SEMA?A knockdown group, N = 10 per group, repeated twice **p ::; 0.01. (C) H&E and CD31 staining in tumor sections from DA-3 wild type, scramble shRNA and SEMA?A shRNA knockdown tumor sections. Significance is indicated *p;::: 0.01.

40 [] l) \·J ~Iam bic !iliRI\ \ ·I'E\1> A [] D \-l \7A shR'A t-.0-l'f-\b 0 30 "''\I 15 "E ... "~ ;:;= 20 e I d. "" ~ 10 ~ 0.5 "' -f. ;:::; ~ X (.; 0 E 0 GrO\IIh Mcdaa IPS VEGF·A VECIF·B

B E 30 12 "E """ I .., 20 = o.s ~ e ~ 0.6 G- 10 ~ 0.-1 ,. -r '-' t2 0.2 0 E 0 CirO\\Ih Media Ll~ EGF P<.iF c F 50 6 40 u 5 •• "E ,.~ c 30 4 ;;:"" e d. 20 ""<; 3 ;: "- 2 ;: IU -c / I () "'5 0 \lfQ\\Ih ~ ledm ll~ 'icrpincl Sc:rpin fl

Figure 6 Macrophages from mice bearing SEMA7A shRNA knockdown tumors produce decreased levels of angiogenic molecules. ELISA of peritoneal macrophages from scramble shRNA DA-3 and SEMA7A shRNA knockdown DA-3 tumor bearers cultured with and without LPS for protein levels of: (A) CXCL2/MIP-2 ; (B) CXCL 1; and (C) MMP-9, N = 16. qPCR of peritoneal macrophages from scramble shRNA DA-3 and SEMA7A shRNA knockdown DA-3 tumor bearers for mRNA expression of: (D) VEGFA and VEGFB; (E) EGF and PGF; and (F) Serpine 1 and Serpinfl, N = 6, repeated twice . ..p :::;; 0.01 , *""p :::;; 0.001 .

41 CHAPTER 2. SEMAPHORIN 7A PLAYS AN IMPORTANT ROLE IN BREAST

CANCER GROWTH AND METASTASIS

2.1 Introduction

Semaphorins are a large family of conserved proteins originally

characterized as directional cues in axonal guidance and neurite outgrowth in

neurogenesis [1-3]. Srubsequently, it has been revealed that semaphorins and their receptors carry out roles beyond neurogenesis and serve functions in

immune regulation, extracellular matrix remodeling, organogenesis, and

angiogenesis [3-7]. Studies have identified the expression of Semaphorin 7A

(S EMA?A) in tumor cells , however, few have described a functional role for

SEMA?Ain tumor progression [8-11]. Hence, its contribution to tumor

progression remains relatively unclear in comparison to other vertebrate

semaphorins.

SEMA?A, or CD108w, is a -80 kDa GPI-anchored transmembrane protein

expressed by multiple cell types including: neurons, immune cells, melanocytes, fibroblasts, bone cells , and tumor cells [12]. This protein can be shed from the

cellular membrane by action of ADAM-17(TACE) [13]. Both anchored and soluble forms of SEMA?A have been shown to bind to PlexinC1 and beta-1 integrin

(CD29) [14-17]. The latter activates the MAPK and FAK pathways in a model of

experimental autoimmune encephalomyelitis causing an increase in

proinflammatory cytokine gene transcripts and proteins. Our group has

42 demonstrated that DA-3 murine mammary tumor cells exhibit high levels of

SEMA7A at both the transcript and protein le vel. Inhibition of DA-3-derived

SEMA7A resulted in decreased angiogenesis, causing the retardation of tumor

growth in vivo [15]. However, the role of SEMA7A in the metastasis of solid tumors is unclear.

~ has been shown that activation of the PI3K/AKT pathway leads to the

expression of SEMA7A [17, 18]. Gi ven that the PI3KIAKT pathway plays an

important role in tumor cell survival and metastasis [19, 20], we investigated the

connection between SEMA7A and the PI3K/AKT pathway in breast cancer[17]. ~

is well established that TGF- ~ plays an important role in the Epithelial to

Mesenchymal Transition (EMT), which has become a hallmark for the de­

differentiation of the normal mammary epithelium into tumor cells [21 , 22].

However, within the context of breast cancer, it is largely unknown if TGF-~ is

involved in the expression of SEMA7 A . In addition to TGF-~ mediated PI3K/AKT

activation, Morote eta/. have shown in endothelial cells that hypoxia can induce the expression ofSEMA7A through the Hypoxia Responsive Elements (HREs) in

its promoter [23].

In this study, we characterized SEMA7A expression in both human and

mouse breast cancer cell lines and found that TGF- ~ and hypoxia can increase

SEMA7A expression in these cells. Utilizing shRNA and genetic deletions of

SEMA7A, we determined that inhibition oftumor-derived and host derived

SEMA7A in 4T1 breast cancer model results in decreased tumor growth and

43 metastasis. Our study shows that SEMA7A plays a critical functional role in the

growth and metastasis of highly aggressive mammary tumor cells.

2.2 Materials and Methods

2.2.1 Mice and cell lines

Female BALB/c mice (8-12 week-olds) were obtained from Charles River

Laboratories, and SEMA7A-1- mice generated by Dr. A.L. Kolodkin (Johns

Hopkins University, Baltimore, MD ), were purchased from Jackson Laboratories.

Using a speed congenic approach [24, 25] SEMA7A-1- mice were backcrossed to

a BALB/c background, reaching 99.9% of desired BALB/c background. Mice were housed and used according to the National Institutes of Health guidelines,

under protocols approved by Florida Atlantic University Institutional Animal Care

and Use Committee. EpH4 mammary cells were provided by Dr. Jenifer

Prosperi, Indiana University School of Medicine-South Bend, IN. EpH4 cells, 4T1

and 4T1-LUC (Perkin Elmer) cells were grown in complete DMEM media (DMEM with 10% FBS). MCF-10A, BT-474, T-47D, MCF-7, MDA-MB468, BT-20,

HC1937, CA 1 a and MDA-MB231 (American Type Culture Collection, Manassas,

VA, USA) were grown and maintained in DMEM containing 5% FBS as described

previously [26, 27]. Female BALB/c or SEMA7A-I- BALB/c mice were inoculated

in the lower right ventral quadrant with 5 x 105 luciferase transfected 4T1

mammary tumor cells .. Bioluminescent Imaging studies of the 4T1 -LUC were

done up to 3-weeks post-tumor cell implantation. For 4T1 and 4T1 -LUC tumor

bearing mice, tissues were collected at 42-days post-tumor cell implantation.

44 2.2.2 RNA isolation and real-time reverse transcriptase-polymerase chain

reaction

Total RNA was extracted from murine or human tumor cells, using the

RNeasy Protect Mini Kit (Qiagen) according to manufacturer's instructions.

Briefly, eDNA was synthesized using Quantitech Reverse Transcription Kit

(Qiagen) and gene expression was detected by SYBR Green real-time PCR

analysis using SYBR RP qPCR primers (Qiagen, proprietary primers, sequence

not disclosed) from SABioscience (Qiagen). The mRNA levels of gene of interest were normalized to j3 -actin, GAPDH or HSP90ab mRNA levels. PCR cycles followed the sequence: 10 min at 95°C of initial denaturation; 15 sees at 95°C;

and 40 cycles of 1 min each at 60°C for annealing . The samples were amplified

using the Stratagene Mx30050 cycler.

2.2.3 Flow cytometry studies

Ki67 antibodies (Biolegend) were used to determine cellular proliferation

by flow cytometry as per manufacturer's protocol. Cell signaling pathways were

also determined by flow cytometry. 4T1 mammary tumor cells were treated wi th

Wortmannin for PI3K/AKT inhibition, and total and phosphoAKT (Cell Signaling) were assessed by flow cytometry using manufacturer's Methanol Intracellular

Staining protocol. 50,000 cells were acquired and total and phophoPI3/AKT were determined, using a FACS Calibur (BD) flow cytometer, followed by

analysis using FloJo software (Tree Star, Inc.).

45 2.2.4 Silencing of SEMA7A in 4T1 murine mammary tumor cells

Semaphorin 7 A gene silencing in 4T1 -LUC mammary tumor cells was

achieved using RNA interference via short hairpin RNA (Qiagen). To confirm

gene knockdown, real time quantitative polymerase chain reaction (q-PCR)

(Qiagen) was performed using the SEMA7A specific primers according to

manufacturer's protocol. Cells were passaged and selected until at least a 5-fold

decrease in the SEMA7Agene expression was achieved when compared to the

scramble control. In non-luciferase 4T1 cells, SEMA7A gene silencing in 4T1

mammary tumor cells was achieved using RNA interference via short hairpin

RNA (Mirimus). Cells were transfected with an shRNA plasmid system using

Avalanche transfection reagent (EZ-Biosystems). An optimized short hairpin RNA

algorithm was used to select the top three shRNA targeting sequences. The

shRNA vector also expressed a GFP reporter protein [28]. The vectors allow for

direct vi sual confirmation of shRNA-mirE expression as they constitutively

express the shRNA, fluorescent marker and puromycin selection marker from a

single transcript that is driven by the CMV promoter. As a negative control, we

used multiple control clones with the shRNA against Renilla Firefly Luciferase.

Gene knockdown was confirmed by q-PCR (Qiagen) using the SEMA 7 A specific

primers according to manufacturer's protocol. The results of gene expression were then confirmed by determination for the SEMA7A protein.

2.2.5 AFM Cell Stiffness Measurements

Cell stiffness measurements were acquired on livi ng 4T1 cells. The bare

AFM tip was lowered onto the cell surface at 4 IJm/s [29]. The acquired force-

46 indentation curves of the cells were fit to a model initially proposed by Hertz to

estimate the Young's modulus assuming that the cell is an isotropic elastic solid

and the AFM tip is a rigid cone [30] . The model is as follows:

K 4 F - a 2 - 2(1 - v );rtan e

where Fis the applied force, a the indentation, K the Young 's modulus, 8 the

angle formed by the indenter and the plane of the surface (55°) and v, Poisson

ratio (0.5). Young's modulus was obtained by least square analysis of the force-

indentation curves using Igor Pro software.

2.2.6 Wounding assay

4T1 LUC shRNA control or 4T1 -LUC SEMA?A shRNA KD mammary tumor cells were cultured under optimal conditions using DMEM culture media with 10% FBS until -80% confluency was achieved. Wound was generated,

morphology and migration was assessed at 0, 6 and 12 hours post-wounding .

2.2.7 Statistical An alysis

Results are expressed as means .:!: standard deviation. Statistical

analyses were performed using GraphPad Prism 6 software (LaJolla, CA).

Statistical comparisons were performed using an unpaired 2-tailed Student t test, with significance at p < 0.05. Nonparametric analysis was used to determine tumor growth and metastasis. For the survival analysis, the Kaplan-Meier method was used.

47 2.3 Results

2.3.1 SEMA7A expression is increased in metastatic breast cancer cells

To determine the expression of SEMA?A in breast cancer, quantitative

RT-PCR was performed in human breast cancer cell lines with varying potential for metastasis. Highly metastatic human breast cancer cell lines, MDA-M8-

231 and CA 1a , expressed the highest levels of S EM A 7 A with lower levels of

expression in MCF-7A , a cell line that exhibits decreased metastatic potential

(Fig. 7 A). In contrast, non-tumorigenic breast cells, MCF1 OA , expressed the

lowest levels of SEMA?A (Fig. ?A). Furthermore, we characterized expression of

SEMA7A in murine breast cancer cell lines with varying degrees of metastatic

potential. We found that EpH4 cells, a non-tumorigenic cell line derived from

spontaneously immortalized mouse mammary gland epithelial cells [31],

expressed the least SEMA?A at both mRNA and protein levels (Fig. 78-C). In

contrast, a cell line comparable to the human MDA-M8231 cells, murine 4T1

mammary tumor cells , expressed the highest levels of SEMA?A. Non-and poorly­

metastatic murine cell lines, 67NR and 4T07, expressed intermediate amounts of

SEMA7A at both mRNA and protein levels (Fig. 78-C). We found that 4T1 cells

had heterogeneous expression of SEMA7 A and it was observed that non­

adherent 4T1 cells had very high expression of SEMA7 A relative to adherent

cells (Supplemental Fig. 1 ). Gi ven that PI3KIAKT has been shown to modulate

SEMA7Aexpression, we treated human MCF10A cells with either 0.5 ~ M or 1

~ M Wortmannin, an irreversible PI3/AKT inhibitor, prior to stimulation with TGF- p

(5ng/ml). This resulted in a decreased expression of SEMA?A in the treated

48 groups as determined by qRT-PCR (Fig. 70). Similarly, pretreatment of murine

EpH4 cells with Wortmannin blocked the induction of SEMA7A expression in a

dose-dependent manner (Fig. 7E). We next examined the effect of TGF-~ on the

phosphorylation of PI3K/AKT proteins in the presence/absence of Wortmannin by flow cytometry using the 4T1 mammary tumor cells. While there were no

significant differences in total AKT expression in cultures between untreated and those treated with Wortmannin, a decrease in mean fluorescence intensity could

be seen when serine is phosphorylated at position 473 (Fig. 7F) resulting in

decreased SEMA7A production. Our results demonstrate that SEMA7A is highly

expressed in both human and murine breast cancer cells. Further, we show that

TGF - ~ signaling via the PI3K/AKT pathway can elicit the expression of SEMA7A

in mammary cells.

2.3.2 Hypoxia induces expression of SEMA7A in murine mammary cell

lines and inhibition of HIF-1 a decreases SEMA7A

Hypoxia is a common hallmark of solid tumors such as breast tumors [32 ,

33]. It is characterized as being an imbalance between intracellular oxygen

delivery and oxygen consumption[34]. Morote-Garcia et al., reported that hypoxia

induction of HIF-1a induces SEMA7A expression in endothelial cells [23].

However, it was still unknown what physiological processes can promote the

aberrant upregulation of SEMA7 A in tumor cells. Here we investigated the role of

hypoxia in driving the expression ofSEMA7A in mammary cells. EpH4 cells and

4T1 were grown in optimal conditions and then placed in a hypoxic chamber

containing less than 1% 0 2 using 5% C02- Nitrogen gas or normoxic conditions

49 for up to 24 hours. Cell-free supernatants were then analyzed for SEMA?A

protein by ELISA. In a time-dependent manner, both EpH4 and 4T1 cells showed

increased SEMA? A production compared to normoxic controls (Fig. SA).

PI3KJAKT signaling has been shown to regulate HIF1-a expression during

hypoxic conditions [35]. This occurs when AKT is phosphorylated at Ser473 and

its downstream target, HIF-1 a, is activated. To understand the role of the

PI3KJAKT pathway in the upregulation of SEMA?Aduring hypoxia, we pretreated

4T1 cells with PI3K inhibitor L Y294002 (25 IJM) prior to exposure to hypoxic

conditions. The PI3K iinhibitor significantly (p5:0.001) blocked the induction of

SEMA?A by hypoxia in 4T1 cells by 70% (Fig. 88). To further delineate the

specific role of HIF-1 a. in inducing SEMA?A, 4T1 cells were treated with the

inhibitor Chetomin, which can disrupt the binding of HIF1 - a to transcriptional

coactivator p300. Pre-treatment of 4T1 cells wi th Chetomin significantly

(ps0.001 ) blocked production of SEMA?A in hypoxic conditions (Fig. SC ). Using

an intermediate-expressing murine breast tumor cell line, 4T07, we induced

activation of HIF1 - a using a hypoxia mimic, Cobalt (II) Chloride (CoCI2). We

analyzed the expression of HIF1- a by flow cytometry after treating 4T07 cells with CoCI2 (100 IJM) and found a significant increase in the levels of HIF1 - a (Fig.

9A), wi th a two-fold increase in SEMA?A expression (Fig. 98). Treatment with

Chetomin reduced expression of SEMA? A in murine 4T07 tumor cells stimulated with CoCI2 (Fig. 9C). These results indicate that SEMA?A expression in

mammary cells can be induced through a hypoxic stimuli and inhibition ofthe

PI3KJAKT pathway blocks this induction.

50 2.3.3 SEMA7A gene knock down in 4T1 mammary tumor cells results in

morphologic and functional ch anges

We have previously reported that tumor-derived SEMA?A induces the

production of angioge·nic molecules in macrophages [15]. To understand the role

of SEMA?A in tumor cells, SEMA?A gene was knocked down in 4T1 mammary tumor cells. The effectiveness of gene knockdown was assessed by qRT-PCR.

Greater than 6-fold gene knockdown was observed in shRNA transfected 4T1-

LUC mammary tumor cells (Fig. 10A). Suppression ofSEMA?A gene in

mammary tumor cells resulted in increased cell-to-cell contact in 4T1-LUC

mammary tumor cells compared to 4T1 scramble control (Fig. 1 08). Silencing of

SEMA?A induced an epithelial-like morphology in 4T1-LUC cells with growth of tumor cells in compact clusters. Semaphorins are known to regulate cell

migration and tumor cell migration is dependent upon morphological changes

and cell association with extracellular matrix, and release of metalloproteases

[36]. The expression of vimentin and CD44 was decreased in 4T1-LUC-shRNA­

SEMA7A knock-down (kd) cells. Fibronectin and E-cadherin expression

remained unchanged, while desmoplakin was increased (Fig.1OC ). Silencing of the SEMA 7 A gene decreased the expression of TGF -P1 (Fig. 100 ). Matrix

metalloproteinases MMP-2, -9, -10 and -13 were also significantly (p ~0.001 )

decreased in SEMA?A silenced 4T1 mammary tumor cells (Fig . 1 DE). We next transfected murine 4T1 tumor cells with a plasmid encoding for the rat SEMA?A

gene, which has 90% homology with the murine SEMA?A. We achieved an 18- fold increase in rat SEMA? A expression, which correlated wi th a doubling in

51 expression of only mesenchymal MMPs: MMP3 and MMP13 (Supplemental Fig.

2). As cell migration may also be dependent on integrins, we explored the effect

of SEMA7Agene silencing on integrin expression. There was a significant

(p$0.001) decrease in the expression of its receptor integrin ~ 1. Also decreased was integrin ~ 3 , integrins a S and a. 7 (Fig . 1 OF). Our results show that a strong

linkage between the expression of SEMA 7A and the expression of mesenchymal

markers and MMPs. We continued our study with the gene-silencing approach to

evaluate the role of SEMA7A in tumor cell function.

SEMA7A gene silencing increases stiffness of 4T1 cells

It is known that cancerous cells are less stiff compared to normal cells [37,

38]. Atomic force microscopy (AFM) measurements were acquired to determine the role of SEMA7A in mediating cell stiffness. In these studies, the AFM

cantilever was used as a microindenter, probing the cell less than 1 mm using

applied forces of less than 1 nN as to not damage the cell. Figure 11 A shows

representative force-indentation curves acquired for 4T1 shRNA control and 4T1-

LUC-shRNA-SEMA7A knock-down cells. Following SEMA7Aknockdown, 4T1

cells indented less for equivalent applied forces. Each force-indentation curve was fitted to the Hertz's model. Histograms in Figure 118 reveal the data

distribution of the Young's modulus values for both cell types. The average

Young's modulus value calculated for the 4T1 mammary tumor cells was 3.7±0.3

kPa (Fig. SC). Following SEMA7A knockdown, this value increased to 7.5±1 kPa,

indicating an increase in the measured cell stiffness.

52 2.3.4 SEMA7A gene silencing decreases proliferation and migration of 4T1

cells

Given that tumor cell proliferation is a critical factor in determining patient

outcomes, we assayed the expression of a commonly used clinical proliferation

marker, ki67. We found a decrease of nearly half the expression of ki67 in 4T1 -

LUC cells that had been silenced for the SEMA7A gene (Fig. 12A-B). To test the

effect of SEMA7 A gene silencing on cell motility, wound healing assay was

employed. Wounding assay revealed decreased tumor cell migration by

SEMA? A silenced tumor cells at both 6 and 12 hours post-wounding (Fig. 12C­

D). These findings suggest that SEMA?A may also play a role in tumor cell

motility.

2.3.5 SEMA7A gene silencing reduces tumor growth and metastasis and

increases survival

Since SEMA?A gene silencing resulted in decreased tumor cell

proliferation and migration, we wanted to further investigate if decreased

SEMA? A would retard tumor growth in mammary tumor bearing mice. Mice

injected with either 4T1 -LUC scramble control or 4T1 -LUC-SEMA7A-shRNA-kd

mammary tumor cells were imaged for luciferase bioluminescence at days 4, 11

and 18. At day 4 post tumor cell implantation, there was no significant difference

in tumor growth between the two groups. However, by days 11 and 18 post­ tumor cell implantation, there was a reduction (p=.00001) in tumor growth in mice

injected with 4T1-LUC-SEMA 7A -shRNA-kd tumor cells (Fig. 13A). This was

quantified by luciferase signal detected by photons/sec (Fig . 138). More

53 importantly, an increase in survival (p=0.0001 ) was observed in mice bearing

SEMA7 A gene silenced mammary tumors (Fig. 13C). Since we observed

increased survi val in mice bearing SEMA7A gene silenced tumors, we next

determined if this translated into decreased metastasis. Lungs are one of the first organs that are infiltrated and colonized by metastatic breast tumors. Thus,

lungs from 4T1 -LUC scramble control or 4T1 -LUC-7A-shRNA-kd mammary tumor bearers were analyzed for metastasis by luciferase signal detection, India

Ink and H&E staining. Detection of the luciferase signals revealed a steep

reduction of tumor cell signal in the lungs of 4T1 -LUC SEMA 7A-shRNA

mammary tumor bearers (Fig. 130). Gross morphologic and histological

examination of lungs stained by India Ink from the two groups revealed very few

metastatic foci in 4T1-LUC 7 A-shRNA mammary tumor bearers (Fig. 13E).

Enumeration of the metastatic foci in the lungs revealed >30 metastatic foci in

4T1-LUC scramble control compared to <5 metastatic foci in lungs of 4T1-LUC

7 A-shRNA-kd mammary tumor bearers (Fig. 13E). We determined that inhibition

of tumor-derived SEMA7A in 4T1 cells decreased tumor growth and metastasis.

2.3.6 Ablation of host-derived SEMA7A decreases rate of tumor growth

and metastasis and increases survival

Given that host cells, such as immune cells, also express SEMA7A we

questioned if host-deriived SEMA7A could also contribute to tumor progression.

Wild-type or SEMA7A deficient female BALB/c mice were inoculated with 4T1-

LUC cells and bioluminescent imaging was performed for 28 days. 4T1 -LUC tumor-bearing mice have a decrease in tumor growth rate (Fig. 14A-B) and

54 increased survi val (Fig. 14C), but more importantly, a decrease in metastasis to the lungs was also observed (Fig. 140).

We proceeded to test if ablation of host derived SEMA 7 A could synergize with gene silencing of tumor-derived SEMA7A. In this approach, we used the wild-type 4T1 cells from early passage that maintained heterogeneous

populations. We achieved an 80% reduction in SEMA7A expression (Fig. 15A) in two subsets of 4T1 cells using an optimized microRNA backbone targeting the 5'

end of the SEMA7AmRNA (4T1-SEMA7A-shRNA1 ) and another targeting the 3'

end of the SEMA7A mRNA (4T1-SEMA7A-shRNA2.) As a control, we used cells

expressing shRNA targeting the Renilla luciferase gene (4T1-Renilla-shRNA), which maintained equal expression to the 4T1 wild-type cells (data not shown).

Subsequently, the SEMA7A shRNA cells or Renilla shRNA control cells were

implanted into either wild-type or SEMA 7 A-1- female BALB/C mice. Silencing of

SEMA7A in 4T1 cells resulted in reduced tumor growth and ablation of host­

derived SEMA7A further synergized to reduce tumor growth rate (Fig. 158), and

metastasis to the lung (Fig. 15C-D). Our results show that ablation of host­

derived SEMA7Aand tumor-derived SEMA7Acan significantly improve

outcomes in our murine breast cancer models.

2.4 Discussion

The objective of this study was to delineate the role of SEMA7A in breast

cancer. We accomplished this objective by: 1) characterizing the expression of

SEMA7A in murine and human cell lines, 2) determining the effect of TGF - ~ , the

55 PI3KIAKT axis and hypoxia in modulating SEMA7A expression, and 3) assessing

both in vitro and in vivo effects of SEMA7A inhibition in mammary tumors.

We first determined the range of SEMA7A expression in various human and

murine breast cell lines. The non-tumorigenic line, MCF10A, had the lowest

expression of SEMA7A (Fig. 7 A) whereas the aggressive MCF1 OCA 1 a cell line

had 1000 times greater expression. So et al., [39] reported that MCF1 OCA 1 a

cells have increased phosphorylation levels pErk, pAkt, Stat3 and Pak4, and we

show concurrently there was an increase in SEMA7A expression. We also

showed that the murine counterparts of MCF1 OA , the EpH4 cells, had very low

expression of SEMA7A. Allegra et al., [9] showed that EpRAS cells, generated via a RAS mutation, expressed high levels of SEMA7A. These findings show that

both pAKT and RAS/Erk activation can induce the expression of SEMA7A. It would be beneficial to determine if specific mutations in these pathways lead to

differential SEMA7A expression.

Morote-Garcia et al. showed the presence of HREs in the SEMA7A

promoter [23]. We are the first to show that hypoxia can also induce the

expression of SEMA?A in mammary cells. The frequency of these HREs could

also be a determining factor in the disparate expression of SEMA 7 A between

EpH4 cells and 4T1. We continued by exploring the link between hypoxic stimuli

and PI3K/AKT activation given that it's been shown that AKT activation increases

HIF-1 a activity[35]. By blocking phosphorylation of AKT we were able to reduce the induction of SEMA7A by hypoxia (Fig. 88), showing that the cross talk

between hypoxia-derived signals and AKT are important for SEMA7A

56 expression. To verify to what extent HIF-1 ex. plays a role in this pathway, we used the inhibitor Chetomin. In both 4T1 and 4T07 cells, Chetomin was very efficient

at blocking the expression of SEMA 7 A. Our findings demonstrate that hypoxic

stimuli are a critical factor in the induction of SEMA7Ain mammary cells.

Inhibition of tumor-derived SEMA7A lessened the malignant potential of 4T1

cells. In accordance wi th our studies, Saito et al.,[40] has shown that shRNA

inhibition of SEMA7A in oral squamous cell carcinoma (OSCC) cells resulted in

decreased cellular proliferation, which was attributed to down-regulation of

cyclins. Additionally, Saito et al., [40] showed that inhibition of SEMA 7 A

decreased invasiveness of oral squamous cell carcinoma cells and secretion of

matrix metalloproteases. Our biophysical studies conducted on the 4T1 cells

revealed an increase in cell stiffness when the SEMA7A gene is silenced. A

softer cell can spread more easily on a substrate, thus facilitating migration.

Thus, the stiffening following SEMA 7 A knockdown is likely to promote a less

migratory cell phenotype. This indeed was observed in our in vivo model. In

vivo, BALB/c mice inoculated with SEMA7A silenced 4T1 -LUC cells showed a

decrease tumor growth rates and delayed onset of metastatic disease compared to mice bearing 4T1 -LUC cells.

We also evaluated the contribution of host-derived SEMA7A using genetic

deletion of SEMA7A in BALB/c mice. We found that tumor growth was reduced in

SEMA7A-/- 4T1-LUC tumor bearing mice (Fig. 14A-B). There was also a

decrease in metastases to the lung at day 42 post-tumor implantation (Fig. 140-

E). Ma et al. similarly showed in a syngeneic model of melanoma, that ablation of

57 host-derived SEMA7A was sufficient to decrease the rate of metastasis [8]. They

also reported that anti body mediated neutralization of SEMA7 A had similar

effects. The heterogeneous 4T1 model, SEMA7A-/- tumor-bearing also held a

difference until day 28 but that difference was negated at day 35. It is possible that the 4T1 cells started to produce levels of SEMA7A that overpowered the

effects of ablating host-derived SEMA7A. When combined, inhibition of tumor­

derived SEMA7A and host-derived SEMA7A resulted in the reduced tumor

growth and metastasis.

Recently, Black et al. corroborated the necessity of studying SEMA7A in the context of breast cancer as a prognostic parameter of metastasis and poor

survival in breast cancer patients [1 0]. Using two models, we have elucidated a

role for SEMA7A in regulating mammary tumor progression, warranting

subsequent studies to survey the downstream molecular effects of SEMA 7A that

lead to tumor growth and metastasis. Overall, our collective results support our

hypothesis that SEMA7A expression plays a functional role in promoting breast

cancer growth and metastasis. Our findings postulate a novel role for SEMA7A in

breast cancer that may lead to further findings of prognostic and therapeutic value.

58 A 2000

0 g> ~ 1600 ...0 ~ 1200 ~ E 800

.,j 400

B .. 250 20 :;"' 200 4; ~ ~ 16 ~; 150 .s. 12 ...,.e 4; (I)

0 MCF10A --··=,....,..,.,.-

0 va.-.tcle Vehic.~ TGfb (5ngfml) • tGFbiS..o/ml) - WRTMNt500nM)­ WRTMN(600 nM) • WRTMN ll!oO nM,- WRTMNC750nM) • F 4T1 G 4T1

~ ~10 ~·· ~ 5 ~ .[] i ,[{J ., 0 ., ••' ..= ...... •• ·•' ...... OMSO W0!1mannln 1 ~M To;tW·AKT Pho&pho--AKTSSW73 - l.Hrealed - Wortrrcannn1j.IM Figure 7 SEMA7A is highly expressed in metastatic human and murine cell lines and TGF-B induces SEMA7A expression via AKT siqnalinq. A-8 ) 1x1 06 cells were grown to -75% confluency, trypsinized and lysed for RNA extraction then assayed for SEMA7A gene expression by quantitative real time PCR C) Cell free supernatants were collected from murine breast cell lines and assayed by ELISA. D-E) 1x105 MCF-10A or EpH4 cells were pre-treated for 12 hours in serum-free media with Wortmannin (0.5 or 1 uM) or DMSO vehicle, 5% FBS was then added and then cultured in 1 ml of complete media with 5ng/ml of rmTGF-beta-1 or vehicle for 24 hours. F-G) 4T1 cells were treated with a PI3KIAKT inhibitor, Wortmannin for one hour. PI3KI AKT phosphorylation was assayed one hour later by flow cytometry and cell-free supernatants were assayed for S EMA7A at 24 hrs. p-value (**) !S 0.01 , (***) <0.001

59 A 60 ... 50

- 40 ~ .E 30 .s01 < 20 ..... < 10 ::Ew Ill 0 0 12 18 24 lime in Hypoxia (hrs) 8 ::1 60 . 4T1 E .....g. 40 < :;c 20 ::Ew Ill 0 Normoxia Hypoxia Hypoxia LY294002 c (25 1-JM ) ::J 80 . 4T1 ~ 80 c :;..... 40 ~ 20 w Ill 0 1-tfpoxla + + + Oletomn (150nM) + O!etorrin (300 nM) +

Figure 8 Hypoxia induces SEMA7A expression in mammary cells. A) 1x1 06 4T1 or EpH4 cells were grown to confluency, then incubated under 1% oxygen hypoxic conditions or normoxic conditions. B) 4T1 cells were grown to 100% conftuency in reduced serum condition, treated with LY294002 for 4 hours , incubated under 1% oxygen hypoxic conditions or normoxic conditions. C) 1x1 06 4T1 cells were treated with Chetomin or control for 6 hours and then incubated under 1% oxygen hypoxic conditions or normoxic conditions for 24 hours. Cell-free supernatants were collected at specific timepoints and assayed for SEMA?A by ELISA. p-value (***) <0.001

60 A 4T07

l! c: :. 0 0

...... ' .. ~ .•. ..· B HIF-1a

c Growth Media :i ~ 1.2 a:::i E 5 0.8 ct ., ~ ~ 0.4 ~.!! Ill~ 0.0 CoO, (100)JM) + + + Oletomn (75nM) - + O>etomn (150 nM) - +

Figure 9 HIF-1a production correlates with SEMA7A expression in 4T07 cells. A) 4T07 cells were grown to 100% confluency in reduced serum conditions for 12 hours, then stimulated wi th CoCI2 or control for 24 hours, and cells were harvested for HIF-1a determination by intracellular flow cytometry staining. B) 4T07 cells were grown to 100% confluency in reduced serum condition, treated wi th CoCI2 or vehicle for 24 hours, cells were then lysed and analyzed for SEMA7Aexpression by qPCR. C) 1x106 4T07 cells were treated with or without

61 A •T1-l.UC SEMA7A lhRNA KD

c D

~ 1.2 = ~ 8 0.8 i ; 0.4 ~ .!! !I...... ,.,~ ! 0.0 Ii E

MMP2 MMP3 MMP9 MMP10 MMP11 MMP13 11gb1 Ugb3 11ga5 11gboV ltga7

Figure 10 Gene silencing of SEMA7A in 4T1-LUC cells decreases expression of mesenchymal and pro-metastatic genes Chetomin for 6 hours and then stimulated by CoCL2 for 24 hours, cells were then lysed and analyzed for SEMA7A by qPCR. p-value (**) ~ 0.01, (***) ~0 . 001 A) Gene expression of 4T1-LUC scramble control cells and 4T1-LUC SEMA 7A shRNA silenced cells was assayed by qPCR. (B) Morphology was observed by phase contrast imaging. (C-F) Gene expression of 4T1-LUC sublines was analyzed for differential gene expression by qPCR. p-value (**) ~ 0.01 , (***) ~0.001

62 A 4T1 -LUC 4T1 ·LUC SEMA7A ohRNA 400 ohRNA I

5 10 15 Young's modulus (kPa) c

4T1 ·LUC S8AA7A ohRNA I

Young's modulus (kPa)

Figure 11 SEMA7A alters tumor cell stiffness. A) Representative force-indentation curves from AFM cell stiffness measurements acquired for 4T1 -LUC scramble shRNA control cells and 4T1- LUC 6-fold shRNA SEMA7 A gene knockdown. Fitted curves derived from the Hertz model are overlaid on the raw data. AFM measurements were acquired at 3JOC at a constant cantilever retraction rate, applied force and contact time. B) Data distribution of Young's modulus values for 4T1 -LUC scramble shRNA control cells (white; n=35) and 4T1-LUC 6-fold shRNA SEMA?Agene knockdown cells (blue; n=29). C) Average of Young's modulus values for stiffness measurements from B. The error is the SEM. p-value (**) ~ 0.01

63 A B 140 120 ::- 100 ~ 4T1·LUC 80 4T1·U.IC .nRNA contt04 ~ - 4T1·U.C: SB&A7A ahA !'fA KO ... 60 cD ~ i' 40 20 0 ...... 4T1 -LUC 4T1 -LUC 4T1 -LUC shRNA SEMA7A Control shRNAKD c D ! 140 !!iTu . 4T1·LUCshRNAControl 0 120 ~i ~ ~ 100 .r:.e-" eo

.t:.i~• eo 0 0 i 40 " 20 !!! ~ ... ~ 0 ~~ 0 12 l:l Hours

Ohn Jhrs 11hn

Figure 12 Silencing of SEMA7A gene in 4T1-LUC mammary tumor decreases cell motility and proliferation. A-B) Proliferation of S EMA?A expressing and shRNA silenced cells were measured by Ki67 intracellular staining. C-D) Motility of 4T1 -LUC scramble control cells and 4T1-IIuc SEMA?AshRNA was assayed using a wound healing assay and measured as percentage wound closure. p-value (***) s <0.001

64 A 4T1- 4T1- B Scramble SEMA7A - H1-SEMA7A.shRNA shRNA shRNA - 411-SCRAMBLE•shRNA

5 10 15 20 25 DAYS

- 4T1-SEMA7A-shRJiA - 411-SCRAMBLE-shRNA

0 20 40 60 60 100 D E DAYS

I j~ ~ "';~ '§;l:..lL \ / ' . . ... uc-____.. ~1L. .... _.,.. -~ -- -

Figure 13 Inhibition of tumor-derived SEMA7A decreases tumor growth rate and metastasis in 4T1-LUC tumor-bearing mice. A-B) Mice representative of 14 mice/group at 4 different time points are shown with quantification oftumor-specific bioluminescence. C) Kaplan-Meier survival curve of tumor bearing mice. D) At day 42 post tumor implantation, animals inoculated with SEMA7A shRNA silenced 4T1 cells showed decrease metastasis compared to the scramble shRNA control 4T1 cells as determined by quantification of bioluminescent signal and E) India Black staining of the lungs. (n=14). p-value (***) ~ 0.001 , (****) ~0.0001

65 A Wild.(ypt 7A-KO 8ALB/t BALB/t B

SEMA7M

Wlld-lype IIAI.Bio 6•10' 0 - Oay3 p•.OOI

14•10 ' 0 g ...2 2•1010 Day7 ti '

0 10 20 30 DAYS to' Oay14 ' c SEMATA-1<0 8A1.81c - Wild-typo IIAI.Bic ;;. to' ~ Oay21 .,. ...i.

0 20 40 60 80 OAYS

D Wlld,type SEMA7A·KO BALB/c BALB/c f

Lung f ~~~~ lL_ J ...... - ..,.,...u_a UtUtAMO-MUI.e

Figure 14 Genetic ablation of host-derived SEMA7A decreases tumor growth rate and metastasis in 4T1-LUC tumor-bearing mice. A-B) Mice representative of 15 mice/group at 4 different time points are shown with quantification oftumor-specific bioluminescence. C) Kaplan-Meier survival curve of tumor bearing mice. D) At day 44 post tumor implantation, metastasis was determined by quantification of bioluminescent signal from the lungs. (n=15). p-value (***) !S 0.001

66 A B 3,000 ~ 4Tt 4 ReniiJ..ehRNA-8ALBie

- 4Tt~SEMA7A.thRNA.1·6A1.Sic 4T1-SEMA711-ahRNA2-BIILS/c i 2,000 --- 4l'l RenHia·ahRNA.7AK0 .. - -- 4TI-SEMA7l\-ahRNA2> 7AKO § 4TI·SEMA71\-ahRNA 1- 71\KO ~ ~ 1,000 t:!

0 7 14 21 28 3S Days

80 • • T1-Rtnllla.shRAA u • •T1·SEMA7A-shRNA1 ~ 60 D 4T1-SEMA7A-IhRNA2 " i 40 ; 20 ::IE .. 0

Figure 15 Inhibition of host-derived and tumor-derived SEMA7A decreases tumor growth rate arn d metastasis in 4T1 tumor-bearing mice. A) Gene silencing of S EMA7A in 4T1 cells. B) Caliper measurements of tumor volume at 5 different times points. C-D) At day 42 post tumor implantation, lungs were excised and metastatic foci were quantified. (n=15). p-value (**) :::;0 .01 , (***) :::; 0.001 .

67 A DAPI SEMA7A

B

4T1 NON-ADH 4T1 VERV-ADH c

~~ 1.4 a:: c 1.2 E ~- 1 4. 0 & 0.8 ~ .~!!.. 0.6 ~,; 0.4 (/) ~ 0.2 .. 0 4T1 NON-ADH 4T1 VERY-ADH Supplemental Figure 1 Non-adherent 4T1 cells expressed high levels of SEMA7 A compared to very-adherent 4T1 cell. A) 4T1 tumor cells were grown in optimal conditions to -75% confluency and stained for SEMA?A mRNA expression using RNAish. B) 4T1 cells were selected for 1 0 passages based on detachment time upon TrypleExpress disassociation from standard cell cultured coated polystyrene plates. C) 4T1 sublines cells were then lysed and analyzed for SEMA?A expression. p-value ~ (**) <0.01

68 A ••• ~~ 20 a:'E E~- 16 <(0&,.._c."' 12 <( .2::- 8 ai~ 1/) a:C> 4 0 Empty-Vector SEMA7A.OE B 2.5 •• z-<(~ a:~ 2 E c 1.5 .... 6Cia: d: ~ ~ 1 ~.! :::!!& 0.5 0 Empty-Vector SEMA7A.OE c C> > ";:1.,_ -c: 3 •• C>a: 2.5 <(-a:"' Zl:' 2 cr-.. 1.5 E c: '?:I"' a..O 0.5 ~ ~ 0 Empty-Vector SEMA7A.OE

Supplemental Figure 2 Exogenous overexpression of SEMA7A in 4T1 cells. A) 4T1 cells were transfected with a plasmid encoding for full-length rat SEMA7A using Avalanche transfection reagent, 1x106 cells were grown to -75% confluency, trypsinized, lysed for RNA extraction and then assayed for exogenous SEMA7A. B-C) Endogenous MMP-13 and MMP-3 gene expression was assayed by qPCR. p-value (**) :s:0.01 , (***) ::;: 0.001.

69 CONCLUSIONS

Preventing the metastatic process is crucial in enhancing the survival of

cancer patients as metastasis contributes to majority of deaths in breast cancer

patients. We have recently discovered that a member of the Semaphorin

neuronal developmental family, Semaphorin ?A (SEMA?A) is overexpressed in

human breast cancer tissue. We hypothesized that SEMA7 A expression has

a direct effect on mammary tumor cells and modulates immune responses to enhance tumor growth and metastasis. The objective of this study was to:

1) delineate the role oftumor-derived SEMA?Aon immune system ; 2) determine the factors leading to SEMA?A gene expression in tumor cells; 3) use mammary tumor models with differential SEMA?A expression to determine its role in

enhancing tumor growth and metastasis; and 4) assess SEMA?A as a viable therapeutic target. SEMA?A is a pleiotropic molecule downstream of the

PI3KIAKT pathway, and is associated with inflammation. Our studies show that

inhibition ofSEMA?A in a preclinical breast cancer model leads to decreased tumor growth and metastatic potential. We reported that mammary tumor-derived

SEMA?A can alternatively activate macro phages to promote angiogenesis. Our

laboratory has been studying a specific member, Semaphorin ?A (SEMA?A), in

both human breast cancer patients and murine models of the disease.

We discovered that DA-3 mammary tumor cells express high levels of

SEMA?A and that tumor-derived SEMA? A induces monocytes to secrete pro-

70 angiogenic chemokines to enhance tumor growth. Decreased SEMA7A limits tumor growth and production of angiogenic CXCL-2/MIP-2 and VEGF-A by

monocytes. SEMA 7A is a knoW7 chemoattractant for immune cells and induces the production of pro-iinflammatory IL-8, IL-6, and IL-1-beta. Given we and other

show evidence for a direct effect of SEMA 7 A on immune cells, it could be further

proposed to determine the role of SEMA7A in modulating immune responses

during mammary tumor progression. Thus, the in vivo effect of tumor-derived

SEMA7Aon myeloid and T-cell populations in mice bearing SEMA7A expressing

and those bearing SEMA7AshRNA silenced mammary tumors may be

characterized. And further, how these populations are altered in SEMA7A-1- mice

before and after tumor inoculation.

In our initial ap[proach to characterize SEMA7A in breast cancer, we have

characterized the gene expression profile of SEMA7A in breast cancer lines,

quantitative RT-PCR was performed in human breast cancer cell lines with varying potential for metastasis. The highly metastatic cell lines: MDA-MB-231

and CA1a cells expressed the highest levels ofSEMA7A in comparison to non­ tumorigenic breast cell line, MCF1 OA, which shows lowest expression of

SEMA7A. To further investigate the role of SEMA7A in breast cancer, a

syngeneic murine orthotopic model breast cancer is used. The 4T tumor model

is a preclinical model of breast cancer derived from a spontaneous mammary tumor in BALB/c mouse. We compared SEMA7A expression to EpH4 cells, a

nontumorigenic cell line derived from spontaneously immortalized mouse

mammary gland epithelial cells which expressed the lowest amount of SEMA7A

71 at mRNA and protein levels (Fig. 18 & 1C). The highest level ofSEMA7Awas found in the more metastatic 4T1 subline. We found that the levels of SEMA7A

expression associated with the degree of metastatic potential. The highest

SEMA7A expression was again seen highest in the metastatic 4T1 tumors.

Based on these results we hypothesized that SEMA7A plays a role in tumor

progression and metastasis.

The mechanisms controlling SEMA7Aexpression during tumor

progression are largely unknown. We described how hypoxia can induce the

expression of SEMA7A, and that the PI3K/AKT pathway is vital for this induction to occur. Semaphorins are known to regulate cell migration and tumor cell

migration is dependent upon morphological changes and cell association with

extracellular matrix, and release of metalloproteases. We used shRNA to gene

silence SEMA7A expression to assess the contribution of tumor-derived

SEMA7A to tumor growth and metastasis. To study the role of SEMA7A we

generated 4T1 murine mammary tumor cells that were either silenced for the

SEMA7A or expressed a Renilla control vector. Using the SEMA7A -specific

hairpin shRNA we achieved a greater than 1 0-fold knockdown in SEMA 7 A gene

expression. In vitro, silencing of SEMA7A reduced the migration and proliferation

of 4T1 cells. SEMA7A silencing decreased the expression of metastasis

promoting MMPs and mesenchymal proteins. SEMA7A-1- Mice bearing 4T1-

SEMA7A-silenced tumor cells showed a further decrease in tumor growth rate

and decreased metastasis. Our study shows that inhibition of both host and

72 tumor-derived SEMA7A can limit tumor metastasis and improve prognosis in a

murine model of metastatic breast cancer.

Given our study shows that inhibition SEMA7A does indeed play a role in

breast cancer progression, it warrants subsequent studies to survey the

downstream molecular effects of SEMA7A leading to tumor growth and

metastasis. For example, one could determine if SEMA7A affects spontaneous

mammary tumor formation. The transgenic mammary tumor model, MMTV­

PyMT closely resembles human breast cancer as these mice spontaneously

develop tumors without a need for tumor cell implantation. As SEMA 7 A may play

a role in tumorigenesis, a transgenic MMTV-PyMT-SEMA 7A knockout mice

could be generated to determine if development of spontaneous tumors and

metastasis is affected. Furthermore, it is still unclear how binding ofSEMA7A to

other proteins either in cis or trans could lead to a signaling cascades that serve

as effectors in aiding tumor cell growth and metastasis. We could speculate,

given the effect of SEMA7A on inflammatory proteins and MMPS, that the

MAPKinase pathway may be a critical downstream player following expression of

SEMA7A. The potential role of SEMA7A in the Epithelial to Mesenchymal

Transition may be delineated by immunofluorescence staining following TGF ­

beta or EGF stimulation of SEMA7 A gene silenced cells or controls to compare

expression and localization of epithelial-related proteins such as, but not limited:

E-cadherin, N-cadherin, Claudin-1, Z0-1 , Vimentin, Snail, Twist, Slug,

TCF8/ZEB1 and ~ - Catenin.

73 Cancer remains a dreaded disease and metastasis is a leading cause of

death in spite of significant improvements in diagnosis and treatments.

Microenvironment at the primary tumor site composed of tumor and host derived factors and the immune cell infiltrates plays a major role in whether a tumor has the potential to metastasize. Delineating the factors involved in metastasis is

crucial in limiting tumor growth and preventing metastasis related deaths. Our

discovery of SEMA7 A that could play a critical role in generating a

microenvironment conducive for tumor growth and metastasis may lead to targeted therapies.

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