The Role of and their Receptors in

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Mohamad Mostafa Abdelhamid Elbaz

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2016

Dissertation Committee:

Dr. Ramesh Ganju, Ph.D., Advisor

Dr. Sujit Basu, M.D., Ph.D.

Dr. Kalpana Ghoshal, Ph.D.

Dr. Xue-Feng Bai, M.D., Ph.D.

Copyright by

Mohamad Mostafa Abdelhamid Elbaz

2016

Abstract

Breast cancer is a heterogeneous disease that has different biological and clinical behaviors. It represents the second cause of cancer deaths among US women. Cannabinoids can be classified into endocannabinoids, and phytocannabinoids. These cannabinoids act on different receptors such as receptor-1 (CB1R), -2

(CB2R), Transient receptor potential vanilloid type-2 (TRPV2), etc. In this thesis, we have examined the anti-tumor role of an important phytocannabinoid,

Cannabidiol (CBD), and a specific CB2R agonist synthetic cannabinoid, JWH-

015. We also studied the role of CB2R activation in cancer cells as well as immune cells and finally we analyzed the role of TRPV2 in improving the efficacy of chemotherapeutic drugs.

CBD is a non-psychotropic cannabinoid compound. Its anti-tumor role and mechanism are not well studied especially in triple-negative breast cancer

(TNBC). In the present study, we analyzed CBD’s anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time- that CBD significantly inhibits epidermal growth

ii

factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP-2 and MMP-9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and in different mouse model systems. Analysis of molecular mechanisms revealed that

CBD significantly inhibits the recruitment of tumor-associated in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction of the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of

GM-CSF and CCL3 , which are important for recruitment and activation. In summary, our study shows -for the first time- that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibition

EGF/EGFR signaling and modulating the tumor microenvironment (TME). These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including

TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

TRPV2 is a cation channel that is triggered by agonists like CBD.

Chemotherapy still the first line for the treatment of triple negative breast cancer

(TNBC) patients, however, TNBC cells usually gain rapid resistance and unresponsiveness against chemotherapeutic drugs. In this study, we analyzed

iii

TRPV2 role in enhancing chemotherapeutic efficacy in TNBC. First, we found that TRPV2 is significantly up-regulated in primary and metastatic breast cancer compared to normal breast tissues. Second, we observed that TNBC patients with higher TRPV2 expression and committed to chemotherapy have significantly higher overall and recurrence free survival compared to patients with lower

TRPV2 expression. We also showed that TRPV2 overexpression significantly increased (DOX) uptake and efficacy. We showed that TNBC cells that are subjected to combination treatment (CBD+DOX) have less viability compared to DOX-treated cells. Analysis of molecular mechanisms showed higher levels of cleaved PARP and caspase-3 in combination treatment compared to DOX alone. Further studies, revealed that CBD enhanced the uptake of DOX into TNBC cells. Importantly, we show that CBD effects are

TRPV2-mediated and TRPV2 downregulation or interference by its dominant negative form inhibits DOX-mediated cytotoxicity. In vivo studies showed that

(CBD+DOX) mice group has significantly reduced tumor weight and enhanced cleaved (caspase-3/PARP) levels compared to DOX group. This study suggests

TRPV2 agonists as adjuvant therapy to improve the anti-tumor chemotherapeutic efficacy and suggest TRPV2 as a prognostic marker for TNBC patients who receive chemotherapy for their treatment plan.

Cannabinoid receptor-2 (CB2R) is an integral part of the . It is upregulated in the primary breast cancer lesions and highly

iv expressed in different types of immune cells. However, its functional role in breast tumorigenesis is not well understood. The present study was aimed at evaluating the mechanistic anti-tumor role of CB2R activation on breast cancer cells and immune cells within the breast TME. First, we analyzed the anti- tumorigenic mechanisms of CB2R activation in ERα- and ERα+ breast cancer cells. Our studies showed that CB2R specific agonist (JWH-015) inhibited EGF and IGF-I-induced migration and invasion of ERα+ and ERα- breast cancer cells.

At the molecular level, JWH-015 inhibited EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and MMP-9/MMP-2. Interestingly, we found that JWH-015 significantly reduced breast cancer growth in vivo and the tumors that were derived from CB2R agonist treated mice showed reduced activation of EGFR and IGF-IR and their downstream targets compared to control group. Since CB2R is highly expressed in immune cells, we assessed the role of

CB2R activation on modulation of immune cells present in tumor stroma. We observed increased tumor weight, more myeloid derived suppressor cells

(MDSCs) (CD11b+/Gr-1+) and less CD3+/CD8+ cells in orthotopically injected

CB2R knock out mice compared to wild type mice. Furthermore, we found that

JWH-015-treated wild type mice had reduced tumor growth and metastasis, more

CD3+/CD8+ cells and less MDSCs within the tumor stroma. For the first time, we show that CB2R activation might suppress breast tumor growth and metastasis through novel mechanisms of inhibiting EGF/EGFR and IGF-I/IGF-IR signaling

v axes on tumor cells and modulating the immune cells’ compositions within the breast TME.

Overall, our studies showed that cannabinoids such as CBD and JWH-015 exert strong anti-tumor activities against breast cancer. They both inhibit hallmark signaling pathways such as EGF/EGFR and IGF-I/IGF-IR pathways. In addition, these drugs have TME modulating potential through different mechanisms which eventually enhance the anti-tumor immunity against breast cancer cells. Finally, we show that CBD, upon activating TRPV2 channel, increases the uptake capacity of breast cancer cells and makes these cancer cells more susceptible to the anti-tumor chemotherapeutic drugs.

vi

Dedication

This document is dedicated to my mother Thanaa Hussein, father Mostafa Elbaz,

my sisters Reham, Shereen and Nariman.

vii

Acknowledgment

I would like to thank my family who sacrifice a lot to help me through all of my life. Special thanks to my mother who passed away and I really miss her and to my father for his continuous sacrifice and support.

I am very grateful and I would like to thank my advisor Dr. Ramesh Ganju.

I thank him for his continuous strong guidance and encouragement during all

Ph.D years. His guidance helped me a lot in all the time of research and writing of this thesis. He always encourages me and makes me believe in myself. I also thank him for giving me a great opportunity to work as a member of his amazing team. I greatly appreciate every valuable advice and every righteous direction he gives till now. I have really learnt from him how to be strong if you face any problem whether in the research or in general life.

I would like to greatly thank my committee members, Dr. Sujit Basu for his insightful comments and encouragement, remarkable advises, sincere valuable guidance and unstoppable support, Dr. Kalpana Ghoshal for her aspiring guidance, immense knowledge and for continuous help and support in every aspect scientifically and personally, and Dr. Xue-Feng Bai, for priceless advises, stimulating scientific discussions, great support and brilliant suggestions during viii my years of Ph.D. I am also grateful to all of my mentors and MCDB program directors, Dr. David Bisaro and Dawn Chandler for their continuous supervision and valuable advises. I want to send special thanks to all the past and present

Ganju lab members for helping me learning new approaches and techniques and for their assistance and their valuable scientific discussions. I thank them all for all the fun we have had during our collaborative work. I wish to all of them great success in their academic life.

ix

Vita

May 2001-2006...... Pharmacy School-Helwan University-Egypt.

June 2006-2011...... …Master degree- Pharmacology Department,

Helwan University-Egypt.

Dec. 2011-Now...... Graduate Research Associate, Department

of Pathology, The Ohio State University.

Publications

1. Elbaz M, Nasser MW, Ravi J et al. Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway; Novel anti-tumor mechanisms of in breast cancer. Molecular Oncology, 2015; 9(4): 906-19.

2. Nasser MW*, Wani NA*, Ahirwar DK, Powell CA, Ravi J, Elbaz M et al. Receptor for Advanced Glycation End products (RAGE) mediates S100A7- induced breast cancer growth and metastasis via modulating tumor microenvironment. Cancer Research, 2015; 15; 75(6):974-85.

3. Elbaz M*, Mohd W. Nasser*, Ramesh K. Ganju. Conditioning the solid tumor microenvironment through inflammatory and S100 family . Cancer letters 2015; 365 (1): 11-22. * Equal first author.

x

4.Elbaz M, Grace Amponsah, Ramesh K. Ganju, Mohd W. Nasser. S-100 proteins. Encyclopedia of cancer. Invited book chapter 5. Ravi J, Elbaz M, Nissar A. Wani, Mohd W. Nasser, Ramesh K. Ganju. Cannabinoid receptor-2 agonist inhibits macrophage induced EMT in non-small cell by downregulation of EGFR pathway. Molecular (In press). 6. Elbaz M, Mohd W. Nasser, Ravi J, Nissar A. Wani Ramesh K. Ganju. Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGF and IGF-I/IGF- IR pathways. (In submission). 7. Elbaz M, Mohd W. Nasser, Konstantin Shilo Ramesh K. Ganju. Augmentation of chemotherapeutic drug efficacy; new role of Transient Receptor Potential Vanilloid type 2 (TRPV2) in triple negative breast cancer. (In submission). 8. Nasser MW, Wani NA, Wilkie T, Elbaz M et al., Ganju RK. S100A7 accelerates breast cancer growth and metastasis through STAT3 pathway. (In submission).

9. Ahirwar DK, Nasser MW, Leone G, Elbaz M, et al., Ganju RK. The role of stromal CXCL12 in breast cancer metastasis. (In submission).

10. Ahirwar DK, Nasser MW, Wani N, Eblaz M, et al., Ganju R. Slit2 blocks breast cancer metastasis by inhibiting tumor associated macrophages. (In submission).

Field of study

Major Field: Molecular, Cellular and Developmental Biology.

Area of Emphasis: Cancer Biology.

xi

Table of contents

Abstract...... ii

Acknowledgement...... vii

Vita...... x

Publications...... x

Fields of Study...... xi

Table of contents……………..………………………………………………..………xii

List of figures...... xix

List of tables...... xxiv

List of important abbreviations...... xxv

Chapter 1: Breast cancer………………………………………...... 1

1.1 Introduction...... 1

1.2 Classification of Breast cancer subtypes...... 2

1.2.1 Classification based on histological grade...... 2

1.2.2 Classification based on histological types...... 3

1.2.3 Classification based on molecular subtype...... 5

1.2.4 Functional classification of breast cancer...... 7

xii

1.3 Breast cancer metastasis...... 8

1.3.1 Metastatic cascade...... 8

1.3.2 Invasion...... 9

1.3.3 Migration...... 10

1.4 Tumor microenvironment and breast cancer...... 11

1.5 Tumor microenvironment cells...... 11

1.5.1 Cancer associated (CAFs)...... 11

1.5.2 Tumor-associated macrophages (TAMs)...... 12

1.5.3 Other leukocytes...... 13

1.5.4 Endothelial cells...... 14

1.5.5 Adipose cells...... 15

1.6 Current treatments of breast cancer...... 16

1.7 Concluding remark...... 20

Chapter 2: Broad-spectrum anti-tumor roles of cannabinoids...... 22

2.1 Introduction...... 22

2.2 Classification of cannabinoids based on source of production...... 26

2.2.1 Endogenous cannabinoids...... 26

2.2.2 Phytocannabiboid...... 28

2.2.3 Synthetic cannabinoids...... 28

2.3 Classification of cannabinoids based on chemical structure...... 29

2.3.1 Classical cannabinoids...... 29

2.3.2 Non-Classical cannabinoids...... 29

xiii

2.3.3 Aminoalkylindole...... 29

2.3.4 ...... 30

2.3.5 Others...... 30

2.4 Receptor expressions and their relation to the tumorigenicity...... 31

2.5 Cannabinoids and their anti-tumor properties...... 33

2.5.1 Cannabinoids and the programmed cell death...... 33

2.5.2 Cannabinoids and tumor cell proliferation...... 34

2.5.3 Cannabinoids and the anti-angiogenic potential...... 35

2.5.4 Cannabinoids and the anti-invasive properties...... 36

2.5.5 Anti-migratory potential of cannabinoids...... 37

2.5.6 Effect of cannabinoids on tumor cell adhesion...... 38

2.5.7 Effect of cannabinoids on tumor growth and metastasis...... 38

2.6 Cannabinoid Receptor-Independent Effects...... 39

2.7 Concluding remark and future directions...... 40

Chapter 3: novel anti-tumor mechanisms of Cannabidiol in breast cancer...... 43

3.1 introduction...... 43

3.2 Materials and methods...... 44

3.2.1 Reagents and antibodies...... 44

3.2.2 Cell culture...... 45

3.2.3 Cell proliferation assay...... 45

3.2.4 Chemotactic assays...... 45

3.2.5 Immunofluroscence...... 46

xiv

3.2.6 Flow-cytometry...... 46

3.2.7 Gelatin zymography...... 46

3.2.8 Luciferase reporter assay...... 47

3.2.9 Colony forming assay...... 47

3.2.10 Western blotting...... 47

3.2.11 Mouse xenograft models...... 48

3.2.12 Wound healing assay...... 48

3.2.13 Real Time PCR...... 49

3.2.14 Immunohistochemistery (IHC)...... 49

3.2.15 Statistical analysis...... 49

3.3 Results...... 50

3.3.1 CBD inhibits breast cancer cell proliferation, migration and

invasion...... 50

3.3.2 CBD modulates EGF/EGFR signaling...... 54

3.3.3 CBD inhibits tumor growth in breast cancer mouse

models...... 58

3.3.4 CBD inhibits metastasis of breast cancer cells to the lung...... 61

3.3.5 CBD inhibits tumor growth and metastasis through inhibition of

macrophage recruitment to tumor sites...... 64

3.4 Discussion...... 70

xv

3.5 Conclusion...... 76

Chapter 4: TRPV2 is a novel prognostic marker and therapeutic target for TNBC

patients………………………………………………………………...... 77

4.1 Introduction...... 77

4.2 Materials and methods...... 79

4.2.1 Reagents and antibodies...... 79

4.2.2 Cell culture...... 79

4.2.3 Western blot...... 79

4.2.4 Immunohistochemical (IHC) analyses...... 80

4.2.5 Mouse models...... 80

4.2.6 Plasmid constructs...... 80

4.2.7 Doxorubicin uptake...... 81

4.2.8 Apoptosis assay...... 81

4.2.9 Cell viability assay...... 81

4.2.10 Colony forming assay...... 81

4.2.11 Calcium imaging...... 81

4.2.12 Statistical analysis...... 82

4.3 Results...... 82

4.3.1 TRPV2 is highly expressed in primary, metastatic triple negative

breast cancer tissues...... 82

xvi

4.3.2 TRPV2 upregulation is associated with better prognosis in ERα-

and TNBC patients who receive chemotherapy...... 85

4.3.3 Overexpression of TRPV2 increased DOX uptake and efficacy....88

4.3.4 CBD enhances the cytotoxic activity and the apoptotic potential

of chemotherapeutic agents...... 90

4.3.5 TRPV2 blocking abrogates CBD-mediated DOX uptake and

apoptotic activation...... 94

4.3.6 CBD improved the anti-tumor chemotherapeutic efficacy in vivo.96

4.4 Discussion...... 98

4.5 Conclusion...... 102

Chapter 5: Novel role of CB2R in tuning breast tumor microenvironment,

EGF/EGFR and IGF-I/IGF-IR pathwa……………………...... 103

5.1 Introduction...... 103

5.2 Materials and method...... 105

5.2.1 Reagents and antibodies...... 105

5.2.2 Cell culture...... 106

5.2.3 Western blot...... 106

5.2.4 Chemotactic Assays...... 106

5.2.5 Gelatin zymography...... 107

5.2.6 Luciferase reporter assay...... 107

5.2.7 Mouse models...... 107

5.2.8 Flow-cytometry...... 108

xvii

5.2.9 Statistical analysis...... 108

5.3 Results...... 109

5.3.1 CB2R activation inhibits EGF-induced tumorigenic events in ERα-

breast cancer cells...... 109

5.3.2 CB2R activation inhibits IGF-I-induced tumorigenic events in ERα+

and ERα- breast cancer cells...... 111

5.3.3 JWH-015 inhibits EGF/EGFR and IGF-I/IGF-IR axes in orthotopic

immunodeficient mouse model...... 115

5.3.4 CB2R knock out mice has higher tumorigenic potential due to

change in tumor microenvironment immune cells’ composition..118

5.3.5 CB2R activation inhibits tumor growth and metastasis through

modulation of the tumor microenvironment...... 121

5.4 Discussion...... 123

5.5 Conclusion...... 128

Chapter 6: Concluding remarks and future directions……….…………………...130

References...... 136

xviii

List of figures

Fig.1 Schematic representation of different subtypes of breast cancer based on

its histological type classification...... 4

Fig.2 Schematic representation of different subtypes of breast cancer based on

its molecular classification...... 7

Fig.3 Schematic representation of different subtypes of cells within the tumor

microenvironment and their role in the tumorigenic process...... 16

Fig.4 Schematic representation of different types of cannabinoids based on their

production source and chemical structure...... 31

Fig.5 CBD inhibits proliferation of breast cancer cells...... 51

Fig.6 CBD inhibits EGF-induced breast cancer cell proliferation and colony

formation...... 52

FIG.7 CBD inhibits EGF-induced breast cancer cell migration and invasion...... 53

Fig.8 CBD inhibits EGF-induced wound healing...... 54

Fig.9 CBD inhibits EGF/EGFR signaling...... 55

xix

Fig.10 CBD inhibits EGF-induced activation of MMPs...... 56

Fig.11 CBD inhibits focal adhesion expression and stress fiber formation...... 57

Fig.12 CBD inhibits breast tumor growth in different mouse model systems...... 59

Fig.13 CBD inhibits proliferation and vascularization of breast tumor and

suppresses EGF/EGFR pathway in different mouse model systems...... 60

Fig.14 CBD inhibits lung metastasis in different mouse model systems...... 62

Fig.15 Quantification of the number of breast cancer lung metastases based on

their size...... 63

Fig.16 CBD inhibits MMPs expression in the tumors of different mouse model

systems...... 64

Fig.17 CBD inhibits macrophage recruitment to the primary breast tumor

microenvironment...... 66

Fig.18 CBD reduced macrophage population in breast tumor microenvironment

in 4T1.2 mouse model...... 66

Fig.19 CBD inhibits macrophage recruitment to the secondary lung

metastases...... 67

Fig.20 CBD did not inhibit total leukocyte count and population in

mice...... 68

Fig.21 CBD inhibits human monocytic cell line (THP-1) recruitment towards

cancer cell conditioned medium...... 68

xx

Fig.22 CBD inhibits macrophage recruitment through modulation of breast

cancer cell profile...... 70

Fig.23 Schematic diagram shows a putative anti-tumor mechanism of action of

CBD...... 75

Fig.24 TRPV2 is highly expressed in primary, metastatic and triple negative

breast cancer tissues...... 84

Fig.25 TRPV2 expression is correlated with better prognosis in ERα- patients

who receive chemotherapy...... 86

Fig.26 TRPV2 expression is correlated with better prognosis in TNBC patients

who receive chemotherapy...... 87

Fig.27 TRPV2 expression is not correlated with better prognosis in ERα+ and

HER2+ patients...... 88

Fig.28 TRPV2 expression is not correlated with better prognosis in ERα+ and

HER2+ patients who receive chemotherapy...... 88

Fig.29 TRPV2 Overexpression enhances uptake of doxorubicin in SUM159

cells...... 89

Fig.30 TRPV2 Overexpression enhances doxorubicin efficacy in TNBC cells....90

Fig.31 CBD potentiates the uptake and the cytotoxicity apoptotic effect of

chemotherapeutic agents...... 92

Fig.32 CBD potentiates the apoptotic effect of chemotherapeutic agents...... 93

Fig.33 TRPV2 pore blocking suppresses CBD and DOX-mediated cytotoxic

effects...... 94

xxi

Fig.34 Dominant negative form of TRPV2 suppresses CBD and DOX-mediated

cytotoxic effects...... 95

Fig.35 TRPV2 downregulation suppresses CBD and DOX-mediated cytotoxic

effects...... 96

Fig.36 CBD improved the anti-tumor chemotherapeutic efficacy in vivo...... 98

Fig.37 CB2R activation inhibits EGF/EGFR signaling in ERα- breast cancer

cells…………………………………………………………………………...110

Fig.38 JWH-015 inhibits EGF-induced migration and invasion in CB2R

dependent manner…………………………………………………………111

Fig.39 JWH-015 inhibits IGF-I/IGF-IR signaling in ERα+ and ERα- breast cancer

cells...... 113

Fig.40 JWH-015 inhibits MMPs secretion in MCF-7 and SUM159

cells...... 114

Fig.41 JWH-015 inhibits IGF-I-induced migration and invasion in CB2R

dependent manner...... 115

Fig.42 JWH-015 suppresses breast cancer growth in vivo by inhibiting

EGF/EGFR and IGF-I/IGF-IR signaling pathways...... 117

Fig.43 CB2R KO mice have higher tumor promoting potential than wild type

mice...... 119

Fig.44 CB2R KO and WT mice analysis of different immune populations in

tumors...... 120

xxii

Fig.45 CB2R KO mice shows less percentages of CD8+ and CD4+ cells in

spleen...... 121

Fig.46 JWH-015 inhibits breast cancer growth and metastasis in immune-intact

mice and modulates the tumor microenvironment...... 123

Fig.47 Schematic representation of the anti-tumor role of CB2R activation in

breast cancer...... 129

Fig.48 Anti-tumor role of JWH-015 and CBD in breast cancer cells...... 131

Fig.49 The role of JWH-015 and CBD in the breast tumor microenvironment..132

xxiii

List of tables

Table-1: Representation of the most commonly used cannabinoids and their

putative molecular target receptors...... 25

Table-2: TRPV2 is highly expressed in malignant and metastatic breast cancer

tissues...... 85

xxiv

List of important abbreviations

α Alpha

β Beta

Ca2+ Calcium

CAFs Cancer Associaed Fibroblasts

CBD Cannabidiol

CBR1 Cannabinoid Receptor1

CB2R Cannabinoid Receptor 2

CSC Cancer Stem Cell

DCs Dendritic Cells

DOX Doxorubicin

ECM Extra-Cellular Matrix

EGF Epidermal

EGFR Epidermal

EMT Epithelial to Mesenchymal Transition

EPCs Endothelial Precursor Cells

xxv

ER receptor

ERK Extracellular Regulated Kinase

FAAH Fatty Acid Amide Hydrolase

FAK Focal Adhesion Kinase g Gram

Her-2 Human receptor 2

IHC Immuno-histochemistry

IGF -like growth factor

IGF-IR Insulin Like Growth Factor Receptor-I

κ kappa

L Liter

μ Micro- m Milli-; meter

M Molar

Met-F-AEA 2-methyl-2′-F-

MMP Matrix Metallo Proteinase

Neu Neutrophil

PARP Poly (ADP-ribose) polymerase

PCD Programmed Cell Death

PR Progesterone receptor

ROS Reactive Oxygen Species

xxvi

RT-PCR Real Time- Polymerase Chain Reaction

STAT3 Signal Transducer and Activator 3

TAM Tumor Associated Macrophage

THC Tetra Hydro

TME Tumor Micro Environment

TMA Tissue Micro Array

TNBC Triple Negative Breast Cancer

TRPV2 Transient Receptor Potential Vanilloid-2

xxvii

Chapter 1: Breast Cancer

1.1 Introduction

Cancer is defined as a wound that never heals. This disease consists of

tumor cells as well as stromal cells that form what is now called the tumor

microenvironment 1. Breast cancer is not a single disease; it is in fact a

heterogeneous disease that has different biological and clinical behaviors 2. It is

actually a collection of different diseases that affects the same anatomical organ

and has different clinical presentations, risk factors, clinical outcomes and

therapy response 2. Breast cancer is one of the most frequent causes of death

among women in United States (US) 3. In US, breast cancer accounts for about

29% of the all cancer types among women 3. It also represents the second cause

of cancer related death among US women 3. Breast cancer originates

from breast tissue, most commonly from the inner lining of milk ducts (ductal

carcinoma) or the lobules that supply the ducts with milk (lobular carcinoma) 4.

Although so many approaches have been developed recently to treat breast

cancer, there are a lot of therapeutic failures, which lead to breast cancer

metastasis and death eventually 1. In this chapter, we will review different

classifications of breast cancer based on their histological grade and type,

1 molecular subtypes as well as functional classification of breast cancer. We will also discuss the tumorigenic events involved in breast cancer metastatic process and how the tumor microenvironment components could affect the tumorigenic potential of breast cancer; finally we will review the recent advances of current treatment available for breast cancer.

1.2 Classification of Breast cancer subtypes:

1.2.1 Classification based on histological grade:

Breast cancer can be classified into subgroups based on its histological grade. The histological grade of breast cancer depends on the degree of differentiation (i.e nuclear pleomorphism and tubule formation) and the index of the proliferation (mitotic index) and the tumor aggressiveness 5.

Classification based on histological grade:

A- Grade 1

In this grade, the tubules represent about more than 75% of the whole breast tumor. The cells are well differentiated, uniform and regular and the nuclei are small. Cells have a lower ability to divide.

B- Grade 2

In this grade, the tubules represent about 10-75% of the whole breast tumor. The cells are moderately differentiated, and there is a moderate increase in cell size and there is variability in morphology between the cells. Cells have moderate ability to divide.

2 C- Grade 3

In this grade, the tubules represent about less than 10% of the whole breast tumor. The cells are poorly differentiated, and there is marked variation in the size of cells and nuclei. Cells have a very high ability to divide.

1.2.2 Classification based on histological type (Fig. 1):

Breast cancer can be classified according to its histological type into 6 :

1- Carcinoma in situ: It is a group of abnormal cells that grow in their normal place. This subtype is classified into 2 major groups:

A- Ductal (DCIS): Very common. It is further classified based on architectural features into 7 :

- Comedo

- Cribriform

- Papillary

- Micropapillary

- Solid

B- Lobular (LCIS): Rare

2- Invasive carcinoma: In this subtype, the cancer cells have invaded and spread to the surrounding breast tissues. This subtype can be further classified into:

A- Ductal (IDC): It is the most common type of breast cancer and represents about 70-80% of all invasive breast cancer cases 8.

B- Lobular

3 C- Ductal/Lobular

D- Mucinous (Colloid)

E- Tubular

F- Medullary

G- Papillary

It is worth noting that the use of molecular classification within IDC is well accepted and recommended for further classification of the invasive carcinoma, however this classification is not very recommended for DCIS 9, 10.

Fig.1 Schematic representation of different subtypes of breast cancer based on its histological type classification.

4

1.2.3 Classification based on molecular subtype:

By means of high throughput microarray, breast cancer has been classified into different molecular subtypes (Fig.2) 11.

A- Luminal Breast cancer

Luminal breast cancer has two distinct subtypes; Luminal A and luminal B breast cancer and refers mostly to positive (ER+) breast cancer subtype 12-16. Luminal A breast cancer is characterized by presence of high expression levels of ER-activated . It is also characterized by lower histological grade, low expression of proliferation related genes and better clinical outcome. It is also human epidermal growth factor receptor-2 (Her-2) negative subtype. Luminal B breast cancer shows higher histological grade and higher proliferation index and poor prognosis 12-16. Recently, Significant number of

Luminal B tumors showed overexpression of Her-2 16. However, based on recent meta-analysis, data suggested that classification of luminal tumors into two subtypes based on the proliferation index might be arbitrary 17. Notably, there is subtype of luminal that shows ER negative (ER-) transcriptomic features 18.

B- ER negative breast cancer

ER negative (ER-) breast cancer are classified into 3 subtypes:

1- Normal breast-like

In this subtype, HER-2 is not present and progesterone receptor (PR) is of unknown pattern. It is enriched in the genes that usually expressed in adipose

5 tissues. Some researchers claimed that this subtype is just artifact and it is just contamination of normal breast tissues 16, 19.

2- HER2

This subtype overexpresses HER-2 and the genes that are related to

HER-2. This is characterized by higher aggressiveness than normal breast-like subtype 2.

3- Basal like

This subtype is characterized by overexpression of the genes that upregulated in normal basal and myoepithelial breast cells such as cytokeratin

(5,17), nestin, epidermal growth factor receptor (EGFR) and CD44 2. Basal like breast cancer shows higher histological grade, mitotic index and necrotic zone in the center of the tumor 20-23. Breast tumors that are developed through breast cancer-1 (BRCA1) show similar immunohistochemical and morphological features to basal like breast cancer. Therefore, researchers have engineered mouse models with BRCA1 mutation and it interestingly has recapitulated all features of basal like breast cancer 24-29.

C- Molecular apocrine breast cancer:

It is similar to HER-2 breast cancer subtype, however it shows activation of the androgen receptor signaling transduction pathway 30, 31.

D- breast cancer subtype

This subtype is characterized by the upregulation of the interferon- regulated genes such as STAT1 11.

6 E- Claudin-low breast cancer subtype

The transcriptomic features of this subtype showed stem cell like properties 32, 33.

It is very important to mention that the use of both histological and molecular classification gives more clarified diagnosis and greater response to therapy.

Fig.2 Schematic representation of different subtypes of breast cancer based on its molecular classification.

1.2.4 Functional classification of breast cancer:

Breast cancer cells can be classified based on its function:

A- Normal breast cancer cells (Majority)

B- Breast cancer stem cells (very few)

7 Recently, breast cancer stem cells (CSCs) have been developed as another important area of research. CSC theory states that small number of cells is responsible for tumor progression and initiation and the rest of tumor cells has limited tumorigenic ability 6. It is worth noting that the markers that are used to identify CSCS and normal mammary stem cells (MaSCs) are the same 34. These markers were successfully isolated from the primary tumors and cancer cell lines and used as a useful tool to study the cancer stem cells.

1.3 Breast cancer metastasis

Metastasis of breast cancer cells is the most common cause of death among western women due to functional damaging of distant vital organs such as lung and liver. The recent implementation of adjuvant therapy and the early diagnosis of breast cancer have significantly reduced the mortality 35.

1.3.1 Metastatic cascade

This process consists of series of sequential steps and the failure in one of them will inhibit the whole process 36. The process starts with the local invasion of the tumor cells to its surrounding tissues. After that, the tumor cells intravasate the blood and/or lymphatic vessels. Through these vessels, the tumor cells could disseminate to distant organs. When reach to the target organ, the tumor cells undergo cell cycle arrest and then adhere to the capillary bed of the target organ.

After that, the tumor cells extravasate the organ parenchyma and proliferate and induce the to this organ. In the meantime, the tumor cells evade the immune response of the host in order to survive and flourish. If all steps are

8 done successfully, then the tumor cells could make a secondary metastatic lesion in a distant organ 37, 38.

1.3.2 Invasion:

The first step of breast cancer metastasis is the invasion of the tumor cells. In order to invade, first, cell-to-cell adhesion as well as adhesion of the tumor cells to the extracellular matrix (ECM) should be altered.

The Cadherin family plays a major role in cell to cell adhesion and the breast cancer metastasis in general 39. E-cadherin is important for cell-to-cell junction while downregulation of it is important for making outgrowth of the breast cancer cells. Downregulation or mutation of E-cadherin, which lead to functional loss, is well documented to improve the breast cancer metastasis 40-42. N- cadherin is the other family member that is associated with the mesenchymal phenotype of cancer cells 43. Epithelial-to-mesenchymal transition (EMT) process is known to enhance cancer progression and metastasis through enhancing tumor cell invasion and degradation of the ECM 44, 45. Upregulation of N-cadherin and downregulation of E-cadherin is observed in many epithelial cancers during the process of invasion. The loss of adhesion between epithelial cancer cells is associated with the loss of E-cadherin while adhesion of cancer cells to stromal cells and subsequent invasion of the stroma is associated with the upregulation of N-cadherin 46.

ECM components include laminin, , fibronectin, vitronectin, fibrinogens and others 39. Degradation of ECM is an important step to allow the

9 invasion and penetration of the cancer cells. This degradation is mainly done by urokinase plasminogen activator system (uPA) as well as matrix metalloproteinases (MMPs) 47, 48. MMPs are known to mediate ECM proteolysis at the invasion front of breast cancer cells 49. Interestingly, researchers showed that downregulation of uPA reduced MMP-9 expression 49. Other proteins such as and heparanase have been reported to participate in the process of

ECM degradation and invasion of breast cancer cells 49-52.

1.3.3 Migration:

Cancer cells need to go distant sites, to do that, cancer cell should have the ability to migrate either as a single cell or in a coordinate manner 53. EMT plays an important role in the migration process. In this process, losing of epithelial marker such as E-cadherin and upregulation of mesenchymal markers such as N-cadherin and vimentin would occur simultaneously. After the EMT process and the loss of adhesion, the polarity of cancer cells changes from apical-basal to front-rear and the migration initiates through cytoskeleton remodeling 54.

Interestingly, there is another type of movement called amoeboid movement in which cells are rounded, not elongated and -like which is the case of mesenchymal movement. Cancer cells, in this case, move through the pores and deformations that are present in ECM without actual degradation of the ECM 53, 55, 56. In this type of migration, cells squeeze and push themselves and generate the mechanical force through actin-myosin contractions 55, 57.

10 1.4 Tumor microenvironment and breast cancer

A well-known theory (seed and soil) theory has been proposed by

Stephen Paget. This theory refers to the tumor cells as (a seed) that can grow if only finds its good soil (tumor microenvironment) 58. Tumor microenvironment consists of different types of cells including immune cells, fibroblasts, endothelial cells, lymph and blood vessels as well as ECM (Fig.3). All these components affect not only the breast cancer growth, they also affect breast cancer metastasis 59-61. Cancer cells interact with tumor microenvironment components and finally turn their mode from (in situ) to metastatic mode 62.

Importantly, breast cancer cells tend to metastasize mainly to the bones and lung more than any other sites in the body such as liver or brain 63. This might be explained because breast cancer cells highly express (C-X-

C Motif) Receptor-4 (CXCR4) however, its ligand Chemokine (C-X-C Motif) ligand-12 (CXCL12) is highly expressed in the metastatic sites especially lung and bones 64, 65.

1.5 Tumor microenvironment cells

1.5.1 Cancer associated fibroblasts (CAFs)

They have the ability to promote breast cancer growth through different ways such as secreting different factors such as transforming growth factor beta-

1 (TGF-β1), CXCL12, and vascular endothelial growth factor (VEGF). They have the ability to enhance secretion of 17 beta- dehydrogenase (E2DH), hence increase estradiol (E2) level which leads to faster growth of ER+ breast

11 cancer 66. Interestingly, the expression level of -6 (IL-6) in CAFS is about 100 fold more if compared to normal associated fibroblasts (NAFs) which participates in enhancing the EMT process in ER+ breast cancer cells 67.

CAFs also play an important role in promoting the invasion and metastasis of breast cancer cells 1. CAFs play a crucial role in the transition of (DCIS) into

IDC through increasing of the expression of MMP-14 and the activity of MMP-9.

Not only that, they also secrete CXCL-12 which recruits endothelial progenitor cells (EPCs) which results in enhancing of tumor angiogenesis 68. CAFs also secret Chemokine (C-C motif) ligand-2 (CCL2), which increases cancer stem cell population and recruits macrophages and promotes intravasation process 69.

CAFs also secrete CCL5, which recruits CD4+FOXP3+ regulatory T-cells (T- regs), which play a major role in promoting lung metastasis 70. Importantly, many studies showed that targeting CAFs helps in induction the efficacy of the chemotherapy and endocrine therapies during breast cancer treatment 71-73.

1.5.2 Tumor-associated macrophages (TAMs)

One important component of tumor microenvironment is macrophages which have been shown to be recruited by non-malignant breast cancer cells then they induce angiogenesis and malignant transformation 74. TAMs show the ability to induce a pre-metastatic niches through interaction with breast cancer cells and endothelial cells therefore they promote the metastatic dissemination of breast cancer cells 75.

CD34+ progenitor cells from bone marrow are the precursors of

12 macrophages. They differentiate into pro- then monocytes then reside into tissues and differentiate into tissue macrophages. It is important to note that macrophages are prominent in the stroma of all malignant tumors 76. Most of

TAMs have M2 phenotype. TAMs play a very important role in tumorigenesis.

They promote breast cancer growth, migration, invasion and angiogenesis 1.

Infiltrating TAMs are correlated with high tumor grade, lower overall and disease free survival and poor prognosis 1. Recent studies showed that TAMs highly express CCL18 which plays a major role in enhancing the invasion and the metastasis of breast cancer cells through enhancing the clustering and promoting the adherence to the ECM and improving EMT 77, 78. In addition, TAMs affect the metastatic process through modulation of the micro RNAs (miRs). For example, TAMs increase breast cancer cell invasiveness through exosome- mediated delivery of oncogenic miRs such as miR-223 79.

1.5.3 Other leukocytes

There are other types of leukocytes, which have tumor promoting abilities.

For example, CD4+ T-reg cells were reported to be highly infiltrated in the tumors of spontaneous breast cancer mouse model and depletion of these T-regs has resulted in inhibition of tumor growth 80. In addition, T-reg cells have been found to be important in the spread of Erbb2-transformed carcinoma cells through nuclear factor-κB (RANK)/ nuclear factor-κB ligand (RANKL) mediated mechanism 70.

Another type of cells is CD8+ cytotoxic T-cells. The number of infiltrating

13 cytotoxic T-cells has been shown to be positively correlated with patient survival

81. Interestingly, some studies showed that chemotherapeutic drugs could change the tumor microenvironment. One study showed that patients who received neoadjuvant chemotherapy showed higher percentages of myeloid cells and CD8+ T-cells and lower percentage of CD4+ T-cells 1. Interestingly, it has been shown that although cytotoxic drugs kill tumor cells, they increase their secretion of colony stimulating factor-I (CSF-I), which is important in macrophage recruitment and associated with poor prognosis. Therefore, co-administration of chemotherapeutic drugs with CSF-I blockers could be a successful strategy which involves killing of cancer cells, inhibition of macrophage recruitment and increasing of CD8+ cytotoxic cells infiltration in the tumor tissues 1, 82.

1.5.4 Endothelial cells

In addition to their crucial roles in the angiogenesis process, endothelial cells play other major roles in breast cancer growth and metastasis. For example, human umbilical endothelial cells (HUVECs) enhance the proliferation of pre- neoplastic breast cells MCF10AT1-EIII8, which suggests that endothelial cells might play a role in breast cancer initiation step 83. In addition, it has been reported that chemotherapeutic drugs induce the endothelial cells to produce

TNF-α, which in turn induces expression of CXCL1/2 in cancer cells. This would increase the chemotaxis of CD11b+/Gr1+ myeloid derived suppressor cells

(MDSCs) to the tumor stroma. These attracted cells also produce S100A8/9, which is well known to enhance cancer cell survival 84.

14 1.5.5 Adipose cells

Adipocytes, preadipocytes and adipose-derived stem cells are the major components of adipose tissue around the breast cancer cells. Solid evidences have shown that cancer-associated adipose (CAA) tissue plays an important role in breast cancer tumorigenesis. For example, it has been reported that collagen

VI is highly expressed in CAA and involved in progression of mammary carcinogenesis 85, 86. In addition, adipose tissue can promote breast cancer growth and metastasis through secretion of different cytokines such as CXCL12,

IL-6 and IL-8. It can also induce EMT process in cancer cells through Platelet- derived growth factor (PDGF) secretion 1. Moreover, CAA increases the resistance of breast cancer radiation therapy 1. Therefore obesity is considered as a poor prognostic factor in breast cancer patients 1.

15

Fig.3. Schematic representation of different cells within the tumor microenvironment and their role in the tumorigenic process.

1.6 Current treatments of breast cancer:

A- Chemotherapy:

It remains the first line of treatment for breast cancer patients. This includes , anthracyclins, platinum compounds and other chemotherapeutic agents.

B- HER-2 targeted therapy

HER-2 is upregulated in about 15 - 25% of primary human breast cancers.

It is associated with poor prognosis and aggressive pattern of breast cancer 87.

16 Therefore, antibodies have been developed to target HER-2 such as and used for HER-2 positive breast cancer patients either alone or in combination with other chemotherapeutic agents 87. Scientists have also developed small-molecule kinase inhibitors to target HER-2, such as . Lapatinib binds to the ATP-binding region of HER-2, and prevents self- phosphorylation and activation 88.

Although HER-2 targeted therapy has shown promising anti-tumor efficacy, 70% of breast cancer patients still develop tumor progression or recurrence due to development of its resistance 89, 90. Recently, there are other molecules have been developed to target HER-2 such as the antibody-drug conjugate (ADC) trastuzumab-DM1. This strategy combines trastuzumab with a fungal toxin DM1 (Emtansine) 91, 92. This therapeutic option allows the targeted delivery of the potent chemotherapy drug DM1 to HER-2+ breast cancer 93.

Another monoclonal antibody that has been developed against HER-2 receptor is . It binds to a different extracellular portion of HER-2 receptor than trastuzumab and it also blocks HER-2 dimerization 87. Other small molecule inhibitors of HER-2 include , , AK-285 and ARRY-380 87.

C- HER-3 targeted therapy

ErbB3 is an important activator of phosphoinositide 3-kinase (PI3K) mediated signaling in ErbB1, ErbB2 and receptor (MET) dependent tumors. Reactivation of ErbB3 is a crucial step for cancers to become resistant to ErbB-targeted inhibitors 94. Therefore, recently HER-3 monoclonal

17 antibodies, such as MM-121, have been developed to block its activity and to inhibit the binding to its ligand and eventually inhibits HER-3 signaling 95, 96.

D- DNA repair targeted therapy

The poly (adenosine diphosphate [ADP])-ribose) polymerases (PARPs) are a family of enzymes that is involved in gene transcription, DNA repair and apoptosis in human cells 97-99. The most important molecule in this family is

PARP-1, which plays a key role in single-stranded DNA base-excision repair.

Inhibition of PARP-1 leads to accumulation of single strand DNA breaks and subsequently double strand breaks will be formed at the replication forks and induce cell death and apoptosis 87, 97-99. The breaks in normal cells are repaired due to the presence of tumor-suppressor proteins BRCA1 and BRCA2 however, in tumor cells, which lacks BRCA1/2, inhibition of PARP leads to apoptosis and tumor cell death 100, 101. Therefore, PARP inhibitors have been developed as a therapeutic option such as iniparib, , veliparib and 87.

E- m-TOR/PI3K

The mammalian target of rapamycin (mTOR) plays an important role in cell cycle progression, and is inhibited by rapamycin. mTOR is part of the kinase family of PI3K 102. Hyperactivation of mTOR as well as PI3K have been observed in various cancer types 103. Therefore, rapamycin and other analogues have been analyzed for the treatment of different cancers. Currently some mTOR inhibitors such as temsirolimus and everolimus inhibitors have been approved for treatment of renal cell carcinoma, breast cancer and other cancer types 87, 94, 104.

18 F- Insulin-like growth factor receptor targeted therapy

Overexpression of both Insulin-like growth factors I and II (IGF-I and IGF-

2) and IGF-R occur during fetal development and in different types of cancers 105.

As such, IGF-R is a promising for antibody directed therapy. Examples of

IGF-IR monoclonal antibody inhibitors that entered clinical trials include , , and 87. However, one major side effect associated with IGF-IR inhibitors is hyperglycemia due to their tendency to bind to the insulin receptors 87.

G- Heat shock protein-90 (HSP-90) targeted therapy

HSP-90 is an important molecular chaperone protein which helps in folding and stabilization of the proteins which are vital to cancer cell survival such as EGFR, VEGFR, BRAF, HER2, and MET 106. Cancers that express higher levels of HSP-90 are associated with aggressive phenotype and very poor prognosis. Therefore, HSP-90 is a perfect target for cancer treatment 107. Several different HSP-90 inhibitors have been developed for clinical trials such as tanespimycin, retaspimycin and ganetespib 87.

H- Histon deacetylase targeted therapy

Hypoxia-inducible factor-1 alpha (HIF-1 α) is a transcriptional factor that regulates multiple cellular signaling pathways and is involved in promotion of angiogenesis by increasing VEGF expression 87. Histon deacetylase (HDAC) inhibitors have been shown to inhibit HIF-1 α. This gives a great option for cancer patients especially those who upregulate VEGF 87. Valproic acid, ,

19 entinostat, and are examples of HDAC inhibitors that are developed for clinical trials. However, serious side effects such as myelosuppression and thrombocytopenia are usually associated with these inhibitors 87.

1.7 Concluding remark

Breast cancer, as we discussed here, is not just one disease. It is a complex disease that has different classifications and different molecular subtypes. This disease includes not only the tumor cells, it includes also immune cells and other stromal cells which participate actively in the tumorigenic process and make it even more complex. Treatment of breast cancer has changed dramatically in the last decade. Several targeted therapies have been developed and they improved the patients’ outcomes. One important challenge in treatment of metastatic breast cancer is that, it remains incurable. The main problem is that the cancer cell develops different ways to evade the action of these drugs especially when there is a redundancy in the biological process, which is targeted by these drugs. This will give difficulty to use only one agent for treatment of breast cancer. One strategy is to use combination therapies, however, combination therapy should be chosen very carefully in order to achieve maximum efficacy and minimum toxicity.

In fact we have to stop dealing with breast cancer as only just one disease for every patient. Treatment should be categorized based on each subtype and may personalized for every individual breast cancer patient. We have to tailor our therapeutic strategy based on the presence of different molecular targets that

20 upregulated, hyperactivated or downregulated. Indeed, so much work is needed to disentangle the complexity of the treatment networks to give the best option for every patient 87.

21 Chapter 2: Broad-spectrum anti-tumor roles of cannabinoids

2.1 Introduction

Cannabinoid receptors and their ligands are active areas of research since several decades. Cannabinoids can be classified into phytocannabinoids, endogenous or synthetic cannabinoids according to their production sources, whereas cannabinoid receptors are categorized mainly to cannabinoid receptor-1

(CB1R) and cannabinoid receptor-2 (CB2R) 108. Additionally, the metabolizing enzymes that degrade the endogenous cannabinoids are also part of the cannabinoid research because it would affect eventually the activity as well as the bioavailability of these compounds 109. In this chapter, we are reviewing the most common cannabinoids, their classification and their affinity towards their molecular targets. We are also reviewing here the anti-tumor roles of different cannabinoids on different cancer types and how the cannabinoid receptor expression could be predictive of prognosis and predictive of effective use of their specific ligands. We are also reporting here the non-cannabinoid receptor mediated actions of some cannabinoids. This article suggests the use of

22 cannabinoids as an adjuvant strategy for cancer patients but this after performing further extensive pre-clinical studies and studying the exact cannabinoid roles on immune cell populations in the tumor microenvironment.

Cannabinoids have been used long time ago for pain relief through using their source, Sativa (marijuana). The medicinal use of these cannabinoids is highly limited due to the psychotropic potential of these compounds especially Δ9- (Δ9-THC) which is the main active constituent of marijuana 110. Since 1940, after the discovery of Δ9-THC, researchers have tried to discover more cannabinoids and understand their functions and mechanism of actions in different diseases. Cannabinoids are terpenophenolic compounds that exert their functions through binding to cannabinoid receptors (CB1R and CB2R). Both receptors are Gαi protein- coupled receptors and have about 44% overall 111. CB1R receptor is mainly expressed in central nervous tissues such as cerebellum and hippocampus whereas CB2R receptor is expressed mainly peripherally and in immune cells such as B, T lymphocyte, NK cells and myeloid cells 111-113. Binding of cannabinoids to CB1R receptor mediates their analgesic, temperature and locomotion control properties and play a major role in cannabinoid-mediated psychoactive effects 108, 114, 115. CB1R is also expressed in peripheral neural and non-neural tissues such as spleen, uterus, testis and vascular endothelium 116.

Endocannabinoid system consists of the endogenous cannabinoids and their receptors and the enzymes that metabolizes these cannabinoids 109. This system

23 has been extensively studied as a molecular target for the therapy of different diseases such as glaucoma, multiple sclerosis, obesity, Parkinson's disease and

Alzheimer's disease 113. In recent years, researchers analyzed the possible role of the cannabinoids and their receptors in cancer. Several studies from different groups have proved that many synthetic cannabinoids, endocannabinoids as well as phytocannabinoid compounds show strong anti-tumor effects both in vitro and in vivo in different cancer types such as breast, lung, pancreatic, prostate and skin cancers 112. In fact, cannabinoids are not only useful as anti-tumor drugs, but they have advantage owing to their palliative effects. For example cannabinoids can be used to inhibit the nausea that is associated with anti-tumor chemotherapeutic drugs, in addition they can relieve the pain and insomnia and stimulate the appetite which are all associated with cancer patients 117, 118. In this chapter, we are focusing on the role of different cannabinoids and their receptors on several tumorigenic events such as cancer cell proliferation, migration, invasion, chemotaxis, adhesion, signaling, etc. and the possibility of the future use of these drugs as a therapeutic strategy in different cancer types.

Cannabinoids can be classified based on their production source and their chemical structures into different categories (Fig.4). Table 1 shows most common cannabinoids and their molecular target receptors.

24 Cannabinoid Receptor Cannabinoid Receptor

2-arachidonoyl- CB1R/CB2R HU-308 CB2R selective glycerol (2-AG) agonist 108, 119 agonist 108, 119

Anandamide CB1R agonist 108, JWH-133 CB2R selective

(AEA) 119 agonist 108

Docosatetraenyl CB1R agonist 108, JWH-015 CB2R selective ethanolamide 120 agonist 108

Palmitoyl- CB2R agonist 108, R-(+)-WIN CB1R/CB2R 55,212-2 ethanolamide 120 Nonselective

(PEA) agonist 108, 119

Homo-γ- CB1R agonist 108, HU-210 CB1R/CB2R linoenylethanolami 120 Nonselective de agonist 108, 119

Δ9- CB1R/CB2R Δ8- CB1R/CB2R tetrahydrocannabi tetrahydrocann nol (Δ9-THC) agonist 108, 119 abinol (Δ8- agonist 108 THC)

Cannabigerol CB1R/CB2R JWH-139 CB2R selective

(CBG) agonist 121 agonist 108

Continued

Table-1: Representation of the most commonly used cannabinoids and their putative molecular target receptors.

25 Table-1 Continued

Cannabichromene TRPA1 agonist CP55940 CB1R/CB2R

(CBC) 122 Nonselective

agonist 108, 119

cannabidiol (CBD) CB1R R-(+)- CB1R agonist

antagonist/CB2R methanandamid 108, 119

inverse e

agonist/TRPV2

agonist 123

AM251 CB1R antagonist AM281 CB1R

108 antagonist 108

2.2 Classification of cannabinoids based on source of production

2.2.1 Endogenous cannabinoids

Endocannabinoids are endogenous cannabinoids that are produced by the animal body. They are bioactive lipids, which show a range of different activities mediated basically by binding to CB1R, CB2R and other target receptors 124-126. They are biosynthesized by postsynaptic cells and bind to their receptors in a retrograde manner 127. Several studies have also shown that endocannabinoids exert their effects through cannabinoid receptors independent manner 128. Endocannabinoids act as retrograde messengers or

26 neuromodulators which affect the release of several neurotransmitters in different neural tissues 129. They have been shown that they play crucial roles in insulin sensitivity, inflammation, fat and energy metabolism 130.The most well-known endocannabinoid is anandamide (AEA), which is synthesized from arachidonic acid and acts as a ligand for CB1R receptor. Another important endocannabinoid is 2-arachidonoylglycerol (2-AG), which has been shown to act as a ligand for

CB1R and CB2R and synthesized from phosphatidylinositol-4,5-bisphosphate

(PIP2) 131-133. Palmitoyl-ethanolamide is an endocannabinoid that acts as CB2R ligand and co-synthesized with AEA in most tissues 108. Homo-γ- linoenylethanolamide and docosatetraenylethanolamide are other endocannabinoids that act on CB1R 108. , sleeping hormone, is another putative endocannabinoid which has similar behavioral functions of AEA 134.

Higher levels of AEA and 2-AG have been found in different types of cancers such as meningioma, glioblastoma, colon carcinoma, adenoma, and prostate cancer 120, 135-138.

There is a strong correlation between the presence of the metabolizing enzymes of the endocannabinoids, fatty acid amide hydrolase (FAAH) or

Monoacylglycerol lipase (MAGL), and the endocannabinoids’ localized concentrations and therefore their effect on the tissues. Pharmacological inhibition or knockdown of MAGL impairs prostate cancer aggressiveness due to elevated levels of 2-AG 139. Elevated FAAH expression in prostate cancerous tissues has been observed compared to normal prostate tissue samples 140.

27 FAAH inhibition has been reported to enhance anandamide anti-tumorigenic effects in non-small lung cancer through inhibition of EGF/EGFR axis 109. In contrast, pancreatic ductal adenocarcinoma patients have a direct correlation between FAAH/MAGL higher levels and higher survival rates 141.

2.2.2 Phytocannabiboid

They are part of the cannabinoid family and they constitute the major components of . These compounds are produced by the trichomes and concentrated in the form of viscous resin. Cannabidiol (CBD),

Cannabibol (CBN) as well as Δ9-THC are the most prevalent phyotocannabinoids 142. Δ9-THC and Δ8-THC binds to CB1R and CB2R with similar affinities, Δ9-THC acts as CB1R/CB2R antagonist and CB1R receptor partial agonist 143, 144. CBD has been shown to have affinity to many receptors other than cannabinoid receptors such as TRPV2 and 5HT1A 123. CBN has affinity towards both CB1R and CB2R, however it has relatively higher affinity towards CB2R 145, 146. Other natural phytocannabinoids have also been characterized such as (THCV), (CBDV), Monoethyl Ether (CBGM), cannabivarin (CBV)

(CBC) and cannabigerol (CBG).

2.2.3 Synthetic cannabinoids

These are cannabinoids that have affinity towards the cannabinoid receptors but they have been synthesized chemically in the laboratories. These

28 compounds have shown both anti-tumor and pro-tumor abilities based on cancer type, concentration, duration of treatment and route of administration 108.

2.3 Classification of cannabinoids based on chemical structures

Cannabinoids have been classified based on their chemical structures into different groups 147.

2.3.1 Classical cannabinoids

These are cannabinoids that are extracted from Cannabis Sativa or the synthetic analogues of these phytocannabinoids such as Δ9-THC, Δ8-THC, HU-

210 and desacetyl-L-nantradol. By terms of replacing the pentyl side chain of Δ8-

THC with dimethylheptyl group, HU-210 has been provided higher selectivity towards CB1R. In addition, by modifying THC, researchers have synthesized

CB2R selective agonists that work in nano-molar concentrations such as L-

759633, L-759656, JWH-133 and HU-308 148-150.

2.3.2 Non-classical cannabinoids

These are cannabinoids of AC-bicyclic and ADC-tricyclic analogs. One important molecule in this category is CP55940, which has both CB1R/CB2R similar affinities. CP47497 and CP55244 are also members of this family 108.

2.3.3 Aminoalkylindole

These are aminoalkylindole compounds that have cannabimimetic properties. The most well-known compound in this category is R-(+)-WIN55212 which has affinity for both CB1R/CB2R but with more selectivity for CB2R.

29 Importantly, some other compounds from different families show higher affinity towards CB2R such as L-768242 and JWH-015 151.

2.3.4 Eicosanoids

The most famous member of this group is anandamide (AEA), which is found originally in mammalian brain and other tissues. This group also includes

AEA analogs such as . Its R (+) isomer has a very high selectivity towards CB1R 152. 2-arachidonoylglycerol (2-AG) is also a member of this group with similar CB1R/CB2R affinities. Arachidonyl cyclopropylamide

(ACPA) and arachidonyl-2-chloroethylamide (ACEA) are other members of that family 108.

2.3.5 Others

This group are diarylpyrazole compounds that show antagonistic potential to the cannabinoid receptors 153 such as SR144528, which is a CB2R blocker and SR141716A, which is CB1R antagonist 108. Analogs of SR141716A such as

AM281 and AM251 that also have CB1R antagonistic activity are other members of this group 108.

30 Fig.4 Schematic representation of different types of cannabinoids based on their production source and chemical structure.

2.4 Receptor expressions and their relation to the tumorigenicity:

A very important consideration is the expression of cannabinoid receptors in the tumor cells versus the normal cells. An interesting study by Ryberg et al showed that CB1R has alternative spliced isoforms (CB1Ra and CB1Rb), which could lead to difference in its function in normal and cancerous tissues 154.

The relation of cannabinoid receptors expression with tumor status and patient outcome has been explored in several settings. These studies showed that the correlation of cannabinoid receptors’ expression to the cancer patient prognosis and patient outcome might be dependent on the cancer type. For example, astrocytomas show that 70% of the tumors express CB1R or CB2R, and CB2R expression level correlates with higher tumor malignancy in this cancer type 149. Similarly, in gliomas, a higher expression of CB2R relative to

31 CB1R is associated with higher tumor grade 155.

Higher expression of CB1R has been found in mantle cell lymphoma; furthermore, higher CB1R and CB2R expressions in non-Hodgkin lymphoma have been reported compared to reactive lymph nodes 156. In contrast, lower

CB1R expression has been found in colon carcinoma compared to normal colon mucosa 157.

In breast cancer, a correlation has been observed between CB2R expression and the expression of estrogen and progesterone receptor as well as

HER-2 and histological grade of the breast tumors 139.

CB1R expression level in tumor tissue is also associated with the severity of disease at the diagnosis and outcome stages 158. For example, CB1R expression in the androgen-sensitive and androgen-independent human prostate cancer cell lines has been reported to be higher than its expression in normal prostate epithelial human cells 159. Furthermore, CB1R expression is upregulated in prostate cancer specimens and is associated with higher tumor grades 160.

Moreover, High expression of CB1R in pancreatic cancer patients is correlated with shorter overall survival 141. In the contrary, higher expression of

CB1R and CB2R is correlated with better prognosis in hepatocellular carcinoma patients 161.

32 2.5 Cannabinoids and their anti-tumor properties

2.5.1 Cannabinoids and the programmed cell death

Induction of apoptosis is one of the most common mechanisms to fight against cancer cells 162. Interestingly, cannabinoids have been shown to induce apoptosis through several mechanisms. For example, one important pro- apoptotic lipid that has been reported to be upregulated by CB1R or CB2R activation is ceramide through stimulation of its de novo synthesis. This phenomenon has been shown in various cancer types including leukemia, pancreatic cancer, glioma and colorectal cancer 163-165. Triggering of sphingomyelin degradation through CB1R activation has been also reported to be another pro-apoptotic mechanism of CB1R 166.

THC has been reported to trigger apoptosis through CB1R activation which in turn leads to inhibition of PI3K-AKT and RAS-MAPK 167. Importantly,

CB1R activation has been associated with inhibition of anti-apoptotic proteins such as survivin, which is known to be overexpressed in human cancers and associated with poor prognosis 168, 169. CB1R activation has been reported to downregulate survivin through inhibition of C-AMP-dependent protein kinase A signaling 139. Using CB1R agonists such as AM-356 has further confirmed the opposite correlation between CB1R and survivin through showing reduction of survivin levels upon CB1R activation 157.

THC has also been shown to induce apoptosis through caspase activation and induction of translocation of BAD to mitochondria 170, 171. Reduction of

33 mitochondrial membrane potential and oxygen consumption, cytochrome-c release and increase of the production of mitochondrial hydrogen peroxide are other common pathways of cannabinoid-induced apoptosis 170-173.

2.5.2 Cannabinoids and tumor cell proliferation:

Various mechanisms have confirmed the ability of cannabinoids to inhibit the proliferation of tumor cells. These mechanisms include the inhibition of cAMP/protein kinase A and adenylyl cyclase, downregulation of EGFR expression, inhibition of EGFR activation, inhibition of the activity and downegulation of and vascular endothelial growth factor receptors 174-179. Interestingly, AEA, through CB1R mediated manner, has the ability to inhibit proliferation of breast cancer cells through prolactin receptor downregulation, inhibition of the receptor trk and cAMP-dependent

PKA pathway 174-179. Furthermore, AEA has been reported to inhibit EGF-induced proliferation of prostate cancer cells through EGFR downregulation and induction of G1 arrest 177.

WIN-55,212-2, a CB1R/CB2R agonist, has also been reported to decrease prostate cancer cell proliferation, inhibit androgen receptor, VEGF and prostate-specific (PSA) expression 159. However, cannabinoids have reverse action in other cancer types. It has been reported that cannabinoids induce EGFR signal transactivation and increase cancer cell proliferation in glioma and lung carcinoma 180.

34 2.5.3 Cannabinoids and the anti-angiogenic potential

Cannabinoids, through binding to their receptors, have been shown to reduce tumor growth by inhibiting tumoral vascular density. Several CB1R and

CB2R agonists such as JWH-133, WIN-55,212-2, THC and HU-210 have shown antiangiogenic action by inhibiting endothelial cell migration and survival 181.

Lower percentage of CD31+ cells has been observed after cannabinoid treatment in different cancer types including melanoma, glioma and lung cancer 174, 181-183.

Interestingly, a stable analog of AEA named Met-fluoro-anandamide (Met-F-

AEA), has been reported to reduce the length and number of endothelial cell spheroids, inhibit tube formation and angiogenesis in chick chorioallantoic membrane in vivo model 184.

Cannabinoids also have the ability to reduce the expression of proangiogenic factors. For example, It has been shown that Met-F-AEA reduced the expression level of VEGFR-I in thyroid cancer 176. CB2R specific agonist

(JWH-133) has been shown to inhibit vascular hyperplasia through downregulation of VEGF and other proangiogenic factors including hypoxia inducible factor-1α (HIF-1α), midkine, inhibitor of differentiation-3 (Id-3), -2 (Ang-2) and Tie-1 in different cancer types 174, 181, 185, 186. However,

JWH-133 upregulates type-I procollagen 1α chain, which is an important MMP substrate during the angiogenesis process 187. Interestingly, the release of VEGF has also been shown to be suppressed by THC in non-small lung cancer 183.

35 2.5.4 Cannabinoids and the anti-invasive properties

Invasion potential is one important characteristic of cancer cell that allows it to metastasize into distant organs, which ultimately the main cause of cancer related death 188. Matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 are secreted by cancer cells to facilitate their invasion through degradation of components of the basement membrane such as collagen type IV and laminin

188. Several cannabinoids have been reported to inhibit the invasion of the tumor cells through modulation of MMP system. Importantly, cannabinoids have shown strong anti-invasive potential in different cancer types. For example, MMP-2 expression has been reduced in cervical cancer cells after being treated with

THC and methanandamide 189. In glioma xenografts, JWH-133 as well as CBD have also been reported to reduce MMP-2 expression in vivo and impair tumor vascularization 181, 190. In addition, MMP-2 expression in endothelial cells is inhibited after Met-F-AEA treatment 184. Similarly, THC, AM-356 and Met-F-AEA have been reported to inhibit MMP-2 expression 184, 189, 191.

The proteolytic activity of MMPs is inhibited by tissue inhibitor of metalloproteinases (TIMPs). Surprisingly, cannabinoids’ effect on TIMP-1 expression is dependent on the cancer type 187. For example, cannabinoids, upon stimulation of CB1R and CB2R, upregulate TIMP-1 expression in cervical and lung cancer and show anti-invasive properties 189. However, TIMP-1 is downregulated by THC in glioma cells and in glioblastoma multiforme patients’ samples 192. In addition, TIMP-1 is downregulated by JWH-133 treatment of

36 glioma cells xenograft in nude mice 192. Interestingly, AM-356, through activation of TRPV1, has anti-invasive role through upregulation of TIMP-1 in lung cancer and cervical carcinoma cells 189, 193.

2.5.5 Anti-migratory potential of cannabinoids

Chemoattractants are very crucial for triggering the migration of cancer cells. Important candidates of these chemoattractants are EGF and CXCL-12 194.

THC has been shown to inhibit EGF-induced migration of NSCLC cells which might be through inhibition of AKT and mitogen-activated protein kinases (MAPK) activity 183. CB2R activation has also been reported to cross talk with CXCR4 and inhibits CXCL-12 mediated chemotaxis of breast cancer cells 195. The chemoattractants secreted from mast cells are another cause of cancer cell migration. Interestingly, 2-AG and WIN-55,212-2 have been reported to inhibit mast cells-induced migration through CB1R dependent mechanism 196.

Cannabinoids, such as HU-210, AEA and docosatetraenylethanolamide (DEA) have been reported to block -induced migration of colon carcinoma cells, however, JWH133, a CB2R agonist, shows no effect 119. The anti-migratory effect of Met-F-AEA through CB1R has been reported to be through inhibition of RhoA and GTPase and induction of RhoA translocation to the cytosol which leads to actin cytoskeleton alterations 197.

CBD, a non-psychoactive phytocannabinoid, has been shown to inhibit glioma cell migration though receptor independent mechanism 198. Furthermore,

CBD has been shown to inhibit breast cancer cell migration through inhibition of

37 EGF/EGFR pathway 199.

2.5.6 Effect of cannabinoids on tumor cell adhesion

Matrix proteins such as cadherins, selectins and integrins are very important for adhesion of cancer cells to the ECM. This adhesion is an integral part of the migration and metastatic process of cancer cells to distant organs 200.

CB1R activation by Met-F-AEA has been reported to inhibit human breast cancer cells’ adhesion to collagen type IV which is an important ECM component, however, no effect on adhesion to fibronectin and laminin has been reported 201.

Met-F-AEA has also been shown to inhibit the affinity of integrin for collagen through inhibition of the phosphorylation of Src and focal adhesion kinase (FAK)

201. Interestingly, HU-210, through CB1R dependent manner, indirectly inhibits

FAK phosphorylation through induction of the phosphorylation of FAK-related non-kinase (FRNK) 202-205.

Cannabinoids can also influence another adhesion molecules of cell adhesion molecules (CAMs) family. WIN-55,212-2 has been shown to inhibit interleukin 1 (IL-1)-induced up-regulation of vascular cell adhesion molecule-1

(VCAM-1) and the intercellular cell adhesion molecule-1 (ICAM-1) in a cannabinoid receptor-independent mechanism 206. Moreover, WIN-55,212-2 inhibits IL-1-induced activation of Nf-κB which is a transcription factor and important regulator of several adhesion molecules 206.

2.5.7 Effect of cannabinoids on tumor growth and metastasis

Since cannabinoids show strong anti-proliferative, anti-invasive, anti-

38 migratory and anti-adhesion properties against cancer cells, so it is expected that these compounds show anti-tumor growth and anti-metastatic potentials as well.

Interesting in vivo studies for cannabinoids have been performed and showed promising anti-tumorigenic effects of several cannabinoids.

THC has been reported to inhibit breast cancer growth and metastasis in breast cancer animal model 207. Furthermore JWH-133 as well as JWH-015 inhibit the tumor growth and lung metastasis of different breast cancer cell lines injected in mice 195, 208. WIN55,212-2 has also inhibited the tumor growth, the number of lung metastatic nodules and suppressed the angiogenesis in immune- deficient mouse model injected by human breast cancer cells 208.

In addition, CBD has shown strong anti-tumor growth and anti-metastatic ability against both human or murine breast cancer cell lines through different mechanisms 199, 209, 210. AM-356, a CB1R agonist, has also been shown to decrease the number and size of lung metastatic nodules from breast cancer cell line 201.

2.6 Cannabinoid Receptor-Independent Effects

Some cannabinoids exert their effects in a cannabinoid receptor non- dependent manner 211. AEA has been reported to induce cell death in lymphoma, uterine cervix carcinoma, neuroblastoma through vanilloid receptors 212, 213.

Furthermore, methenandamide (AM-356) inhibits TIMP1, which is involved in cancer cell invasion, through its action on TRPV1 189.

The pro-apoptotic ability of AEA in cholangiocarcinoma has been shown to

39 be through ceramide accumulation and FAS and recruitment to the lipid rafts in a non-cannabinoid receptor dependent manner 214. COX-2 is another important protein that participates in the CB-receptor independent anti-tumor effects of cannabinoids. For example AEA induces apoptosis in COX-2 highly expressing keratinocytes, however AEA shows little effect on keratinocytes and colon carcinoma cells with low expression of COX-2 215. Furthermore, AEA pro- apoptotic effect in neuroglioma has been shown to be mediated through COX-2 and this apoptotic effect was not changed after using cannabinoid receptor blockers 216. Interestingly, AEA has been reported to induce apoptosis through induction of intracellular calcium influx that activates COX-2 and in turn cytochrome c is released and caspase-3 is activated subsequently in a vanilloid receptor dependent manner 213.

Although CBD is a phytocannabinoid, it has no strong activity towards

CB1R or CB2R. CBD has been reported to inhibit breast cancer and glioma cells tumorigenic potential through mechanisms that are independent on cannabinoid or TRPV1 receptors 198, 210, 217. Another non-cannabinoid dependent mechanism of cannabinoids, especially CBD, is the ability to interfere of binding of (LPI) to GPR55 and therefore inhibits breast cancer cells’ proliferation in a cannabinoid receptor non-dependent manner 218.

2.7 Concluding remark and future directions

We have presented in this chapter that cannabinoids have broad- spectrum anti-tumor activities. They inhibit cancer cell proliferation, migration,

40 invasion, and adhesion in vitro. They also proved their efficacy in vivo through inhibition of tumor growth and metastasis in different animal models and in different cancer types. However, some cannabinoids show no effects or opposite effects in some studies. This might be due to the differential sensitivity of different cancer tissues to different types of cannabinoids. The effect of cannabinoids can also depend on the tumor grade and the appropriate dose for every cancer types.

Also, the route of administration might play an important role to decide the appropriate bioavailability of cannabinoids. All of these aspects should be extensively and carefully studied to reach an appropriate conclusion of the treatment regimen needed for each cancer type. It is also highly recommended if these cannabinoids will be studied in higher animal models like cats, dogs and monkeys before going to human clinical trials. Cannabinoids such as CBD can be also used as a triggering factor or sensitizing agent for other anti-tumor therapies. This will be extremely useful because in this case we will get the benefit from the already established anti-tumor therapies (chemo and/or radiotherapies), in addition we will get the benefit from the cannabinoids as anti- tumor agents and as palliative agents to counteract the side effects of the chemo and radiotherapy in cancer patients. Another consideration is that the tumor microenvironment could greatly affect the anti-tumor potential of cannabinoids.

Therefore, it is very urgent to study the exact role of cannabinoid receptors and their ligands on different immune cell populations to come up with a broader understanding of how these ligands will do if they are used clinically. Finally, a lot

41 of these ligands show effects that are non-cannabinoid receptor mediated. These effects should be exactly defined to design new cannabinoid receptor ligands that have all good aspects of the non-cannabinoid receptor mediated anti-tumor effects and avoid the bad ones.

42 Chapter 3: novel anti-tumor mechanisms of Cannabidiol in breast cancer.

3.1 Introduction

Breast cancer, because of its heterogeneous nature, is classified into different subtypes 13. TNBC is one of the most aggressive subtypes and is characterized by loss of expression of ER, PR, and Her-2/neu 219. TNBC is known to be unresponsive to estrogen receptor antagonists and Her2 antibody therapies and resistant to its standard chemotherapy 219, 220.

Cannabinoids can be classified into phyto-cannabinoids that are derived from Cannabis Sativa, endogenous cannabinoids that are synthesized inside animal tissues, and synthetic cannabinoids that are produced in laboratories 221.

CBD is a member of the cannabinoid family and one of the constituents of

Cannabis sativa. CBD, interestingly, has no psychotropic activity. CBD acts as an inverse agonist for CB2R and an antagonist for CB1R. Additionally, CBD acts as an agonist for TRPV1, TRPV2, TRPA1, PPARγ and 5HT-1A receptors. Moreover, it acts as an antagonist for TRPM8 and GPR55 receptors 123. Recently it was reported that CBD down-regulates metastatic factor (ID1) and up-regulates pro- differentiation factor (ID2) 209, 222. CBD has also been reported to induce programmed cell death in different cancer cells 210, 221, 223. However, CBD’s

43 molecular mechanism of action, its effect on tumor microenvironment and its anti- tumor effect against TNBC are not fully characterized.

TNBC cells are known to highly express basal markers like EGFR and cytokeratin 5/6 224 . Higher expression of EGFR in TNBC is associated with poor survival rates 225. EGFR inhibitors have been used for many solid tumors treatment, inevitably they fail due to development of drug resistance 226. The tumor microenvironment plays a pivotal role in tumor growth and metastasis 227.

TAMs are well known to contribute principally in tumor progression 227. There are two different forms of macrophages (M1 and M2); M1 are anti-tumorigenic macrophages while M2 have pro-tumorigenic ability 228.

In the present study, we document inhibitory properties of CBD on growth and metastatic properties of breast cancer cell lines including TNBC in vitro and in vivo. Furthermore, we show that CBD inhibits EGF-induced proliferation, migration/invasion, and activation of ERK and AKT signaling pathways. We also show that CBD inhibits breast cancer growth and metastasis through modulation of the tumor microenvironment. This study provides insight into novel CBD- mediated anti-tumorigenic/metastatic mechanisms.

3.2 Materials and methods

3.2.1 Reagents and antibodies

Cell culture reagents were purchased from Gibco Laboratories (Grand Island,

NY). The following reagents and antibodies used in this study were purchased from different sources: Cannabidiol (Sigma Aldrich); human/murine EGF

44 (Peprotech); GAPDH, AKT, p-EGFR/EGFR, and p-ERK/ERK (Santa Cruz); Ki67,

CD11b, CD206, CD31, and F4/80 from (NeoMarkers); and p-AKT from (Cell

Signaling). For flow-cytometery studies all antibodies were purchased from

(Biolegend). For lung fixation Bouin’s reagent was purchased from (Sigma

Aldrich).

3.2.2 Cell culture

Human TNBC cell line SUM159 229 was kindly provided by (Dr. Sarmila

Majumder, The Ohio State University) and 4T1.2, a subclone of murine TNBC cell line 4T1 cells 230, 231 was kindly provided Dr. Robin Anderson 232. SCP2, a subclone of MDA-MB-231 cells, was kindly provided by Dr. Joan Massagué 233.

MVT-1 cell line was obtained from Dr. Johnson 234. RAW264.7 cell line was purchased from American Type Culture Collection (ATCC). The identity of these cells was verified regularly on the basis of cell morphology. Cells were cultured in

DMEM containing 10% fetal bovine serum (FBS), 5 units/mL penicillin, and 5

ᵒ mg/mL streptomycin (Corning Cellgro) and grown in 5% CO2 incubator at 37 C.

3.2.3 Cell proliferation assay

Cells were seeded in 96 well plates and treated with different concentrations of

CBD with or without EGF (100 ng/ml) for 48 hours in SFM and subjected to MTT assay (Roche) according to manufacturer’s protocol.

3.2.4 Chemotactic assays

45 Chemotactic assays were performed using transwell chambers (Corning-Costar).

Briefly, cells were pretreated with CBD or vehicle. Top chambers were loaded with (1.5×105 cells for migration assay) or (2×105 cells for invasion assay) in serum-free medium (SFM) and bottom chambers contained SFM in presence or absence of EGF or cancer cells conditioned media. Cells that migrated were stained and counted 235.

3.2.5 Immunofluroscence

Immunofluorescence was performed as described earlier, briefly cells were seeded in 4 well glass chamber slides, incubated with primary antibodies vinculin or phalloidin overnight at 4°C. After that, cells were stained with Alexa Fluor- 594 or 488 conjugated secondary antibodies and visualized using Olympus FV1000 confocal microscope 109.

3.2.6 Flow-cytometry

Single cell suspensions were prepared from tumors as described 195, 208, 236. Cells were incubated with anti-CD11b APC, anti-F4/80 PE, and anti-CD206 (Alexa

Flour-488) for 1 hour then fixed with 2% paraformaldehyde and acquired on a BD

FACS caliber, then analyzed using Flowjo software.

3.2.7 Gelatin zymography

Gelatin zymography for collected conditioned media was performed as described earlier 109 . Briefly, supernatants from the treated cells were concentrated using centrifugal filter units (Millipore) and then run on zymogram gel (Novex) by

46 electrophoresis. The proteins were then renatured and developed and the bands stained for visualization according to the manufacturer’s protocol (Life

Technologies).

3.2.8 Luciferase reporter assay

We used NF-kB luciferase reporter assay (Promega) to determine NF-kB activity as described previously 109. Briefly, NF-kB luciferase constructs containing either wild type or NF-kB vector were transfected in the pretreated cells using lipofectamine 2000 (Invitrogen). For internal control, we co-transfected cells with

Renilla luciferase vector. 24 h after transfection, EGF (100 ng/ml) was added and then incubated for another 24 h. Cells were lysed and luciferase assay was performed according to manufacturer’s protocol.

3.2.9 Colony forming assay

One thousand cells were seeded in 60 mm2 plates in DMEM supplied with 10%

FBS. The next day the media were changed to DMEM with 3% FBS and were incubated for 6 days with or without (EGF 100 ng/ml). Cells were fixed, stained and clones were counted 109.

3.2.10 Western blotting

Cells were plated and lysed in lysis buffer (RIPA). Tumor samples were processed and lysed for further analysis. Equal conc. of total proteins were loaded on 4-12% SDS–polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (BioRad) and blocked for 1 h with 5% milk.

47 Membranes were then incubated with primary antibody overnight, then incubated for 1h at RT with secondary antibodies. The membranes then were stained developed using a chemiluminescence system (ECL Amersham Biosciences) and exposed to X-ray film (Kodak).

3.2.11 Mouse xenograft models

Female Balb/C and FVB mice were purchased from (Charles River Laboratories

Inc.). Tumors were formed by orthotopically injecting 4T1.2 (1x105) or MVT-1

(5x105) cells into the 4th mammary glands. When tumors became palpable, mice were randomized and injected peri-tumorally with CBD (10 mg/kg) or vehicle on alternate days for 3 weeks. Tumors were measured every week with external calipers and tumor volume was calculated according to the formula tumor volume

= (length x width2 / 2) 195, 236.

3.2.12 Wound healing assay

Wound healing experiment has been performed as described earlier 237. Briefly,

Cells were treated with CBD or vehicle for 24 h. Monolayers were scratched with a sterile 200 µL micropipette tip, washed, and incubated in media supplemented with 0.1% FBS in the presence or absence of CBD or vehicle and EGF (100 ng/ml). After another 24 h, cells were fixed and photographed.

48 3.2.13 Real Time PCR

RT-PCR and IHC were performed as described earlier 208, 236. RNA has been isolated from tumor tissues using TRIzol reagent (Invitrogen). C-DNA has been synthesized and Reverse transcriptase PCR (RT-PCR) reaction has been performed using RT-PCR kits (Applied Biosystem). Genes’ expression has been analyzed after normalization to GAPDH using 2-ΔΔCT method.

3.2.14 Immunohistochemistery (IHC)

Samples from mice tumor xenografts were embedded in OCT (Tissue-Tek) and stained using standard IHC techniques according to the manufacturer's recommendation (Vector Laboratories), using different primary antibodies. Slides were stained with the corresponding secondary antibodies and detected using light microscope.

3.2.15 Statistical analysis

Results were represented as mean ± SD. Student’s t test was used to compare vehicle and CBD-treated groups. P<0.05 was considered to be statistically significant. For all graphs, * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001.

49 3.3 Results

3.3.1 CBD inhibits breast cancer cell proliferation, migration and invasion.

Cell proliferation is an important characteristic of cancer cells to survive and form a tumor mass 109. EGF/EGFR axis plays an important role in proliferation of breast cancer cells 226; therefore, we performed MTT assay to evaluate CBD’s effect on cancer cell viability. We found that CBD induces a strong dose-dependent decrease in proliferation of SUM159 and SCP2 human

TNBC as well as 4T1.2 murine cell lines after 48 hours incubation (Fig.5 A-C).

We chose lower concentrations (CBD, 3 and 6 µM) that have lower direct effect on proliferation for further studies. We tested the anti-proliferative ability of low doses of CBD in presence of EGF. As shown in figures 6-A and 6-B, CBD significantly inhibits EGF-induced proliferation in SUM159 and 4T1.2 cells respectively. In order to test the ability of CBD to inhibit cancer cell survival and proliferation to form single cell clones, we performed colony-forming assay. We found that CBD significantly inhibited the number of cancer colony forming cells

(SUM159 and 4T1.2) respectively after EGF stimulation (Fig.6 C-D).

50 Fig.5 CBD inhibits proliferation of breast cancer cells. SUM159 (A), 4T1.2 (B) and SCP2 (C) cells were treated with CBD (3-15) µM for 48 hours and then subjected to MTT assay. Measurements were plotted as % of control.

Migration and invasion are two important characteristics of cancer cells to metastasize and form secondary tumors 238. When there is a gradient of extracellular stimuli like EGF, cells will undergo cytoskeletal rearrangement associated with an increase in cell adhesion, resulting in directional migration 239.

Therefore, we evaluated the ability of CBD to inhibit EGF-induced migration of

4T1.2 and SUM159. CBD (6 µM) significantly inhibited EGF-induced migration in

51 these cell lines (Fig.7 A-B). To confirm, we analyzed CBD effect in wound healing assay. As expected, CBD 6µM inhibits EGF induced wound closure in

MDA-MB231 and SUM159 cell lines (Fig.8 A-B).

Fig.6 CBD inhibits EGF induced breast cancer EGF-induced cell proliferation and colony formation. SUM159 (A) or 4T1.2 (B) cells were treated with vehicle or CBD (3 and 6) µM with or without EGF (100 ng/ml) for 48 hours and subjected to MTT assay. Data represent fold change of proliferation after 48 h (Day 2) relative to basal level of proliferation (Day 0). Colony forming assay was performed for SUM159 (C) or 4T1.2 (D) cells that were treated with vehicle or CBD with or without EGF (100 ng/ml).

52

The ECM and the basement membrane are barriers of cancer cells that hinder migration and invasion, so we were interested to examine CBD’s modulatory effect on cancer cell invasion through ECM. We found that CBD (6

µM) inhibited EGF-induced invasion of SUM159 cells and 4T1.2 cells through

ECM and basal membrane coated filters (Fig.7 C-D).

FIG.7 CBD inhibits EGF induced breast cancer EGF-induced cell migration and invasion. SUM159 (A) or 4T1.2 (B) cells were treated with CBD 6 µM for 48 hours and subjected to transwell migration assay. SUM159 (C) or 4T1.2 (D) cells were treated with CBD 6 µM for 48 hours and subjected to transwell invasion assay. Number of migrated or invaded cells were counted and plotted as % of control.

53 Fig.8. CBD inhibits EGF-induced wound healing. (A) MDA-MB231 or (B) SUM159 cells were treated with vehicle or CBD for 24 h and wound scratches were induced and incubated for another 24 h in presence or absence of EGF (100ng/ml). % of wound closure 24 h after wound scratching has been quantified.

These results suggest that CBD -even at low concentrations- has the ability to inhibit the proliferative, migratory and invasive ability of TNBC cells induced by EGF.

3.3.2 CBD modulates EGF/EGFR signaling.

EGFR signaling pathways are known to activate many cellular targets that are important for cancer cell survival, migration, and invasion. Both AKT and

ERK are downstream targets of the EGF/EGFR pathway and they are crucial for and metastasis 240. NF-kB activation is well known to promote the migration and invasion of TNBC cells 241. Furthermore, there is a correlation between high levels of NF-kB activation and EGFR overexpression in breast cancer 242. Therefore, we examined the ability of CBD to inhibit NF-kB signaling.

54 As shown in figure 9-A, CBD 6µM inhibited EGF-induced translocation of NF-kB into the nucleus in SUM159 cells compared to vehicle-treated cells. To further understand the molecular mechanism by which CBD modulates the EGF/EGFR pathway, we treated SUM159 with CBD 6 µM for 48 hours and then we stimulated the cells with EGF (100ng/ml) for 15 and 30 minutes. As shown, CBD

(6 µM) inhibited EGF-induced phosphorylation of EGFR, AKT and ERK in

SUM159 (Fig. 9-B).

Fig.9 CBD inhibits EGF/EGFR signaling. (A) SUM159 cells were treated with vehicle or CBD 6µM in presence or absence of EGF and subjected to NF-kB luciferase reporter assay. (B) SUM159 cells were treated with CBD 6µM, stimulated with EGF for 15 or 30 minutes then cell lysates were used for western blot analysis for the indicated proteins.

55 EGF has also been reported to induce tumor cell invasion through induction of matrix metalloproteinase MMP-9 and MMP-2 secretion 243, 244. In order to understand whether CBD-mediated inhibition of invasion is due to CBD effect on MMPs, we treated cells with CBD and analyzed MMPs activity in cancer cell-conditioned media by Zymography. As shown, CBD has reduced MMP-2 and

MMP-9 activities in SUM159 and 4T1.2 (Fig.10 A-B).

Fig.10 CBD inhibits EGF-induced activation MMPs. SUM159 (A) or 4T1.2 (B) cells were treated with CBD 3 or 6 µM in presence of EGF (100ng/ml) for 48 hours and conditioned media were used for gelatin zymography.

Cancer cells make protrusions to help them in adhesion and migration, and these protrusions are rich in actin filaments and adhesion molecules 245. We examined the effect of CBD on EGF-induced actin stress fiber and focal adhesion formation. By immunofluorescence, we showed that CBD 6 µM inhibits

EGF-induced actin stress fiber formation as shown by changes of Phalloiden expression and focal adhesion formation as detected by changes in Vinculin expression in SUM159 cells (Fig.11).

56

Fig.11 CBD inhibits focal adhesion expression and stress fiber formation. Confocal microscopy visualization of SUM159 cells treated with vehicle or CBD 6µM and stimulated with EGF (100 ng/ml) and stained for phalloidin (red), vinculin (green) and DAPI (blue).

These results indicate that CBD can inhibit EGF-induced tumorigenic properties of cancer cells through inhibition of the activation of EGFR, AKT, ERK and NF-kB signaling; in addition it blocks MMPs secretion, and suppresses phalloiden expression and actin stress fiber formation induced by EGF.

57 3.3.3 CBD inhibits tumor growth in breast cancer mouse models.

To confirm the in vitro data, we evaluated CBD ability to inhibit breast cancer growth in vivo. We found that CBD inhibited tumor growth, in 4T1.2 mouse model, as shown by reduction in tumor volume (Fig. 12-A) and weight

(Fig. 12-C) compared to control group. By IHC staining, we observed a dramatic decrease in the tumor cell proliferation, tumor vascularization and p-EGFR expression in CBD-treated group (Fig. 13-A). To further investigate CBD’s mechanism of tumor suppression, we used the tumor lysates to examine CBD effect on signaling pathways in vivo. CBD treated tumors showed decreased phosphorylation of AKT and ERK proteins (Fig. 13-C).

58

Fig.12 CBD inhibits breast tumor growth in different mouse model systems. Tumor volume measurements of 4T1.2 (A) or MVT-1 (B) mouse models were assessed every week for control and treated groups. Tumor weight of various experimental groups was determined in 4T1.2 (C) or MVT-1 (D) mouse models. Representative photographs showing tumors dissected from various experimental groups of 4T1.2 (E) or MVT-1 (F) mouse models.

We confirmed in vivo results using another highly aggressive mouse cell line (MVT-1). CBD treatment (10 mg/kg) significantly reduced tumor volume and weight in MVT1-1 model (Fig. 12-B and 12-D, respectively). We also analyzed

Ki67, CD31 and p-EGFR levels by IHC staining. CBD-treated tumors had decreased proliferative activity, vessel formation and p-EGFR expression (Fig.

13-B). Furthermore, CBD-treated group also showed less activation of AKT, and

ERK proteins in MVT-1 tumor lysates compared to the control group (Fig. 13-D).

59

Fig.13 CBD inhibits proliferation and vascularization of breast tumor and suppresses EGF/EGFR pathway in different mouse model systems. Representative photomicrographs of immunostaining with Ki67, CD31 and phospho-EGFR (p-EGFR) of tumors of control and CBD-treated groups in 4T1.2 (A) or MVT-1 (B) mouse models. Western blot images of 4T1.2 (C) or MVT-1 (D) tumors showing the expression of phospho-ERK or AKT (p-ERK, p-AKT) and total ERK and AKT (ERK, AKT) proteins in control and CBD- treated groups.

60 These results suggest that CBD has the potential to inhibit tumor growth through suppression of tumor cell proliferation, angiogenic potential and inhibition of the activation of EGFR, AKT and ERK proteins.

3.3.4 CBD inhibits metastasis of breast cancer cells to the lung.

Breast cancer patients’ survival is limited in part by the development of distant metastases 246. To study efficacy of CBD in inhibition of metastasis, two highly aggressive breast cancer cell lines (4T1.2 and MVT-1) were employed.

The 4T1.2 subclone is known for its propensity to metastasize to the lung, with rates superior to that of its parental cell line (4T1) 231. After 3 weeks of treatment,

CBD-treated groups showed significantly less number of metastatic nodules and less total lung weight in 4T1.2 and MVT-1 mouse models than control groups

(Fig.14 A-F) and (Fig.15 A-D).

61

Fig.14 CBD inhibits lung metastasis in different mouse model systems. Representative lung images and H&E staining photomicrographs were taken of control and CBD-treated groups of 4T1.2 (A) and MVT-1 (B) mouse models. The number of metastatic lung nodules was counted for 4T1.2 (C) and MVT-1 (D) mouse models of control and CBD-treated groups. Total lung weight was determined for 4T1.2 (E) and MVT-1 (F) mouse models of control and CBD-treated groups.

62

Fig.15 Quantification of the number of breast cancer lung metastases based on their size. Number of lung metastases that are >1mm (A) or <1mm (B) in 4T1.2 mouse model. Number of lung metastases that are >1mm (C) or <1mm (D) in MVT-1 mouse model.

Since tumor cells secrete MMPs which enhance their ability to invade and metastasize to distant organs 247, we analyzed MMP-2 and MMP-9 expressions in tumor lysates of CBD-treated and control groups. The CBD-treated group had significantly lower expression of MMP-2 and MMP-9 in both 4T1.2 and MVT-1 tumors (Fig.16 A-B), suggesting that the anti-metastatic effects of CBD are likely mediated through decreased MMP-2 and MMP-9 secretion by the tumor cells.

63 Fig.16 CBD inhibits MMPs expression in the tumors of different mouse model systems. RT-PCR quantification was performed for MMP-9 and MMP-2 in 4T1.2, 18S rRNA primers were used for loading control purpose (A) and MVT-1 (B) mouse models in control and CBD-treated tumors.

3.3.5 CBD inhibits tumor growth and metastasis through inhibition of macrophage recruitment to tumor sites.

Since TAMs are known to modulate tumor angiogenesis, cancer cell proliferation, and invasion 248, we investigated CBD’s effect on macrophage populations within the breast tumor microenvironment. First, we examined breast cancer xenografts for total (F4/80+) and M2 (Arginase-1+) macrophages by IHC.

64 CBD-treated tumors showed decreased F4/80 and Arginase-1-positive cells compared to control group (Fig.17 A-B). Next, we analyzed macrophage populations in the primary tumors by flow-cytometry. The CBD-treated tumors have significantly smaller percentages of total macrophage (CD11b+, F4/80+) in

MVT-1 and 4T1.2 tumors (Fig. 17-C and Fig.18 A-B, respectively). Decreased percentage of M2 (F4/80+, CD206+) macrophages was detected in CBD-treated tumors of MVT-1 tumor-bearing mice (Fig. 17-D).

65

Fig.17 CBD inhibits macrophage recruitment to the primary breast tumor microenvironment. Representative IHC photomicrographs of control and CBD-treated tumors of 4T1.2 (A) and MVT-1 (B) mouse models using F4/80 and Arginase-1 antibodies. Percentages of CD11b+/F4/80+ (C) and CD206+/F4/80+ (D) cells in control and CBD-treated tumors in MVT-1 mouse model.

Fig.18 CBD reduced macrophage population in breast tumor microenvironment in 4T1.2 mouse model. (A) Representative flow-cytometry image showing percentages of CD11b+/ F4/80+ cells in control and CBD-treated tumors in 4T1.2 mouse model. (B) Quantification of flow-cytometry data.

66 Macrophages facilitate tumor angiogenesis, ECM breakdown, and cancer cell motility, which are crucial components of the metastatic process 249.

Furthermore, macrophages are needed for metastatic cancer cell survival and formation of pre-metastatic niche 250. Therefore, to investigate CBD effect on macrophage populations within secondary lung metastases, we analyzed F4/80 and arginase-1 expressions in the lungs of vehicle- and CBD-treated groups by

IHC staining. As expected, CBD-treated group had less F4/80 and Arginase-1 expression in the lung metastases compared to vehicle-treated groups (Fig.19 A-

B). Importantly, we have not found difference in the total number of leukocytes or percentage monocyte populations in peripheral blood of control and CBD-treated group (Fig.20 A-B).

Fig.19 CBD inhibits macrophage recruitment to the secondary lung metastases. Representative IHC photomicrographs of lungs of control and CBD-treated groups of 4T1.2 (A) and MVT-1 (B) mouse models using F4/80 and Arginase-1 antibodies.

67

Fig.20 CBD did not inhibit total leukocyte count and monocyte population in mice. (A) Peripheral blood has been collected, heparinized and red blood cells have been lysed and the total leukocyte count has been determined. (B) Leukocytes were stained with F4/80 and CD11B antibodies and the percentage of total monocytes has been calculated by FACS analysis.

In order to understand why there is a reduction of macrophage population within the tumor, we treated SUM159 or 4T1.2 cells with CBD 6 µM or vehicle for

48 hours, and the collected conditioned media were used as a chemo-attractant to test migration of THP-1 or RAW264.7 cells, respectively, towards cancer cells.

We observed a significant reduction in the number of migrated THP-1 or

RAW264.7 cells towards CBD-conditioned medium compared to the control- conditioned medium (Fig.21 and Fig.22 C).

Fig.21 CBD inhibits human monocytic cell line (THP-1) recruitment towards cancer cell conditioned medium. Relative THP-1 cells migration towards SFM (Con) or the conditioned media of vehicle-treated (CM) and CBD-treated (CM-CBD) SUM-159 cancer cells.

68

We hypothesized that CBD might modulate cytokine production from tumor cells, which in turn affects macrophage recruitment towards the cancer cells. To test this hypothesis, we performed cytokine array analysis, using the previously mentioned conditioned media. Interestingly, we found that CBD- treated 4T1.2 cells secreted less CCL3, GM-CSF and MIP-2 proteins compared to vehicle-treated cells (Fig.22 A-B).

These results suggest that CBD modulates cytokine production from tumor cells, which leads to less recruitment of total macrophages and M2 macrophages into the primary and secondary tumor sites. This partially explains the ability of CBD to modulate the breast tumor microenvironment, which helps in inhibition of tumor progression and metastasis to distant organs.

69 Fig.22 CBD inhibits macrophage recruitment through modulation of breast cancer cell cytokine profile. (A) 4T1.2 cells were treated with vehicle or CBD for 48 hours and conditioned media were used for cytokine profiling. (B) Quantification of cytokine array data using Image-J software for the affected cytokines. Data represent protein levels relative to loading controls. (C) Relative RAW 264.7 cells migration towards SFM (Con) or the conditioned media of vehicle-treated (CM) and CBD-treated (CM-CBD) 4T1.2 tumor cells.

3.4 Discussion

Aggressive breast cancer subtypes are characterized by high proliferative and metastatic index and very poor prognosis. TNBC is one of the highly aggressive breast cancer subtypes that constitutes 12-24% of all breast cancer subtypes and shows poor relapse-free and overall survival 219. In contrast to other breast cancer subtypes, TNBC lacks molecular targets amendable to specific therapeutic agent 251. Chemotherapy still is the first line of treatment for

70 TNBC, however rapid development of resistance and the poor response rate are common 220, thus highlighting an urgent need for novel therapeutic therapies.

Cannabinoids have been used in cancer therapy owing to their ability to modulate pain, as well as their anti-inflammatory, cell growth inhibition, and apoptotic effects 252. CBD is a phytocannabinoid that constitutes about 40% of cannabis sativa extract. Although CBD exerts strong anti-tumorigenic activity in various cancer types, CBD’s anti-tumor effect on highly aggressive and metastatic breast cancer cells including TNBC subtype and its influence on the

EGF/EGFR pathway and the tumor microenvironment still needs to be clarified.

In the present study, we investigated the molecular basis of CBD’s anti-tumor activity. We showed that CBD inhibits breast cancer growth and metastasis, especially the TNBC subtype. To our knowledge, we discovered for the first time that CBD has the ability to inhibit EGF-induced tumorigenesis. We also showed – for the first time – that CBD has the ability to inhibit breast cancer growth and metastasis through modulation of the tumor microenvironment.

The EGF/EGFR axis plays an important role in cancer cell survival, growth, metastasis and angiogenesis 253, 254. EGFR is known to be overexpressed in TNBC. Current EGFR inhibitors have been shown to get rapid resistance in various cancer cell types 226. Therefore, it is important to identify novel drugs that can target this axis in order to inhibit cancer growth and metastasis mediated by the EGF/EGFR pathway, especially in TNBC patients. In our present study, we showed that CBD suppressed the activation of EGF/EGFR

71 signaling transduction pathways in highly aggressive and metastatic breast cancer cells including TNBC subtype in vitro. These pathways involve two key molecules (ERK and AKT), which are known to be important survival molecules.

This explains the ability of CBD to inhibit EGF-induced proliferation, migration, and invasion. This finding is especially important because it is well known that

EGFR overexpression is associated with poor prognosis, especially with TNBC patients 225.

Aberrant activation of EGF/EGFR signaling activates the NF-kB pathway, which in turn has a tumor pro-survival effect with resistance to chemotherapy 255.

Here, we reported that CBD inhibits EGF-induced activation of NF-kB. This effect might be highly useful, as it is clear that inhibition of EGF-induced activation of

NF-kB should be an important anti-tumor target and it explains as well the ability of CBD to inhibit EGF-induced breast cancer cell proliferation. It is also known that NF-kB has an important role in breast cancer metastasis through multi-target effects. It was reported that NF-kB can induce CXCR-4 expression which is a well-known receptor needed for breast cancer cell migration through binding to its ligand (SDF-1alpha) 256. It was also shown that NF-kB activation is required for EMT process and inhibition of NF-kB signaling prevented EMT and significantly reduced metastatic potential of breast cancer cells 257. Interestingly,

NF-KB activation has been shown to promote bone metastasis through GM-CSF induction 258 which it might explain the lower level of secreted GM-CSF from

72 CBD-treated cancer cells and suggests that CBD may play a role in inhibition of breast cancer metastasis to bone.

Adhesion molecules are known to connect actin stress fibers to ECM, and therefore they help in regulation of cancer cell migration 238. Reorganization of the actin cytoskeleton can lead to stress fiber assembly which in turn modulates the migration and invasion of cancer cells 259. Metalloproteinases are well-known to hydrolyze ECM and basement membrane components and activate invasion and metastasis of cancer cells 260. Our results show that CBD inhibits actin stress fibers and focal adhesion formation. Furthermore, we showed that CBD downregulates MMP-2 and MMP-9 secretion in TNBC cell lines and tumor lysates. These findings provide insights into the ability of CBD to inhibit breast cancer cell migration and invasion and decreased number of metastatic lung nodules in CBD-treated mice.

Our in vivo experiments confirmed the inhibitory activity of CBD on tumor growth and metastasis at 10 mg/kg doses. This finding is in accordance to

McAllister et al, who showed that CBD reduced breast cancer metastasis through down-regulation of ID-1 and up-regulation of ID-2 209. The lack of significant tumor weight reduction in their study might be due to use of lower doses of CBD

(5mg/kg) and possibly due to variable sensitivity of different breast cancer cell lines to CBD. In our study, the inhibitory effects of CBD on breast cancer xenografts resulted in decrease of lung metastases, decrease in tumor cell proliferation (as measured by Ki67 expression) and tumor vascularity (as

73 measured by cell vessel density, CD31 expression). Moreover, CBD-treated tumors showed less activation of EGFR, AKT and ERK proteins, which is in keeping with our in vitro findings that CBD inhibits the EGF/EGFR signaling transduction pathway and its downstream targets.

The tumor microenvironment is well known to modulate TAMs have been shown to activate EGFR signaling and facilitate angiogenesis, degradation of

ECM, and invasion of tumor cells 261. M2 macrophages have been known to inhibit adaptive immune response and enhance stromal invasion through production of EGF and VEGF. Furthermore, M2 macrophages secrete IL-17 and various MMPs, which improve cancer cell stromal invasion 262. Our study shows that CBD inhibits total macrophage and M2 macrophage populations within both primary tumors and secondary tumor metastatic sites. This might be due to inhibition of the recruitment of macrophages to the tumor site which has been confirmed by showing significantly less number of migrated mouse macrophage cell line (RAW 264.7) towards cancer cell conditioned media that was treated with CBD. This finding suggests that CBD may change the cytokine profile secreted from the cancer cells leading to decreased recruitment of macrophages to the tumor sites. We confirmed that by analyzing the affected cytokines secreted from 4T1.2 cell line after CBD treatment. We found that CBD inhibits

CCL3 and GM-CSF secretion. This finding confirms our hypothesis as CCL3 is an important chemokine that is involved in macrophage recruitment and enhancement of lung metastasis 263. Furthermore, high GM-CSF is associated

74 with CCL18+ macrophages which induces cancer metastasis and reduces patient survival 78.

Taken together, these finding show that CBD can suppress the activation of EGF/EGFR signaling transduction pathway and its downstream targets AKT,

ERK and NF-kB. It is likely that CBD, through acting on its receptors, changes the cytokine secretion of cancer cells (less GM-CSF, CCL3). In turn, decreased recruitment of macrophages to the tumor microenvironment suppresses angiogenesis and inhibits the invasive potential of tumor cells. Eventually tumor cells display decreased proliferative and metastatic ability (Fig.23).

Fig.23 A diagram shows a putative anti-tumor mechanism of action of CBD.

75

3.5 Conclusion

Overall, these results suggest CBD as a potent anti-tumor drug with anti- proliferative, anti-migratory, and anti-invasive properties. These results also suggest a cross-talk between EGFR and one of the receptors that CBD acts on.

Furthermore, CBD has a tumor microenvironment modulating property which suggests an important role of CBD receptors on changing the cytokine profile within the tumor microenvironment. This study advocates the use of CBD in breast cancer patients especially those with highly aggressive and metastatic cancer cells including TNBC patients, and those who have resistance to conventional EGFR therapy.

76 Chapter 4: TRPV2 is a novel prognostic marker and therapeutic target for TNBC

patients

4.1 Introduction

Breast cancer is one of the most frequent causes of death among women in United States 264. Excluding non-melanoma skin cancers, breast cancer accounts for 23% of all cancers in women worldwide 264. TNBC is one of the most aggressive breast cancer subtypes that is characterized by loss of ER, PR as well as Her2/neu expression 265. TNBC is known to be unresponsive to ER,

PR antagonists or trastuzumab therapies. The standard therapy for TNBC using taxanes, anthracyclins, and has been found to be therapeutically resisted in TNBC patients 266-270.

Studies about transient receptor potential (TRP) channels are emerging as a new field of research regarding tumor growth and metastasis 271. TRP channels are proteins consisting of six transmembrane segments 272. There are six families of TRP channels and there are similarities between these families in sequence homology and permeability to cations however, they respond differently to different external stimuli and local environment 272. These channels can be

77 considered as molecular sensors that play many roles in different physiological and pathological conditions 272.

TRPV2 responds to noxious heat as well as to cell membrane stretch and osmolarity changes 273. TRPV2 channel is activated by agonists such as Δ9- tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) 274, 275. Activation of

TRPV2 has been reported to be induced by growth factors leading to PI-3K- dependent and independent TRPV2 translocation towards the plasma membrane

276, 277. In this study we used CBD, a non-psychotropic cannabinoid, which has been shown to exert antiproliferative and antimetastatic properties in breast cancer cell lines 190, 209, 222 as a TRPV2 agonist.

Previous studies showed that TRPV4 enhances the uptake ability of aminoglycoside antibiotics and the mutation of the functional pore region abrogates this uptake 278. There is no much known about the role of TRPV2 in breast cancer. Therefore, in the present study, we analyzed -for the first time-the differential TRPV2 expression levels in normal, malignant, metastatic breast cancer and TNBC tissues. We also analyzed the importance of TRPV2 as a prognostic marker for TNBC patients. Furthermore, We analyzed the efficacy of

TRPV2 activation in enhancing the anti-tumor activity of chemotherapeutic drugs and how it can affect its apoptotic potential in vitro and in vivo. Finally, we analyzed the role of TRPV2 and its splice variant S-TRPV2 in modulating the uptake of chemotherapeutic drugs in TNBC cells.

78 4.2 Materials and methods

4.2.1 Reagents and antibodies

Cell culture reagents have been obtained from Gibco Laboratories (Grand Island,

NY). The following reagents and antibodies were purchased from different sources: CBD (Sigma Aldrich), Tranilast (Cayman), cleaved PARP and cleaved caspase-3 antibodies (); GAPDH antibody (Santa Cruz), Ki67 antibody (NeoMarkers) and TRPV2 antibody (Sigma).

4.2.2 Cell culture

SUM159 cells were obtained from Dr. Sarmila Majumder, The Ohio State

University. 4T1 and MDA-MD231 cell lines were purchased from American Type

Culture Collection (ATCC). Cells were cultured in DMEM containing 10% heat- inactivated fetal bovine serum (FBS), 5-units/mL penicillin, and 5 mg/mL streptomycin 279.

4.2.3 Western blot

Cells were plated and lysed in lysis buffer (RIPA). Tumor samples were processed and lysed for further analysis. Equal concentrations of total proteins were loaded on 4-12% SDS–polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (BioRad) and blocked for 1 h with 5% milk.

Membranes were then incubated with primary antibody overnight, and then incubated for 1 h at RT with secondary antibodies. The membranes then were stained developed using a chemiluminescence system (ECL Amersham

Biosciences) and exposed to X-ray film (Kodak).

79 4.2.4 Immunohistochemical (IHC) analyses

Tumors samples were fixed, and embedded in paraffin. Primary antibodies has been used against Ki67 (1:100), CD31 (1:100) and TRPV2 (1:100) for overnight incubation at 4°C. Vectastain Elite ABC reagents with avidin DH:biotinylated horseradish peroxidase H complex with 3,3′-diaminobenzidine (Polysciences) and Mayer hematoxylin (Fisher Scientific) has been used for detection of the bound primary antibodies.

4.2.5 Mouse models

Female NU/NU nude mice were purchased from (Charles River Laboratories

Inc.). Tumors were induced by orthotopic injection of tumor cells in 4th mammary glands as following: SUM159 (5 x 106) cells. When tumors became palpable, mice were randomized, and injected once per week for 4 weeks with CBD (5 mg/kg) or vehicle peri-tumorally, then with doxorubicin 5 mg/kg I.P after two hours 195, 236.

4.2.6 Plasmid constructs

Whole sequence TRPV2 overexpressing plasmid has been purchased from

(Origene), whereas short TRPV2 plasmid, which lacks the pore and sixth transmembrane region, S-TRPV2 has been kindly provided from Dr.Kojima-

Shizuoka University, Japan 280. In addition small interfering RNA (SiRNA) either non-targeting or TRPV2 Si-RNA have been purchased from (Dharmacon) company.

80 4.2.7 Doxorubicin uptake

Cells are treated with CBD 5 μM for 2 hours followed by DOX (5 μM) for 30 minutes. Cells are then trypsinized and washed and analyzed using a BD FACS caliber, then plotted for analysis.

4.2.8 Apoptosis assay

Detection of apoptosis in cells was performed using APO green TUNEL assay

(Bio-Tool). Cells were fixed, permeabilized and treated 1h with recombinant TdT enzyme and APO-Green labeling Mix. After that, cells were detected using fluorescent microscope (Olympus).

4.2.9 Cell viability assay

10,000 cells were seeded in 96 well plates for 24 h and then treated with different conc. of DOX with vehicle or CBD 5 M for 24h in SFM and subjected to MTT assay (Roche) according to manufacturer’s protocol 199.

4.2.10 Colony forming assay

Colony forming assay has been performed as described earlier 190

4.2.11 Calcium imaging

Intracellular Ca2+ concentration in SUM159 and 4T1 cells was measured using single cell Ca2+ imaging and the fluorescence Ca2+ indicator dye Fluo-4-AM.

Cells were loaded with 5 μM Fluo-4-AM in Krebs-Ringer-Hepes (KRH) buffer

(125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 6 mM glucose, 1.2 mM MgCl2, and

81 25 mM Hepes, pH 7.4) for 40 min at room temperature. For Ca2+ measurement,

cells were exposed to KRH buffer containing 1.2 mM Ca2+ with TRPV2 agonist

CBD 10 μM for 30 seconds then treated with TRP blocker (ruthenium red 20 μM)

for 1 min and captured by Zeiss LSM780 confocal microscope at room

temperature. Digital Ca2+ image analysis was performed using Image-J.

4.2.12 Statistical analysis

Results were represented as mean ± SD. Student’s t test was used to compare

vehicle and treated groups. P<0.05 was considered to be statistically significant.

For all graphs, * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001.

For TMA correlation studies, Log rank test has been used.

4.3 Results

4.3.1 TRPV2 is highly expressed in primary, metastatic triple negative

breast cancer tissues.

In order to analyze the differential expression pattern of TRPV2 in normal

versus primary or secondary metastatic tissues, we setup a tissue microarray

(TMA) of 100-breast tissue. Figures 24A and 24B and Table 2 show that

TRPV2 is significantly highly expressed in primary and metastatic breast cancer

tissues as well as TNBC tissues compared to normal breast tissues.

Furthermore, we analyzed the expression of TRPV2 in a TMA constructed only

for TNBC tissues and we observed strong or moderate expression of TRPV2 in

82 most of TNBC tissues (Fig.24 D-E). In addition, we observed high expression of

TRPV2 in different ERα- and TNBC cell lines (Fig.24 C).

83

Fig.24 TRPV2 is highly expressed in primary, metastatic and triple negative breast cancer tissues. (A) Representative image of IHC staining showing TRPV2 expression in normal, TNBC, malignant, metastatic breast tissues. (B) Quantitation of TRPV2 expression of 100 tissues including normal, malignant and metastatic breast tissues. Data are represented as % of negatively versus positively TRPV2 expressing tissues. (C) Western blot image showing TRPV2 expression in different human and murine ERα- breast cancer cell lines. (D) Representative image of IHC staining showing different grades of TRPV2 expression in the TMA of TNBC that has been used in the study. (E) Quantitation of TRPV2 expression in TMA constructed for only TNBC tissues.

These results indicate that TRPV2 is highly expressed in primary and metastatic and TNBC tumor versus normal breast tissues.

84 Type/Stain Low or no staining High staining Total Malignant Frequency 16 33 49

Percentage 32.65% 67.35% 50% Metastatic Frequency 12 27 39

Percentage 30.77% 69.23% 39.8% Normal Frequency 9 1 10

Percentage 90% 10% 10.2% Total Frequency 37 61 98

Percentage 37.76% 62.24% 100%

Table-2. TRPV2 is highly expressed in malignant and metastatic breast cancer tissues. The association between TRVP2 expression and tissue type was tested using Fisher’s exact test. The results showed that normal tissue has significantly different expression compared to malignant and metastatic tissues (p=0.0015). Higher proportion of the normal tissues didn’t have TRPV2 expression.

4.3.2 TRPV2 upregulation is associated with better prognosis in ERα- and

TNBC patients who receive chemotherapy.

In order to analyze the correlation between cancer patient survival and

TRPV2 expression, we used publically available Kaplan Meier plotter 281, we found that breast cancer patients , especially ERα- breast cancer patients, who express higher level of TRPV2 have significantly higher recurrence free survival than patients with lower TRPV2 expression level (Fig.25 A). In order to analyze the involvement of TRPV2 in the chemotherapy-associated prognosis of breast cancer patients, we used Kaplan Meier datasets and we analyzed the recurrence free survival of the breast cancer patients who received or not chemotherapeutic agents. As shown in Figure 25B, breast cancer patients in general and ERα-

85 breast cancer patients specifically with higher expression of TRPV2 have better prognosis than those who have lower expression of TRPV2. Interestingly, this applies only for patients who received chemotherapy. In contrast, no difference in prognosis between breast cancer patients with higher or lower TRPV2 expression that are not subjected to chemotherapy. For ERα+ or HER+ breast cancer patients, TRPV2 expression has not been correlated with any significant difference in prognosis between patients who express higher or lower TRPV2 level whether they receive chemotherapy or not (Fig.27 and Fig.28).

Fig.25 TRPV2 expression is correlated with better prognosis in ERα- patients who receive chemotherapy. (A) Kaplan Meier blot showing recurrence free survival of high/low expressing TRPV2 breast cancer patients. (B) Kaplan Meier blot showing recurrence free survival of high/low expressing TRPV2 breast cancer patients who received or not chemotherapy medication.

86 By means of TMA constructed only for TNBC tissues, we analyzed the survival of TNBC patients in correlation of TRPV2 expression. We observed that patients with strong TRPV2 expression and admitted to chemotherapy plan, have significantly higher recurrence free survival (RFS), P= 0.039, as well as higher overall survival (OS), P= 0.085, than those with lower or moderate expression of

TRPV2 (Fig. 26 A-B). These results suggest that TRPV2 expression is a good prognostic marker especially in ERα- patients who receive chemotherapy in their treatment plan.

Fig.26 TRPV2 expression is correlated with better prognosis in TNBC patients who receive chemotherapy. Recurrence free survival (RFS) (C) and overall survival (OS) (D) of TNBC patients of high/low TRPV2 expression in the TMA of TNBC that has been used in the study.

87

Fig.27 TRPV2 expression is not correlated with better prognosis in ERα+ and HER2+ patients. Kaplan Meier blots showing recurrence free survival of high/low expressing TRPV2 breast cancer patients.

Fig.28 TRPV2 expression is not correlated with better prognosis in ERα+ and HER2 + patients who receive chemotherapy. Kaplan Meier blots showing recurrence free survival of high/low expressing TRPV2 breast cancer patients who received or not chemotherapy medication.

4.3.3 Overexpression of TRPV2 increased DOX uptake and efficacy.

In order to analyze whether TRPV2 expression level within the cells would affect their ability to uptake chemotherapeutic drugs, we overexpressed TRPV2

88 in SUM159 (Fig.29 B), then we performed drug uptake experiment. As shown,

TRPV2 overexpressing cells showed higher uptake of doxorubicin (DOX) in both basal level and after CBD treatment compared to empty vector-transfected cells

(Fig.29 A and C).

Fig.29 TRPV2 Overexpression enhances uptake of doxorubicin in SUM159 cells. (A) SUM159 cells were transfected with empty vector (SUM-vec) or with full-length TRPV2 plasmid (SUM-TRPV2) and these cells were treated 0.5μM DOX for 30 min with or without CBD for 2 hours. Cells were collected for FACS analysis to detect DOX positive cells. (B) WB for TRPV2 overexpression in SUM-vec and SUM-TRPV2 cells. (C) Quantification of FACS data for DOX uptake in SUM-vec and SUM-TRPV2 cells.

We also analyzed the effect of TRPV2 overexpression in DOX-mediated cytotoxicity. We found that TRPV2 overexpression in TNBC breast cancer cell lines SUM159 and MDA-MB231 has enhanced DOX-mediated cytotoxicity compared to empty vector-transfected cells (Fig.30 A-B). These results suggests

89 that uptake of the chemotherapeutic drugs is triggered, in part, by TRPV2 channel. Furthermore, higher levels of TRPV2 expression are associated with higher efficacy of chemotherapeutic agents.

Fig.30 TRPV2 Overexpression enhances doxorubicin efficacy in TNBC cells. (A) SUM-Vec or SUM- TRPV2 cells were treated different concentration of DOX for 24 hours and subjected to MTT assay. (B) MDA-MB231-Vec or MDA-MB231-TRPV2 cells were treated different concentration of DOX for 24 hours and subjected to MTT assay.

4.3.4 CBD enhances the cytotoxic activity and the apoptotic potential of chemotherapeutic agents.

It has been shown previously that CBD acts as selective agonist for

TRPV2 in HEK293 and glioma cell lines using calcium mobilization studies 275,

282. TRP channels have been previously reported to participate in selective introduction of xenobiotics into target cells 278, 282. DOX is an chemotherapeutic drug with a small molecular weight and it has a natural fluorescent properties 283 that confers a good option to study chemotherapeutic

90 drug uptake by flowcytometry. To study the potential of TRPV2 activation in induction of chemotherapeutic drug uptake in TNBC, we treated SUM159 and

MDA-MB231 with CBD 5 (µM) for two hours followed by DOX (5 µM) for 30 minutes and we checked for DOX-positive cells by flowcytometric analysis. As shown in Fig. 31 A-B, TNBC cells that were treated with CBD in combination with doxorubicin have higher percentage of DOX positive cells compared with cells treated with DOX only. We also analyzed the apoptotic potential of combination therapy using Tunnel assay. As shown in Figures 31C and 31D,

CBD (5 µM) in combination with DOX have significantly higher percentage of

Tunnel positive cells compared to CBD or DOX alone.

Next, we evaluated whether this increase of DOX uptake is associated with decrease in cell viability. To test that we performed MTT assay of different conc. of DOX alone or in combination with CBD. As shown in Figures 32A and

32B, CBD synergizes the cytotoxic activity of DOX in SUM159 and MDA-MB231

TNBC cells respectively.

91

Fig.31 CBD potentiates the uptake and the cytotoxicity apoptotic effect of chemotherapeutic agents. SUM159 (A) or MDA-MB231 (B) cells were treated with vehicle or CBD for 2h followed by DOX for 30 min then subjected to FACS analysis of DOX uptake. (C) SUM159 cells were treated with CBD 5μM or DOX 0.5μM or combination of CBD and DOX and subjected to tunnel assay. (D) Quantification of the % of tunnel positive cells has been calculated.

In order to test the ability of combination therapy to inhibit cancer cell ability to survive and proliferate and form single cell clones, we performed colony-forming assay. We found that CBD in combination with DOX significantly inhibited the number of the cancer colony forming TNBC cells compared to CBD or DOX alone (Fig.32 C-D).

To analyze the molecular level of that apoptotic effect, we analyzed the levels of activated PARP and caspase-3 which directly involved in apoptosis, we found that CBD upregulated cleaved caspase-3 and cleaved PARP levels when

92 co- administrated with doxorubicin compared to CBD or DOX single treatments

(Fig.32 E).

Fig.32 CBD potentiates the apoptotic effect of chemotherapeutic agents. SUM159 (A) or MDA-MB231 (B) cells were treated with different concentrations of DOX in presence or absence of CBD for 24 hours and subjected to MTT assay. SUM159 (C) or MDA-MB231 (D) cells were treated with 0.5μM DOX in presence or absence of CBD for 6 days in reduced serum media and number of formed colonies has been counted. (E) SUM159 cells were treated with Veh or CBD 5μM or DOX 0.5μM or CBD+DOX for 24 h and the lysates were subjected to western blot analysis to detect the indicated proteins.

93 These results show that TRPV2 activation increases the uptake of chemotherapeutic agents, which in turn associated, with higher apoptotic potential and higher efficacy of chemotherapeutic drugs against TNBC cells.

4.3.5 TRPV2 blocking abrogates CBD-mediated DOX uptake and apoptotic activation.

In order to study whether the CBD’s effects are mediated through TRPV2, we used TRPV2 specific blocker (Tranilast) 284-286. As shown, TRPV2 blocking abrogated DOX uptake that has been increased by CBD treatment (Fig.33 A-B).

On the molecular level, we found that TRPV2 blocker has decreased cleaved

PARP and cleaved caspase-3 to its basal levels (Fig.33 C).

Fig.33 TRPV2 pore blocking suppresses CBD and DOX-mediated cytotoxic effects. (A) SUM159 cells were treated with Vehicle or CBD or Tranilast (TR) or CBD +Tranilast for 2h followed by DOX for 30 min then subjected to FACS analysis of DOX uptake. (B) Quantitation of the % of DOX-positive cells of the uptake experiments of the four groups. (C) SUM159 cells were treated with the indicated drugs and the lysates were collected and immunoblotted against the indicated proteins.

94 We also used a dominant negative form of TRPV2 to check whether it could inhibit intrinsic ability of TRPV2 to induce chemotherapeutic uptake. As shown, overexpression of S-TRPV2, dominant negative form of TRPV2 (DN-

TRPV2), inhibited DOX uptake in basal and CBD-mediated levels (Fig.34 A-B).

Fig.34 Dominant negative form of TRPV2 suppresses CBD and DOX-mediated cytotoxic effects. (A) SUM-Vec or SUM-DNTRPV2 transfected cells were treated 0.5μM DOX for 30 min with or without CBD for 2 hours. Cells were collected for FACS analysis. (B) Quantification of FACS data has been presented.

We have also downregulated TRPV2 by SiRNA transfection and as expected, TRPV2 downregulation inhibited DOX uptake and it inhibited the effect of DOX and CBD on the reduction of colony forming SUM159 cells (Fig.35 A-D).

95

Fig.35 TRPV2 downregulation suppresses CBD and DOX-mediated cytotoxic effects. (A) SUM-NT SiRNA or SUM-TRPV2-SiRNA transfected cells were treated 0.5μM DOX for 30 min with or without CBD for 2 hours. Cells were collected for FACS analysis. (B) Quantification of FACS data has been presented (lower bar graph). (C) Western blot image showing TRPV2 expression in SUM-NT SiRNA and SUM-TRPV2-SiRNA transiently transfected cells. (D) SUM-NT SiRNA or SUM-TRPV2-SiRNA transfected cells have been used for colony formation assay in the presence or absence of DOX and/or CBD and the number of colonies have been counted.

These results suggest that TRPV2 activation is responsible of CBD- mediated effects on chemotherapeutic uptake and subsequent activation of

PARP and caspase-3. These data also suggest that TRPV2 downregulation, blocking or presence of dominant negative form of TRPV2 might inhibit chemotherapeutic drug uptake ability and subsequent cytotoxic effects on TNBC cells.

4.3.6 CBD improved the anti-tumor chemotherapeutic efficacy in vivo.

To examine the ability of CBD, as a TRPV2 agonist, to increase the chemotherapeutic drug efficacy in vivo, we used Nude mice and injected them

96 with SUM159 cells orthotopically in 4th mammary gland. The mice were treated with either vehicle or CBD or DOX or CBD in combination with DOX. As shown,

CBD+DOX group showed significantly lower tumor volume and weight compared to single treatments of CBD or DOX alone (Fig.36 A-C). In order to analyze the molecular level, we used tumor lysates of these tumors for analysis of apoptotic markers. As shown, (CBD+DOX) treated tumors showed higher level of cleaved caspase-3 and cleaved PARP compared to single treatment groups (Fig.36 D).

These results suggest that CBD could enhance the chemotherapeutic efficacy in vivo and inhibit the tumor growth of breast cancer cells.

97

Fig.36 CBD improved the anti-tumor chemotherapeutic efficacy in vivo. (A) Nude mice were orthotopically injected with SUM159 cells and subjected to the indicated treatments for 4 weeks and the tumor volume (A) has been measured every week and the tumor weight (B) of the dissected tumors has been determined for each group. (C) Representative images of dissected tumors from the indicated experimental groups. (D) Tumor lysates from the experimental groups were used for western blot analysis and immunoblotted against the indicated proteins.

4.4 Discussion

TNBC is a very aggressive subtype of breast cancer due to its lack of the hormonal receptor and thus no targeted therapy is available. Till now, chemotherapy is the first choice to treat TNBC, however, resistance, relapse, poor response rate and massive multiple organ toxicity are common problems associated with chemotherapeutic drugs 220. Thus, there is an urgent need for new strategies to increase the efficacy and reduces the toxicity of the chemotherapeutic drugs. One of these strategies is to enhance the sensitivity of

TNBC cells towards the chemotherapeutic drugs. In this study, we analyzed the

98 possibility of exploitation of TRPV2 in enhancing the uptake of chemotherapeutic drugs into TNBC. In the present study, we showed, for the first time, that TRPV2 is highly expressed in malignant and metastatic breast cancer tissues compared to normal breast tissues. We also discovered that higher TRPV2 expression is associated with higher RFS and OS in TNBC patients. We also showed – for the first time- that TRPV2 overexpression or activation enhances the uptake of DOX in TNBC cells and activates several apoptotic pathways. We presented here that

TRPV2 activation could enhance the chemotherapeutic activity in vivo against

TNBC cells. Finally, we showed that the level of chemotherapeutic drug uptake depends on TRPV2 expression level and its splice variant that lack the pore domain region.

The activation of TRPV channels by specific agonists allows large cations to flux 287. The diameter of the pore in TRPV family could vary from 5.4 Å to >6.8

Å in different types of TRPV channels 287, 288. Previous studies showed that neuroblastoma cells that were transfected with TRPV1 and exposed to specific agonist in combination with DOX, have shown higher accumulation of DOX in activated TRPV1-transfected cells 289. Additionally, TRPv4 channel has been reported to increase cellular antibiotic drug uptake 278. Therefore, we hypothesized that TRPV2 as a member of TRP family that shares many features to each other, when activated, a pore conformational change could happen to favor higher uptake of chemotherapeutic drugs into TNBC cells.

99 There is little or no knowledge about TRPV2 role in TNBC. In this study, we observed a dramatic increase of TRPV2 expression in malignant as well as metastatic breast cancer tissues compared to normal breast tissues. Importantly, we found that high TRPV2 expression level is a good prognostic factor for ERα- and TNBC patients who administrate chemotherapeutic drugs and these patients have better RFS and OS compared to those who have lower TRPV2 expression.

These phenomena led us to ask whether or not TRPV2 has a role in improving the efficacy of chemotherapeutic drugs. To address this question, we overexpressed TRPV2 in TNBC. As expected, TRPV2 overexpression enhances the ability of TNBC cells to uptake DOX and this significantly enhances DOX anti- tumorigenic efficacy. Furthermore, we found that CBD, which is a well-known

TRPV2 agonist, has the ability to increase the drug uptake in TNBC cells. We also found that TRPV2 downregulation, blocking or TRPV2 functional interference with its dominant negative form, have significantly inhibited TNBC ability to uptake DOX.

Since DOX induces its anti-cancer effects through activating the apoptotic signaling in cancer cells 290, we analyzed whether TRPV2 activation has a role in enhancing DOX-mediated apoptosis. Expectedly, we found that some apoptotic signals, such as cleaved PARP and cleaved caspase-3, have been significantly up-regulated in cells that are treated with TRPV2 agonist (CBD) in combination with DOX. These effects of CBD have been suppressed when we downregulated

100 TRPV2 and when we used a selective TRPV2 blocker, which confirms that these effects are TRPV2-mediated.

In this regard, clinician can get benefit from the presence of high TRPV2 levels at the tumor sites, therefore TRPV2 activation can be used as a tool to enhance the chemotherapeutic uptake and efficacy specifically at the malignant sites. This observation has been supported by the in vivo data, which showed that the combination therapy (CBD+DOX) had significantly less tumor volume, weight, and higher levels of activated PARP and caspase-3, compared to single treatments.

A short-form TRPV2 (S-TRPV2), which lacks the putative pore-forming region, by means of alternative splicing is produced naturally in human leukemia cells 291. The finding that S-TRPV2 transfection inhibits the translocation of full length of TRPV2 to the plasma membrane has supported strongly suggests S-

TRPV2 as a natural dominant negative form of TRPV2 that can be used as a strong tool to block TRPV2 function 280. We used this tool to analyze whether the presence of S-TRPV2 could have an effect on cancer cell ability to uptake the chemotherapeutic drugs. We found that overexpression of S-TRPV2 has significantly inhibits the uptake ability of TNBC cells. This shows that the uptake ability of cancer cells, regarding TRPV2 channel, depends not only on the expression level of the full length TRPV2 but also depends on the presence of the splice variant which lacks the pore domain. Apparently, both forms have two opposite roles regarding chemotherapeutic drug uptake in TNBC.

101 4.5 Conclusion

This study showed - for the first time - that TRPV2 up-regulation and activation enhances the sensitivity of TNBC cells towards chemotherapeutic drugs in vitro and in vivo. We showed also - for the first time - that TRPV2 might be a good prognostic marker for TNBC patients who receive chemotherapy in their treatment plan. Furthermore, this study revealed that TRPV2 is highly expressed in malignant tissues compared to normal breast tissues; and the level of chemotherapeutic uptake depends, in part, on expression level of TRPV2 as well as its splicing variant S-TRPV2. This study supports the use of TRPV2 agonists as an adjuvant therapy to enhance the efficacy of chemotherapeutic drugs in TNBC.

102 Chapter 5: Novel role of CB2R in tuning breast tumor microenvironment,

EGF/EGFR and IGF-I/IGF-IR pathways.

5.1 Introduction

Cannabinoids, cannabinoid receptors and the enzymes that metabolize the endogenous cannabinoids are the main components of endocannabinoid system. Cannabinoids act primarily on G-protein coupled receptors (GPCRs): cannabinoid receptors 1 and 2 (CB1R and CB2R). Cannabinoids have shown anti-proliferative and anti-invasive potentials in vitro. They have also shown anti- tumor growth and anti-metastatic properties in vivo in different cancer types 182,

199, 208, 237. Previous studies showed that cannabinoids inhibit various growth factor receptors as well as chemokine receptors’ signaling 199, 237. CB2R has previously shown high expression in breast cancer tissues as well as different breast cancer cell lines 208. Synthetic CB2R specific agonists such as JWH-015 and JWH-133 have shown strong anti-tumorigenic properties through different mechanism including inhibition of CXCL12/CXCR4 axis, cyclo-oxygenase-2

(COX-2) expression and induction of apoptosis in different cancer types 195, 208,

237. Although there is a strong evidence of the anti-tumor properties of CB2R

103 agonists, there is not much known about the role of CB2R activation on different growth factor receptor, such as EGFR and insulin-like growth factor 1 receptor

(IGF-IR), mediated tumorigenic events in breast cancer.

Overexpression of EGFR and IGF-IR, lack of hormonal targeted cancer therapy, low survival rate and poor patient prognosis are hallmark features of the -negative (ERα-) breast cancer subtype 224, 225, 251, 265, 292.

The standard therapy for ERα- breast cancer using anthracyclins, taxanes and platinum compounds has shown unresponsiveness and rapid development of resistance 266-270. IGF-IR is predominantly activated in ERα+ as well as ERα- breast cancer subtypes 293, 294. IGF-I has shown a great importance in breast cancer progression through its anti-apoptotic, mitogenic, and invasive potential in both ERα+ and ERα- breast cancer cells 295-297.

Recently, tumor microenvironment has caught the attention of scientists owing to its tumor progression and metastasis modulation potentials 248. The tumor microenvironment consists of tumor cells, endothelial cells, ECM, fibroblasts, blood vessels, lymphatic vessels and different types of immune cells.

Immune cells within the tumor microenvironment include macrophages, T-cells,

B-cells, myeloid derived suppressor cells (MDSCs) and neutrophils. All of these immune cells’ populations have significant roles in either augmenting or suppressing the aggressiveness of tumor cells. CB2R has shown high expression in different immune cell types including macrophages, dendritic cells,

B-cells, T-cells, NK and NK-T cells 111, 298, 299. Interestingly, it has been shown

104 that the formation of B-cells and different T-cell subsets, including CD4+ and

CD8+ T-cells, requires CB2R activation. In addition, CB2R deficient mice showed a lack of these types of cells 298. Although it seems that CB2R might play important roles in immune cells, which eventually affects the tumor microenvironment composition, and in turn the tumor growth and metastasis, the role of CB2R on the immune cells within the breast tumor microenvironment has not been studied yet.

In this study, we analyzed the effect of the CB2R activation on different tumorigenic related events. First, we analyzed the effect of CB2R specific agonist on EGF-induced tumorigenic events in ERα- breast cancer cells. Second we analyzed the effect of CB2R activation on IGF-I induced tumorgenesis in ERα+ and ERα- breast cancer cells. We also studied the effect of CB2R activation on breast cancer growth and metastasis using different mouse model systems.

Finally, by using CB2R KO mice as well as CB2R specific agonist, we analyzed the role of CB2R in immune cells within the breast tumor microenvironment and how it can affect the tumorigenic process.

5.2 Materials and methods

5.2.1 Reagents and antibodies

Cell culture reagents were obtained from Gibco Laboratories (Grand Island, NY).

The following reagents and antibodies used in this study were purchased from different sources: JWH-015 (Tocris Bioscience), human EGF and IGF-I proteins

(Peprotech). Antibodies: CB2R (Abcam), p-AKT, p-STAT3 and p-IGF-IR (Cell

105 Signaling); p-ERK/ERK, p-EGFR, GAPDH, STAT3 and AKT (Santa Cruz). All antibodies for flowcytometric studies have been purchased from (Biolegend).

5.2.2 Cell culture

SUM159 cells were obtained from Dr. Sarmila Majumder (The Ohio State

University). MDA-MB 231, T47D and MCF7 cells were purchased from ATCC.

SCP2 cells were kindly obtained from Dr. Joan Massagué 233. Cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 5-units/ml penicillin, and 5-mg/ml streptomycin.

5.2.3 Western blot analysis

Cells were plated in 100 cm2 dishes and lysed in lysis buffer (RIPA). Tumor samples were homogenized and lysed in RIPA buffer for further analysis. 50 mg of proteins were loaded on 4–12% SDS–polyacrylamide gels (Invitrogen) then transferred to nitrocellulose membranes (BioRad) and blocked with 5% milk.

Membranes were incubated overnight with primary antibody, washed, and incubated for 1h at RT with horseradish peroxidase conjugated secondary antibody. The membranes were then washed and stained using a chemiluminescence system (ECL Amersham Biosciences) and exposed to X-ray film (Kodak).

5.2.4 Chemotactic Assays

Chemotactic assays were performed using transwell chambers (Costar 8.0 mm pore size) as described previously 190, 208, 237. Briefly, serum starved cells were pretreated with JWH-015 or vehicle for 24 h. Top chambers were loaded with

106 cells in serum free medium (SFM) and bottom chambers contained SFM in the presence or absence of chemo-attractants. Cells that migrated or invaded across the membrane were counted by fixing in 37% formaldehyde and 25% glutaraldehyde in PBS and stained with 0.1% crystal violet in PBS for 30 minutes.

Migrated/invaded cells per membrane were detected by light microscopy, counted in 5 fields and the percentage of migration determined.

5.2.5 Gelatin zymography

Gelatin zymography for collected conditioned media was performed as described earlier 109 .

5.2.6 Luciferase reporter assay

We used NF-kB luciferase reporter assay (Promega) to determine NF-kB activity as described previously 109, 190.

5.2.7 Mouse models

Female FVB, C57BL/6 and nude (NU/NU) mice were purchased from (Charles

River Laboratories Inc.). CB2R knock out (KO) mice with C57BL/6 background were purchased from (Jackson lab.). Tumors were formed by orthotopically injecting tumor cells in the 4th mammary glands as following: MVT-1 (1x105) cells in FVB mice, SUM159 (5 x 106) cells in nude (NU/NU) and EO771 (1x105) cells in

WT or CB2R KO C57BL/6 mice. For CB2R agonist experiments, mice were randomized when tumors became palpable, and then injected peri-tumorally with

107 JWH-015 (10 mg/kg) or vehicle on alternate days. Tumors were measured every week with external calipers and tumor volume was calculated according to the formula tumor volume = (length x width2 x 0.52) 195, 236. For WT/CB2R KO experiment, tumors were measured every week for 4 weeks with external calipers and tumor volume was calculated as mentioned previously.

5.2.8 Flow-cytometry

Single cell suspensions were prepared from tumors as described 236. For myeloid cells staining, cells were incubated with anti-CD11b APC, anti-Gr-1 (Alexa Flour-

488) and anti-F4/80 PE. For lymphoid cells staining, cells were incubated with anti-CD3 APC, anti-CD8 PE, and anti-CD4 (Alexa Flour-488). Anti-CD49b (Alexa

Flour-488) has been used for detection of natural killer (NK) cells. After incubation for 1 hour, cells were fixed with 2% paraformaldehyde and acquired on a BD FACS caliber, then plotted for analysis

5.2.9 Statistical analysis

Results were represented as mean ± SD. Student’s t test was used to compare vehicle and treated groups. P<0.05 was considered to be statistically significant.

For all graphs, * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001.

108 5.3 Results

5.3.1 CB2R activation inhibits EGF-induced tumorigenic events in ERα- breast cancer cells.

In this study, we analyzed CB2R and EGFR expression in ERα- and ERα+ breast cancer cells. As shown, EGFR is highly expressed in different ERα- cell lines compared to ERα+ breast cancer cell lines however; CB2R is highly expressed in both breast cancer subtypes (Fig.37 A). Thereafter, we used

SUM159 cells as a representative of ERα- breast cancer cells 229 and MCF-7 as a representative of ERα+ breast cancer cells 300.

To analyze the possible role of CB2R activation on EGF/EGFR pathway, we studied the effect CB2R specific agonist (JWH-015) 195 on EGF-induced tumorigenic events in ERα- cell line (SUM159). We found that JWH-015 inhibits

EGF-induced migration and invasion of SUM159 cells (Fig.37 B-C). NF-kB activation is crucial for cancer cells to promote their migration and invasive potential especially for ERα- cells 241, 256. Moreover, EGFR overexpression strongly correlates with NF-kB activation in breast cancer 242. Therefore, we analyzed the effect of CB2R agonist on NF-kB activation by using luciferase reporter assay. As shown, JWH-015 inhibits translocation of NF-kB active units into the nucleus (Fig.37 D). To test the specificity of JWH-015 towards CB2R, we used CB2R-specific antagonist (SR144528), and we found that CB2R blocking is significantly abrogated JWH-mediated inhibition of migration and invasion of

SUM159 cells after EGF stimulation (Fig.38 A-B).

109 To further analyze the molecular mechanism, we investigated CB2R agonist’s effect on EGF/EGFR axis. As shown, JWH-015 inhibits EGFR activation and its downstream targets STAT3, AKT and ERK (Fig.37 E).

Fig.37 CB2R activation inhibits EGF/EGFR signaling in ERα- breast cancer cells. (A) Western blot image shows expression of EGFR and CB2R proteins in different ERα-and ER+α breast cancer cell lines. GAPDH was used as a loading control. (B) SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell migration assay. (C) SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell invasion assay. Number of migrated or invaded cells were counted and plotted as % of control. (D) SUM159 cells were treated with vehicle or JWH 5µM in presence or absence of EGF and subjected to NF-kB luciferase reporter assay. (E) SUM159 cells were treated with JWH-015, stimulated with EGF for 15 or 30 minutes then cell lysates were used for western blot analysis for the indicated proteins.

110 Fig.38 JWH-015 inhibits EGF-induced migration and invasion in CB2R dependent manner. SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell migration (A) or invasion assay (B) in presence or absence of EGF (100 ng/ml) and specific CB2R blocker SR-144528.

These results indicate that CB2R triggering has the ability to inhibit EGFR activation and its downstream targets and therefore suppresses EGF-induced tumorigenic events in ERα- breast cancer cells.

5.3.2 CB2R activation inhibits IGF-I-induced tumorigenic events in ERα+ and ERα- breast cancer cells.

ERα+ breast cancer cells are associated with hyperactivation of IGF-IR

301. IGF-IR activation is, in turn, associated with increased invasion, secretion of metalloproteinases (MMPs) and activation of EMT process in ERα+ cells 296.

ERα- breast cancer cells are also associated with hyperactivation of IGF-IR 294.

IGF-IR activation and phosphorylation is associated with poor prognosis in many breast cancer subtypes including ERα+ and ERα- subtypes 302. Therefore, we analyzed IGF-IR expression in ERα+ and ERα- breast cancer cells. As shown,

111 IGF-IR is highly expressed in different ERα+ cell lines especially MCF-7 cells; it is also expressed in ERα- breast cancer cells but it might be at lower levels

(Fig.39 A). Therefore, in this study, we analyzed the role of CB2R activation on

IGF-I-induced tumorigenic events in ERα+ as well as ERα- breast cancer cells. In

MCF-7 cells (ERα+), we found that JWH-015 inhibits IGF-I-induced migration and invasion (Fig.39 B-C). We subsequently analyzed the molecular mechanism through investigation CB2R agonist’s effect on IGF-I/IGF-IR pathway. As shown,

JWH-015 inhibits the activation of IGF-IR and its downstream target AKT (Fig.39

D).

112

Fig.39 JWH-015 inhibits IGF-I/IGF-IR signaling in ERα+ and ERα- breast cancer cells. (A) Western blot image shows expression of IGF-IR in different ERα- and ERα+ breast cancer cell lines. GAPDH was used as a loading control. (B) MCF-7 cells were treated with JWH-015 for 24 hours and subjected to transwell migration assay. (C) MCF-7 cells were treated with JWH-015 for 24 hours and subjected to transwell invasion assay. Number of migrated or invaded cells were counted and plotted as % of control. (D) MCF-7 cells were treated with JWH-015, stimulated with IGF-I for 15 or 30 minutes then cell lysates were used for western blot analysis for the indicated proteins. (E) SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell migration assay. (F) SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell invasion assay. Number of migrated or invaded cells were counted and plotted as % of control. (G) SUM159 cells were treated with JWH-015, stimulated with IGF-I for 15 or 30 minutes then cell lysates were used for western blot analysis for the indicated proteins

113

Furthermore, by using zymogram, we found that JWH-015 inhibits IGF-I- induced secretion of metalloproteinase-9 (MMP-9) in MCF-7 cells (Fig.40 A).

Fig.40 JWH-015 inhibits MMPs’ secretion in MCF-7 and SUM159 cells. (A) MCF-7 cells were treated with vehicle or JWH 5 or 7.5 µM in presence or absence of IGF-I and the collected conditioned media was subjected to gelatin zymography. (B) SUM159 cells were treated with vehicle or JWH 5 or 7.5 µM in presence or absence of IGF-I and the collected conditioned media was subjected to gelatin zymography.

We also investigated the role of JWH-015 on IGF-I induced tumorigenic events in SUM159 cells (ERα-). Similarly, we found that JWH-015 inhibits IGF-I- induced migration and invasion (Fig.39 E-F). JWH-015 also inhibits the activation of IGF-IR and its downstream target AKT in SUM159 cells (Fig.39 G).

Furthermore, JWH-015 inhibits IGF-I-induced secretion of metalloproteinase-2

(MMP-2) (Fig.40 B). To confirm the specificity of JWH-015 towards CB2R in these studies, we used a CB2R-specific antagonist (SR144528), and we found that CB2R blocking has significantly abrogated JWH-potential in inhibition of migration and invasion induced by IGF-I in MCF-7 and SUM159 cells (Fig.41 A-

D). These results indicate that CB2R activation has the ability to inhibit IGF-IR activation and IGF-I-induced secretion of MMP-9/MMP-2, which might explain the inhibitory effect of JWH-015 on IGF-I-induced migration and invasion in both

ERα+ and ERα- cells.

114 Fig.41 JWH-015 inhibits IGF-I-induced migration and invasion in CB2R dependent manner. SUM159 cells were treated with JWH-015 for 24 hours and subjected to transwell migration (A) or invasion (B) assays in presence or absence of IGF-I (100 ng/ml) and SR-144528. MCF-7 cells were treated with JWH- 015 for 24 hours and subjected to transwell migration (C) or invasion (D) assays in presence or absence of IGF-I (100 ng/ml) and SR-144528.

5.3.3 JWH-015 inhibits EGF/EGFR and IGF-I/IGF-IR axes in orthotopic immunodeficient mouse model.

In order to analyze the anti-tumorigenic potential of CB2R specific agonist in vivo, we injected immune-deficient (nude mice) orthotopically with SUM159 cells. After the tumor became palpable, the mice were treated, through peri-

115 tumoral injection, with either vehicle or JWH-015 (10 mg/Kg) for 3 weeks. The tumors were then harvested for analysis. As shown, JWH-treated group has less tumor volume and tumor weight compared to vehicle-treated group (Fig.42 A-B).

We analyzed tumor lysates to find out the ability of CB2R activation to inhibit

EGFR and IGF-IR activation in vivo and found that the JWH-treated group has less activation of EGFR, IGF-IR, STAT3, AKT and ERK proteins compared to control group (Fig.42 D).

116

Fig.42 JWH-015 suppresses breast cancer growth in vivo by inhibiting EGF/EGFR and IGF-I/IGF-IR signaling pathways. (A) Tumor volume measurements of orthotopically injected nude mice were assessed every week for control and treated groups. (B) Tumor weight of vehicle-treated or JWH-treated nude mice was determined at the euthanasia day. (C) Representative photographs showing tumors dissected from control and treated groups. (D) Western blot images of the tumors of the control and treated groups showing the expression of phospho-EGFR, phospho-STAT3 phospho-ERK and AKT (p-EGFR, p-IGF-IR, p-STAT3, p-ERK and p-AKT) and total EGFR, IGF-IR, STAT3, ERK and AKT (EGFR, IGF-IR, STAT3, ERK and AKT).

These results suggest that CB2R activation has the ability to inhibit breast cancer growth through inhibition of EGF/EGFR as well as IGF-I/IGF-IR pathways and their downstream targets.

117 5.3.4 CB2R knock out mice has higher tumorigenic potential due to

change in tumor microenvironment immune cells’ composition.

Since CB2R is highly expressed in immune cells, we want to analyze whether CB2R has a significant role on the immune cells within the breast tumor microenvironment 303. Therefore, we used CB2R knock out (KO) mice to study the role of endogenous cannabinoid on CB2R that is present on host immune cells and to analyze how this might affect the tumorigenic process (Fig.43 A). We injected wild type (WT) and CB2R KO mice orthotopically with murine breast cancer cell line (E0771) in the mammary fat pad and we observed the tumor growth in a 4-week period. Interestingly, CB2R KO group had higher tumor volume (Fig.43 B), and tumor weight (Fig.43 C) compared to WT mice.

Subsequently, we analyzed different immune cells’ populations within

CB2R KO and WT mice. CB2R KO tumors showed significantly lower percentage of CD3+/CD8+ cells and significantly higher percentage of CD11b+/Gr-1+ cells

(Fig.43 E-F).

118

Fig.43 CB2R KO mice have higher tumor promoting potential than wild type mice. (A) Genotyping image showing PCR DNA bands of WT, heterozygous and CB2R KO mice (B) Tumor volume measurements of orthotopically injected WT or CB2R KO C57BL/6 mice with EO771 breast cancer cells, were assessed every week. (C) Tumor weight of WT or CB2R mice was determined at the euthanasia day. (D) Representative photographs showing tumors dissected from control and treated groups. Percentages of CD3+/CD8+ (E) and CD11b+/Gr-1+ (F) cells within the tumors of WT and CB2R KO tumors were quantified by flow-cytometry. (G) Western blot image shows the expression of PDL-1 protein in the tumors of WT and CB2R KO mice.

CD3+/CD4+ population is also higher in WT mice, however we have not found any significant difference, between WT and CB2R KO mice, in the percentage of other immune cells (Fig.44 A-E).

119

Fig.44 CB2R KO and WT mice analysis of different immune populations in tumors. Percentages of CD3+/CD4+ (A), CD49 b+ (B), CD3+/CD49 b+ (C), F4/80/CD11b+ (D) and F4/80/CD206+ (E) cells within the tumors of WT and CB2R KO tumors were quantified by flow-cytometry.

CB2R KO mice also showed lower percentage of CD4+ and CD8+ cells within the spleenocyte population compared to WT group (Fig.45 A-D).

120

Fig.45 CB2R KO mice show less percentages of CD8+ and CD4+ cells in spleen. Spleenocytes, of WT and CB2R KO mice, which have been injected orthotopically with EO771 cells, were made into singe cell suspensions and analyzed by FACS analysis for CD4+ (A), CD8+ (B), CD49B+ (C) and F4/80/CD11b+ (D) cells then plotted as a percentage of total leukocytes.

To check the possible role of CB2R on immune check points, we analyzed

PDL-1 expression, and we found that CB2R KO tumors showed higher expression of PDL-1 compared to WT tumors (Fig.43 G). These results suggest that CB2R plays an important in tumor microenvironment modulation through its effect, mainly, on myeloid derived suppressor cells (MDSC) and cytotoxic T-cells.

5.3.5 CB2R activation inhibits tumor growth and metastasis through

modulation of the tumor microenvironment.

To confirm the role of CB2R in immune cells within the tumor microenvironment and its effect on breast cancer growth and metastasis, we used CB2R specific agonist strategy on in vivo immune-intact mice study. We injected FVB mice orthotopically with highly metastatic mouse breast cancer cell line (MVT-1). After the tumor become palpable, the mice were treated, by intra-

121 peritoneal injection, with either vehicle or JWH-015 (10 mg/Kg) for 4 weeks. As shown, the JWH-treated group has less tumor volume and tumor weight compared to the control group (Fig.46 A-B). We also analyzed if CB2R activation has the ability to reduce the metastatic potential of the highly aggressive MVT-1 cells. Interestingly, JWH-015 treatment has significantly reduced the number of lung metastatic nodules compared to vehicle-treated mice (Fig.46 D-E).

To analyze the effect of CB2R activation on immune cell populations within the tumor microenvironment, we performed flow-cytometric analysis of the tumors from control and treated groups. We found that JWH-treated tumors showed a significantly higher percentage of CD3+/CD8+ cells and a significantly lower percentage of CD11b+/Gr-1+ cells (Fig.46 F-G).

122

Fig.46 JWH-015 inhibits breast cancer growth and metastasis in immune-intact mice and modulates the tumor microenvironment (A) Tumor volume measurements of orthotopically injected FVB mice with MVT-1 breast cancer cells, were assessed every week for control and treated groups. (B) Tumor weight of vehicle-treated or JWH-treated FVB mice was determined at the euthanasia day. (C) Representative photographs showing tumors dissected from control and treated groups. (D) Representative photographs showing the lung metastatic nodules of the control and JWH-treated mice. (E) Total number of lung metastatic nodules of the control and JWH-treated mice. Percentages of CD3+/CD8+ (F) and CD11b+/Gr-1+ (G) cells within the tumors of control and JWH-treated tumors were quantified by flow-cytometry in MVT-1 mouse model.

These results confirm the potential role of CB2R activation in the inhibition of tumor growth and metastasis through modulation of immune cell populations within the breast tumor microenvironment.

5.4 Discussion

CB2R is highly expressed in different types of cancers, including breast cancer, in different immune cell types 111, 208, 298, 299. Although CB2R activation

123 has shown significant anti-tumorigenic properties, its functional role in regulating breast cancer is not well understood. In the present study, we elucidated the role of CB2R in breast cancer growth and metastasis. Following CB2R activation, we observed inhibition of breast cancer growth, suppression of EGF/EGFR as well as IGF-I/IGF-IR signaling pathways in vitro and in vivo. Further we found that

CB2R activation significantly inhibited breast cancer growth and metastasis through modulating breast immune cells’ compositions within the breast tumor microenvironment, suggesting that CB2R activation enhances the immune response against the tumor cells.

CB2R is overexpressed in both ERα- and ERα+ breast cancer cells. In addition, EGF/EGFR axis is up-regulated in ERα- breast cancer and plays crucial roles in improving cancer cell proliferation, metastasis, survival as well as tumor angiogenesis 254, 304. IGF-I/IGFR axis is also hyper-activated in ERα+ as well as

ERα- breast cancer subtypes and associated with poor patient prognosis295-297.

Therefore, EGFR and IGF-IR inhibitors have been developed as therapeutic options; however, these inhibitors usually gained rapid resistance development and eventually failed 305, 306 226. It is crucial then to identify other small molecules that can achieve the interruption of these axes by different, possibly indirect, ways. In our present study, we observed that CB2R activation by CB2R specific agonist inhibits EGF/EGFR and IGF-I/IGFR signaling transduction pathways in

ERα- and ERα- breast cancer cells in vitro and in vivo. These pathways involve key molecules such as STAT3, ERK, AKT and NF-kB, which play major roles in

124 cancer cell survival, proliferation, migration and invasion. We also showed that

JWH-015 inhibited IGF-I-induced secretion of MMP-9 and MMP-2, which play significant roles in the invasion of breast cancer cells. These studies are in accordance with previous studies, which showed that CB2R agonists inhibit the tumor growth and metastasis through apoptosis induction and inhibition of EGFR activation in non-small cell lung cancer (NSCLC) 199, 237.

Since endocannabinoids activate CB2R, which is highly expressed in immune cells, the role of CB2R activation, by endocannabinoids, in the tumor stromal immune cells was studied. For this purpose, we used CB2R KO mice as well as CB2R specific agonist strategies. Notably, we found that CB2R KO mice have significantly higher tumor volume and weight compared to WT mice after orthotopic injection with breast cancer cells (E0771). Interestingly, the tumors of

CB2R KO mice showed less cytotoxic T-cells and more MDSCs. Cytotoxic T- cells and MDSCs are important players within the tumor microenvironment.

Cytotoxic T-cells are involved in killing of cancer cells through recognition of specific antigenic peptides as well as MHC-I proteins on the surface of the tumor cells 307-309. MDSCs participate in cancer progression through inhibition of innate and adaptive immunities. High levels of MDSCs are associated with a highly metastatic form of breast cancer 310, 311. These studies suggest that CB2R activation may inhibit breast cancer growth and metastasis by enhancing the immune cell response against the tumor cells. These results are in accordance with previous studies which showed that CB2R is crucial for development of T-

125 cells and the CB2R KO mice have deficiency in development of T-cell subsets.

This study has been confirmed through CB2R specific agonist’s studies in intact immune system mice. We observed that CB2R specific agonist significantly inhibited tumor growth as well as metastasis to the lungs. CB2R agonist-treated tumors showed also higher percentage of cytotoxic T-cells and lower percentage of MDSCs within the tumor stroma, which is consistent with CB2R KO findings.

Endocannabinoids acts on both CB1R and CB2R. Therefore, in order to study the role endocannabinoids on only CB2R that is expressed mainly on immune cells, we use CB2R KO mice as well as CB2R specific agonist strategies. We checked whether CB2R has a significant role in immune cells’ within the breast tumor microenvironment. Notably, we found that CB2R KO mice have significantly higher tumor volume and weight compared to WT mice after orthotopic injection with breast cancer cells (E0771). Interestingly, the tumors of

CB2R KO mice showed less cytotoxic T-cells and more MDSCs. These results are in accordance with previous studies which showed that CB2R is crucial for development of T-cells and the CB2R KO mice have deficiency in development these cell subsets. This indicates that CB2R activation of the host immune cells, possibly through the endogenous cannabinoids inside the animal body, can participate in tumor regression through increasing of cytotoxic T-cells and decreasing MDSC total populations or through affecting their chemotaxis. This explains the lower tumorigenic potential of tumor cells in CB2R KO environment.

This study has been confirmed through CB2R specific agonist’s studies in intact

126 immune system mice (FVB mice). CB2R specific agonist significantly inhibited tumor growth as well as metastasis to the lungs. CB2R agonist-treated tumors showed also higher percentage of cytotoxic T-cells and lower percentage of

MDSCs within the tumor stroma, which is consistent with CB2R KO findings.

Previous studies showed that Δ9-tetrahydrocannabinol (THC) has increased

MDSC population in mice after single dose mice I.P injection312. This might be explained because THC is not only specific to CB2R; it acts on CB1R as well.

Another explanation is that decrease in MDSC has been observed in peripheral sites such as peritoneal cavity not on tumor sites. Furthermore, THC has been administrated as a single dose only. Longer administration of THC over 4 weeks period might show different effect. Recently there is a growing attention towards programmed cell death-1/programmed cell death Ligand-1 (PD-1/PDL-1) immune-inhibitory axis. This axis plays a crucial role in the escape of tumor cells from immune surveillance 313, 314. The interaction between PD-1/PD-L1 has been reported to inhibit T-lymphocyte survival, proliferation and its cytotoxic action and cytokine release ability. This interaction also induces the apoptosis of T-cells and differentiation of CD4+ T-cells into regulatory T-cells and promotes tumor cells’ resistance to cytotoxic T-lymphocyte (CTL) attack 315-319. Interestingly, CB2R KO tumors showed higher expression of PDL-1 compared to WT tumors. This could further explain the higher tumorigenic potential of CB2R KO mice.

127 5.5 Conclusion

CB2R activation inhibits tumor growth whereas CB2R deletion enhances tumor growth in vivo. In addition, CB2R activation inhibits EGFR/IGFR signaling pathways in breast cancer cells. CB2R activation also modulates breast tumor microenvironment by decreasing MDSC population and increasing cytotoxic T- cell population in the tumor stroma (Fig.47 A-B). Overall, CB2R is an important therapeutic target in ERα- and ERα+ breast cancer subtypes and its activation could lead to inhibition of EGFR/IGF-IR signaling pathways and enhanced immune response against breast tumor cells.

128

Fig.47 Schematic representation of the anti-tumor role of CB2R activation in breast cancer. (A) Schematic representation of the direct anti-tumor role of CB2R activation showing the possible cross-talk between CB2R and EGFR and IGF-IR. (B) Schematic representation of the indirect anti-tumor role of CB2R activation on cancer cells showing the effect of CB2R receptor activation on different immune cell populations within the breast tumor microenvironment.

129 Chapter 6: Concluding remarks and future directions

Our data reveal that CBD and the synthetic cannabinoid JWH-015 have anti-cancerous activities in breast cancer. They exert in vitro and in vivo anti- proliferative and anti-metastatic effects through inhibition of EGF/EGFR, IGF-

I/IGF-IR signaling pathways and through activation of TRPV2 channel. JWH-015 exerts its action through activating CB2R. On cancer cells, this leads to inhibition of EGF/EGFR and IGF-I/IGF-IR signaling pathways which makes the cancer cells less able to proliferate and metastasize in vitro and in vivo. In case of immune cells, JWH-015 increases the population of cytotoxic T-cells and decreases MDSCs population. This eventually leads to enhanced anti-tumor microenvironment against breast cancer cells. CBD also inhibits EGF/EGFR signaling pathway, which makes the cancer cells less able to proliferate and metastasize in vitro and in vivo. CBD inhibits the secretion of key cytokines from the breast cancer cells which important for monocyte chemotaxis to the tumor sites. This results in less TAMs in the tumor mass and eventually more enhanced anti-tumor microenvironment against breast cancer cells. CBD also activates

TRPV2, which increases the uptake ability of cancer cells to the

130 chemotherapeutic drugs. This results eventually to less breast cancer growth if

CBD co-administrated with the cancer chemotherapy (Fig. 48-49).

Fig.48 Anti-tumor role of JWH-015 and CBD in breast cancer cells. Schematic diagram shows the activation of CB2R by JWH-015, which in turn leads to less activation of EGFR and IGF-IR and their mediated tumorigenic events. The diagram also shows inhibition of EGFR activation by CBD and how CBD activates chemotherapeutic drug uptake through activating TRPV2 channel on breast cancer cell.

131

Fig.49 The role of JWH-015 and CBD in the breast tumor microenvironment. Schematic diagram shows how JWH-015 modulates the tumor microenvironment by increasing cytotoxic T-cell population and decreasing MDSC population, while CBD modulates the tumor microenvironment by decreasing monocyte recruitment, which leads to less TAMs in the tumor sites. Both JWH-015 and CBD leads to enhanced anti- tumor microenvironment by different mechanisms. EPC: endothelial precursor cells; CAF: cancer associated fibroblasts; DC: dentritic cells; Neu: neutrophils, MDSC: myeloid derived suppressor cells; TAMs: tumor associated fibroblasts.

Although both of JWH-015 and CBD show strong anti-tumor activities, It would be interesting to further study these cannabinoids in more detailed manner to gain better understanding of their anti-tumor properties:

 It will be very interesting to investigate, by co-immunoprecipitation studies, the interaction between cannabinoid receptors (CB2R and TRPV2) and growth

132 factor receptors (EGFR and IGF-IR) to decide whether this interaction is through direct binding or through indirect cross-talk mechanism.

 It would be interesting to study the role of CB2R in immunotherapy since we have observed that the tumors that developed in CB2R KO mice have higher expression of immune checkpoint protein PDL-1. It would be also good to check the effect of CB2R on other checkpoint proteins such as CTLA-4 and whether we can exploit this phenomenon to enhance the current anti-tumor immunotherapy.

 It would be extremely useful if different chemotherapeutic drugs would be tested in combination with different TRPV2 agonists to find out the best combination that has the maximum anti-tumor growth and metastasis potential and analyze the mechanism behind that.

 Since both CBD and JWH-015 inhibited breast tumor growth and metastasis through different mechanisms, it would be interesting to study the effect of combining both drugs and analyze whether they will lead to higher anti- tumor effect or not.

 To study the role of TRPV2 in drug uptake in vivo, it would be preferable to stably knock down or knock out TRPV2 in different breast cancer cell lines and use these stably transfected cells for orthotopic injection in mammary fat pad.

Then we can examine the effect of different chemotherapeutic drugs on the

KD/KO tumor cells versus the empty vector transfected tumor cells.

 Study the role of CBD as well as JWH-015 on modulating the activity of the wild type as well as the mutant forms of EGFR and IGF-IR on breast cancer

133 cells to check whether the effect of these drugs is basically on the wild type forms only or it is also applied for mutant forms of these growth factor receptors.

 Study the role of CBD in vivo after macrophage depletion by liposomal clodronate to analyze whether the anti-tumor effect of CBD has been abrogated or not.

 Study the role of CB2R in cytotoxic T-cell as well as MDSC through their isolation from WT and CB2R KO mice and examine their proliferative, migration and invasion potential and how they can participate in the modulation of cancer cell’s proliferation and chemotaxis potential through co-culturing breast cancer cells with these immune cells that either have or not CB2R.

 Both of our drugs, CBD as well as JWH-015 have shown anti-tumor growth and anti-metastatic potential in in vitro and in vivo mouse model systems.

It will be important to try these compounds for clinical trials, however, they need more characterization of their exact mechanism of action and they need to be tested on higher animals like cats and chimpanzee before going to human clinical trials. Furthermore, it is very important to address which kind of cannabinoid receptors is abundantly expressed in every patient and what is the differential expression of these receptors in different tissues and in different cancer types.

These studies are extremely important in order to predict which type of patient will be more responsive to these compounds.

 Since FAAH, an enzyme that degrades the endocannabinoids, is highly expressed in endothelial cells, in addition, we have preliminary data that show

134 FAAH inhibition has decreased breast cancer growth and metastasis; therefore, it will be useful to study its role in angiogenesis. Also, we have observed an increase in FAAH after M0 to M2 macrophage conversion. This observation provides us an insight to further study the role of FAAH enzyme within the tumor microenvironment.

135 References

1 Mao Y, Keller ET, Garfield DH, Shen K, Wang J. Stromal cells in tumor microenvironment and breast cancer. Cancer and Metastasis Reviews 2013; 32: 303-315.

2 Weigelt B, Geyer FC, Reis-Filho JS. Histological types of breast cancer: how special are they? Molecular oncology 2010; 4: 192-208.

3 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for clinicians 2015; 65: 5-29.

4 Sariego J. Breast cancer in the young patient. The American Surgeon 2010; 76: 1397-1400.

5 Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long- term follow-up. Histopathology 1991; 19: 403-410.

6 Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nature Reviews Cancer 2007; 7: 791-799.

7 Anatomic AoDo, Pathology S. Recommendations for the reporting of breast carcinoma. Human Pathology 1996; 27: 220-224.

8 Li C, Uribe D, Daling J. Clinical characteristics of different histologic types of breast cancer. British journal of cancer 2005; 93: 1046-1052.

9 Network NCC. Prostate cancer. NCCN clinical practice guidelines in oncology. Journal of the National Comprehensive Cancer Network: JNCCN 2004; 2: 224.

10 Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. Journal of clinical oncology 2007; 25: 5287-5312.

136

11 Hu Z, Fan C, Oh DS, Marron J, He X, Qaqish BF et al. The molecular portraits of breast tumors are conserved across microarray platforms. BMC genomics 2006; 7: 96.

12 Weigelt B, Baehner FL, Reis‐Filho JS. The contribution of profiling to breast cancer classification, prognostication and prediction: a retrospective of the last decade. The Journal of pathology 2010; 220: 263-280.

13 Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747-752.

14 Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001; 98: 10869-10874.

15 Sørlie T, Tibshirani R, Parker J, Hastie T, Marron J, Nobel A et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences 2003; 100: 8418-8423.

16 Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. Journal of clinical oncology 2009; 27: 1160-1167.

17 Wirapati P, Sotiriou C, Kunkel S, Farmer P, Pradervand S, Haibe-Kains B et al. Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures. Breast Cancer Res 2008; 10: R65.

18 Westbury CB, Reis‐Filho JS, Dexter T, Mahler‐Araujo B, Fenwick K, Iravani M et al. Genome‐wide transcriptomic profiling of microdissected human breast tissue reveals differential expression of KIT (c‐Kit, CD117) and oestrogen receptor‐α (ERα) in response to therapeutic radiation. The Journal of pathology 2009; 219: 131-140.

19 Peppercorn J, Perou C, Carey L. Molecular subtypes in breast cancer evaluation and management: divide and conquer. Cancer investigation 2008; 26: 1-10.

20 Fulford L, Easton D, Reis‐Filho J, Sofronis A, Gillett C, Lakhani S et al. Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast. Histopathology 2006; 49: 22-34.

137 21 Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Modern Pathology 2006; 19: 264-271.

22 Reis‐Filho JS, Milanezi F, Steele D, Savage K, Simpson PT, Nesland J et al. Metaplastic breast carcinomas are basal‐like tumours. Histopathology 2006; 49: 10-21.

23 Turner N, Reis-Filho J, Russell A, Springall R, Ryder K, Steele D et al. BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene 2007; 26: 2126-2132.

24 Turner N, Reis-Filho J. Basal-like breast cancer and the BRCA1 phenotype. Oncogene 2006; 25: 5846-5853.

25 Foulkes WD, Stefansson IM, Chappuis PO, Bégin LR, Goffin JR, Wong N et al. Germline BRCA1 and a basal epithelial phenotype in breast cancer. Journal of the National Cancer Institute 2003; 95: 1482-1485.

26 Lakhani SR, Jacquemier J, Sloane JP, Gusterson BA, Anderson TJ, van de Vijver MJ et al. Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations. Journal of the National Cancer Institute 1998; 90: 1138-1145.

27 Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S et al. Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clinical Cancer Research 2005; 11: 5175-5180.

28 Liu X, Holstege H, van der Gulden H, Treur-Mulder M, Zevenhoven J, Velds A et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proceedings of the National Academy of Sciences 2007; 104: 12111-12116.

29 McCarthy A, Savage K, Gabriel A, Naceur C, Reis‐Filho J, Ashworth A. A mouse model of basal‐like breast carcinoma with metaplastic elements. The Journal of pathology 2007; 211: 389-398.

30 Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D et al. Identification of molecular apocrine breast tumours by microarray analysis. Breast Cancer Research 2005; 7: P2. 11.

31 Doane A, Danso M, Lal P, Donaton M, Zhang L, Hudis C et al. An estrogen receptor-negative breast cancer subset characterized by a hormonally regulated

138 transcriptional program and response to androgen. Oncogene 2006; 25: 3994- 4008.

32 Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, Hu Z et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome biology 2007; 8: R76.

33 Hennessy BT, Gonzalez-Angulo A-M, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S, Lee J-S et al. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer research 2009; 69: 4116-4124.

34 Malhotra GK, Zhao X, Band H, Band V. Histological, molecular and functional subtypes of breast cancers. Cancer biology & therapy 2010; 10: 955-960.

35 Weigelt B, Peterse JL, Van't Veer LJ. Breast cancer metastasis: markers and models. Nature reviews cancer 2005; 5: 591-602.

36 Poste G, Fidler IJ. The pathogenesis of cancer metastasis. Nature 1980; 283: 139- 146.

37 Hunter KW, Crawford N, Alsarraj J. Mechanisms of metastasis. Breast Cancer Res 2008; 10: S2.

38 Talmadge JE, Fidler IJ. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer research 2010; 70: 5649-5669.

39 Li D-M, Feng Y-M. Signaling mechanism of cell adhesion molecules in breast cancer metastasis: potential therapeutic targets. Breast cancer research and treatment 2011; 128: 7-21.

40 Rothberg BEG, Bracken MB. E-cadherin immunohistochemical expression as a prognostic factor in infiltrating ductal carcinoma of the breast: a systematic review and meta-analysis. Breast cancer research and treatment 2006; 100: 139- 148.

41 Kowalski PJ, Rubin MA, Kleer CG. E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res 2003; 5: R217-R222.

42 Berx G, Cleton-Jansen A, Nollet F, De Leeuw W, Van de Vijver M, Cornelisse C et al. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. The EMBO journal 1995; 14: 6107.

139 43 Kotb AM, Hierholzer A, Kemler R. Replacement of E-cadherin by N-cadherin in the mammary gland leads to fibrocystic changes and tumor formation. Breast Cancer Research 2011; 13: R104.

44 Bonnomet A, Brysse A, Tachsidis A, Waltham M, Thompson EW, Polette M et al. Epithelial-to-mesenchymal transitions and circulating tumor cells. Journal of mammary gland biology and neoplasia 2010; 15: 261-273.

45 Ota I, Li X-Y, Hu Y, Weiss SJ. Induction of a MT1-MMP and MT2-MMP- dependent basement membrane transmigration program in cancer cells by Snail1. Proceedings of the National Academy of Sciences 2009; 106: 20318-20323.

46 Cavallaro U, Christofori G. Cell adhesion and signalling by cadherins and Ig- CAMs in cancer. Nature Reviews Cancer 2004; 4: 118-132.

47 Danø K, Behrendt N, Høyer-Hansen G, Johnsen M, Lund LR, Ploug M et al. Plasminogen activation and cancer. Thromb Haemost 2005; 93: 676-681.

48 Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer 2002; 2: 161-174.

49 Kelly T, Yan Y, Osborne RL, Athota AB, Rozypal TL, Colclasure JC et al. Proteolysis of extracellular matrix by invadopodiafacilitates human breast cancer cell invasion and ismediated by matrix metalloproteinases. Clinical & experimental metastasis 1998; 16: 501-512.

50 Rolli M, Fransvea E, Pilch J, Saven A, Felding-Habermann B. Activated integrin αvβ3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells. Proceedings of the National Academy of Sciences 2003; 100: 9482-9487.

51 Mitchell K, Svenson KB, Longmate WM, Gkirtzimanaki K, Sadej R, Wang X et al. Suppression of integrin α3β1 in breast cancer cells reduces cyclooxygenase-2 gene expression and inhibits tumorigenesis, invasion, and cross-talk to endothelial cells. Cancer research 2010; 70: 6359-6367.

52 Götte M, Yip GW. Heparanase, hyaluronan, and CD44 in cancers: a breast carcinoma perspective. Cancer Research 2006; 66: 10233-10237.

53 McSherry E, Donatello S, Hopkins A, McDonnell S. Molecular basis of invasion in breast cancer. Cellular and molecular life sciences: CMLS 2007; 64: 3201- 3218.

140 54 Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S et al. Epithelial–mesenchymal transition in cancer development and its clinical significance. Cancer science 2010; 101: 293-299.

55 Yilmaz M, Christofori G. Mechanisms of motility in metastasizing cells. Molecular Cancer Research 2010; 8: 629-642.

56 Scully OJ, Bay B-H, Yip G, Yu Y. Breast cancer metastasis. Cancer Genomics- Proteomics 2012; 9: 311-320.

57 Friedl P, Wolf K. Tube travel: the role of proteases in individual and collective cancer cell invasion. Cancer research 2008; 68: 7247-7249.

58 Paget S. The distribution of secondary growths in cancer of the breast. The Lancet 1889; 133: 571-573.

59 Kalluri R, Zeisberg M. Fibroblasts in cancer. Nature Reviews Cancer 2006; 6: 392-401.

60 Folkman J, Kalluri R. Cancer without disease. Nature 2004; 427: 787-787.

61 De Palma M, Venneri MA, Roca C, Naldini L. Targeting exogenous genes to tumor angiogenesis by transplantation of genetically modified hematopoietic stem cells. Nature medicine 2003; 9: 789-795.

62 Coghlin C, Murray GI. Current and emerging concepts in tumour metastasis. The Journal of pathology 2010; 222: 1-15.

63 Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. Journal of Clinical Investigation 2005; 115: 44.

64 Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME et al. Involvement of chemokine receptors in breast cancer metastasis. nature 2001; 410: 50-56.

65 Bruce J, Carter D, Fraser J. Patterns of recurrent disease in breast cancer. The Lancet 1970; 295: 433-435.

66 Adams EF, Newton C, Braunsberg H, Shaikh N, Ghilchik M, James V. Effects of human breast fibroblasts on growth and 17β-estradiol dehydrogenase activity of MCF-7 cells in culture. Breast cancer research and treatment 1988; 11: 165-172.

67 Hugo HJ, Lebret S, Tomaskovic-Crook E, Ahmed N, Blick T, Newgreen DF et al. Contribution of fibroblast and mast cell (afferent) and tumor (efferent) IL-6

141 effects within the tumor microenvironment. Cancer microenvironment 2012; 5: 83-93.

68 Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005; 121: 335-348.

69 Qian B-Z, Li J, Zhang H, Kitamura T, Zhang J, Campion LR et al. CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 2011; 475: 222-225.

70 Tan W, Zhang W, Strasner A, Grivennikov S, Cheng JQ, Hoffman RM et al. Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling. Nature 2011; 470: 548-553.

71 Loeffler M, Krüger JA, Niethammer AG, Reisfeld RA. Targeting tumor- associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. Journal of Clinical Investigation 2006; 116: 1955.

72 Martinez-Outschoorn UE, Goldberg AF, Lin Z, Ko Y-H, Flomenberg N, Wang C et al. Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells. Cancer biology & therapy 2011; 12: 924-938.

73 Sun Y, Campisi J, Higano C, Beer TM, Porter P, Coleman I et al. Treatment- induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Nature medicine 2012; 18: 1359-1368.

74 Lin EY, Pollard JW. Tumor-associated macrophages press the angiogenic switch in breast cancer. Cancer Research 2007; 67: 5064-5066.

75 Robinson BD, Sica GL, Liu Y-F, Rohan TE, Gertler FB, Condeelis JS et al. Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clinical Cancer Research 2009; 15: 2433-2441.

76 Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer research 2006; 66: 605-612.

77 Chen J, Yao Y, Gong C, Yu F, Su S, Chen J et al. CCL18 from tumor-associated macrophages promotes breast cancer metastasis via PITPNM3. Cancer cell 2011; 19: 541-555.

142 78 Su S, Liu Q, Chen J, Chen J, Chen F, He C et al. A Positive Feedback Loop between Mesenchymal-like Cancer Cells and Macrophages Is Essential to Breast Cancer Metastasis. Cancer Cell 2014; 25: 605-620.

79 Yang M, Chen J, Su F, Yu B, Su F, Lin L et al. Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells. Mol Cancer 2011; 10: 541-552.

80 Knutson KL, Dang Y, Lu H, Lukas J, Almand B, Gad E et al. IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice. The Journal of Immunology 2006; 177: 84-91.

81 Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AH et al. Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. Journal of Clinical Oncology 2011; 29: 1949-1955.

82 DeNardo DG, Brennan DJ, Rexhepaj E, Ruffell B, Shiao SL, Madden SF et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer discovery 2011; 1: 54-67.

83 Shekhar MP, Werdell J, Santner SJ, Pauley RJ, Tait L. Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer research 2001; 61: 1320-1326.

84 Acharyya S, Oskarsson T, Vanharanta S, Malladi S, Kim J, Morris PG et al. A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell 2012; 150: 165-178.

85 Iyengar P, Espina V, Williams TW, Lin Y, Berry D, Jelicks LA et al. Adipocyte- derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment. Journal of Clinical Investigation 2005; 115: 1163.

86 Iyengar P, Combs TP, Shah SJ, Gouon-Evans V, Pollard JW, Albanese C et al. Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization. Oncogene 2003; 22: 6408-6423.

87 Tinoco G, Warsch S, Glück S, Avancha K, Montero AJ. Treating breast cancer in the 21st century: emerging biological therapies. Journal of Cancer 2013; 4: 117.

143 88 Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2- overexpressing and trastuzumab-treated breast cancer cells. Cancer research 2006; 66: 1630-1639.

89 Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. Journal of Clinical Oncology 2002; 20: 719-726.

90 Fang L, Barekati Z, Zhang B, Liu Z, Zhong X. Targeted therapy in breast cancer: what’s new. Swiss Med Wkly 2011; 141: w13231.

91 Perez EA, Spano JP. Current and emerging targeted therapies for metastatic breast cancer. Cancer 2012; 118: 3014-3025.

92 Phillips GDL, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody–cytotoxic drug conjugate. Cancer research 2008; 68: 9280-9290.

93 Mathew J, Perez EA. in human epidermal growth factor receptor 2-positive breast cancer: a review. Current opinion in oncology 2011; 23: 594-600.

94 Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. The Lancet 2008; 372: 449-456.

95 Schoeberl B, Faber AC, Li D, Liang M-C, Crosby K, Onsum M et al. An ErbB3 antibody, MM-121, is active in cancers with ligand-dependent activation. Cancer research 2010; 70: 2485-2494.

96 Moyo V, Higgins M, Aravelo-Araujo R, Iannotti N, Charu V, Dhindsa N et al. A randomized, double-blind phase II trial of exemestane with or without MM-121 in postmenopausal women with locally advanced or metastatic estrogen receptor- positive (ER plus) and/or progesterone receptor-positive (PR+), HER2-negative breast cancer. JOURNAL OF CLINICAL ONCOLOGY, vol. 29. AMER SOC CLINICAL ONCOLOGY 2318 MILL ROAD, STE 800, ALEXANDRIA, VA 22314 USA, 2011.

97 Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M et al. Inhibition of poly (ADP-ribose) polymerase in tumors from BRCA mutation carriers. New England Journal of Medicine 2009; 361: 123-134.

144 98 Krishnakumar R, Kraus WL. The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets. Molecular cell 2010; 39: 8-24.

99 Amir E, Seruga B, Serrano R, Ocana A. Targeting DNA repair in breast cancer: a clinical and translational update. Cancer treatment reviews 2010; 36: 557-565.

100 Carey LA, Sharpless NE. PARP and cancer—if it's broke, don't fix it. The New England journal of medicine 2011; 364: 277.

101 Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005; 434: 917-921.

102 Vezina C, Kudelski A, Sehgal S. Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. The Journal of antibiotics 1975; 28: 721-726.

103 Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends in molecular medicine 2007; 13: 433-442.

104 Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. New England Journal of Medicine 2007; 356: 2271-2281.

105 Gao J, Chang YS, Jallal B, Viner J. Targeting the insulin-like growth factor axis for the development of novel therapeutics in oncology. Cancer research 2012; 72: 3-12.

106 Banerji U. Heat shock protein 90 as a drug target: some like it hot. Clinical Cancer Research 2009; 15: 9-14.

107 Pick E, Kluger Y, Giltnane JM, Moeder C, Camp RL, Rimm DL et al. High HSP90 expression is associated with decreased survival in breast cancer. Cancer research 2007; 67: 2932-2937.

108 Chakravarti B, Ravi J, Ganju RK. Cannabinoids as therapeutic agents in cancer: current status and future implications. Oncotarget 2014; 5: 5852.

109 Ravi J, Sneh A, Shilo K, Nasser MW, Ganju RK. FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway. Oncotarget 2014.

110 Adams R. Marihuana: Harvey Lecture, February 19, 1942. Bulletin of the New York Academy of Medicine 1942; 18: 705.

145

111 Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids 1993.

112 Sarfaraz S, Adhami VM, Syed DN, Afaq F, Mukhtar H. Cannabinoids for cancer treatment: progress and promise. Cancer research 2008; 68: 339-342.

113 Bosier B, Muccioli GG, Hermans E, Lambert DM. Functionally selective cannabinoid receptor signalling: therapeutic implications and opportunities. Biochemical pharmacology 2010; 80: 1-12.

114 Piomelli D. The molecular logic of endocannabinoid signalling. Nature Reviews Neuroscience 2003; 4: 873-884.

115 Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990: 561-564.

116 Mackie K. Distribution of cannabinoid receptors in the central and peripheral nervous system. Cannabinoids. Springer, 2005, pp 299-325.

117 Pacher P, Bátkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacological reviews 2006; 58: 389-462.

118 Velasco G, Sánchez C, Guzmán M. Towards the use of cannabinoids as antitumour agents. Nature Reviews Cancer 2012; 12: 436-444.

119 Joseph J, Niggemann B, Zaenker KS, Entschladen F. Anandamide is an endogenous inhibitor for the migration of tumor cells and T lymphocytes. Cancer Immunology, Immunotherapy 2004; 53: 723-728.

120 Bifulco M, Laezza C, Pisanti S, Gazzerro P. Cannabinoids and cancer: pros and cons of an antitumour strategy. British journal of pharmacology 2006; 148: 123- 135.

121 Gauson L, Stevenson L, Thomas A, Baillie G, Ross R, Pertwee R. Cannabigerol behaves as a partial agonist at both CB1 and CB2 receptors. Proceedings 17th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: Saint-Sauveur, QC, 2007, p 206.

122 Romano B, Borrelli F, Fasolino I, Capasso R, Piscitelli F, Cascio M et al. The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis. British journal of pharmacology 2013; 169: 213-229.

146

123 Massi P, Solinas M, Cinquina V, Parolaro D. Cannabidiol as potential anticancer drug. British journal of clinical pharmacology 2013; 75: 303-312.

124 Pertwee RG. Emerging strategies for exploiting cannabinoid receptor agonists as medicines. British journal of pharmacology 2009; 156: 397-411.

125 Starowicz K, Nigam S, Di Marzo V. Biochemistry and pharmacology of endovanilloids. Pharmacology & therapeutics 2007; 114: 13-33.

126 Brown A. Novel cannabinoid receptors. British journal of pharmacology 2007; 152: 567-575.

127 Stella N, Schweitzer P, Piomelli D. A second endogenous cannabinoid that modulates long-term potentiation. Nature 1997; 388: 773-778.

128 Di Marzo V, Melck D, De Petrocellis L, Bisogno T. Cannabimimetic fatty acid derivatives in cancer and inflammation. Prostaglandins & other lipid mediators 2000; 61: 43-61.

129 Christie MJ, Vaughan CW. Neurobiology: Cannabinoids act backwards. Nature 2001; 410: 527-530.

130 Rumińska A, Dobrzyń A. [The endocannabinoid system and its role in regulation of metabolism in peripheral tissues]. Postepy biochemii 2011; 58: 127-134.

131 Schmid HH. Pathways and mechanisms of N-acylethanolamine biosynthesis: can anandamide be generated selectively? Chemistry and Physics of Lipids 2000; 108: 71-87.

132 Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz J-C et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons 1994.

133 Bisogno T, Melck D, Petrocellis L, Marzo V. Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2‐arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin. Journal of neurochemistry 1999; 72: 2113-2119.

134 Cravatt BF, Prospero-Garcia O, Siuzdak G, Gilula NB, Henriksen SJ, Boger DL et al. Chemical characterization of a family of brain lipids that induce sleep. SCIENCE-NEW YORK THEN WASHINGTON- 1995: 1506-1506.

147 135 Petersen G, Moesgaard B, Schmid PC, Schmid HH, Broholm H, Kosteljanetz M et al. Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non‐tumour brain tissue. Journal of neurochemistry 2005; 93: 299-309.

136 Schmid PC, Wold LE, Krebsbach RJ, Berdyshev EV, Schmid HH. Anandamide and other N-acylethanolamines in human tumors. Lipids 2002; 37: 907-912.

137 Nithipatikom K, Endsley MP, Isbell MA, Falck JR, Iwamoto Y, Hillard CJ et al. 2-Arachidonoylglycerol a novel inhibitor of androgen-independent prostate cancer cell invasion. Cancer Research 2004; 64: 8826-8830.

138 Ligresti A, Bisogno T, Matias I, De Petrocellis L, Cascio MG, Cosenza V et al. Possible endocannabinoid control of colorectal cancer growth. Gastroenterology 2003; 125: 677-687.

139 Hermanson DJ, Marnett LJ. Cannabinoids, endocannabinoids, and cancer. Cancer and Metastasis Reviews 2011; 30: 599-612.

140 Endsley MP, Thill R, Choudhry I, Williams CL, Kajdacsy‐Balla A, Campbell WB et al. Expression and function of fatty acid amide hydrolase in prostate cancer. International Journal of Cancer 2008; 123: 1318-1326.

141 Michalski CW, Oti FE, Erkan M, Sauliunaite D, Bergmann F, Pacher P et al. Cannabinoids in pancreatic cancer: correlation with survival and pain. International Journal of Cancer 2008; 122: 742-750.

142 Russo EB. Taming THC: potential cannabis synergy and phytocannabinoid‐ terpenoid entourage effects. British journal of pharmacology 2011; 163: 1344- 1364.

143 Paronis CA, Nikas SP, Shukla VG, Makriyannis A. Δ9-Tetrahydrocannabinol acts as a partial agonist/antagonist in mice. Behavioural pharmacology 2012; 23: 802.

144 Ross RA, Gibson TM, Stevenson LA, Saha B, Crocker P, Razdan RK et al. Structural determinants of the partial agonist‐inverse agonist properties of 6′‐ azidohex‐2′‐yne‐Δ8‐tetrahydrocannabinol at cannabinoid receptors. British journal of pharmacology 1999; 128: 735-743.

145 Mahadevan A, Siegel C, Martin BR, Abood ME, Beletskaya I, Razdan RK. Novel cannabinol probes for CB1 and CB2 cannabinoid receptors. Journal of medicinal chemistry 2000; 43: 3778-3785.

148 146 Petitet F, Jeantaud B, Reibaud M, Imperato A, Dubroeucq M-C. Complex pharmacology of natural cannabivoids: Evidence for partial agonist activity of Δ 9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors. Life sciences 1998; 63: PL1-PL6.

147 Howlett A, Barth F, Bonner T, Cabral G, Casellas P, Devane W et al. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacological reviews 2002; 54: 161-202.

148 Griffin G, Fernando SR, Ross RA, McKay NG, Ashford ML, Shire D et al. Evidence for the presence of CB 2-like cannabinoid receptors on peripheral nerve terminals. European journal of pharmacology 1997; 339: 53-61.

149 Sánchez C, de Ceballos ML, del Pulgar TG, Rueda D, Corbacho C, Velasco G et al. Inhibition of glioma growth in vivo by selective activation of the CB2 cannabinoid receptor. Cancer Research 2001; 61: 5784-5789.

150 Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A et al. Agonist‐inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656 and AM630. British journal of pharmacology 1999; 126: 665-672.

151 Showalter VM, Compton DR, Martin BR, Abood ME. Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. Journal of Pharmacology and Experimental Therapeutics 1996; 278: 989-999.

152 Abadji V, Lin S, Taha G, Griffin G, Stevenson LA, Pertwee RG et al. (R)- methanandamide: a chiral novel anandamide possessing higher potency and metabolic stability. Journal of medicinal chemistry 1994; 37: 1889-1893.

153 Gatley SJ, Lan R, Pyatt B, Gifford AN, Volkow ND, Makriyannis A. Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors. Life sciences 1997; 61: PL191-PL197.

154 Ryberg E, Vu HK, Larsson N, Groblewski T, Hjorth S, Elebring T et al. Identification and characterisation of a novel splice variant of the human CB1 receptor. FEBS letters 2005; 579: 259-264.

155 Calatozzolo C, Salmaggi A, Pollo B, Sciacca F, Lorenzetti M, Franzini A et al. Expression of cannabinoid receptors and neurotrophins in human gliomas. Neurological Sciences 2007; 28: 304-310.

149 156 Islam T, Asplund A, Lindvall J, Nygren L, Liden J, Kimby E et al. High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of Cyclin D1 in mantle cell lymphoma. Leukemia 2003; 17: 1880-1890.

157 Wang D, Wang H, Ning W, Backlund MG, Dey SK, DuBois RN. Loss of cannabinoid receptor 1 accelerates intestinal tumor growth. Cancer research 2008; 68: 6468-6476.

158 Chung SC, Hammarsten P, Josefsson A, Stattin P, Granfors T, Egevad L et al. A high cannabinoid CB 1 receptor immunoreactivity is associated with disease severity and outcome in prostate cancer. European Journal of Cancer 2009; 45: 174-182.

159 Sarfaraz S, Afaq F, Adhami VM, Mukhtar H. Cannabinoid receptor as a novel target for the treatment of prostate cancer. Cancer research 2005; 65: 1635-1641.

160 Czifra G, Varga A, Nyeste K, Marincsák R, Tóth BI, Kovács I et al. Increased expressions of cannabinoid receptor-1 and transient receptor potential vanilloid-1 in human prostate carcinoma. Journal of cancer research and clinical oncology 2009; 135: 507-514.

161 Xu X, Liu Y, Huang S, Liu G, Xie C, Zhou J et al. Overexpression of cannabinoid receptors CB1 and CB2 correlates with improved prognosis of patients with hepatocellular carcinoma. Cancer genetics and cytogenetics 2006; 171: 31-38.

162 Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000; 21: 485-495.

163 Velasco G, Galve-Roperh I, Sánchez C, Blázquez C, Haro A, Guzmán M. Cannabinoids and ceramide: two lipids acting hand-by-hand. Life sciences 2005; 77: 1723-1731.

164 Guzman M. Effects on cell viability. Cannabinoids. Springer, 2005, pp 627-642.

165 Cianchi F, Papucci L, Schiavone N, Lulli M, Magnelli L, Vinci MC et al. Cannabinoid Receptor Activation Induces Apoptosis through α–Mediated Ceramide De novo Synthesis in Colon Cancer Cells. Clinical Cancer Research 2008; 14: 7691-7700.

166 Sánchez C, Galve-Roperh I, Rueda D, Guzmán M. Involvement of sphingomyelin hydrolysis and the mitogen-activated protein kinase cascade in the Δ9- tetrahydrocannabinol-induced stimulation of glucose metabolism in primary astrocytes. Molecular Pharmacology 1998; 54: 834-843.

150 167 Greenhough A, Patsos HA, Williams AC, Paraskeva C. The cannabinoid δ9‐ tetrahydrocannabinol inhibits RAS‐MAPK and PI3K‐AKT survival signalling and induces BAD‐mediated apoptosis in colorectal cancer cells. International journal of cancer 2007; 121: 2172-2180.

168 Sarela AI, Scott N, Ramsdale J, Markham AF, Guillou PJ. Immunohistochemical detection of the anti-apoptosis protein, survivin, predicts survival after curative resection of stage II colorectal carcinomas. Annals of surgical oncology 2001; 8: 305-310.

169 Altieri DC. Validating survivin as a cancer therapeutic target. Nature Reviews Cancer 2003; 3: 46-54.

170 Gustafsson K, Christensson B, Sander B, Flygare J. Cannabinoid receptor- mediated apoptosis induced by R (+)-methanandamide and Win55, 212-2 is associated with ceramide accumulation and p38 activation in mantle cell lymphoma. Molecular pharmacology 2006; 70: 1612-1620.

171 Jia W, Hegde VL, Singh NP, Sisco D, Grant S, Nagarkatti M et al. Δ9- tetrahydrocannabinol-induced apoptosis in Jurkat leukemia T cells is regulated by translocation of Bad to mitochondria. Molecular cancer research 2006; 4: 549- 562.

172 Herrera B, Carracedo A, Diez-Zaera M, del Pulgar TG, Guzmán M, Velasco G. The CB 2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the mitochondrial intrinsic pathway. Experimental cell research 2006; 312: 2121-2131.

173 Athanasiou A, Clarke AB, Turner AE, Kumaran NM, Vakilpour S, Smith PA et al. Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death. Biochemical and biophysical research communications 2007; 364: 131- 137.

174 Casanova ML, Blázquez C, Martínez-Palacio J, Villanueva C, Fernández-Aceñero MJ, Huffman JW et al. Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. Journal of Clinical Investigation 2003; 111: 43.

175 Melck D, Rueda D, Galve-Roperh I, De Petrocellis L, Guzmán M, Di Marzo V. Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer cells. FEBS letters 1999; 463: 235-240.

15 1

176 Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis. The FASEB Journal 2003; 17: 1771-1773.

177 Mimeault M, Pommery N, Wattez N, Bailly C, Hénichart JP. Anti‐proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: Implication of epidermal growth factor receptor down‐regulation and ceramide production. The Prostate 2003; 56: 1-12.

178 De Petrocellis L, Melck D, Palmisano A, Bisogno T, Laezza C, Bifulco M et al. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation. Proceedings of the National Academy of Sciences 1998; 95: 8375- 8380.

179 Melck D, De Petrocellis L, Orlando P, Bisogno T, Laezza C, Bifulco M et al. Suppression of Nerve Growth Factor Trk Receptors and Prolactin Receptors by Endocannabinoids Leads to Inhibition of Human Breast and Prostate Cancer Cell Proliferation 1. Endocrinology 2000; 141: 118-126.

180 Hart S, Fischer OM, Ullrich A. Cannabinoids induce cancer cell proliferation via tumor necrosis factor α-converting enzyme (TACE/ADAM17)-mediated transactivation of the epidermal growth factor receptor. Cancer research 2004; 64: 1943-1950.

181 Blázquez C, Casanova ML, Planas A, del Pulgar TG, Villanueva C, Fernández- Aceñero MJ et al. Inhibition of tumor angiogenesis by cannabinoids. The FASEB journal 2003; 17: 529-531.

182 Blázquez C, Carracedo A, Barrado L, Real PJ, Fernández-Luna JL, Velasco G et al. Cannabinoid receptors as novel targets for the treatment of melanoma. The FASEB journal 2006; 20: 2633-2635.

183 Preet A, Ganju R, Groopman J. Δ9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo. Oncogene 2008; 27: 339-346.

184 Pisanti S, Borselli C, Oliviero O, Laezza C, Gazzerro P, Bifulco M. Antiangiogenic activity of the endocannabinoid anandamide: Correlation to its tumor‐suppressor efficacy. Journal of cellular physiology 2007; 211: 495-503.

152 185 Blázquez C, González-Feria L, Álvarez L, Haro A, Casanova ML, Guzmán M. Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas. Cancer research 2004; 64: 5617-5623.

186 Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP et al. Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells. Journal of Biological Chemistry 2000; 275: 40725-40731.

187 Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP. Growth factor– induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood 2001; 97: 2323-2332.

188 Curran S, Murray G. Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis. European Journal of Cancer 2000; 36: 1621- 1630.

189 Ramer R, Hinz B. Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1. Journal of the National Cancer Institute 2008; 100: 59-69.

190 Elbaz M, Nasser MW, Ravi J, Wani NA, Ahirwar DK, Zhao H et al. Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway; novel anti- tumor mechanisms of Cannabidiol in breast cancer. Molecular Oncology.

191 Blázquez C, Salazar M, Carracedo A, Lorente M, Egia A, González-Feria L et al. Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression. Cancer research 2008; 68: 1945-1952.

192 Blázquez C, Carracedo A, Salazar M, Lorente M, Egia A, González-Feria L et al. Down-regulation of tissue inhibitor of metalloproteinases-1 in gliomas: a new marker of cannabinoid antitumoral activity? Neuropharmacology 2008; 54: 235- 243.

193 Hornebeck W, Lambert E, Petitfrère E, Bernard P. Beneficial and detrimental influences of tissue inhibitor of metalloproteinase-1 (TIMP-1) in tumor progression. Biochimie 2005; 87: 377-383.

194 Roussos ET, Condeelis JS, Patsialou A. Chemotaxis in cancer. Nature Reviews Cancer 2011; 11: 573-587.

195 Nasser MW, Qamri Z, Deol YS, Smith D, Shilo K, Zou X et al. Crosstalk between CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion. PloS one 2011; 6: e23901.

153

196 Rudolph MI, Boza Y, Yefi R, Luza S, Andrews E, Penissi A et al. The influence of mast cell mediators on migration of SW756 cervical carcinoma cells. Journal of pharmacological sciences 2008; 106: 208-218.

197 Laezza C, Pisanti S, Malfitano AM, Bifulco M. The anandamide analog, Met-F- AEA, controls human breast cancer cell migration via the RHOA/RHO kinase signaling pathway. Endocrine-related cancer 2008; 15: 965-974.

198 Vaccani A, Massi P, Colombo A, Rubino T, Parolaro D. Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor‐independent mechanism. British journal of pharmacology 2005; 144: 1032-1036.

199 Elbaz M, Nasser MW, Ravi J, Wani NA, Ahirwar DK, Zhao H et al. Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: Novel anti-tumor mechanisms of Cannabidiol in breast cancer. Molecular oncology 2015; 9: 906-919.

200 Shiozaki H, Oka H, Inoue M, Tamura S, Monden M. E‐cadherin mediated adhesion system in cancer cells. cancer 1996; 77: 1605-1613.

201 Grimaldi C, Pisanti S, Laezza C, Malfitano AM, Santoro A, Vitale M et al. Anandamide inhibits adhesion and migration of breast cancer cells. Experimental cell research 2006; 312: 363-373.

202 Zhou D, Song Z. CB1 cannabinoid receptor-mediated tyrosine phosphorylation of focal adhesion kinase-related non-kinase. FEBS letters 2002; 525: 164-168.

203 Richardson A, Parsons JT. A mechanism for regulation of the adhesion-associated protein pp125FAK 1996.

204 Gervais FG, Thornberry NA, Ruffolo SC, Nicholson DW, Roy S. Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide. Journal of Biological Chemistry 1998; 273: 17102-17108.

205 Sieg DJ, Hauck CR, Schlaepfer DD. Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. Journal of cell science 1999; 112: 2677-2691.

206 Curran NM, Griffin BD, O'Toole D, Brady KJ, Fitzgerald SN, Moynagh PN. The synthetic cannabinoid R (+) WIN 55,212-2 inhibits the interleukin-1 signaling pathway in human astrocytes in a cannabinoid receptor-independent manner. Journal of Biological Chemistry 2005; 280: 35797-35806.

154 207 Caffarel MM, Andradas C, Mira E, Pérez-Gómez E, Cerutti C, Moreno-Bueno G et al. Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition. Mol Cancer 2010; 9: 4598-4599.

208 Qamri Z, Preet A, Nasser MW, Bass CE, Leone G, Barsky SH et al. Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer. Molecular cancer therapeutics 2009; 8: 3117-3129.

209 McAllister SD, Murase R, Christian RT, Lau D, Zielinski AJ, Allison J et al. Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis. Breast cancer research and treatment 2011; 129: 37-47.

210 Ligresti A, Moriello AS, Starowicz K, Matias I, Pisanti S, De Petrocellis L et al. Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. Journal of Pharmacology and Experimental Therapeutics 2006; 318: 1375-1387.

211 Begg M, Pacher P, Bátkai S, Osei-Hyiaman D, Offertáler L, Mo FM et al. Evidence for novel cannabinoid receptors. Pharmacology & therapeutics 2005; 106: 133-145.

212 Maccarrone M, Lorenzon T, Bari M, Melino G, Finazzi-Agrò A. Anandamide induces apoptosis in human cells via vanilloid receptors Evidence for a protective role of cannabinoid receptors. Journal of Biological Chemistry 2000; 275: 31938- 31945.

213 Contassot E, Tenan M, Schnüriger V, Pelte M-F, Dietrich P-Y. Arachidonyl ethanolamide induces apoptosis of uterine cervix cancer cells via aberrantly expressed vanilloid receptor-1. Gynecologic oncology 2004; 93: 182-188.

214 DeMorrow S, Glaser S, Francis H, Venter J, Vaculin B, Vaculin S et al. Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth RECRUITMENT OF Fas AND Fas LIGAND TO LIPID RAFTS. Journal of Biological Chemistry 2007; 282: 13098-13113.

215 Van Dross RT. Metabolism of anandamide by COX‐2 is necessary for endocannabinoid‐induced cell death in tumorigenic keratinocytes. Molecular carcinogenesis 2009; 48: 724-732.

216 Hinz B, Ramer R, Eichele K, Weinzierl U, Brune K. Up-regulation of cyclooxygenase-2 expression is involved in R (+)-methanandamide-induced apoptotic death of human neuroglioma cells. Molecular pharmacology 2004; 66: 1643-1651.

155

217 Massi P, Vaccani A, Ceruti S, Colombo A, Abbracchio MP, Parolaro D. Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. Journal of Pharmacology and Experimental Therapeutics 2004; 308: 838-845.

218 Pineiro R, Maffucci T, Falasca M. The putative cannabinoid receptor GPR55 defines a novel autocrine loop in cancer cell proliferation. Oncogene 2011; 30: 142-152.

219 Bosch A, Eroles P, Zaragoza R, Viña JR, Lluch A. Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research. Cancer treatment reviews 2010; 36: 206-215.

220 Yu K-D, Zhu R, Zhan M, Rodriguez AA, Yang W, Wong S et al. Identification of prognosis-relevant subgroups in patients with chemoresistant triple-negative breast cancer. Clinical cancer research 2013; 19: 2723-2733.

221 Shrivastava A, Kuzontkoski PM, Groopman JE, Prasad A. Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy. Molecular cancer therapeutics 2011; 10: 1161- 1172.

222 McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez P-Y. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Molecular cancer therapeutics 2007; 6: 2921-2927.

223 Marcu JP, Christian RT, Lau D, Zielinski AJ, Horowitz MP, Lee J et al. Cannabidiol enhances the inhibitory effects of Δ9-tetrahydrocannabinol on human glioblastoma cell proliferation and survival. Molecular cancer therapeutics 2010; 9: 180-189.

224 Gazinska P, Grigoriadis A, Brown JP, Millis RR, Mera A, Gillett CE et al. Comparison of basal-like triple-negative breast cancer defined by morphology, immunohistochemistry and transcriptional profiles. Modern Pathology 2013; 26: 955-966.

225 Park HS, Jang MH, Kim EJ, Kim HJ, Lee HJ, Kim YJ et al. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer. Modern Pathology 2014.

226 Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors—impact on future treatment strategies. Nature reviews Clinical oncology 2010; 7: 493-507.

156

227 Mantovani A, Sica A. Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Current opinion in immunology 2010; 22: 231-237.

228 Dijkgraaf EM, Heusinkveld M, Tummers B, Vogelpoel LT, Goedemans R, Jha V et al. Chemotherapy alters monocyte differentiation to favor generation of cancer- supporting M2 macrophages in the tumor microenvironment. Cancer Research 2013; 73: 2480-2492.

229 Grigoriadis A, Mackay A, Noel E, Wu PJ, Natrajan R, Frankum J et al. Molecular characterisation of cell line models for triple-negative breast cancers. BMC genomics 2012; 13: 619.

230 Jin L, Simone B, Sano Y, Lim M, Zhao S, Savage J et al. Decreasing the metastatic potential in Triple Negative Breast Cancer through the miR-17 cluster. Cancer Research 2012; 72.

231 Lelekakis M, Moseley JM, Martin TJ, Hards D, Williams E, Ho P et al. A novel orthotopic model of breast cancer metastasis to bone. Clinical & experimental metastasis 1999; 17: 163-170.

232 Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, Tavaria MD et al. Genomic Analysis of a Spontaneous Model of Breast Cancer Metastasis to Bone Reveals a Role for the Extracellular Matrix1 1 Department of Defense Breast Cancer Research Program grants DAMD17-98-1-8144 (RL Anderson) and DAMD17-01-1-0371 (MD Tavaria), Susan G. Komen Breast Cancer Foundation predoctoral fellowship (EK Sloan), and NIH/National Cancer Institute grant ROI CA90291 (RL Anderson). Molecular Cancer Research 2005; 3: 1-13.

233 Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. The Journal of clinical investigation 2005; 115: 44-55.

234 Pei XF, Noble MS, Davoli MA, Rosfjord E, Tilli MT, Furth PA et al. Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice. In Vitro Cellular & Developmental Biology-Animal 2004; 40: 14-21.

235 Wani NA, Nasser MW, Ahirwar DK, Zhao H, Miao Z, Shilo K et al. CXC motif chemokine 12/CXC chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment. Breast Cancer Research 2014; 16: R54.

157 236 Nasser MW, Qamri Z, Deol YS, Ravi J, Powell CA, Trikha P et al. S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways. Cancer Research 2012; 72: 604-615.

237 Preet A, Qamri Z, Nasser MW, Prasad A, Shilo K, Zou X et al. Cannabinoid Receptors, CB1 and CB2, as Novel Targets for Inhibition of Non–Small Cell Lung Cancer Growth and Metastasis. Cancer Prevention Research 2011; 4: 65-75.

238 Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. Current opinion in cell biology 2005; 17: 559-564.

239 Xie H, Pallero MA, Gupta K, Chang P, Ware MF, Witke W et al. EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway. Journal of cell science 1998; 111: 615-624.

240 Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK. Targeting the EGFR signaling pathway in cancer therapy. Expert opinion on therapeutic targets 2012; 16: 15-31.

241 Singel SM, Batten K, Cornelius C, Jia G, Fasciani G, Barron SL et al. Receptor- interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways. Breast Cancer Research 2014; 16: R28.

242 Biswas DK, Iglehart JD. Linkage between EGFR family receptors and nuclear factor kappaB (NF‐κB) signaling in breast cancer. Journal of cellular physiology 2006; 209: 645-652.

243 Kim S, Choi JH, Lim HI, Lee S-K, Kim WW, Cho S et al. EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line. Cellular signalling 2009; 21: 892- 898.

244 Xu J-W, Li Q-Q, Tao L-L, Cheng Y-Y, Yu J, Chen Q et al. Involvement of EGFR in the promotion of malignant properties in multidrug resistant breast cancer cells. International journal of oncology 2011; 39: 1501.

245 Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2007; 1773: 642-652.

158 246 Jin U-H, Lee S-O, Pfent C, Safe S. The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis. BMC Cancer 2014; 14: 498.

247 Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Seminars in cancer biology, vol. 10. Elsevier, 2000, pp 415-433.

248 Place AE, Jin Huh S, Polyak K. The microenvironment in breast cancer progression: biology and implications for treatment. Breast Cancer Res 2011; 13: 227.

249 Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nature Reviews Cancer 2004; 4: 71-78.

250 Gil-Bernabé AM, Ferjančič Š, Tlalka M, Zhao L, Allen PD, Im JH et al. Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice. Blood 2012; 119: 3164-3175.

251 Nogi H, Kobayashi T, Suzuki M, Tabei I, Kawase K, Toriumi Y et al. EGFR as paradoxical predictor of chemosensitivity and outcome among triple-negative breast cancer. Oncology reports 2009; 21: 413-417.

252 Pisanti S, Malfitano AM, Grimaldi C, Santoro A, Gazzerro P, Laezza C et al. Use of cannabinoid receptor agonists in cancer therapy as palliative and curative agents. Best Practice & Research Clinical Endocrinology & Metabolism 2009; 23: 117-131.

253 Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R et al. ErbB receptors: from oncogenes to targeted cancer therapies. The Journal of clinical investigation 2007; 117: 2051-2058.

254 Capdevila J, Elez E, Macarulla T, Ramos FJ, Ruiz-Echarri M, Tabernero J. Anti- epidermal growth factor receptor monoclonal antibodies in cancer treatment. Cancer treatment reviews 2009; 35: 354-363.

255 Tanaka K, Babic I, Nathanson D, Akhavan D, Guo D, Gini B et al. Oncogenic EGFR signaling activates an mTORC2–NF-κB pathway that promotes chemotherapy resistance. Cancer discovery 2011; 1: 524-538.

256 Helbig G, Christopherson KW, 2nd, Bhat-Nakshatri P, Kumar S, Kishimoto H, Miller KD et al. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem 2003; 278: 21631-21638.

159

257 Huber MA, Azoitei N, Baumann B, Grünert S, Sommer A, Pehamberger H et al. NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. Journal of Clinical Investigation 2004; 114: 569-581.

258 Park BK, Zhang H, Zeng Q, Dai J, Keller ET, Giordano T et al. NF-κB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF. Nature medicine 2006; 13: 62-69.

259 Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Reviews Cancer 2003; 3: 362-374.

260 Jacob A, Jing J, Lee J, Schedin P, Gilbert SM, Peden AA et al. Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells. Journal of cell science 2013; 126: 4647-4658.

261 Schedin P, O’Brien J, Rudolph M, Stein T, Borges V. Microenvironment of the involuting mammary gland mediates mammary cancer progression. Journal of mammary gland biology and neoplasia 2007; 12: 71-82.

262 Talmadge JE, Donkor M, Scholar E. Inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer and Metastasis Reviews 2007; 26: 373-400.

263 Wu Y, Li Y-Y, Matsushima K, Baba T, Mukaida N. CCL3-CCR5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process. The Journal of Immunology 2008; 181: 6384-6393.

264 Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: a cancer journal for clinicians 2013; 63: 11-30.

265 Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor (ER)‐negative, progesterone receptor (PR)‐negative, and HER2‐negative invasive breast cancer, the so‐called triple‐negative phenotype. Cancer 2007; 109: 1721-1728.

266 Bayraktar S, Glück S. Molecularly targeted therapies for metastatic triple- negative breast cancer. Breast cancer research and treatment 2013: 1-15.

267 Hudis CA, Gianni L. Triple-negative breast cancer: an unmet medical need. Oncologist 2011; 16: 1-11.

160 268 Liedtke C, Mazouni C, Hess KR, André F, Tordai A, Mejia JA et al. Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. Journal of Clinical Oncology 2008; 26: 1275-1281.

269 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA et al. Triple- negative breast cancer: clinical features and patterns of recurrence. Clinical cancer research 2007; 13: 4429-4434.

270 Pogoda K, Niwińska A, Murawska M, Pieńkowski T. Analysis of pattern, time and risk factors influencing recurrence in triple-negative breast cancer patients. Medical Oncology 2013; 30: 1-8.

271 Gkika D, Prevarskaya N. TRP channels in prostate cancer: the good, the bad and the ugly? Asian Journal of Andrology 2011; 13: 673-676.

272 Montell C, Birnbaumer L, Flockerzi V, Bindels RJ, Bruford EA, Caterina MJ et al. A unified nomenclature for the superfamily of TRP cation channels. Molecular cell 2002; 9: 229-231.

273 Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A -receptor homologue with a high threshold for noxious heat. Nature 1999; 398: 436-441.

274 Morelli MB, Nabissi M, Amantini C, Farfariello V, Ricci‐Vitiani L, di Martino S et al. The transient receptor potential vanilloid‐2 cation channel impairs glioblastoma stem‐like cell proliferation and promotes differentiation. International Journal of Cancer 2012; 131: E1067-E1077.

275 Qin N, Neeper MP, Liu Y, Hutchinson TL, Lubin ML, Flores CM. TRPV2 is activated by cannabidiol and mediates CGRP release in cultured rat dorsal root ganglion neurons. The Journal of Neuroscience 2008; 28: 6231-6238.

276 Kanzaki M, Zhang Y-Q, Mashima H, Li L, Shibata H, Kojima I. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nature cell biology 1999; 1: 165-170.

277 Penna A, Juvin V, Chemin J, Compan V, Monet M, Rassendren F-A. PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane. Cell calcium 2006; 39: 495-507.

278 Karasawa T, Wang Q, Fu Y, Cohen DM, Steyger PS. TRPV4 enhances the cellular uptake of aminoglycoside antibiotics. Journal of cell science 2008; 121: 2871-2879.

161 279 Nasser MW, Wani NA, Ahirwar DK, Powell CA, Ravi J, Elbaz M et al. RAGE mediates S100A7-induced breast cancer growth and metastasis by modulating the tumor microenvironment. Cancer research 2015; 75: 974-985.

280 Nagasawa M, Nakagawa Y, Tanaka S, Kojima I. Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages. Journal of cellular physiology 2007; 210: 692-702.

281 Györffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast cancer research and treatment 2010; 123: 725-731.

282 Nabissi M, Morelli MB, Santoni M, Santoni G. Triggering of the TRPV2 channel by cannabidiol sensitizes glioblastoma cells to cytotoxic chemotherapeutic agents. Carcinogenesis 2013; 34: 48-57.

283 Eramo A, Ricci-Vitiani L, Zeuner A, Pallini R, Lotti F, Sette G et al. Chemotherapy resistance of glioblastoma stem cells. Cell Death & Differentiation 2006; 13: 1238-1241.

284 Aoyagi K, Ohara-Imaizumi M, Nishiwaki C, Nakamichi Y, Nagamatsu S. Insulin/phosphoinositide 3-kinase pathway accelerates the glucose-induced first- phase insulin secretion through TrpV2 recruitment in pancreatic beta-cells. Biochem J 2010; 432: 375-386.

285 Hisanaga E, Nagasawa M, Ueki K, Kulkarni RN, Mori M, Kojima I. Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic β-cells. Diabetes 2009; 58: 174-184.

286 Mihara H, Boudaka A, Shibasaki K, Yamanaka A, Sugiyama T, Tominaga M. Involvement of TRPV2 activation in intestinal movement through nitric oxide production in mice. The Journal of Neuroscience 2010; 30: 16536-16544.

287 Owsianik G, Talavera K, Voets T, Nilius B. Permeation and selectivity of TRP channels. Annu Rev Physiol 2006; 68: 685-717.

288 Voets T, Nilius B. The pore of TRP channels: trivial or neglected? Cell Calcium 2003; 33: 299-302.

289 Santoni G, Farfariello V. TRP channels and cancer: new targets for diagnosis and chemotherapy. Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune, Endocrine & Metabolic Disorders) 2011; 11: 54-67.

162

290 Wang S, Konorev EA, Kotamraju S, Joseph J, Kalivendi S, Kalyanaraman B. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms intermediacy of H2O2-and p53-dependent pathways. Journal of Biological Chemistry 2004; 279: 25535-25543.

291 Erler I, Hirnet D, Wissenbach U, Flockerzi V, Niemeyer BA. Ca2+-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat. Journal of Biological Chemistry 2004; 279: 34456-34463.

292 Dey N, R Smith B, Leyland-Jones B. Targeting basal-like breast cancers. Current Drug Targets 2012; 13: 1510-1524.

293 Bhargava R, Beriwal S, McManus K, Dabbs DJ. Insulin-like growth factor receptor-1 (IGF-1R) expression in normal breast, proliferative breast lesions, and breast carcinoma. Applied Immunohistochemistry & Molecular Morphology 2011; 19: 218-225.

294 Litzenburger BC, Creighton CJ, Tsimelzon A, Chan BT, Hilsenbeck SG, Wang T et al. High IGF-IR Activity in Triple-Negative Breast Cancer Cell Lines and Tumorgrafts Correlates with Sensitivity to Anti–IGF-IR Therapy. Clinical Cancer Research 2011; 17: 2314-2327.

295 Hankinson SE. Circulating levels of sex and prolactin in premenopausal women and risk of breast cancer. Hormonal Carcinogenesis V. Springer, 2008, pp 161-169.

296 Walsh LA, Damjanovski S. IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-b1 resulting in epithelial to mesenchymal transition. Cell Commun Signal 2011; 9: 10.

297 Davison Z, de Blacquière GE, Westley BR, May FE. Insulin-like growth factor- dependent proliferation and survival of triple-negative breast cancer cells: implications for therapy. Neoplasia 2011; 13: 504-515.

298 Ziring D, Wei B, Velazquez P, Schrage M, Buckley NE, Braun J. Formation of B and subsets require the cannabinoid receptor CB2. Immunogenetics 2006; 58: 714-725.

299 Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990; 346: 561-564.

163 300 Rondón-Lagos M, Verdun Di Cantogno L, Marchiò C, Rangel N, Payan-Gomez C, Gugliotta P et al. Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis. Mol Cytogenet 2014; 7: 8.

301 Aaltonen KE, Rosendahl AH, Olsson H, Malmström P, Hartman L, Fernö M. Association between insulin-like growth factor-1 receptor (IGF1R) negativity and poor prognosis in a cohort of women with primary breast cancer. BMC cancer 2014; 14: 794.

302 Law JH, Habibi G, Hu K, Masoudi H, Wang MY, Stratford AL et al. Phosphorylated insulin-like growth factor-i/insulin receptor is present in all breast cancer subtypes and is related to poor survival. Cancer Research 2008; 68: 10238- 10246.

303 Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K et al. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science 2005; 310: 329-332.

304 Zhang Z, Song T, Jin Y, Pan J, Zhang L, Wang L et al. Epidermal growth factor receptor regulates MT1-MMP and MMP-2 synthesis in SiHa cells via both PI3- K/AKT and MAPK/ERK pathways. International Journal of Gynecological Cancer 2009; 19: 998-1003.

305 Pitts TM, Tan AC, Kulikowski GN, Tentler JJ, Brown AM, Flanigan SA et al. Development of an integrated genomic classifier for a novel agent in colorectal cancer: approach to individualized therapy in early development. Clinical Cancer Research 2010; 16: 3193-3204.

306 Ekyalongo RC, Mukohara T, Kataoka Y, Funakoshi Y, Tomioka H, Kiyota N et al. Mechanisms of acquired resistance to insulin-like growth factor 1 receptor inhibitor in MCF-7 breast cancer cell line. Investigational new drugs 2013; 31: 293-303.

307 Baxevanis CN, Papamichail M. Characterization of the anti-tumor immune response in human cancers and strategies for immunotherapy. Critical reviews in oncology/hematology 1994; 16: 157-179.

308 Matsumura M, Fremont DH, Peterson PA, Wilson IA. Emerging principles for the recognition of peptide by MHC class I molecules. Science 1992; 257: 927-934.

309 Long EO. Intracellular traffic and antigen processing. Immunology today 1989; 10: 232-234.

164

310 Diaz-Montero CM, Salem ML, Nishimura MI, Garrett-Mayer E, Cole DJ, Montero AJ. Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin– chemotherapy. Cancer Immunology, Immunotherapy 2009; 58: 49-59.

311 Markowitz J, Wesolowski R, Papenfuss T, Brooks TR, Carson III WE. Myeloid- derived suppressor cells in breast cancer. Breast cancer research and treatment 2013; 140: 13-21.

312 Hegde VL, Nagarkatti M, Nagarkatti PS. Cannabinoid receptor activation leads to massive mobilization of myeloid‐derived suppressor cells with potent immunosuppressive properties. European journal of immunology 2010; 40: 3358- 3371.

313 Okazaki T, Honjo T. The PD-1–PD-L pathway in immunological tolerance. Trends in immunology 2006; 27: 195-201.

314 Keir ME, Liang SC, Guleria I, Latchman YE, Qipo A, Albacker LA et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. The Journal of experimental medicine 2006; 203: 883-895.

315 Tseng S-Y, Otsuji M, Gorski K, Huang X, Slansky JE, Pai SI et al. B7-DC, a new molecule with potent costimulatory properties for T cells. The Journal of experimental medicine 2001; 193: 839-846.

316 Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB et al. Tumor- associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nature medicine 2002; 8: 793-800.

317 Wang L, Pino-Lagos K, de Vries VC, Guleria I, Sayegh MH, Noelle RJ. Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+ CD4+ regulatory T cells. Proceedings of the National Academy of Sciences 2008; 105: 9331-9336.

318 Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD- L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proceedings of the National Academy of Sciences 2002; 99: 12293-12297.

319 Tsushima F, Yao S, Shin T, Flies A, Flies S, Xu H et al. Interaction between B7- H1 and PD-1 determines initiation and reversal of T-cell anergy. Blood 2007; 110: 180-185.

165