Crosstalk Between Cannabinoid Receptors and Epidermal

CROSSTALK BETWEEN CANNABINOID RECEPTORS AND EPIDERMAL

GROWTH FACTOR RECEPTOR IN NON-SMALL CELL LUNG CANCER

DISSERTATION

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

in the Graduate School of The Ohio State University

JANANI RAVI

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2015

Dissertation Committee:

Dr. Ramesh Ganju, Advisor

Dr. Kalpana Ghoshal

Dr. Xianghong Zou

Dr. Sujit Basu

Janani Ravi

2015

Abstract

(i) The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anticancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and

NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-

AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and

PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination

ii treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with

FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

(ii) JWH-015, a cannabinoid receptor 2 (CB2) agonist has tumor regressive property in various cancer types. However, the underlying mechanism by which it acts in lung cancer is still unknown. Tumor associated macrophage (TAM) intensity has positive correlation with tumor progression. Also, macrophages recruited at the tumor site promote tumor growth by enhancing epithelial to mesenchymal (EMT) progression. In this study, we analyzed the role of JWH-015 on EMT and macrophage infiltration by regulation of

EGFR signaling. JWH-015 inhibited EMT in NSCLC cells A549 and also reversed the mesenchymal nature of CALU-1 cells by downregulation of EGFR signaling targets like

ERK and STAT3. Also, in vitro co-culture experiments of A549 with M2 polarized macrophages provided evidence that JWH-015 decreased migratory and invasive abilities which was proved by reduced expression of FAK, VCAM1 and MMP2. Furthermore, it decreased macrophage induced EMT in A549 by attenuating the mesenchymal character by downregulating EGFR and its targets. These results were confirmed in an in vivo subcutaneous syngenic mouse model where JWH-015 blocks tumor growth and also iii inhibits macrophage recruitment and EMT at the tumor site which was regulated by

EGFR pathway. Finally, JWH-015 reduced metastatic lesions in a tail vein syngenic mouse model. These data confer the crosstalk between CB2 and EGFR receptors, leading to anti-proliferative and anti-tumorigenic effects, thus enhancing our understanding of the therapeutic efficacy of JWH-015 in NSCLC.

Dedication

This document is dedicated to my mother Rajeswari, father Ravi, sister Pavithra and

grandparents.

Acknowledgement

First and foremost, I would like to thank my parents who have made me the person I am today. I am indebted to my father and mother for their sacrifice and support. I owe all my accomplishments to them.

I would like to thank my advisor Dr. Ramesh Ganju. I thank him for his continuous support, guidance and encouragement over the past three years. He has shared his passion for science with me and encouraged me to think the big picture. I also thank him for all the valuable advice and questions he has asked in the past years, which prepared me for any challenge I may face in the future.

I would like to thank my committee members, Dr. Kalpana Ghoshal, Dr. Sujit Basu and

Dr. Xianghong Zou, for their time and guidance. I am grateful to MCDB program director, Dr. David Bisaro for giving me this opportunity.

I thank all the past and present Ganju lab members, Nissar A Wani, Mohd W Nasser,

Catherine A Powell, Mohamad Elbaz and Amita Sneh for helping me learn new techniques and for their assistance. I am grateful to all the members in the lab for their valuable, fun discussions and friendship.

I would like to thank all my friends in Columbus for making my years pleasant and memorable.

Vita

May 2006 ...... CSI Bain School

June 2010 ...... B.Tech Biotechnology, SVCE

Sep 2010 to present ...... Graduate Research Associate, Department

of Pathology, The Ohio State University

Publications

1. Nasser MW, Qamri Z, Deol YS, Ravi J et al. S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways. Cancer Research, 2012;

72(3):604-15.

2. Manchanda PK, Kibler AJ, Zhang M, Ravi J et al. Vitamin D receptor as a therapeutic target for benign prostatic hyperplasia. Indian J Urol., 2012; 28(4):377-81.

3. Ravi J et al. FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway.

Oncotarget, 2014; 5(9): 2475-86.

vii

4. Ravi J*, Chakravarti B*, et al. Cannabinoids as therapeutic agents in cancer: current status and future implications. Oncotarget, 2014; 5(15):5852-72. (Co- first author)

5. Elbaz M, Nasser MW, Ravi J 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; (in press).

6. Nasser MW*, Wani NA*, Ahirwar DK, Powell CA, Ravi J, et al. Receptor for

Advanced Glycation End products (RAGE) mediates S100A7-induced breast cancer growth and metastasis via modulating tumor microenvironment. Cancer

Research, 2015; (in press).

7. Ravi J et al. Tumor associated macrophages induce EMT in NSCLC which is inhibited by synthetic cannabinoid JWH-015 by regulating the EGFR pathway. (In submission).

8. Nasser MW*, Wani NA*, Ravi J et al. S100A7 accelerates breast cancer growth and metastasis through STAT3 pathway. (In submission).

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

viii

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgement ...... vi

Vita ...... vii

Publications ...... vii

Fields of Study ...... viii

List of figures ...... xvi

List of tables ...... xix

List of important abbreviations ...... xx

Chapter 1 ...... 1

Lung cancer ...... 1

1.1 Introduction ...... 1

1.2 Origin of lung cancer...... 2

1.3 Classification of lung cancer based on molecular subtypes ...... 2

1.4 Targeted therapy in lung cancer ...... 6

1.5 Lung cancer metastasis...... 9

1.6 Role of microenvironment in lung cancer progression and metastasis ...... 12

Chapter 2 ...... 15

Cannabinoids as therapeutic agents in cancer: Current status ...... 15

2.1 Introduction ...... 15

2.2 Cannabinoid and its receptor ...... 17

2.3 Endogenous cannabinoids ...... 18

2.4 Phytocannabinoids ...... 19

2.5 Synthetic cannabinoids ...... 20

2.6 Cannabinoid receptor mediated signaling in cancer ...... 22

2.7 Role of cannabinoids in regulation of cancer growth ...... 24

2.7.1 Cannabinoids and breast cancer ...... 24

2.7.2 Cannabinoids and prostate cancer ...... 29

2.7.3 Cannabinoids and lung cancer ...... 30

2.7.4 Cannabinoids and skin cancer ...... 31

2.7.5 Cannabinoids and pancreatic cancer ...... 31

2.7.6 Cannabinoids and bone cancer ...... 32

2.7.7 Cannabinoids and glioma ...... 33

2.7.8 Cannabinoids and lymphoma ...... 36

2.7.9 Cannabinoids and oral cancer ...... 36

2.7.10 Cannabinoids and head and neck cancer ...... 37

2.7.11 Cannabinoids and thyroid carcinoma ...... 37

2.8 Role of cannabinoids in pro-metastatic mechanisms like angiogenesis, migration

and invasion...... 38

2.9 Role of cannabinoids in cancer metastasis ...... 40

2.10 Role of cannabinoids in stemness and cancer ...... 40

2.11 Role of cannabinoids in energy metabolism and cancer ...... 41

2.12 Role of cannabinoids in immune environment and cancer ...... 43

Chapter 3 ...... 49

FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway ...... 49

3.1 Introduction ...... 49

3.2 Materials and methods ...... 53

3.2.1 Reagents and antibodies ...... 53

3.2.2 Cell culture ...... 53

3.2.3 Cell proliferation assay ...... 53

3.2.4 Clonogenic assay ...... 54

3.2.5 Chemotaxis and wound healing assays ...... 54

3.2.6 FAAH small interfering RNA ...... 55

3.2.7 Immunofluorescence ...... 55

3.2.8 Gelatin zymography ...... 55

3.2.9 Luciferase reporter assay ...... 55

3.2.10 Western blotting ...... 56

3.2.11 Cell cycle analysis ...... 56

3.2.12 Apoptosis assay ...... 57

3.2.13 Mouse xenograft model ...... 57

3.2.14 Real Time PCR ...... 57

3.2.15 Tissue Microarray (TMA) and Immunochemical (IHC) analyses ...... 57

3.2.16 Statistical analysis...... 58

3.3 Results ...... 58

3.3.1 Primary lung cancer tissues and NSCLC cell lines express CB1 and FAAH .. 58

3.3.2 FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines ...... 60

3.3.3 FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines ...... 62

xii

3.3.4 FAAH inhibition enhances Met-F-AEA mediated inhibition of EGFR signaling

in NSCLC cell lines ...... 67

3.3.5 FAAH inhibition enhances Met-F-AEA induced cell cycle arrest and apoptosis

at later stage ...... 70

3.3.6 FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor

growth in vivo by downregulating EGFR signaling ...... 72

3.4 Conclusion ...... 76

3.5 Discussion ...... 79

Chapter 4 ...... 81

Synthetic cannabinoid agonist JWH-015 inhibits macrophage induced EMT in non-small cell lung cancer by downregulation of EGFR signaling ...... 81

4.1 Introduction ...... 81

4.2 Materials and methods ...... 84

4.2.1 Cell culture ...... 84

4.2.2 Reagents and antibodies ...... 84

4.2.3 Real Time Reverse Transcription PCR ...... 85

4.2.4 Immunochemical (IHC) analyses ...... 85

4.2.5 Clonogenic assay ...... 85

4.2.6 Chemotaxis and wound healing assays ...... 85

4.2.7 Immunofluorescence ...... 86 xiii

4.2.8 Western blotting ...... 86

4.2.9 Mouse xenograft model ...... 87

4.2.10 Tail vein syngenic mouse model ...... 87

4.2.11 Flow cytometry ...... 87

4.2.12 Statistical analysis...... 87

4.3 Results ...... 88

4.3.1 CB2 and EGFR are expressed in NSCLC patients and cell lines ...... 88

4.3.2 JWH-015 inhibits EGF induced EMT in A549 cells ...... 89

4.3.3 JWH-015 promotes mesenchymal to epithelial transition in CALU-1 cells .... 92

4.3.4 JWH-015 inhibits M2 macrophage induced EMT in A549 cells ...... 97

4.3.5 JWH-015 prevents lung colonization of ED1 cells in in vivo tail vein syngenic

mouse model ...... 101

4.3.6 JWH-015 inhibits NSCLC tumor growth in vivo in a subcutaneous mouse

model ...... 103

4.3.7 JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of

tumor cells by downregulation of EGFR signaling ...... 106

4.4 Conclusion ...... 109

4.5 Discussion ...... 113

Chapter 5 ...... 114

xiv

Future directions ...... 114

References ...... 117

Appendix- Journal’s license terms ...... 151

List of figures

Figure 1: Molecular pathways in lung cancer...... 3

Figure 2: Potential molecular targets and therapeutic agents...... 7

Figure 3: Ten driver mutations in 60% (252/422) of lung adenocarcinomas...... 9

Figure 4: Signaling interactions within specific tissues at sites of non-small cell lung cancer metastasis...... 11

Figure 5: Micro-environmental interactions influencing cancer growth, tissue remodeling, angiogenesis and migration and invasion into tissues...... 13

Figure 6: Cannabinoids and their classification...... 20

Figure 7: Cannabinoid mediated signaling in cancer cells...... 23

Figure 8: Modulatory effect of cannabinoids on hormone sensitive breast cancer cells. 26

Figure 9: Modulatory effect of cannabinoids on HER-2 +ve and Triple –ve breast cancer cells...... 28

Figure 10: EGFR signaling pathway in lung cancer...... 51

Figure 11: NSCLC cell lines and primary lung cancer tissues express CB1 and FAAH. 59

Figure 12: FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines...... 60

Figure 13: FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines ...... 62

xvi

Figure 14: FAAH inhibition enhances wound healing activity of Met-F-AEA in A549 cells...... 63

Figure 15: FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines...... 64

Figure 16: FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines...... 65

Figure 17: FAAH inhibition enhances Met-F-AEA mediated inhibition of NF-kB in

NSCLC cell lines...... 67

Figure 18: FAAH inhibition enhances Met-F-AEA mediated inhibition of EGFR signaling in NSCLC cell lines...... 69

Figure 19: FAAH inhibition enhances Met-F-AEA induced cell cycle arrest and apoptosis at later stage...... 71

Figure 20: FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo...... 73

Figure 21: FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo by downregulating EGFR signaling...... 75

Figure 22: CB2 and EGFR are expressed in NSCLC patients and cell lines...... 88

Figure 23: JWH-015 inhibits EGF induced signaling in A549 cells...... 89

Figure 24: JWH-015 inhibits EGF induced EMT in A549 cells...... 91

Figure 25: JWH-015 inhibited EGF induced migration and invasion in CALU1 cells.. . 94

Figure 26: JWH-015 promotes mesenchymal to epithelial transition in CALU-1 cells.. 95

Figure 27: JWH-015 inhibits EGF induced signaling in CALU1 cells...... 96

xvii

Figure 28: M1 to M2 conversion of THP-1 cells...... 98

Figure 29: JWH-015 inhibits the M2 macrophage proliferation and migration in A549 cells...... 99

Figure 30: JWH-015 inhibits M2 macrophage induced EMT in A549 cells...... 100

Figure 31: JWH-015 inhibits proliferation in ED1 cells...... 102

Figure 32: JWH-015 inhibits NSCLC metastasis in in vivo mouse model...... 103

Figure 33: JWH-015 inhibits NSCLC growth in subcutaneous mouse model...... 105

Figure 34: JWH-015 decreases macrophage recruitment to tumor site...... 107

Figure 35: JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of tumor cells by downregulation of EGFR signaling...... 108

xviii

List of tables

Table 1: Lung cancer molecular subtypes based on high SOE...... 4

Table 2: Lung cancer molecular subtypes based on medium or low SOE...... 5

Table 3: Cannabinoid’s structure and its role in different physiological processes...... 44

Table 4: Role of cannabinoid in different cancers and its associated signaling...... 45

xix

List of important abbreviations

CBD Cannabidiol

CBR1 Cannabinoid Receptor1

CBR2 Cannabinoid Receptor 2

CDK Cyclin Dependent Kinase

CSC Cancer Stem Cell

ECM Extra-Cellular Matrix

EGFR Epidermal Growth Factor Receptor

EMT Epithelial to Mesenchymal Transition

ERK Extracellular Regulated Kinase

FAAH Fatty Acid Amide Hydrolase

FAK Focal Adhesion Kinase

IHC ImmunoHisto Chemistry

Met-F-AEA 2-methyl-2′-F-Anandamide

MMP Matrix Metallo Proteinase

NSCLC Non-Small Cell Lung Cancer

PCD Programmed Cell Death

ROS Reactive Oxygen Species

RT-PCR Real Time- Polymerase Chain Reaction

STAT3 Signal Transducer and Activator 3

TAM Tumor Associated Macrophage

THC Tetra Hydro Cannabinol

TME Tumor Micro Environment

TMA Tissue Micro Array

VCAM1 Vascular Cell Adhesion Molecule 1

xxi

Chapter 1

Lung cancer

1.1 Introduction

Lung cancer is estimated to be one of the most fatal cancer related malignancies in the world and fatality is often associated with drug resistance complications. Thus, there is an immediate requirement to develop innovative ways for the treatment of this disease (1-2).

Lung cancer is one of the major causes of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, contributes to about

85% of cases with possibility of advanced stages in two-thirds of NSCLC patients. It is a metastatic form of cancer which is the primary cause of cancer related deaths in the

United States. Though tobacco smoking is the major cause of lung cancer, about 15-20% of cases are attributed to non-smokers and involve the activation of various signaling pathways for tumor development (3). Adenocarcinoma and squamous cell carcinoma, the two most common histological subtypes of lung cancer are categorized as NSCLC. Poor prognosis and chemotherapeutic resistance which may be due to modulation of key cell signaling mechanisms pose major concerns (1-2). Tumor heterogeneity in NSCLC leads to poor patient outcome and drug resistance complications (4-5).

1.2 Origin of lung cancer

Though smoking accounts for most causes of lung cancer, there are various factors involved leading to this disease:

 Tobacco smoke

 Exposure to cooking fumes

 Inherited genetic susceptibility

 Occupational and environmental exposure

 Hormonal factors

 Pre-existing lung disease

 Oncogenic viruses

 Chromosomal aberrations

 Mutagenesis

 DNA methylation (6)

1.3 Classification of lung cancer based on molecular subtypes

The major signaling pathway in lung cancer is the EGFR pathway. It plays crucial role in cell growth, survival, differentiation, invasion and metastasis. Based on the important signaling pathways, lung cancer is characterized into several subtypes as shown in Fig.1.

Figure 1: Molecular pathways in lung cancer. The major pathways are EGFR (blue),

KRAS (yellow), EML4-ALK (orange), P53/BCL (purple). AKT/PI3K pathway (green) and the MAPK pathway (red). Reprinted from PLoS ONE 7(2):e31906. doi:10.1371/journal.pone.0031906 (3).

West et al. categorized lung cancer subtypes based on the oncogenes/tumor suppressor genes associated with the disease and their potential to cause therapeutic intervention.

Table 1 shows oncogenes/tumor suppressor genes with high SOE (Strength Of Evidence) 3 and Table 2 denotes oncogenes/tumor suppressor genes with medium or low SOE. High

SOE genes serve as the dominant genes in disease onset, progression and therapeutic intervention as compared to low SOE genes.

Table 1: Lung cancer molecular subtypes based on high SOE. Reprinted from PLoS

ONE 7(2):e31906. doi:10.1371/journal.pone.0031906 (3).

Table 2: Lung cancer molecular subtypes based on medium or low SOE. Reprinted from

PLoS ONE 7(2):e31906. doi:10.1371/journal.pone.0031906 (3).

Subtype 1 refers to aberrations in the EGFR signaling pathway.

Subtype 2 involves mutations in the K-ras gene, a family of small GTPases required for cellular functions. 5

Subtype 3 is characterized by aberrations in the EML4-ALK oncogene which is a relatively newly discovered oncogene.

Subtype 4 harbors c-MET gene aberrations.

Subtype 5 refers to aberrations in the AKT/PI3K pathway which plays a role in cell cycle and survival.

Subtype 6 involves aberrations in the vascular endothelial growth factor (VEGF) pathway which is very important signaling protein in vasculogenesis and angiogenesis.

ROS-1 mutation is new discovery that is categorized into subtype 7.

Subtype 8 involves epigenetic alterations which are changes in DNA and nucleosome affecting gene expression.

Aberrations in the insulin like growth factor (IGF) axis contribute to subtype 9 (3).

1.4 Targeted therapy in lung cancer

Various targeted therapies have been discovered and used in NSCLC, especially EGFR tyrosine kinase inhibitors (EGFR-TKI) as EGFR is a very important receptor related to

NSCLC progression and metastasis. Other than EGFR-TKI, there are various individualized targeted drugs related to other molecules like PI3K, AKT, RAS, etc. which have been in use for the NSCLC patients. This kind of treatment benefits patients with identified key carcinogenic mutations and hence treated well using these novel therapeutic targets. A summary of the molecular targets and their therapeutic agents are shown in Fig.2.

Figure 2: Potential molecular targets and therapeutic agents. EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FGFR, fibroblast growth factor receptor; HER, human epidermal growth factor receptor; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PI3K (PIK3CA), phosphatidylinositol 3-kinase; VEGFR, vascular endothelial growth factor receptor.

Reprinted with permission from Springer Science. Front Med. 2013 Jun;7(2):157-71. doi: 10.1007/s11684-013-0272-4 (7) .

Driver mutations are mutations that provide selective growth advantage to cancer cells.

These mutations play important role in cancer progression and metastasis.

The Lung cancer mutation consortium determined ten important driver mutations which were studied from 1000 patients. These mutations are responsible for resistance to therapy (Fig.3).

They are:

KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog),

EGFR (epidermal growth factor receptor),

ALK fusion (anaplastic lymphoma kinase fusion gene),

BRAF (v-raf murine sarcoma viral oncogene homolog B1),

PIK3CA (PI3K) (phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha), MET amp (met protooncogene amplification),

HER2 (human epidermal growth factor receptor-2),

MEK1 (MAP2K1) (mitogen-activated protein kinase kinase 1),

NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog) and

AKT1 (v-akt murine thymoma viral oncogene homolog 1) (7).

Figure 3: Ten driver mutations in 60% (252/422) of lung adenocarcinomas. Data were obtained from a report by the Lung Cancer Mutation Consortium. Reprinted with permission from Springer Science. Front Med. 2013; 7(2):157-71. doi: 10.1007/s11684-

013-0272-4 (7).

1.5 Lung cancer metastasis

Lung is an organ that is more exposed to toxic substances due to inhalation process.

Thus, it has complex microenvironment which is composed of vascularization and oxygenation. This creates a unique tumor-stromal interaction network and various factors, ultimately leading to distant metastasis. Several key stages are involved in metastasis:

 Local invasion of tumor through basement membrane

 Intravasation into lymph nodes

 Extravasation into distant tumor site

 Pre-metastatic niche at the metastatic site

 Micrometastases and macrmetastases formation

Lung cancer has been shown to metastasize to various organs. % represents percentage of patients with lung cancer metastasis to distant organs:

1. Liver (33–40%)

2. Brain (15–43%)

3. Kidney (16–23%)

4. Adrenal glands (18–38%)

5. Bone (19–33%) and

6. Abdominal lymph nodes (29%)

Around 40% of patients are diagnosed with distant metastasis in NSCLC (Fig. 4). Hence, treatment options are required which prevent metastasis to various sites apart from elimination of primary tumor. Treatment of brain metastasis is more difficult as drugs have to pass the blood-brain barrier. Hence, there is search for new drugs that overcome these difficulties.

Figure 4: Signaling interactions within specific tissues at sites of non-small cell lung cancer metastasis. Preferential metastatic sites are brain, bone, liver and adrenal gland.

Reprinted with permission from Springer Science. Cancer Treat Rev.2014; 40:558-66. doi:10.1016/j.ctrv.2013.10.001 (8).

1.6 Role of microenvironment in lung cancer progression and metastasis

Tumor-stromal interactions are crucial in tumor progression and metastasis. Epithelial-to- mesenchymal transition (EMT), an important phenotype change and hypoxia accelerate this process. The tumor stroma consists of various cell types involved in the tumor microenvironment (TME) and the extra-cellular matrix (ECM):

 Cancer associated fibroblasts (CAF)

 Mesenchymal stem cells (MSC)

 Tumor associated macrophages (TAM)

 Endothelial cells

 B cells

 T cells

 NK cells

 Myeloid derived suppressor cells (MDSC)

 Bone marrow derived cells (BMDC)

 Adipocytes

These cell types are involved in homing or secretion of various cytokines and growth factors, which are tumor promoting or tumor inhibiting.

Figure 5: Micro-environmental interactions influencing cancer growth, tissue remodeling, angiogenesis and migration and invasion into tissues. Reprinted with permission from Springer Science. Cancer Treat Rev.2014; 40:558-66. doi:10.1016/j.ctrv.2013.10.001 (8).

(i) Angiogenic factors like: PDGF, FGF-2, FGF-6, IL-6, IL-8 VEGF and angiopoietin-1

(ii) Cytokine/cytokine-receptor pairs like: monocyte-chemotactic protein (MCP)/CCR2,

HMGB1/RAGE, SDF1 (CXCL12)/CXCR4, SCF/c-Kit, VEGF/VEGFR and HGF/c-Met

(iii) Growth factors like: endothelin-1 (EDN1), VEGF-A, PDGF-C, osteopontin, IL-8 and

CXCL1

(iv)Macrophage secreting factors like: cyclooxygenase-2 (COX2), matrix metalloproteinase-9 (MMP9), PDGF-B, VEGFA, HGF, cathepsin-K and urokinase-type plasminogen activator with MMP9/uPA are involved in malignant development of tumor, thus leading to metastasis (Fig. 5) (8).

Chapter 2

Cannabinoids as therapeutic agents in cancer: Current status

2.1 Introduction

Cannabis sativa plant has been used for several hundreds of years both recreationally and medicinally. Centuries ago, the Chinese medicine refers to cannabis plant for pain-relief and hallucination. It contains 3 major classes of bioactive molecules; flavanoids, terpenoids and more than 60 types of cannabinoids (9).

Cannabinoids are the active compounds of this marijuana plant. But, the use of cannabinoids is in question because of their phsychotropic and addictive issues The most active constituent of this plant is ∆9-tetrahydrocannabinol (∆9-THC), elucidated between

1940s and 1960s (10). This discovery has opened the way to identification of the molecular action of various cannabinoids and the cannabinoid receptors. Evidence shows that smoking of cannabis preparations caused cancer of the respiratory and oral tracts or, at least, potentiated tobacco smoke-induced damages (11).

Cannabinoid is a family of complex chemicals (terpenophenolic compounds) that exert most of their actions by binding to and activating specific Gαi protein-coupled receptors named as cannabinoid receptor, CB1 (Central receptor) and CB2 (Peripheral receptor)

15 respectively (12-13). CB1 and CB2 have been cloned and characterized from mammalian tissues, the main difference between them being their tissue expression pattern (14).

CB1 receptors are ubiquitously located, with their highest presence found in the central nervous system (basal ganglia, hippocampus, cerebellum and cortex) where they mediate cannabinoid psychoactive effects (15-16). CB1 receptors are also present in peripheral nerve terminals, as well as in extra-neural tissues such as testis, uterus, vascular endothelium, eye, spleen, ileum and in adipocytes (16). CB2 receptor expression is mostly restricted to particular elements of the immune system (enriched area of B lymphocyte) (17-18). The human CB2 receptor shows 68% amino acid homology with the CB1 receptor in the trans-membrane domains and a 44% overall homology (17).

Cannabinoid receptors and their endogenous ligands termed as the endocannabinoid system have been used as putative molecular targets for the treatment of various diseases, including neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease,

Huntington’s disease, etc.), neuropathic and inflammatory pain, glaucoma, multiple sclerosis, cardiovascular disorders and obesity etc (13).

Recently cannabinoid’s role has been explored in the area of cancer research. Cancer is caused by uncontrolled proliferation of cells and the ability of these cells to invade into other tissues and spread. Anti-cancer agents function as apoptotic, cell cycle defective or

DNA damage agents. A major discovery in cancer in cannabinoid use in cancer treatment is its ability in targeted killing of tumors.

Several preclinical studies suggest that ∆9-THC, other naturally occurring cannabinoids, synthetic cannabinoid agonists and endocannabinoids have anti-cancer effects in vitro against lung carcinoma, gliomas, thyroid epithelioma, lymphoma, skin carcinoma, uterine carcinoma, breast cancer, prostate carcinoma, pancreatic cancer and neuroblastoma (12).

These findings were also supported by in vivo studies and the majority of effects of cannabinoids are mediated via CB1 and CB2. The transient receptor potential vanilloid type 1 (TRPV1) has been described as an additional receptor target for several cannabinoids. In addition, the palliative effects of cannabinoids include inhibition of nausea and emesis which are associated with chemo- or radiotherapy, appetite stimulation, pain relief, mood elevation and relief from insomnia in cancer patients.

Synthetic THC (Marinol, Dronabinol) and its derivative nabilone (Cesamet), as well as

Sativex, have been approved in several countries to control nausea and cancer-related pain in cancer patients undergoing chemotherapy (19-20). In this review article we focused on the role of cannabionds in different cancer types and the respective signaling pathways.

2.2 Cannabinoid and its receptor

Cannabinoids can be classified into three groups based on their source of production; endogenous cannabinoids (endocannabinoids), phytocannabinoids and synthetic cannabinoids (Fig.6) and their putative molecular targets (CB1 or CB2 receptor or

TRPV1) have been identified (Table 3). The central and most of the peripheral effects of cannabinoids rely on CB1 receptor activation.

2.3 Endogenous cannabinoids

Endogenous cannabinoids which are produced in our body include lipid molecules containing long-chain polyunsaturated fatty acids, amides, esters and ethers that bind to

CB1 or CB2 receptors. Several pharmacological evidences show that endocannabinoids also exert biological effects through non-CB1/CB2 receptors (21). Endocannabinoids mainly act as neuromodulators or retrograde messengers which affect the release of various neurotransmitters in the peripheral and neural tissues (22). They also play important role in inflammation, insulin sensitivity, and fat and energy metabolism.

Inhibition of endocannabinoids may be a tool in reducing the prevalence of metabolic syndrome(23).

Two of the best characterized endocannabinoids are N-arachidonoylethanolamine (AEA- anandamide) and 2- arachidonoylglycerol (2-AG) which affect our mood, appetite, pain sensation, inflammation response, and memory (15, 24).

Anandamide which was isolated from porcine brain in 1992 was shown as the first brain metabolite, to function as a ligand for CB1, (15). 2-arachidonoyl-glycerol which was isolated from canine gut acts through both CB1 and CB2 receptors (24-25). Palmitoyl- ethanolamide, or N-(2-Hydroxyethyl) hexadecamide (N-acyl-ethanolamide) is co-

18 synthesized with anandamide in all tissues and acts through CB2 (26-28). Other unsaturated fatty acid ethanolamides like Docosatetraenylethanolamide and Homo-γ- linoenylethanolamide act as agonists for the neuronal CB1 receptor (28-29). Another putative endogenous cannabinoid, oleamide, or cis-9-octadecenoamide, has also been isolated and shown to have similar actions to anandamide in the behavioral rodent tests

(30).

2.4 Phytocannabinoids

Phytocannabinoids are only known to occur naturally in significant quantity in the cannabis plant, and are concentrated in a viscous resin that is produced in glandular structures known as trichomes. ∆9-THC, cannabidiol (CBD) and cannabinol (CBN) are the most prevalent natural cannabinoids (31).

∆9-THC binds with similar affinities for both CB1 and CB2 receptors at submicromolar concentration. It behaves as a CB1 receptor partial agonist and CB1/CB2 receptor antagonist (32). ∆8-THC has similar affinities for CB1 and CB2 receptors as like ∆9-THC

(33).

Other common cannabinoids are cannabidiol (CBD), Cannabigerol (CBG),

Cannabichromene (CBC), Cannabicyclol (CBL), Cannabivarin (CBV),

Tetrahydrocannabivarin (THCV), Cannabidivarin (CBDV), Cannabichromevarin

(CBCV), Cannabigerovarin (CBGV), Cannabigerol Monoethyl Ether (CBGM). 19

Figure 6: Cannabinoids and their classification. This figure illustrates how cannabinoids are divided into three main categories according to their availability in nature.

2.5 Synthetic cannabinoids

Synthetic cannabinoids have been extensively used as a pharmacological agent, both in vitro and in vivo, to obtain more detailed insight of cannabinoid action, in order to evaluate their potential clinical use. They showed both antineoplastic and protumoral activity, depending on type of agonist, target tissues, route of administration, doses and duration of the treatment (34-35). Synthetic cannabinoids are classified on the basis of chemical structure of molecules and they are capable of a more selective activation of cannabinoid receptor (36).

20 a) Classical cannabinoids: Compounds isolated from the plant C. sativa or synthetic analogs of these compounds fall into this category. HU-210, Δ9-THC, Δ8-THC and desacetyl-L-nantradol are synthetic cannabinoids which behave as CB1/CB2 receptor agonists (lack of CB1/CB2 selectivity. The most psychotropic component of the C.sativa plant is Δ9-THC which shows affinity for both the cannabinoid receptors. Increased affinity of HU-210 is due to replacing pentyl side chain of Δ8-THC with a dimethylheptyl group. Other CB2-selective agonists that have been synthesized by structurally modifying

THC molecule are JWH-133, JWH-139, and HU-308 and L-759633 and L-759656 which was effective in nanomolar range (37-39).

b) Nonclassical Cannabinoids: These are a family of AC-bicyclic and ACD-tricyclic cannabinoid analogs. Furthermore bi-cyclic analog, CP55940, an important cannabinoid agonist has similar affinity for CB1 and CB2 receptors. Also, it is highly potent in vivo.

CP55244 and CP47497 are other cannabinoids that fall in this category.

c) Aminoalkylindoles: These are a family of aminoalkylindoles with cannabimimetic properties. R-(+)-WIN55212 is the most well known compound in this series. It exhibits high affinity for both cannabinoid receptors, but more selective for CB2. It has similar pharmacological effects like THC in vivo. JWH-015 and L-768242 also show affinity towards CB2 than R-(+)-WIN55212 (40).

21 d) Eicosanoids: Anandamide, which is an endogenous cannabinoid ligand was originally discovered in mammalian brain and other tissues and acts similar to THC.

Methanandamide, its R-(+)-isomer is nine times more CB1 specific than the S-(+)-isomer

(41). 2-arachidonoylglycerol, another well studied endocannabinoid has both CB1 and

CB2 affinities. Other compounds are arachidonyl-2-chloroethylamide (ACEA) and arachidonylcyclopropylamide (ACPA).

e) Others: These represent diarylpyrazole compounds which function antagonistic to cannabinoid receptors (42). SR141716A is a potent CB1 antagonist and SR144528 is a

CB2 antagonist (43-44). AM251 and AM281 are analogs of SR141716A which block

CB1 receptor-mediated effects.

2.6 Cannabinoid receptor mediated signaling in cancer

The widespread distribution of cannabinoid receptors (CB1/2,TRPV1) regulate a variety of central and peripheral physiological functions, including neuronal development, neuromodulatory processes, energy metabolism as well as cardiovascular, respiratory, reproductive functions. CB1/2 receptors are also responsible for proliferation, motility, invasion, adhesion and apoptosis of cancer cells both in vitro and in vivo (Table 4).

CB1/2 receptor activation leads to various events like affecting Ca2+ and K+ channels, modulation of adenyl cyclase and cyclic AMP (c-AMP) levels in most tissues and models, regulation of members mitogen activated protein kinase family (MAPKs), like

22 extracellular signal regulated kinase-1 and -2 (ERK1/2), p38, MAPK and c-Jun N terminal kinase (JNK) (45-46) as shown in Fig.7.

Figure 7: Cannabinoid mediated signaling in cancer cells. Cannabinoids activate CB1 or

CB2 receptor which in turn modulates diverse signaling targets.

2.7 Role of cannabinoids in regulation of cancer growth

One of the important aspects of an effective anti-tumor drug is its ability to inhibit proliferation of cancer cells. Cancer cells proliferate rapidly in uncontrolled manner.

Also, these cells escape death mechanism which a normal cell undergoes like apoptosis.

Apoptosis is a kind of programmed cell death (PCD) mechanism which involves activation of caspase dependent and independent pathways (47). Cannabinoids have been proved to be anti-proliferative and apoptotic drugs.

This section comprises of the detailed role of cannabinoids in modulation of tumor proliferation, cell cycle and apoptosis in various cancer types.

2.7.1 Cannabinoids and breast cancer

Breast cancer is one of the most common human malignancies and the second leading cause of cancer-related deaths in women, and its incidence in the developing world is on the rise (48-49). It represents approximately 30% of newly diagnosed cancers each year.

It is mainly classified into three main subtypes according to their molecular profiles: hormone receptor-positive, HER2-positive (ErbB2-positive, a member of EGFR family) and triple-negative tumors (50-51). Cannabinoid-based medicines have been useful for the treatment of these three breast cancer subtypes.

CB1 and CB2 receptor expression has been described in different breast cancer tissue and cell line by immunohistochemistry, RT-PCR and western blot. CB1 expression was detected in 14% of human Her-2 positive breast cancer tumor tissue and 28% of human breast carcinoma (34, 52). But no correlation between CB1 expression and ErbB2 expression was found (52). CB1 receptors are also present in different breast cancer cell lines (MCF-7, T-47D, MDA-MB-231, TSA-E1, MDA-MB-468) and in human breast tissues (34, 53-59). By contrast, CB2 immunoreactivity was detected in 72% of human breast tumor tissue and 91% of ErbB2-positive tumor tissue, suggesting a link between

CB2 and ErbB2-expression (52).The expression of CB2 receptor was also analyzed in different breast carcinoma cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB-468,

EVSA-T, SkBr3) and human breast tissues (34, 52-54, 57, 59-60). The putative novel cannabinoid receptor subtype GPR55 was highly expressed in a MDA-MB-231 cells, but it is expressed at lower (30-fold) levels in MCF-7 cells (61).

Cannabinoids modulate the growth of hormone sensitive breast cancer cells as shown in

Fig.8 and 9. JWH-O15 inhibits hormone sensitive breast cancer metastasis by modulating

CXCL12/CXCR4 signaling axis (35, 62). Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis.

Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Breast tumor cells express FAAH abundantly.

Inhibition of FAAH (siRNA-FAAH or FAAH inhibitor URB597) induced cell death by activating nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway and heme oxygenase-1 (HO-1) induction and transcription (63). 25

Anandamide inhibits basal and nerve growth factor (NGF) induced proliferation of MCF-

7 and EFM-19 cells in culture through CB1 receptor and ∆9-THC inhibits 17beta- estradiol-induced proliferation of MCF7 and MCF7-AR1 cells (53, 64-66). ∆9-THC also inhibits cell proliferation of ER-/PR+ breast cancer cells. The effects of anandamide and

∆9-THC were mediated by blocking transition from one phase of cell cycle to another,

G1-S and G2-M respectively (57, 64, 67-68). Cell cycle arrest is responsible for apoptotic cell death. The analog of anandamide, Met-F-AEA reduces MDA-MB-231 proliferation by arresting cells in the S phase of the cell cycle (68).

Figure 8: Modulatory effect of cannabinoids on hormone sensitive breast cancer cells.

Cannabinoids are involved in receptor dependent/independent regulation of various hallmarks of breast cancer like proliferation, migration, invasion, etc. 26

Anandamide inhibits adenylyl cyclase (AC) and thus activating the Raf-1/ERK/MAP pathway in ER+/PR+ breast cancer cells whilst THC activates the transcription factor

JunD to finally execute action towards apoptosis in ER-/PR+ breast cancer cells (57, 64,

67). One study shows that anandamide inhibits proliferation of MDA-MB31 cells by modulating Wnt/β-catenin signaling pathway (69). This effect is occurred by inhibition of the cyclin-dependent kinase CDK2 (58, 68).

Synthetic cannabinoids containing naphthoylindole, JWH-018, JWH-073, JWH-122 and

JWH-210 and of one benzoylindole AM-694 shows anti-estrogenic property in MCF-7 cells (70). Cannabinoid induced signaling in ER+ breast cancer cells is shown in Fig.3.

WIN 55,212-2 and JWH-133 also produce an inhibition of MDA-MB-231 proliferation by blocking the progression trough the cell cycle, G1 to S phase transition and induced apoptosis (34). The anti-proliferative effect of WIN 55,212-2 and JWH-133 is validated in both in xenograft-based and PyMT genetically engineered model of triple-negative breast cancer modulate through the COX-2/PGE2 signaling pathway (34). JWH-015 also reduces breast cancer-induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression.

Figure 9: Modulatory effect of cannabinoids on HER-2 +ve and Triple –ve breast cancer cells. Cannabinoids inhibit key signaling targets in triple negative breast cancer which has worse prognosis in patients.

CBD inhibits AKT and mTOR signaling as well as decreased levels of phosphorylated mTOR and 4EBP1, and cyclin D1. CBD enhances the interaction between beclin1 and

Vps34; it inhibits the association between beclin1 and Bcl-2 (71). LPI stimulates proliferation and this effect was blocked by CBD (61). Thus, cannabinoids along with

COX-2 inhibitors or other chemotherapeutic agents may represent as novel

28 chemopreventive tools for the treatment of breast cancer. Fig.4 shows the effect of cannabinoids on HER-2 and triple negative breast cancer pathway.

2.7.2 Cannabinoids and prostate cancer

Prostate cancer is the most common malignancy among men of all races and is one of the leading causes of cancer death in this population. CB1 and CB2 expression levels were higher in prostate cancer tissues and several cell lines including PC-3, DU-145, LNCaP,

CWR22Rv1, CA-HPV-10 as compared with normal prostate epithelial cells (53, 72-79).

Moreover, the putative cannabinoid receptor GPR55 is also expressed in PC-3 and DU-

145 cells (80). ∆9 –THC, WIN-55,212-2, R(+)-Methanandamide , Cannabidiol (CBD),

Anandamide, JWH-015, HU120, 2-AG and its stable analogue noladin have exerted anti- proliferative, apoptotic and anti-invasive effects in different prostate cancer cells both in vitro and in vivo (53, 76-77, 81-85). ∆9 –THC induced apoptosis via a receptor- independent manner whilst in another study, the same group reported that activation of cannabinoid receptors in PC-3 cells stimulated the PI3K/Akt pathway with sequential involvement of Raf-1/ERK1/2 and nerve growth factor induction (74, 76). Treatment of

WIN-55,212-2 resulted in sustained activation of ERK1/2 and inhibition of AKT, which was associated with the induction of phosphatases (82, 86). CBD also mimicked the same effect in LNCaP cells as WIN-55,212-2 (86). The effects of anandamide in LNCaP,

DU145, and PC3 cells were mediated through down-regulation of epidermal growth factor receptor (EGFR) and accumulation of ceramide (85). 29

JWH-015 triggered a de novo synthesis of ceramide, which induced cell death, followed by JNK (c-Jun N-terminal kinase) activation and Akt inhibition (84). Effects of R(+)-

Methanandamide and JWH-015 were rescued by treatment with SR 144528 in PC-3 cells

(84, 87). Interestingly, (R)-methanandamide was shown to have a mitogenic effect on

LNCaP cells at very low doses (54, 83). FAAH is a serine hydrolase that metabolizes N- acylethanolamines including AEA, OEA and PEA to fatty acids plus ethanolamine. A recent report showed that FAAH is also over-expressed in prostate cancer cells and the inhibition of FAAH can enhance the survival of cancer patient (88-89).

2.7.3 Cannabinoids and lung cancer

Lung cancer has one of the highest mortality rates among cancer-suffering patients.

Cannabinoids (CBs) could halt tumor development without side effects via specific targeting of CB1/CB2 receptor. Studies suggest the involvement of COX-2 and PPAR-γ in CBD’s proapoptotic and tumor-regressive action in A549, H460 cells and primary cells from a patient with lung cancer (90). Moreover, CBD caused up-regulation of COX-

2 and PPAR-γ in tumor tissue and tumor regression in A549-xenografted nude mice (90).

JWH-133 induced anti-proliferative potential in A549 cell line via DNA fragmentation

(91). Recently, we published results on the role of FAAH in regulating the effects of

AEA in NSCLC. We showed that blocking FAAH increases the levels of AEA, which in turn inhibits EGFR signaling pathway, ultimately leading to cell cycle arrest and

30 apoptosis. These results generate a rationale for further in vivo efficacy studies with this compound in preclinical cancer models.

2.7.4 Cannabinoids and skin cancer

Melanoma is the mainly cause of skin cancer–related deaths worldwide. CB1 and CB2 receptors are expressed in normal skin and skin tumors of mice and humans (92).

Activation of CB1/CB2 receptors induced the apoptotic death of tumorigenic epidermal cells, without affecting the nontransformed epidermal cells. WIN-55,212-2 or JWH-133 induced anti-proliferative effect in epidermal cell lines (PDV.C57 and HaCa4) and reduces malignant tumors in nude mice (92). WIN-55,212-2 or JWH-133 induced G1 cell cycle arrest on melanoma cells, via inhibition of p-Akt and hypophosphorylation of the pRb retinoblastoma protein tumor suppressor (92).

2.7.5 Cannabinoids and pancreatic cancer

Pancreatic cancer is one of the most aggressive and devastating human malignancies.

CB1 and CB2 receptors were expressed in normal and pancreatic cancer tissues, analyzed by RT-PCR (93). Cannabinoid receptors on pancreatic cancer cells may affect prognosis and pain status of PDAC patients (93). Cannabinoid administration leads to apoptosis of pancreatic tumor cells via CB2 receptor and ceramide-dependent up-regulation of p8 and

ATF-4 and TRB3 stress–related genes (94). Another study showed that CB1 receptor antagonist AM251–induced cell death in pancreatic MIAPaCa-2 cells occurred via receptor-independent manner (95).

2.7.6 Cannabinoids and bone cancer

Chondrosarcoma and osteosarcoma are the most frequent primary bone cancers (96).

Bone metastases are a frequent complication of cancer and the most frequent type of pain related to cancer. Breast cancer and prostate cancer mainly metastasize to bone which act as a fertile soil for the growth of secondary tumors (97). The skeletal endocannabinoid system plays a significant role in regulating bone mass and bone turnover. The expression levels of CB1 and CB2 receptors were analyzed in bone cancer patient using immunohistochemistry (98). Bone metastatic patient has severe pain so cannabinoids can attenuate pain and hyperalgesia (99). Sativex is the combination of delta-9-tetrahydrocannabinol and cannabidiol, used to treat pain in cancer (99). WIN55,212-2 induces apoptosis in the NCTC-2472 sarcoma cell line and AM1241 produced a reduction in bone loss in bone tumor animal model (NCTC-2472 cell line injected in to femur of mice) (100-101). Effects of subcutaneously administered WIN55,212-2 on weight bearing and mechanical hyperalgesia were consistent with cannabinoid receptor mediated anti-nociception (100). WIN55,212-2 also attenuates tumour-evoked mechanical hyperalgesia following local (intraplantar) administration through activation of CB1 and

CB2 receptors (102). Injection of CP55 940 produced anti-nociceptive properties in the 32 tail flick test and suppressed mechanical hyperalgesia in NCTC-2472 or melanoma B16-

F10 xenografted bone tumor model (103). Indeed, intraplantar administration of AEA reduces mechanical hyperalgesia, URB597 increases AEA levels and decreases hyperalgesia in a model of calcaneous bone cancer pain (98). However, intrathecal administration of either URB597 or MGL (URB602) inhibitors failed to produce antinociception when tested for spontaneous flinches, limb use and weight bearing (104).

Moreover, the CB1 agonist arachidonoyl-2-chloroethylamide (ACEA) produces anti– nociceptive properties following intrathecal administration in this model; ACEA suppressed spontaneous flinches and increased limb use and weight bearing (104).

AM1241 produces significantly reduced bone loss and decreased the incidence of cancer- induced bone fractures (101, 105). Administration of JWH-015 and AM1241 attenuated tumor-evoked tactile allodynia and thermal hyperalgesia by reducing NR2B-dependent activity (105-106). CB2 agonist, JWH-015 reduced breast cancer induced bone pain, bone loss, and breast cancer cell proliferation via cytokine/chemokine suppression in murine mammary cell line implanted into the femur intramedullary space (107). JWH-

015 increased survival without the major side effects of current therapeutic options.

2.7.7 Cannabinoids and glioma

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer, exhibit high resistance to conventional chemotherapies.

In glioblastoma endothelial cells, CB1 and CB2 receptors were present in about 38% and 33

54% of the cells respectively, analyzed by immunohistochemistry. CB2 expression levels were higher in glioblastoma tissues in comparison to CB1. Selective CB2 agonists may become important targets for the treatment of glioma. Cannabinoids inhibit tumor growth in animal models by inducing apoptosis of tumor cells and impairing tumor angiogenesis.

Administration of ∆9-THC and JWH-133 inhibits MMP-2 expression in in vivo model of glioma (108-110). The growth inhibitory effect of these cannabinoids is prevented by blocking ceramide synthesis, and the expression of the stress protein p8 (109-110). Both

∆9-THC and WIN-55,212-2 resulted in sustained activation of ERK1/2 and inhibition of

AKT (111). Furthermore, ∆9-THC induced eukaryotic translation initiation factor 2alpha

(eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the

Akt/mammalian target of rapamycin complex 1 (mTORC1) axis (112). The activation of this pathway was necessary for the antitumor action of cannabinoids in vivo (112). In contrast to that CBD treatment induces apoptosis in glioma cells in vitro and tumor regression in vivo through activation of caspases and reactive oxygen species via receptor-independent manner Furthermore, studies revealed that CBD induced TRPV2- dependent Ca2+ influx which triggers the drug uptake and synergizes with cytotoxic agents to induce apoptosis of glioma cells (113). Authors thought that CBD which do not specifically interact with CB1/CB2 receptors, can modulate the activity of ∆9-THC. On that basis Marcu et al determined the growth inhibitory effect of CBD in combination with ∆9-THC in the U251 and SF126 glioblastoma cell lines (114). Furthermore, the combined treatment of Δ9-THC and temozolomide (TMZ) exert a strong antitumoral 34 action in glioma xenografts by inducing autophagy (115). The submaximal doses of ∆9-

THC and CBD in combination with TMZ produced a strong antitumoral action in both

TMZ-sensitive and TMZ-resistant tumors (115).

Treatment of KM-233 (novel cannabinoid ligand) caused a time dependent change in the phosphorylation profiles of MEK, ERK1/2, Akt, BAD, STAT3, and p70S6K in U87MG human GBM cells (116). At 12mg/kg daily dose of KM-233 for 20 days revealed around

80 % reduction in tumor size in the orthotopic model of U87MG (116). Glioma cells develop resistance to cannabinoid treatment due to the upregulation of Amphiregulin

(EGFR family ligand) and the growth factor midkine (Mdk) (117-118). Amphiregulin expression was associated with increased ERK activation and Mdk mediated its protective effect through ALK which interferes with autophagic cell death (119). The silencing of amphiregulin and Mdk or ALK pharmacological inhibition can overcome drug resistance of glioma to cannabinoids antitumoral action. Furthermore, to improve the efficacy of cannabinoids action, microencapsulation methods were used which facilitates a sustained release of the two cannabinoids for several days (120).

Administration of CBD- and THC-loaded poly-ε-caprolactone microparticles reduced tumor growth, cell proliferation and increased apoptosis in mice bearing glioma xenografts with the same efficacy than a daily local administration of these drugs in solution (120).

2.7.8 Cannabinoids and lymphoma

CB1 and CB2 receptors were over-expressed in mantle cell lymphoma (MCL), and B cell non-Hodgkin lymphoma (121-122). ∆9-THC inhibits cell viability and increased apoptosis both in vitro in EL4 and MCL cells and EL4 tumor bearing mice. In next studies the combination of ∆9-THC and other cytotoxic agents induced apoptosis in leukemia cells by MAPK/ERK pathway (121, 123). In addition R(+)-methanandamide and WIN-55,212-2 induced apoptosis in MCL cells, was associated with ceramide accumulation and p38, depolarization of the mitochondrial membrane, and caspase activation (124). R(+)-methanandamide also induced apoptosis in CLL cells (125). In contrast, cannabinoids decreased cell viability as assessed by metabolic activity. The persistent expression of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 II (LC3 II) and p62, as well as the lack of protection from chloroquine, indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process, distinguishing from classical autophagy (126). Paraptosis-like cell death-a third type of a programmed cell death occurred in response to cannabinoids

(126).

2.7.9 Cannabinoids and oral cancer

Oral cancer is mainly occurs in the mouth including lips, tongue and throat. Smoking, tobacco chewing and alcohol consumption increases the incidence of oral cancer.

Radiation therapy and surgery is the common treatment for oral cancer. ∆9-THC induced apoptosis in oral squamous cell carcinoma (OSCC), a malignant form of oral cancer

(127).

2.7.10 Cannabinoids and head and neck cancer

Marijuana smoking increases the incidence of head and neck cancer in young people but its constituent, cannabinoids have anti-tumor properties. One study reports that moderate marijuana use is associated with reduced risk of HNSCC (128).

2.7.11 Cannabinoids and thyroid carcinoma

Thyroid carcinoma is the most aggressive form which occurs in thyroid gland. IL-12 gene transfer in to anaplastic thyroid carcinoma cell line (ARO) has anti-tumorigenic effect

(129). This effect was observed due to the activation of cannabinoid receptor.

Furthermore they have reported that CB2 agonist JWH-133 and CB1/CB2 agonist WIN-

55,212-2 induced apoptosis in ARO and ARO/IL-12 cells (129). 2-methyl-2'-F- anandamide (Met-F-AEA) also induced apoptosis in thyroid carcinoma cells via activation of p53 and p21 mediated pathway (130).

2.8 Role of cannabinoids in pro-metastatic mechanisms like angiogenesis, migration and invasion

Migration and invasion are characteristic features of cancer cells. Carcinoma cells that are invasive have higher migratory potential which helps them to disseminate into the surrounding tissues and spread to other organs, ultimately leading to metastasis (131).

Angiogenesis, which involves growth of new vasculature has been shown to be closely related to cancer metastasis. Developing novel anti-invasive and anti-angiogenic targets would be more effective in inhibiting metastasis at earlier stage (132).

In breast cancer, Met-F-AEA leads to the inhibition of the focal adhesion kinase (FAK) and RhoA-ROCK pathways (133). JWH-015 reduces CXCL12-induced cell migration and invasion of a highly metastatic MDA-MB-231-derived cell line by inhibiting ERK and cytoskeletal focal adhesion and stress fiber formation (134). Novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8 reduced tumor growth by targeting

VEGF-mediated angiogenesis signaling in MCF-7 and MCF-7 Tam resistant cells (135).

Cannabidiolic acid (CBDA) inhibits migration of MDA-MB-231 cells via inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase,

RhoA (136). Lysophosphatidylinositol (LPI), the putative endogenous ligand for GPR55, also stimulates cell migration and invasion in a MDA-MB-231 cell line and its effect is blocked by pretreatment with cannabidiol (CBD) (61, 137).

Recent study shows that THC and JWH-133 exert anti-proliferative, pro-apoptotic, anti- angiogenic, and anti-invasive effects in both in vitro and in vivo models of ErbB-2 breast 38 cancer by modulating AKT, phospho-S6 ribosomal protein, MMP-2 and MMP-9 expression levels shown in Fig.3 (52, 138-139).

In lung cancer, CBD inhibits invasion of A549 cells both in vitro and in vivo that was accompanied by up-regulation of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and decreased expression of plasminogen activator inhibitor-1 (PAI-1) (140-141). P38 and ERK1/2 were identified as upstream targets for up-regulation of TIMP-1 (141). But recent report suggests that CBD induced TIMP-1 via upregulation of intercellular adhesion molecule-1 (ICAM-1) (142). JWH-133 induced anti-angiogenic potential in

A549 cell line via inhibition of MMP-2 secretion respectively (91).

In skin cancer, treatment of WIN-55,212-2 or JWH-133 caused impairment of tumor vascularization and decreased expression of proangiogenic factors such as VEGF, placental growth factor, and angiopoietin-2 (92).

In glioma, (143), one study reveals that CBD also inhibits angiogenesis by modulating

MMP-2 pathway and Id-1 gene expression in glioblastoma cells (144-145).

Administration of CBD- and THC-loaded poly-ε-caprolactone microparticles reduced tumor growth and angiogenesis in mice bearing glioma xenografts with the same efficacy than a daily local administration of these drugs in solution (120).

2.9 Role of cannabinoids in cancer metastasis

Met-F-AEA, WIN 55,212-2, JWH-133 and JWH-015 inhibit the migration and invasion of MDA-MB231 breast cancer cells to distant sites such as lung (58, 133, 146-148). CBD inhibits cell proliferation and invasion of 4T1 cells (mammary metastatic cell line) and reduces primary tumor volume as well as lung metastasis in 4T1-xenografted orthotopic model of nude mice (149-150). This anti-metastatic effect was mediated by downregulation of Id-1 (a basic helix-loop-helix transcription factor inhibitor), ERK and also by inhibiting the ROS pathway. Furthermore, CBD reduced the number of metastatic foci in 4T1- tail vein injected syngenic model. In lung cancer, JWH-015 and Win55,212-

2 inhibit in vitro chemotaxis, chemoinvasion and in vivo tumor growth and lung metastasis via inhibition of AKT, matrix metalloproteinase 9 expression (MMP-9), but the pretreatment of CB1/CB2 selective antagonists, AM251 and AM630 antagonized their effects (151). ∆9-THC inhibits growth of Lewis lung adenocarcinoma via inhibition of DNA synthesis and it suppresses growth and metastasis of A549 and SW-1573 (human lung cancer cell lines) both in vitro and in vivo due to inhibition of epidermal growth factor–induced phosphorylation of ERK1/2, c-Jun-NH2-kinase1/2 and Akt (152-153).

2.10 Role of cannabinoids in stemness and cancer

Cancer stem cells (CSC) are part of the tumor cell population. Though they might be very less in number, they have the ability to self renew and replicate to produce enormous

40 cancer cell types. CSCs have been shown to be drug resistant with higher invasive and metastatic potential (154).

Studies show that cannabinoid receptors are involved in differentiation of neural progenitors from ectoderm and hematopoietic progenitors from mesoderm. CB1 and CB2 receptor activation modulate proliferation and differentiation of daughter progenitors.

Cannabinoids- HU210, WIN55,212-2, AEA and methAEA induced concentration dependent cytotoxicity in P19 embryonal carcinoma (EC) cells (155). It involved partial regulation by cannabinoid receptors leading to oxidative stress, necrosis coupled with apoptosis. Both CB1 and CB2 receptors are expressed in glioma stem like cells (GSC).

HU-210 and JWH-133 helped in neural differentiation of GSC and blocked GSC mediated gliomagenesis (156). These open further investigation on the function of cannabinoids and the link between stem cell and tumor progression.

2.11 Role of cannabinoids in energy metabolism and cancer

One of the important by-products in energy metabolism is a set of compounds called

Reactive Oxygen Species (ROS) which is produced from mitochondria and consists of

- - H2O2, superoxide O2 , hydroxyl radical O2 , etc. Increased ROS production has been associated with triggering of apoptosis (157).

CBD modulates ERK and reactive oxygen species (ROS) pathways, which lead to downregulation of Id-1 expression. Id-1, an inhibitor of basic helix-loop-helix transcription 41 factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers (149-150). Arachidonoyl cyclopropamide (ACPA) or GW405833

(GW) induced AMPK mediated autophagy in pancreatic adenocarcinoma cells is strictly related to the inhibition of energy metabolism through a ROS-dependent increase of the

AMP/ATP ratio (158). The combination of cannabinoids and gemcitabine, a nucleoside analogue used in cancer chemotherapy, synergistically inhibit pancreatic adenocarcinoma cell growth by a ROS-mediated autophagy induction without affecting normal fibroblasts

(159).

Cannabidiol (CBD)-induced endoplasmic reticulum stress mediated cell death of MDA-

MB231 breast cancer cells, with the coexistence of autophagy and apoptosis (71).

Recently one published report shows that ∆9-THC and ∆8-THC inhibited mitochondrial oxygen consumption rate via receptor independent manner in oral cancer cells (160). In primary lymphocytes, treatment with CBD induced caspase 8 induced apoptosis which was mediated by oxidative stress. Similar result has been reported in glioma cells where

CBD causes oxidative stress and higher enzymatic activities of glutathione reductase and glutathione peroxidase.

In NSCLC cell line H460, agonists AEA, THC and HU-210 modulated the activity of mitochondrial complexes I and II-III, decreasing the mitochondrial membrane potential.

∆9-THC and cannabidiol acted synergistically to inhibit cell proliferation, modulations of the cell cycle and induction of reactive oxygen species and apoptosis as well as specific modulations of extracellular signal-regulated kinase and caspase activities in

42 glioblastoma (114). KM-233 induced mitochondrial depolarization, cleaved caspase 3, significant cytoskeletal contractions, and redistribution of the Golgi-endoplasmic reticulum structures in U87MG human GBM cells (116).

2.12 Role of cannabinoids in immune environment and cancer

Cancer is a type of inflammatory disease, where immune cells infiltrate into the tumor site and secrete factors which enhance the prospects of proliferation, angiogenesis and metastasis (161). Hence, it is important to identify anti-cancer agents that target the immune related cancer environment.

In glioma, WIN-55,212-2 caused accumulation of ceramide which is essential for cell death and it also had anti-inflammatory effects (162). WIN55,212-2, abolished the PGN- activated cell growth which activates a number of inflammatory pathways, including NF-

κB (aggravates tumors) and this effect was reversed by CB1 antagonist AM281 but not by the CB2 antagonist, AM630 (163). Anandamide reduces proliferation and production of cytokines like IL-2, TNF-α and INF-γ in human T lymphocytes by activating CB2 receptor (164). In astrocytoma and glioblastoma cells, WIN55,212-2 inhibited IL-1 mediated activation of adhesion molecules and chemokines like ICAM-1, VCAM-I and

IL-8, which was receptor independent (165). In murine T cells, cannabinol decreased IL-

2 production by inhibiting nuclear factor of activated T-cells (NF-AT) and activator

43 protein-1 (AP-1) (166). In CD8+ T lymphocytes, JWH-133 downregulated SDF-1 induced migration in CB2 receptor dependent manner (167).

Table 3: Cannabinoid’s structure and its role in different physiological processes.

Cannabinoid’s name Structure Role (Abbreviation) and its target Anandamide (AEA) CB1 Analgesic, antiemetic, appetite agonist stimulant, tumour growth inhibitor (168). 2-arachidonoyl-glycerol Analgesic, antiemetic, appetite (2-AG) stimulant, tumour growth CB1/CB2 agonist inhibitor (168). Palmitoyl-ethanolamide neuromodulatory and (PEA) immunomodulatory (169). CB2 agonist Docosatetraenyl neuromodulatory and ethanolamide immunomodulatory (169). CB1 agonist Homo-γ- neuromodulatory and linoenylethanolamide immunomodulatory (169). CB1 agonist Oleamide CB1 agonist neuromodulatory and immunomodulatory (169).

∆9-tetrahydrocannabinol Analgesic, antiemetic, appetite (∆9-THC) stimulant tumour growth CB1/CB2 agonist inhibitor (168).

(Continued.)

(Table 3. continued.)

∆8-tetrahydrocannabinol Anti-tumor agent, inhibitors of (∆8-THC) CB1/CB2 mitochondrial O2 consumption agonist in human sperm, antiemetic, appetite stimulant (160, 170- 172). cannabidiol (CBD), CB1 Anti-tumor agent, attenuate agonist catalepsy, immunosuppressive, inflammatory or anti- inflammatory agent (depends upon used concentration of drug), antipsychotics (173- 177) Cannabigerol (CBG), multiple sclerosis, antiemetic, anti-inflammatory agent, treatment for neurological disorder (178-180)

Cannabichromene anti-inflammatory agent, (CBC), treatment for neurological disorder, hypomotility,

antinociception, catalepsy, and hypothermia (181-183) Tetrahydrocannabivarin Hepatic ischaemia, anti- (THCV), inflammatory (184-185)

Cannabigerovarin Anti-inflammatory (186) (CBGV),

HU-210 Analgesic, multiple sclerosis, CB1/ CB2 Nonselective neuroprotective (168) agonist

CP-55,940 Anti-cancer agent, Analgesic, CB1/ CB2 Nonselective antiemetic, appetite stimulant agonist

(Continued.)

(Table 3. continued.)

R-(+)-WIN 55,212-2 Analgesic, antiemetic, appetite CB1/ CB2 Nonselective stimulant, tumour growth agonist inhibitor, multiple sclerosis (168) JWH-015 Anti-tumor,anti-inflammatory, CB2 selective agonist antiemetic (187)

JWH-133 Neurological disorders, Anti- CB2 selective agonist cancer (91, 188)

JWH-139 Analgesic, antiemetic, appetite CB2 selective agonist stimulant tumour growth inhibitor (168)

HU-308 Tumour growth inhibitor (in CB2 selective agonist glioma, skin carcinoma, lymphoma (168)

CP55940 Analgesic, antiemetic, appetite CB/CB2 agonist stimulant, tumour growth inhibitor, multiple sclerosis (168) R-(+)-methanandamide Analgesic, antiemetic, appetite CB1 agonist stimulant tumour growth inhibitor (168) AM251 CB1 antagonist Metabolic syndrome (189)

AM281 CB1 antagonist Improves recognition loss induced by naloxone in morphine withdrawal mice, various pharmacological property (190-191)

Table 4: Role of cannabinoid in different cancers and its associated signaling.

Cannabinoids Anti-cancer effect and its mechanism of action Anandamide 1)Breast cancer: (blocks G1 - S phase transition)-Regulates Raf- 1/ERK/MAP pathway, Wnt/β catenin signaling 2)Prostate cancer: Regulates EGFR pathway THC 1)Breast cancer: (block G2 - M phase transition)- Activates the transcription factor JunD Anti-tumor action in MMTV-neu mice via inhibition of AKT Anti-invasive effect-modulate MMP-2/MMP-9 pathway 2)Prostate cancer: PI-3/AKT and Raf-1/ERK1/2 pathway Mitogenic effect at low doses. 3)Lung cancer: ERK1/2, JNK and AKT pathway Mitogenic effect at low doses. 4)Glioma: MMP-2pathway, ER stress mediated autophagy 5)Lymphoma: MAPK/ERK pathway

2-AG 1)Breast cancer: Suppression of nerve growth factor Trk receptors and prolactin receptors 2)Prostate cancer: NF-κB/cyclin D and cyclin E, Suppression of nerve growth factor Trk receptors and prolactin receptors. 3)Glioma: Inhibition Ca(2+) influx 4)Bone cancer: Attenuates mechanical hyperalgesia HU120 1)Prostate cancer: AKT pathway

WIN-55,212-2 1)Breast cancer: Regulates COX-2/PGE2 signaling pathway 2)Prostate cancer: Sustained activation of ERK1/2 3)Skin cancer: Inhibits pro-angiogenic growth factor, AKT and pRB pathway 4)Glioma: Ceramide and NF-Κb pathway 5)Lymphoma: Ceramide and p38 pathway

(Continued.)

(Table 4. continued.)

R-(+)-MET 1)Breast cancer: decreased phosphorylation of focal adhesion– associated protein kinase and Src and tyrosine kinases involved in migration and adhesion 2)Prostate cancer: mitogenic effect at low dose

JWH-133 1)Breast cancer: inhibition of AKT-Regulate COX-2/PGE2 signaling pathway 2)Lung cancer: MMPs pathway 3)Skin cancer: G1 arrest-AKT pathway

Met-F-AEA 1)Breast cancer: S phase cell cycle arrest Regulates FAK/Src and RhoA-ROCK pathways

JWH-015 1)Breast cancer: CXCR-4/CXCL12 pathway 2)Prostate cancer: JNK/AKT signaling pathway

∆9-THC 1)Breast cancer: mitogenic effect in cells expressing low levels of CB1/CB2 receptors. 2)Prostate cancer: PI3K/Akt and Raf-1/ERK1/2 pathway Mitogenic at low doses 3)Lung cancer: EGFR/ERK1/2, c-Jun-NH2-kinase1/2, and Akt pathway. Mitogenic at low doses 4)Glioma: MMP-2 pathway

CBD 1)Breast cancer: ER stress/ERK and reactive oxygen species (ROS) pathways 2)Prostate cancer: ERK1/2 and AKT pathways 3)Lung cancer: up-regulation of TIMP-1 Cox-2 and PPAR-γ regulation 4)Cervical cancer: Up-regulation of TIMP1

CBDA 1)Breast cancer: PKA/RhoA pathway

AME1241 1)Bone cancer: Anti-nociception

Chapter 3

FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-

small cell lung cancer by downregulating the EGF/EGFR pathway

3.1 Introduction

The cannabinoid family is categorized into endogenous cannabinoids (produced inside the body), phytocannabinoids (plant derived) and synthetic cannabinoids which activate the specific G-protein coupled receptors CB1 and CB2. CB1 receptor is mainly expressed in the brain and CNS whereas CB2 receptor is expressed in immune system (14, 17, 152).

The use of cannabinoid agonists as anti-cancer agents has proven successful in various in vitro and in vivo cancer models such as glioma, breast, prostate, colon, leukemia and lymphoid tumors (34, 192-194). They have been shown to modulate various cell survival pathways such as the extracellular signal-related kinase (ERK), phosphoinositide 3- kinase (PI3K), p38 mitogen-activated protein kinase (p38 MAPK), protein kinase B

(AKT) and ceramide pathways (168, 195-196).

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the two well characterized endocannabinoids which are endogenous ligands for the cannabinoid receptors. Although endocannabinoids were initially studied for their neurological and psychiatric effects, there is increasing evidence of their contribution to inflammation and tumorigenesis

(197-198). AEA, which is mainly synthesized from phospholipids, is inactivated by enzyme fatty acid amide hydrolase (FAAH) mediated hydrolysis to arachidonic acid

(AA) and ethanolamine (EA), whereas 2-AG is hydrolyzed into AA and glycerol (88,

199-202). Thus, the effects of the endocannabinoids are profoundly affected by their enzyme mediated hydrolysis. Moreover, inactivation of FAAH activity has been shown to potentiate the anti-tumorigenic effects of AEA in prostate cancer (203). However, the exact roles of FAAH and its regulation of AEA activity have not been elucidated in the context of tumorigenicity in NSCLC. In our work, we focus on AEA, an endogenous cannabinoid agonist specific for the CB1 receptor and the effect of FAAH inhibition on the activity of AEA.

The genetic abnormalities associated with lung cancer are attributed to alterations in the signaling pathways which are targets for drug therapies. Most of these stimulatory signaling pathways are driven to a malignant phenotype characterized by uncontrolled proliferation and an apoptosis escape mechanism.

Epidermal growth factor receptor (EGFR) is a family of four Receptor tyrosine kinases

(RTKs) EGFR (ERBB1, HER1), ERBB2 (HER2, Neu), ERBB3 (HER3) and ERBB4

(HER4) (204-205). EGFR dysregulation is associated with multiple cancer types including malignant transformations and metastasis (206). EGFR overexpression and signaling pathway gene mutations play a vital role in lung tumorigenesis (Fig. 10).

Recent evidence suggests that cancer cells undergo escape mechanisms to defend against the host system by activation of alternative growth signaling pathways (153).

Figure 10: EGFR signaling pathway in lung cancer. Reprinted from Oncotarget. Nov

2010; 1(7): 497–514 (206).

The cell cycle in eukaryotes is regulated by a family of cyclins and cyclin dependent kinases (CDKs), which are members of protein kinase complexes. Each complex consists of a cyclin (regulatory subunit) which binds to a CDK (catalytic subunit) to form an active cyclin-CDK complex that gets activated at various checkpoints during the cell division cycle (207-208).

Several studies indicate that cell cycle markers are mutated in most malignant cancers and might lead to Programmed Cell Death (PCD), where cells undergo suicide program

(82, 207-208). Apoptosis is a type of PCD which involves the activation of caspases and

DNA fragmentation (209-211). Cell cycle dysregulation and resistance to apoptosis are often attributed to abnormal EGFR signaling (204, 212). Hence, identification of novel receptors expressed in tumor cells that target against EGFR activation will be a promising strategy against NSCLC.

In our present study, we analyzed the effect of AEA on lung tumorigenesis when FAAH is inhibited. We show that Met-F-AEA in combination with URB597 reduces NSCLC growth in vitro and in vivo. Our results reveal that the combination treatment inhibits the activation of EGFR and its downstream signaling targets. These findings suggest the possibility of exploring the components of endocannabinoid system as a novel therapeutic target for NSCLC treatment.

3.2 Materials and methods

3.2.1 Reagents and antibodies

Met-F-AEA and URB597 were purchased from Sigma Aldrich. Antibodies used were P-

AKT, caspase-9, PARP, CDK4 (Cell Signaling), P-ERK, ERK, AKT, GAPDH, P-EGFR,

EGFR (Santa Cruz), cyclin D1, Ki67 (Neomarkers) and FAAH (Cayman Chemicals).

3.2.2 Cell culture

Human NSCLC cell lines- A459, A549, CALU1, H1299 and H460 were obtained from

ATCC (American Type Culture Collection) and cultured in DMEM or RPMI-1640

(Corning Cellgro), supplemented with 10% heat inactivated fetal bovine serum (FBS), 5 units/mL penicillin, and 5 mg/mL streptomycin (Corning Cellgro). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere incubator.

3.2.3 Cell proliferation assay

Cells were seeded at a density of 5000 cells per well in 96 well plates and allowed to grow for 24h. Briefly, cells were treated with Met-F-AEA (10µM), URB597 (0.2µM) or in combination. Cell viability was measured using the MTT assay (Roche) as described in the supplier’s protocols, based on the absorbance reading at 570nm with respect to the control.

3.2.4 Clonogenic assay

Cells were seeded at low density in complete media (1000 cells per well in six well plates) and treated with vehicle, Met-F-AEA/URB597 or in combination for six days.

After the treatment period, cells were washed with PBS and fixed with 4% formaldehyde for 20min, washed again, stained with 0.1% crystal violet and individual clones were manually counted under the microscope.

3.2.5 Chemotaxis and wound healing assays

For the migration assay, 8µm transwell plates (Corning-Costar) were used. Briefly, cells were seeded in the upper chamber and chemoattractant EGF (100ng/ml) was added to the lower chambers as previously described. 12 hours after EGF stimulation, cells that migrated to the lower chamber were fixed, stained using Hema stain and counted. For the invasion assay, pre-coated Matrigel invasion chambers (BD Falcon) were used. After 24 hours of stimulation with EGF similar to that in migration assay, invaded cells were stained and counted.

For wound healing assay, cells were grown to 80% confluence in complete media.

Monolayers were wounded by scratching with a sterile plastic 200 µL micropipette tip, washed, and incubated in the presence of vehicle, Met-F-AEA/URB597 or in combination and EGF (100 ng/ml). After 18 h, cells were photographed using a low- magnification phase-contrast microscope.

3.2.6 FAAH small interfering RNA

H460 cells were transfected with FAAH siRNA (Dharmacon) using Lipofectamine, as per the manufacturer’s recommendations. Scrambled non targeting siRNA was used as control. 36h after transfection, cells were treated with either Met-F-AEA or vehicle and subjected to MTT and western blot analysis

3.2.7 Immunofluorescence

Cells were seeded in 8 well chamber slides, treated, fixed and incubated with primary antibodies phalloidin or vinculin overnight at 4°C. After washing, cells were stained with

Alexa Fluor- 488 or 594 conjugated secondary IgG antibodies and visualized under

Olympus FV1000 Filter confocal microscope.

3.2.8 Gelatin zymography

Gelatin zymography was used to determine MMP activity. Briefly, supernatants of treated cells were collected, concentrated using centrifugal filter units (Millipore) and run on Novex zymogram gel. The gel was then renatured and developed to visualize the bands as per the manufacturer’s protocol (Life Technologies).

3.2.9 Luciferase reporter assay

NF-kB activity was determined using NF-kB luciferase reporter assay (Promega). To determine the luciferase reporter activity, NF-kB luciferase constructs containing either the wild type or NF-kB vector were transfected in the pre-treated cells using 55 lipofectamine. For internal control, cells were co-transfected with Renilla luciferase vector. 24h after transfection, EGF (100ng/ml) was added and then incubated for another

24h. Then, the cells were lysed to perform the luciferase assay as per the manufacturer’s protocol.

3.2.10 Western blotting

Cells were washed, lysed and protein estimation was performed using Bradford assay.

Aliquots of cellular lysates (50µg) were electorphoresed on a 4-12% Novex SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk for an hour at room temperature. The membranes were then probed overnight with specific primary antibody (1:1000) overnight at 4°C. After washing thrice with 1X TBST, blots were exposed to secondary antibody (anti-mouse or anti-rabbit IgG-HRP, 1:2000) for an hour, washed thrice and detected using ECL chemiluminescence detection system (Thermo

Scientific).

3.2.11 Cell cycle analysis

Cells were trypsinized, washed with 1X PBS and fixed with 70% ethanol overnight at

4°C. Then, the cells were spun down, washed twice and incubated with 20µg/ml propidium iodide and 10 µg/ml RNAse for 30 min, washed and the DNA content was analyzed by flow cytometry.

3.2.12 Apoptosis assay

Apoptosis was detected in cells using Click-iT TUNEL assay kit (Life Technologies).

Cells were washed, fixed and treated with TdT enzyme for 1h. Then, the cells were stained with AlexaFluor 594 dye-labeled reaction buffer for 30min and detected under fluorescent microscope (Olympus).

3.2.13 Mouse xenograft model

H460 cells (2x106) in 100µl PBS were injected subcutaneously into the left flank of each male nude mouse. Once the tumors reached palpable size, they were treated with Met-F-

AEA (5mg/kg), URB597 (1mg/kg) or in combination for 3 weeks. Tumor volume was calculated using the formula vol. = length*(width)2/2.

3.2.14 Real Time PCR

RNA was isolated from tissues using TRIzol reagent (Invitrogen). Reverse transcriptase

PCR (RT-PCR) reaction was done using RT-PCR kits (Applied Biosystem, CA).

Expression of genes analyzed by q-PCR was normalized to GAPDH using the 2-

ΔCT method.

3.2.15 Tissue Microarray (TMA) and Immunochemical (IHC) analyses

Tissue microarrays from paraffin-embedded formalin fixed lung cancer tissue specimens were obtained from the Human Tissue Resource Network, Department of Pathology, The

Ohio State University. Samples were stained with FAAH antibody and CB1 antibody and detected using a kit (Vector Laboratories).

Samples from tumor xenografts of mice were dissected, embedded in OCT (Tissue-Tek) and stained using standard immunohistochemistry techniques as per the manufacturer's recommendation (Vector Laboratories), using the primary antibody. Slides were stained with secondary antibodies and detected.

3.2.16 Statistical analysis

Results were represented as mean ± SD which were analyzed using Student’s two-tailed t test. A value of P<0.05 was considered to be statistically significant.

3.3 Results

3.3.1 Primary lung cancer tissues and NSCLC cell lines express CB1 and FAAH

Anandamide is known to mediate its effects through cannabinoid receptor CB1 (192,

210). Hence, the expression of FAAH and CB1 were assessed in the NSCLC cell lines-

A459, A549, CALU1, H460 and H1299 (Fig 11A). All the cell lines expressed FAAH and CB1 as detected by Western Blot. Also, we utilized tissue microarray (TMA) to detect levels of FAAH and CB1 in cancer patients. Eleven of fifty seven (19.3%) lung adenocarcinomas showed high cytoplasmic expression of FAAH (Fig 11B, C). Twenty two of thirty five (62.9%) lung adenocarcinomas showed high cytoplasmic expression of

CB1 (Fig 11D, E).

A B C

A549

H460

A459

CALU1 H1299 FAAH CB1 GAPDH

D E

Figure 11: NSCLC cell lines and primary lung cancer tissues express CB1 and FAAH.

(A) NSCLC cell lines were subjected to immunoblot analysis to determine the expression of CB1 and FAAH. GAPDH served as loading control. Representative photomicrographs of IHC staining of FAAH expression in lung adenocarcinoma (B) and respiratory epithelia (C). Representative photomicrographs of IHC staining of CB1 expression in lung adenocarcinoma (D) and respiratory epithelia (E).

3.3.2 FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines

A B 120 120 100 100 * * 80 * 80 * 60 60

40 40 Cell Viability (%) Viability Cell 20 (%) Viability Cell 20 0 0 Control MetF Inh MetF+Inh Control MetF Inh MetF+Inh C D 120 120 100 100 * 80 80 ** * * 60 60 * ** 40 40

20 20 Colony Colony Formation (%) 0 Colony Formation (%) 0 Control MetF Inh MetF+Inh Control MetF Inh MetF+Inh

Figure 12: FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines. A549 (A) and H460 (B) cells were serum starved for 24h and treated with control, Met-F-AEA (MetF, 10µM), FAAH inhibitor URB597(Inh, 0.2µM) or

MetF+Inh and analyzed for viability by MTT assay. 1000 individual A549 (C) and H460

(D) cells were plated and subjected to colony formation by treating with control, Met-F-

AEA (MetF, 10µM), FAAH inhibitor URB597 (Inh, 0.2µM) or MetF+Inh for six days.

The colonies were then fixed, stained and counted. P<0.05 (*) and P<0.005 (*) as calculated by Student’s t test.

Proliferation is one of the characteristic features of cancer cells to grow and multiply (64,

85, 213). To analyze whether inhibiting FAAH can enhance the activity of Met-F-AEA

(synthetic analogue of anandamide), we evaluated the potential of Met-F-AEA in combination with FAAH inhibitor- URB597 as a possible therapeutic target.

We treated the NSCLC cell lines- A549 and H460 with either FAAH inhibitor- URB597 or Met-F-AEA or in combination and observed the effects after 24h. MTT assay showed a significant decrease in cell viability in the combination treatment when compared to

Met-F-AEA alone (Fig 12A, B).

Also, we performed clonogenic assay which measures the ability of single cell to form clones. Combination treatment significantly reduced the number of colonies when compared to Met-F-AEA or URB597 alone in both the cell lines (Fig 12C, D).

To enhance the effect of the endocannabinoid Met-F-AEA, we used the siRNA approach to knockdown FAAH in H460 cells. To analyze the efficiency of knockdown, the transfected cells were subjected to Western Blot analysis, which showed reduced expression of FAAH (Fig 13A). Cells transfected with FAAH siRNA were more sensitive to Met-F-AEA treatment than the control siRNA transfected cells (Fig 13B).

These data suggest that Met-F-AEA in combination with URB597 inhibits cell growth in vitro.

A B 140 ControlVector FAAHFAAH- siRNA Vector FAAH-siRNA 120 FAAH 100 80 * GAPDH 60 40 Cell Viability (%) Viability Cell 20 0 Control MetF Control MetF

Figure 13: FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in

NSCLC cell lines. (A) H460 cells were transfected with 100pmol of either the non targeting control (vector) or FAAH-siRNA (Dharmacon) using Lipofectamine 2000

(Invitrogen) for 36h and the expression of FAAH in siRNA and non targeted cells were evaluated by Western blot. (F) H460 cells which were transfected with either FAAH siRNA or non targeted control (vector) for 36h were treated with Met-F-AEA for 24h and subjected to MTT assay. P<0.05 (*) as calculated by Student’s t test.

3.3.3 FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines

Migration and invasion are required for cancer cells to spread within the specific tissue and form the characteristic features of angiogenesis and metastasis (131). The

62 extracellular matrix (ECM) acts as a barrier towards this cell motility process.

Chemoattractants like EGF provide a directed migration for cancer cells (131, 204, 212).

Control MetF Inh MetF+Inh

0 h

18 h

Figure 14: FAAH inhibition enhances wound healing activity of Met-F-AEA in A549 cells. Cells were plated in six-well plates and pre-treated with control, Met-F-AEA

(MetF, 10µM), URB597 (Inh, 0.2µM) or MetF+Inh for 24h. The monolayer was wounded by scoring a scratch with a sterile 200µl plastic tip, and then washed and fed with media containing EGF (100ng/ml). After 18h, cells were photographed using a low magnification phase contrast microscope and the migratory ability was assessed qualitatively.

We further analyzed the effects of Met-F-AEA in combination with URB597 on EGF induced chemotaxis and observed significant inhibition in EGF induced migration (Fig

14, 15A, B) and invasion (Fig 15C, D) when compared to Met-F-AEA or URB597 alone in A549 and H460 cells.

A B 2500 2500 Control ControlControl 2000 MetF 2000 MetFMetF Inh InhInh MetF+Inh ** MetF+InhMetF+Inh 1500 1500 *

1000 1000 No. of cells cells of No. migrated No. of cells cells of No. migrated 500 500

0 0 EGF - - - - + + + + EGF - - - - + + + +

C D 1250 1000 Control Control MetF MetF 1000 Inh 750 Inh MetF+Inh MetF+Inh 750 * 500 *

500 No. of cells cells of No. invaded 250 cells of No. invaded 250

0 0 EGF - - - - + + + + EGF - - - - + + + +

Figure 15: FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines. A549 (A) and H460 (B) cells were treated with control,

Met-F-AEA (MetF, 10µM), FAAH inhibitor URB597 (Inh, 0.2µM) or MetF+Inh for 24h and subjected to EGF (100ng/ml)-induced migration using transwell plates. A549 (C) and

H460 (D) cells were treated with control, Met-F-AEA (MetF, 10µM), FAAH inhibitor

URB597 (Inh, 0.2µM) or MetF+Inh for 24h and subjected to EGF (100ng/ml)-induced invasion (continued.) (Fig. 15 continued.) assay using transwell plates coated with matrigel. Invaded cells were counted. P<0.05 (*) as calculated by Student’s t test.

To migrate through the ECM, cancer cells possess extended protrusions that are rich in actin filaments and adhesion molecules (214). We investigated the effects of Met-F-AEA when FAAH is inhibited on EGF induced actin stress fiber and focal adhesion formation.

A B

Control MetF Inh MetF+ Inh DAPI

MMP2

Vinculin Phalloidin

MetF+ Inh Control MetF Inh

Figure 16: FAAH inhibition enhances the anti-migratory and anti-invasive activities of

Met-F-AEA in NSCLC cell lines. (A) Confocal microscopy visualization of A549 cells treated with control, Met-F-AEA (MetF, 10µM), FAAH inhibitor URB597 (Inh, 0.2µM) or MetF+Inh for 24h and stimulated with EGF (100ng/ml) and stained for (contd.)

(Fig.16 contd.) phalloidin (red), vinculin (green) expression and DAPI (blue). (B) A549

(upper panel) and H460 (lower panel) cells were treated with control, Met-F-AEA (MetF,

10µM), FAAH inhibitor URB597(Inh, 0.2µM) or MetF+Inh for 48h and the supernatants were collected, concentrated and run on zymogram gels to detect the active form of

MMP2.

We observed that cells treated with the combination treatment significantly inhibited

EGF induced actin and vinculin expressions more effectively than Met-F-AEA or

URB597 alone.

Further examination with the confocal microscopy revealed the decreased presence of migratory structures such as lamellipodia in the cells treated with the combination treatment when compared to control (Fig 16A).

Tumor invasion begins with protrusion of the ECM and basement membrane. The matrix metalloproteinase (MMP) family has been shown to be responsible for degradation of the

ECM, which is one of the initial steps in metastasis. Higher expressions of MMP2 and

MMP9 have also been correlated with poor prognosis in early stages of lung adenocarcinoma (215).

In our present study, we observed a significant decrease in secretion of MMP2 by zymogram when cells were treated with combination treatment, Met-F-AEA along with

URB597 (Fig 16B).

These data suggest that the Met-F-AEA in combination with URB597 inhibits cell migration and invasion in NSCLC cell lines.

3.3.4 FAAH inhibition enhances Met-F-AEA mediated inhibition of EGFR signaling in NSCLC cell lines

A B 5 5 Control Control 4 MetF 4 MetF Inh Inh MetF+Inh 3 MetF+Inh 3 * 2 2 *

1 1

Relative Relative Luciferase Units Relative Luciferase Units 0 0 EGF - - - - + + + + EGF - - - - + + + +

Figure 17: FAAH inhibition enhances Met-F-AEA mediated inhibition of NF-kB in

NSCLC cell lines. A549 (A) and H460 (B) cells were treated with control, Met-F-AEA

(MetF, 10µM), FAAH inhibitor URB597(Inh, 0.2µM) or MetF+Inh for 24h and transfected with either wild-type or NF-kB plasmid for 24h, stimulated with EGF

(100ng/ml) for additional 24h, lysed and analyzed for luciferase activity. Renilla luciferase vector served as internal control. P<0.05 (*) as calculated by Student’s t test.

NF-kB is involved in invasion and metastasis in various cancer types (216). Previous studies have shown that NF-kB activation is involved in EGFR mediated lung tumor progression (216-217). Therefore, we sought out to determine if FAAH inhibition can enhance Met-F-AEA mediated inhibition of EGF induced NF-kB activation by luciferase reporter assay. Cells were transfected with either vector or NF-kB plasmid and the translocation of NF-kB into the nucleus was studied in the presence or absence of EGF.

Met-F-AEA in combination with URB597 treated cells underwent significantly reduced

EGF induced NF-kB translocation into the nucleus when compared to Met-F-AEA or

URB597 alone (Fig 17A, B).

The EGFR signaling pathway activates diverse cellular targets which are crucial for cell proliferation, survival, angiogenesis, migration and adhesion and are often dysregulated in cancer cells. AKT and ERK are important survival molecules that are essential for

EGF induced cell growth and motility (153, 204, 212).

To understand the molecular mechanism by which FAAH inhibition enhances Met-F-

AEA and regulates EGFR pathway, we treated the cells with Met-F-AEA together with

URB597, induced with EGF and observed reduction in the tyrosine phosphorylation of

EGFR, serine phosphorylation of AKT and tyrosine phosphorylation of ERK which are the immediate downstream targets of EGFR pathway when compared to Met-F-AEA or

URB597 alone (Fig 18A, B).

To further validate that FAAH inhibition enhances the anti-tumorigenic effects of Met-F-

AEA, H460 cells were transfected with FAAH siRNA, treated with Met-F-AEA or 68 vehicle, stimulated with EGF and analyzed by Western blotting. Cells transfected with

FAAH siRNA and treated with Met-F-AEA showed reduced expression levels of P-AKT and P-ERK when compared to Met-F-AEA alone (Fig 18C).

A B Con MetF Inh MetF+ Inh C Con MetF Inh MetF+ Inh Vector siRNA EGF(min) 0 15 0 15 0 15 0 15 EGF (min) 0 15 0 15 0 15 0 15 EGF (min) 0 15 0 15 0 15 0 15 P-EGFR P-EGFR P-ERK

T-EGFR T-EGFR T-ERK

P-ERK P-ERK P-AKT

T-ERK T-ERK T-AKT

P-AKT P-AKT GAPDH

Control + + - - + + - - T-AKT T-AKT MetF - - + + - - + +

Figure 18: FAAH inhibition enhances Met-F-AEA mediated inhibition of EGFR signaling in NSCLC cell lines. A549 (A) and H460 (B) cells were treated with control,

Met-F-AEA (MetF, 10µM), FAAH inhibitor URB597(Inh, 0.2µM) or MetF+Inh for 24h, stimulated with EGF (100ng/ml) for 0 and 15 min and subjected to Western blot to determine expression of P-EGFR, P-ERK, P-AKT and T-EGFR, T-ERK, T-AKT. (C)

H460 cells which were transfected with FAAH siRNA or non targeted vector for 36h were treated with either Met-F-AEA or control for 24h, stimulated with EGF (100ng/ml) for 0 and 15 min and analyzed by Western blotting to determine expression levels of P-

ERK, P-AKT and T-ERK, T-AKT. P<0.05 (*) as calculated by Student’s t test.

Taken together, these results indicate that Met-F-AEA in combination with URB597 downregulates EGFR receptor activation and also significantly inhibits the downstream signaling targets of EGFR pathway.

3.3.5 FAAH inhibition enhances Met-F-AEA induced cell cycle arrest and apoptosis at later stage

Cell cycle dysregulation is frequently associated with cancer growth. EGFR activation leads to tumor cell survival, aberrant cell cycle and evasion of apoptosis, ultimately leading to resistance to cytotoxic therapies (153, 204, 212). Hence, we sought to assess whether Met-F-AEA in combination with URB597 can induce cell cycle arrest leading to apoptosis.

Cells were treated with control, Met-F-AEA, URB597 or combination treatment of Met-

F-AEA with URB597 for 48h and subjected to cell cycle analysis.

We observed significant cell cycle arrest in the G0/G1 phase in the combination therapy treated cells when compared to Met-F-AEA alone (Fig 19A).

A Control MetF Inh MetF + Inh

G0/G1: 68.3 G0/G1: 77.4 G0/G1: 77.1 G0/G1: 82.7 S: 15.1 S: 11.4 S: 12.3 S: 8.3 G2/M: 15.6 G2/M: 10.1 G2/M: 9.6 G2/M: 7.8

B Control MetF Inh MetF + Inh

C 1 2 3 4 D 1 2 3 4

Pro-Casp9 Pro-Casp9

Pro-PARP Pro-PARP

Cdk4 Cdk4

Cyclin D1 Cyclin D1

GAPDH GAPDH

Figure 19: FAAH inhibition enhances Met-F-AEA induced cell cycle arrest and apoptosis at later stage. (A) A549 cells were treated with control, MetF, 10µM, URB597

(Inh, 0.2µM) or MetF+Inh for 48h, stained with PI and analyzed by flow cytometry. (B)

Representative images of TUNEL positive cells in H460 cells. A549 (C) and H460 (D) cells were pre-treated with control (1), Met-F-AEA (2), URB597(3) or MetF+Inh (4) for

48h and subjected to Immunoblot analysis to determine the expression of cell cycle markers. GAPDH is loading control. P<0.05 (*) as calculated by Student’s t test.

Met-F-AEA in combination with URB597 increased the percentage of apoptotic cells as shown by TUNEL assay (Fig 19B) and significantly inhibited pro-caspase9 and pro-

PARP.

Apoptosis was also induced by cell cycle arrest, as we observed significant decrease in cyclin D1 and CDK4, which are essential for G1/S phase progression (Fig 19C, D).

These results show that Met-F-AEA together with URB597 induced apoptosis and is mediated by cell cycle blockade.

3.3.6 FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo by downregulating EGFR signaling

To evaluate the tumor suppressive effects of Met-F-AEA in combination with URB597 in vivo, we determined the anti-tumorigenic potential of the combination treatment on H460 cells in nude mouse model.

We induced tumors by injecting H460 cells subcutaneously into the right flank of male nude mice. When the tumors reached a palpable size, we treated them with ethanol control, Met-F-AEA, URB597 or Met-F-AEA in combination with URB597 every third day for three weeks.

Tumor volume was monitored throughout the treatment period (Fig 20A).

A B 1400 1.6 1200 Control 1.4 MetF 1.2 1000 * Inh 1 800 ** MetF+Inh 0.8 600 0.6 400 Tumor Volume (mm3) Volume Tumor 0.4

* (g) weight Tumor 200 0.2 0 0 wk1 wk2 wk3 Control MetF Inh MetF+Inh

C Control MetF Inh MetF+Inh

Figure 20: FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo. (A) H460 cells were subcutaneously injected in nude mice and palpable tumors were treated with control, Met-F-AEA (MetF, 5mg/kg), FAAH inhibitor URB597

(Inh, 1mg/kg) or MetF+Inh every third day for 3 weeks. Tumor volume was assessed periodically and calculated using the formula= length x (width) 2 /2. (B) Tumor weight measured from various experimental groups. (C) Representative tumors dissected from various experimental groups. (D) Representative photomicrographs of H&E staining of tumors extracted from various experimental groups. P<0.05 (*) and P<0.005 (**) as calculated by Student’s t test.

We observed a dramatic decrease in tumor formation in the combination regime treated mice as compared to control (Fig 20B, C).

H&E staining revealed that the combination regime treated tumors were less aggressive and necrotic than the control tumors (Fig 20D). n addition, there was a significant decrease in the expression of proliferation marker Ki67 and hence, a reduced mitotic index in Met-F-AEA and URB597 combination treated tumors as compared to Met-F-AEA or URB597 treated tumors alone (Fig 21A, B).

To further determine the mechanism by which the tumors are inhibited, we isolated the tumor xenografts from the nude mice and extracted protein and RNA from them.

The Met-F-AEA and URB597 combination treated tumors showed lesser phosphorylation of EGFR, ERK and AKT compared to Met-F-AEA or URB597 treated tumors alone (Fig 21C).

A Control MetF B C

MetF Inh 90 Con 80 P-EGFR 70 T-EGFR 60 50 P-AKT Inh MetF+Inh 40 *

cells/highfield 30 T-AKT + 20 P-ERK

Ki67 10 0 T-ERK Con MetF Inh MetF +Inh D 700 600 MMP9 600 MMP2Inh 500 500 400 400 300 300 * 200 200 * * 100

100 ** * Relative Relative Expression 0 Relative Expression 0 * Control MetF Inh MetF+Inh Control MetF Inh MetF+Inh

Figure 21: FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo by downregulating EGFR signaling. (A) Representative photos of Ki67 staining of tumors extracted from various experimental groups. (B) No. of Ki67 positive cells using bright field microscopy in each experimental group and the average was calculated. (C) Xenograft tumors isolated were subjected to Western blot analysis to determine expression of P-EGFR, P-ERK, P-AKT and T-EGFR, T-ERK, T-AKT.

Control, Met-F-AEA (MetF), FAAH inhibitor URB597(Inh) or MetF+Inh. (D) Xenograft tumors were subjected to Real Time PCR to determine expression of MMP2 and MMP9.

P<0.05 (*) and P<0.005 (**) as calculated by Student’s t test.

Also, real time PCR analysis revealed that MMP2 and MMP9 levels were significantly downregulated in the URB597 treated tumors and Met-F-AEA in combination with

URB597 treated tumors when compared to the control (Fig 21D), confirming with our in vitro findings.

3.4 Conclusion

Cannabinoids and their derivatives are being focused on cancer treatments because of their association with pain modulation, cell growth inhibition, anti-inflammation, tumor regression, cell cycle arrest and apoptosis.

The endocannabinoid system is involved in a complex array of signaling pathways which might be receptor dependent or independent (34, 192-194). Though recent studies have shown that endocannabinoids exert potential anti-tumor effects in various cancer cells

(19), not much is known about their effects in lung cancer. Here, we report, for the first time that Met-F-AEA and FAAH inhibitor URB597 significantly inhibited NSCLC growth. We have also explored the mechanisms by which Met-F-AEA in combination with URB597 inhibits NSCLC tumor growth.

In our present study, we found that FAAH and cannabinoid receptor CB1 are expressed in lung cancer patient samples as well as in NSCLC cell lines. Increased FAAH expression has been associated with poor patient survival in prostate and breast cancer

(203, 218). Also, overexpression of FAAH was found to be sufficient to increase migration and invasion in prostate cancer cells (203).

Our results show that Met-F-AEA that binds to CB1 does not have significant anti- tumorigenic effects in vitro and in vivo. This might be due to the conversion of AEA by enzyme FAAH into metabolites, leading to limitation in the concentration and action of the ligand.

Previous reports show that AEA is converted into arachidonic acid (AA) and ethanolamine (EA) by FAAH enzyme (88, 202). AA is metabolized by COX2 and other enzymes to form prostaglandin (PGE2) and epoxyeicosatetraenoic acid (EE) (219-220).

These secondary metabolites have been shown to enhance tumor growth and metastasis in various cancer types (220-221).

Also, there are reports which suggest that methanandamide increases murine lung tumor growth by modulation of prostaglandin (PGE2) production and COX2 expression(220).

Therefore, in our study, we blocked FAAH pharmacologically (by using FAAH inhibitor

URB597) and genetically (by siRNA approach) to enhance the anti-tumorigenic effects of Met-F-AEA. Our results show, for the first time, that Met-F-AEA, together with

URB597 exerts anti-proliferative effects on NSCLC in vitro and in vivo.

In our study, we have shown that Met-F-AEA in combination with URB597 inhibits

EGFR phosphorylation and downregulates EGFR mediated signal transduction pathways involving AKT and ERK, which are key cell survival molecules, in vitro and in vivo. This is important because EGF/EGFR axis is known to regulate cancer cells to proliferate and migrate to distant sites.

Furthermore, aberrant EGFR expression and function lead to highly aggressive lung tumors, ultimately causing poor patient survival and increased resistance to conventional chemotherapeutic drugs (204, 206). Thus, it would be interesting to identify novel targets that have growth inhibiting effects by targeting the EGFR pathway. In our present study, we observed that Met-F-AEA in combination with URB597 inhibits stress fiber and focal adhesion formations.

Focal adhesions have been shown to connect the ECM to actin stress fibers thus re- organizing the matrix and regulating cell migration (131). They are the main subcellular macromolecules that form close contacts between cells and the ECM. They play an important role in cell growth and migration. Transformation of epithelial cells into invasive carcinomas also depends on reorganization of the actin cytoskeleton that leads to stress fiber assembly (222). Furthermore, the Met-F-AEA in combination with URB597 78 reduces EGF induced invasion and also downregulates MMP2 secretion, which mediates the invasiveness of cancer cells by aiding them to degrade the ECM and metastasize

(215).

Our results show that Met-F-AEA in combination with URB597 causes G0/G1 cell cycle arrest mediated apoptosis, which is shown by reduction in G1/S phase checkpoint markers Cyclin D1 and CDK4 and apoptotic markers caspase-9 and PARP. This is important because in cancer, there exists an imbalance between cell proliferation and apoptosis which leads to tumor progression.

Also, uncontrolled cellular growth due to aberrant EGFR signaling leads to dysregulation of the cell cycle, which involves G1/S checkpoint markers that are responsible for cell cycle progression (207, 223).

Furthermore, apoptosis, a programmed cell death mechanism (224), is usually associated with cell cycle arrest. Hence, our results suggest the ability of Met-F-AEA in combination with URB597 as an apoptosis inducing agent, controlling the cell survival/death cycle.

3.5 Discussion

Overall, the results of our study suggest that the activity of the endocannabinoid anandamide increases when FAAH is inhibited, leading to enhanced anti-proliferative, anti-migratory and anti-invasive effects.

We have also shown that Met-F-AEA in combination with URB597 crosstalks with EGF receptor to inhibit its activation, subsequently leading to downregulation of its signaling targets.

These implicate that Met-F-AEA along with URB597 can be used as an effective therapeutic strategy for the treatment of EGFR overexpressing NSCLC. This is especially imperative considering the resistance of NSCLC to various chemotherapeutic drugs and its poor prognosis.

Chapter 4

Synthetic cannabinoid agonist JWH-015 inhibits macrophage induced EMT in non-

small cell lung cancer by downregulation of EGFR signaling

4.1 Introduction

The genetic abnormalities associated with lung cancer are attributed to alterations in the signaling pathways which are targets for drug therapies. Most of these stimulatory signaling pathways are driven to malignant phenotype characterized by uncontrolled proliferation and apoptosis escape mechanism.

Epidermal growth factor receptor (EGFR) is a family of four Receptor tyrosine kinases

(RTKs) EGFR (ERBB1, HER1), ERBB2 (HER2, Neu), ERBB3 (HER3) and ERBB4

(HER4) (204). EGFR is frequently overexpressed or mutated in NSCLC and was the first mutation identified to be more aberrated in non-smoking lung cancer patients. Hence,

EGFR has been considered as a predicted biomarker in NSCLC patients which led to rise of several TKI (Tyrosine Kinase Inhibitors) (225). Targeting EGFR would lead to better treatment of lung cancer in future.

Epithelial to Mesenchymal Transition (EMT) is a dynamic, significant event in cancer progression. EMT is correlated with malignant properties like migration, invasion, 81 evasion of apoptosis, stemness, metastasis, etc. in vitro and in vivo. E-cadherin dysfunction contributes to poor patient prognosis in lung cancer. Also, EMT markers have been clinically associated with pathology of NSCLC (226).

EGFR is frequently attributed to dysfunction in tumors of epithelial origin, leading to

EMT like features. In NSCLC cell line A549, EGF stimulates EMT by inducing a morphological change, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers N-cadherin, fibronectin and vimentin. Loss of E-cadherin leads to disruption of cell-cell junctions, thus making the cells more motile and migratory.

MMPs (matrix metalloproteinases) are secreted which degrade the ECM (extra-cellular matrix), thus allowing the cells to become more invasive, ultimately leading to metastasis to distant organs (227-228). Reports suggest that EMT can lead to acquired resistance to conventional EGFR-TKI chemotherapeutic drugs, thus increasing the difficulty in lung cancer treatment.

The tumor microenvironment (TME) comprises of variety of cell types like cancer associated fibroblasts (CAF), natural killer (NK) cells, tumor associated macrophages

(TAM), myeloid derived suppressor cells (MDSC), endothelial cells, etc. These infiltrated cells secrete various factors which play crucial role in cancer progression,

EMT and metastasis. Emerging targets focus on the interplay between cancer cells and their microenvironment (229-230).

Macrophages are involved in regulation of tissue homeostasis, inflammation and are associated with several pathological diseases. TAM density is inversely correlated with 82 patient prognosis in various cancer types. Also, TAMs have been related to angiogenesis, invasion, metastasis and immune modulation in different carcinomas (231).

Reports suggest that M2 macrophages induce EMT by regulating TLR4/IL-10 signaling in pancreatic cancer cells (232). In hepatoma cells, activated macrophages enhance the migratory and invasive properties, leading to decreased E-cadherin levels (233). Also, macrophages secreted activators for EGFR and STAT3 which enhanced the invasiveness in breast tumors (234). These confirm a strong crosstalk between macrophages and tumor progression, mainly through stimulation of EMT.

The cannabinoid family is categorized into endogenous, synthetic and phytocannabinoids which activate specific G-protein coupled receptors- CB1 and CB2. CB1 is mainly expressed in the brain and CNS whereas CB2 is expressed in immune system. The use of cannabinoid agonists as anti-cancer agents has proven successful in various in vitro and in vivo cancer models such as glioma, breast, prostate, colon, leukemia and lymphoid tumors. They have been shown to modulate various cell survival pathways such as ERK,

PI3K, p38 MAPK, AKT and ceramide pathways (195).

JWH-015 is a synthetic CB2 agonist which possesses anti-proliferative and anti-invasive effects in various cancer types (84, 194, 235). Although JWH-015 is involved in modulating various signaling pathways, not much is known about how it regulates EGFR signaling in NSCLC.

In our present study, we show that JWH-015 has anti-migratory and anti-invasive effects in NSCLC cells. Also, we hypothesize that JWH-015 inhibits recruitment of macrophages to the tumor site by inhibiting EMT. This may be possible through downregulation of EGFR signaling by JWH-015 at the tumor site which might be activated by EGF secreted by the macrophages. Therefore, we prove our hypothesis that there exists a crosstalk between CB2 and EGFR by performing in vitro co-culture experiments as well as in vivo.

4.2 Materials and methods

4.2.1 Cell culture

Human NSCLC- A549 cells (ATCC) were cultured in DMEM (Corning Cellgro). Murine lung cancer ED1 cells (kindly provided by Dr. Ethan Dmitrovsky), CALU1 and THP1 cells (ATCC) were cultured in RPMI-1640 (Corning Cellgro). The media were supplemented with 10% FBS (Corning Cellgro) and 1% penicillin/ streptomycin

(Corning Cellgro).

4.2.2 Reagents and antibodies

JWH-015 was purchased from Tocris Bioscience. Antibodies used were P-AKT, E- cadherin (Cell Signaling), P-ERK, ERK, AKT, GAPDH, P-EGFR, EGFR, VCAM-1,

STAT3 (Santa Cruz), N-cadherin, P-FAK (Abcam), P-STAT3 (BD Biosciences).

4.2.3 Real Time Reverse Transcription PCR

RNA was isolated from tissues using TRIzol reagent (Invitrogen). Reverse transcriptase

PCR (RT-PCR) reaction was done using RT-PCR kits (Applied Biosystem, CA).

Expression of genes analyzed by q-PCR was normalized to GAPDH using the 2-

ΔCT method.

4.2.4 Immunochemical (IHC) analyses

4.2.5 Clonogenic assay

1000 cells per well were seeded and treated with vehicle, JWH-015 for six days with

EGF (100ng/ml) or conditioned media from M2-TAMs. After the treatment period, cells were washed and stained with 0.1% crystal violet and individual clones were counted.

4.2.6 Chemotaxis and wound healing assays

For migration assay, 8µm transwell plates (Corning-Costar) were used. Briefly, cells were seeded in upper chamber and EGF (100ng/ml) or conditioned media from M2 macrophages were added to lower chambers (237, 239). 12 hours later, cells that migrated to lower chamber were fixed, stained using Hema stain and counted. For

85 invasion assay, pre-coated Matrigel invasion chambers (BD Falcon) were used. After 24 hours of stimulation similar to migration assay, invaded cells were stained and counted.

For wound healing assay, cells were grown to 80% confluence in complete media.

Monolayers were wounded by scratching with a sterile plastic 200 µL micropipette tip, washed, and incubated in the presence of vehicle or JWH-015 and EGF (100 ng/ml).

After 36 h, cells were photographed using a low-magnification phase-contrast microscope.

4.2.7 Immunofluorescence

Cells were seeded in 8 well chamber slides, treated, fixed and incubated with primary antibodies overnight at 4°C. After washing, cells were stained with Alexa Fluor- 488 or

594 conjugated secondary IgG antibodies and visualized under Olympus FV1000 Filter confocal microscope.

4.2.8 Western blotting

Cells were washed, lysed and protein estimation was performed using Bradford assay.

Aliquots of cellular lysates (50µg) were electrophoresed on Novex SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk for an hour at room temperature. The membranes were probed overnight with specific primary antibody (1:1000) overnight at 4°C. After washing thrice with 1X TBST, blots were exposed to secondary antibody (1:2000) for an hour, washed thrice and detected using

ECL chemiluminescence (Thermo Scientific). 86

4.2.9 Mouse xenograft model

EDI cells (3x106) in 100µl PBS were injected subcutaneously into the left flank of each syngenic 7 week old male FVB mouse. Once the tumors reached palpable size, they were treated with JWH-015 (7.5mg/kg), for 3 weeks and sacrificed.

4.2.10 Tail vein syngenic mouse model

ED1 cells (1x106) in 100µl PBS were injected by tail vein in syngenic 7 week old male

FVB mice. After a week of injection, mice were treated with ethanol control or JWH-015

(7.5mg/kg) for 3 weeks. After treatment, lungs were isolated and surface metastatic lesions were counted.

4.2.11 Flow cytometry

For FACS, single cell suspensions from tumor infiltrating cells were blocked with 1%

FBS in PBS and incubated with anti-F4/80 PE, anti-CD11b APC, and anti-CD206 Alexa

Fluor 488 for 1h. Then, the cells were washed and analyzed with FACS Caliber using

CellQuest software (BD Biosciences).

4.2.12 Statistical analysis

Results were represented as mean ± SD which were analyzed using Student’s two-tailed t test. A value of P<0.05 was considered to be statistically significant.

4.3 Results

4.3.1 CB2 and EGFR are expressed in NSCLC patients and cell lines

JWH-015 has been shown to exert its effects by activating CB2 receptor (194, 235). To understand the function of this ligand and how it activates CB2 and affects EGFR pathway, we checked for the expression of CB2 and EGFR in lung cancer cell lines-

A549, CALU-1 and ED1.

A A549 CALU1 EDI

CB2

EGFR

GAPDH

B CB2 C EGFR

Figure 22: CB2 and EGFR are expressed in NSCLC patients and cell lines. (A) NSCLC cell lines- A549, CALU1 and ED1 were subjected to immunoblot to determine expression of CB2 and EGFR. GAPDH is loading control. Kaplan Meyer plotter showing overall survival rate of lung cancer patients expressing (B) CB2 and (C) EGFR in lungs.

Both the receptors were expressed in these cells (Fig. 22A). Also, the publically available datasets like Kaplan Meyer plotter (240) show that increased CB2 (Fig 22B) and EGFR

(Fig 22C) levels in lung cancer patients are associated with poor overall survival.

4.3.2 JWH-015 inhibits EGF induced EMT in A549 cells

A B C J E J+E 300 Control 250 JWH 0h Control+EGF 200 JWH+EGF

150 ** 100 **

No. ofNo.colonies 36h 50

Figure 23: JWH-015 inhibits EGF induced signaling in A549 cells. (A) 1000 individual

A549 cells were subjected to colony formation assay by treating with control or JWH-

015 (5μM) for six days. Colonies were stained and counted. (B) Cells were pre-treated with control or JWH-015 (5μM) for 12h. Monolayer was wounded by a scratch, washed and fed with media containing EGF (100ng/ml). After 36h, plates were photographed.

P<0.01 (**) as calculated by Student’s t test.

To study the effect of JWH-015 on EGF mediated proliferation, we performed clonogenic assay, which is the ability of single cell to grow into a colony. JWH-015 potently decreased the number of colonies formed in absence as well as presence of EGF in A549 cells (Fig. 23A).

Cell migration is initiated by protrusion into the dense ECM and moving through this membrane with the help of actin rich structures in cell membranes. This movement is highly enhanced by secretion of chemoattractants like EGF which activate cancer cells

(131). JWH-015 inhibited EGF directed cell migration in A549 as shown by wound healing assay (Fig. 23B).

Cancer progression begins with dissemination of tumor cells from the extra-cellular matrix (ECM) into other distant sites. The initial event involves detachment of cells due to regulation of cell adhesion molecules (CAM) like cadherins, integrins, selectins and immunoglobulin superfamily (241). To test how JWH-015 affects the ability of cancer cells to detach from the tumor site and become more motile, we treated A549 cells with

JWH-015 for 48h and checked for markers involved in ECM regulation like FAK,

VCAM1 and MMP2.

Focal adhesion kinase (FAK) is a tyrosine kinase that is activated by ECM-integrin binding. Activated FAK is involved in various cellular functions involved in tumorigenesis like cell adhesion, migration, invasion and evasion of apoptosis (241-242).

Vascular cell adhesion molecule-1 (VCAM-1), an adhesion molecule that belongs to the immunoglobulin superfamily promotes adhesion of tumor cells to vascular endothelial 90 cells which ultimately leads to tumor angiogenesis and metastasis (243). JWH-015 significantly inhibited p-FAK and VCAM-1 (Fig. 24A).

A549 is a human lung adenocarcinoma cell line with epithelial characteristics. It has been shown that the ligand EGF induced EMT in A549, thus converting it from epithelial to fibroblastic, mesenchymal phenotype (244).

A B C J1 J2 J3 J4 C E J J+E

P-FAK E-CADHERIN

GAPDH GAPDH

VCAM-1 N-CADHERIN

GAPDH GAPDH

C SNAIL SLUG 450 200 400 Control Control JWH JWH 350 EGF 150 EGF 300 JWH+EGF JWH+EGF 250 * 100 200 150 *

100 50 Relative expression Relative 50 expression Relative 0 0

Figure 24: JWH-015 inhibits EGF induced EMT in A549 cells. (A) A549 cells were pre- treated with control (C) or JWH-015 at 1, 2, 3 and 4μM (J1, J2, J3, J4) for 48h and subjected to Immunoblot to determine expression of invasive markers. A549 cells were pre-treated with control or JWH-015 (2.5μM) for 24h, stimulated with EGF (100ng/ml) for 48h and subjected to Western blot (B) or (contd.) (Fig.24 contd.) Real time PCR (C) to determine expression of EMT markers. Control (C), JWH-015 (J), EGF (E), JWH-

015+EGF (J+E). P<0.05 (*) as calculated by Student’s t test. 91

EMT is marked by downregulation of epithelial markers like E-cadherin and upregulation of mesenchymal markers like N-cadherin, Snail, Slug and Vimentin. Also, loss of E- cadherin leads to loss of adherent junctions that exist between cells.

The mesenchymal property helps the cells disperse and invade the basement membrane, thus becoming more migratory (229).

To study the effect of JWH-015 on EMT progression, we induced A549 cells with EGF to stimulate EMT in the presence or absence of JWH-015 and analyzed various EMT markers. As expected, EGF induced EMT by upregulation and downregulation of mesenchymal and epithelial markers respectively.

Also, JWH-015 inhibited EGF induced EMT by reversing the EMT process. We observed decrease in mesenchymal markers like N-cadherin, Snail, Slug and increase in epithelial markers like E-cadherin (Fig. 24B, C).

These results confirm our hypothesis that JWH-015 inhibits EMT by downregulation of

EGFR pathway in A549 cells.

4.3.3 JWH-015 promotes mesenchymal to epithelial transition in CALU-1 cells

CALU-1 is a human lung adenocarcinoma cell line with fibroblastic, spindle shaped morphology, characteristic of mesenchymal property (245). We further investigated the role of JWH-015 on EMT by assessing whether it can revert the mesenchymal phenotype to epithelial morphology.

We initially checked for the effect of JWH-015 on proliferation of cells. JWH-015 inhibited EGF induced proliferation as shown by colony formation assay (Fig. 25A).

A crucial step in EMT progression is the ability of cells to elongate, migrate and invade the ECM barrier by degradation of various enzymes secreted by the membrane. This migratory-invasive feature is the hallmark for metastasis. JWH-015 potently inhibited

EGF induced migration (Fig. 25B, D) and invasion (Fig. 25C) in CALU-1 cells.

Also, we observed reduced secretion of MMP-2 in the presence of JWH-015 as shown by zymogram (Fig. 26A). MMPs are matrix metalloproteinases involved in ECM degradation. In early stage lung adenocarcinoma, higher expression of MMP-2 is involved in worse prognosis (215).

These results were strengthened by inhibition of other invasive markers like FAK and

VCAM-1 (Fig. 26B).

B Control 120 Control A 180 Control 120 C JWH JWH JWH 160 EGF 100 EGF EGF 100 140 JWH+EGF JWH+EGF JWH+EGF * 120 cells 80 * 80 100 * 60 60 80 40

60 40 cells invaded

% % No. ofNo.colonies

40 % migrated 20 20 20 0 0 0

D C J E J+E

36h

Figure 25: JWH-015 inhibited EGF induced migration and invasion in CALU1 cells. (A)

1000 individual CALU1 cells were subjected to colony formation assay by treating with control or JWH-015 (5μM) for six days. Colonies were stained and counted. CALU1 cells were treated with control or JWH-015 (5μM) for 48h and subjected to EGF

(100ng/ml)-induced migration (B), invasion (C) Number of cells migrated or invaded were stained and counted. (D) Cells were pre-treated with control or JWH-015 (5μM) for

36h. Monolayer was wounded by a scratch, washed and fed with media containing EGF

(100ng/ml). After 36h, plates were photographed. P<0.05 (*) as calculated by Student’s t test.

A B C J5 J10 C C J5 J10 C J1 J5 J10 P-FAK N-CADHERIN MMP2 GAPDH GAPDH VCAM-1 GAPDH

D SNAIL SLUG 300 450 250 E-CADHERIN * 400 250 350 200 200 300 250 * 150 150 ** 200 100 100 150 100 50 50

Relative expression Relative 50

Relative expression Relative Relative expression Relative 0 0 0 Control J5 J10 Control J5 J10 Control J5 J10

Figure 26: JWH-015 promotes mesenchymal to epithelial transition in CALU-1 cells.

(A) CALU1 cells were treated with control or JWH-015 at 1, 5 and 10μM (J1, J5, J10) for 48h and supernatants were concentrated and run on zymogram gels. (B) CALU1 cells were pre-treated with control or JWH-015 at concentrations of 5 and 10μM (J5, J10) for

48h and subjected to Immunoblot to determine expression of P-FAK and VCAM-1.

GAPDH is loading control. CALU1 cells were pre-treated with control or JWH-015 at 5 and 10μM (J5, J10) for 48h and subjected to Immunoblot (C) or Real time PCR (D) to determine expression of EMT markers. P<0.05 (*) and P<0.01 (**) as calculated by

Student’s t test.

To understand the molecular mechanism related to MET reversion, we treated CALU-1 cells with JWH-015 and checked for various EMT markers. We observed that JWH-015 inhibits and reverses the mesenchymal property of this cell line by increasing expression of epithelial markers like E-cadherin and decreasing mesenchymal markers like N- cadherin, Snail and Slug (Fig. 26C, D). These results confirmed our findings that JWH-

015 attenuates the mesenchymal phenotype of CALU-1 cells.

Control JWH-015

P-EGFR

T-EGFR

P-ERK

T-ERK

0 5 15 30 0 5 15 30 EGF (min)

Figure 27: JWH-015 inhibits EGF induced signaling in CALU1 cells. CALU1 cells were pre-treated with control or JWH-015 (5μM) for 48h, stimulated with EGF (100ng/ml) for

0, 5, 15m and subjected to Immunoblot to determine expression of P-EGFR and P-ERK.

T-EGFR and T-ERK served as loading control.

Since we are interested in regulation of EGF induced EMT by JWH-015, we studied the signaling molecules involved in EGFR pathway. Activation of this pathway leads to onset of various events like cancer proliferation, migration, invasion and survival. Extra- cellular regulated kinase (ERK) is important downstream target of EGFR (246). We treated CALU-1 cells with JWH-015 for 24h and then stimulated the cells with EGF at various time points to check the activation of EGFR targets. We found that JWH-015 decreased the phosphorylation of EGFR and ERK (Fig. 27).

4.3.4 JWH-015 inhibits M2 macrophage induced EMT in A549 cells

Emerging therapies focus on the tumor microenvironment (TME) as a vital component of cancer progression and metastasis. TME is composed of different kinds of cell types which penetrate into the tumor site and influence changes in ECM by secreting or recruiting tumorigenic factors (229, 247). Macrophages, which reside in most tissues play important role in tumor-stromal interactions. TAMs are a unique population in TME that foster tumorigenesis and metastasis by secreting factors that increase invasion, alter ECM composition, cause immune suppression, imbalance homeostasis, etc. (232, 248).

Macrophage activation can be classified into two types- M1 (classical) and M2

(alternative). LPS, IFN-γ trigger macrophages into M1 state, whereas, IL-4, IL-10 trigger macrophages into M2 state. M1 macrophages defend the host against infections and act as tumor suppressors. M2 macrophages suppress M1 mediated functions and promote cancer progression and angiogenesis (231). Also, macrophage derived growth factors and 97 cytokines alter the composition of tumor population, exhibiting a strong paracrine loop.

Reports suggest that macrophage activation and secretion of factors into the tumor site enhances migration, invasiveness, promoting EMT of cancer cells (232-233).

300 ARGINASE-I

250 *

200

150

100 Relative expression Relative 50

0 THP1 THP1-M2

Figure 28: MI to M2 conversion of THP-1 cells. THP-1 cells were treated with PMA

(100nM) together with IL-4 (20ng/ml) (THPI-M2) for 24h and were subjected to Real

Time PCR to determine expression of M2 marker Arg-1. P<0.05 (*) as calculated by

Student’s t test.

To investigate the effect of macrophage activation and secretion of factors that affect the tumor progression, we performed indirect co-culture assays with human monocyte cell line THP1 and lung adenocarcinoma cell line A549 (247). We stimulated THP1 cells with PMA together with IL-4 for 24h before indirectly co-culturing with A549 cells. IL-4

98 induced THP1 cells exibit M2 property (tumor inducing macrophages) which was verified by higher expression of M2 marker Arginase-I (Fig. 28). Co-culture of conditioned media (CM) of M2 macrophages with A549 cells enhanced proliferative

(Fig. 29A) and migratory (Fig. 29B, C) abilities of A549 cells which were inhibited by treatment of A549 with JWH-015.

A B C 300 Control 120 Control 120 Control JWH JWH JWH 250 M2 CM 100 M2 CM 100 M2 CM JWH+M2 CM JWH+M2 CM * JWH+M2 CM * 200 80 80

60 150 * 60

100 40 40 No. ofNo.colonies % invaded cells % invaded 20 50 % cells migrated 20

0 0 0 Figure 29: JWH-015 inhibits M2 macrophage proliferation and migration in A549 cells.

(A) 1000 individual A549 cells were subjected to colony formation assay by treating with control or JWH-015 (5μM) in the presence of M2 CM for six days. Colonies were stained and counted. A549 cells were treated with control or JWH-015 (5μM) for 48h and subjected to M2-polarized TAM CM-induced migration (B) and invasion (C). Number of cells migrated or invaded were stained and counted. P<0.05 (*) as calculated by Student’s t test.

A Control JWH M2 CM JWH+M2 CM C Control JWH M2 CM JWH+M2 CM P-ERK MMP2

B Control JWH M2 CM JWH+M2 CM T-ERK P-FAK P-STAT3 VCAM-1 T-STAT3 GAPDH

SNAIL SLUG D Control JWH M2 CM JWH+M2 CM E 250 Control 250 Control JWH JWH N-CAD M2 CM 200 200 M2 CM JWH+M2 CM JWH+M2 CM * GAPDH 150 150 * 100 100

50 50

Relative expression Relative Relative expression Relative 0 0

Figure 30: JWH-015 inhibits M2 macrophage induced EMT in A549 cells. (A) A549 cells were treated with control or JWH-015 (5μM) for 24h in the presence of M2 CM, then conditioned media was replaced by fresh media for another 48h and the supernatants were concentrated and run on zymogram gels. A549 cells were pre-treated with control or

JWH-015 (5μM) for 24h, stimulated with M2 CM for 48h and subjected to Immunoblot to determine expression of P-FAK and VCAM-1 (B), EGFR signaling pathway like P-

STAT3, P-ERK (C) and EMT markers (D) and also subjected to Real time PCR (E).

GAPDH is loading control. JWH-015 (JWH), M2-polarized TAM CM (M2 CM).

P<0.05 (*) as calculated by Student’s t test.

100

We hypothesize that macrophages secrete factors that activate the EGFR pathway, which promote tumorigenesis of cancer cells by stimulating EMT process. To test our idea, we performed co-culture experiments of M2 macrophages with A549 cells and checked for

EGFR targets. We observed that M2 macrophages activated pSTAT3, pERK in A549 cells which were inhibited by treatment of A549 with JWH-015 (Fig. 30C).

These data show that macrophages secrete ligands/factors that stimulate EGFR signaling in lung cancer cells that is markedly inhibited by JWH-015 treatment.

To further validate our data, we checked for EMT markers in the M2-macrophage-A549 co-culture experiment. M2 macrophages promoted EMT in A549 cells which was attenuated by JWH-015. This was proved by downregulation of mesenchymal markers like Snail, Slug and N-cadherin after JWH-015 treatment (Fig. 30D, E).

Hence, M2 macrophages activated EGFR pathway, thus inducing EMT in A549 cells which was inhibited by JWH-015.

4.3.5 JWH-015 prevents lung colonization of ED1 cells in in vivo tail vein syngenic mouse model

Metastasis is a complex process with multiple cascade of events (241). Reports suggest that EMT is involved in various steps of metastasis like invasion, degradation of ECM, etc (228). To study the effect of JWH-015 on cancer metastasis, we extended our in vitro findings to tail vein model in FVB mice.

101

250

200

150 *

100 No. ofNo.colonies 50

0 Control JWH-015 Control+EGF JWH+EGF

Figure 31: JWH-015 inhibits proliferation in ED1 cells. 1000 individual ED1 cells were plated and subjected to colony formation assay by treating with control or JWH-015

(5μM) in the presence or absence of EGF (100ng/ml) for six days. Colonies were stained and counted. P<0.05 (*) as calculated by Student’s t test.

To test whether JWH-015 exerts tumor suppressive effects in this cell line, we initially performed EGF induced proliferation (Fig. 31) which was significantly inhibited by

JWH-015.

Mouse ED1 lung cancer cells were injected intravenously into immunocompetent FVB mice. After a week of injection, mice were treated with ethanol control or JWH-015 (7.5 mg/kg) (n=5), intraperitoneally for 3 weeks and surface lung metastases were identified

(Fig. 32A) and the metastatic lesions were counted.

102

A B 12 Control JWH 10 8 6 * 4

lung nodules lung 2 No. ofNo.metastatic 0 Control JWH

Figure 32: JWH-015 inhibits NSCLC metastasis in in vivo mouse model. ED1 cells were injected by tail vein in FVB mice and after a week, mice were treated with control or

JWH-015 (7.5mg/kg) for 3 weeks. After treatment, their lungs were isolated (A) and metastatic lesions were counted (B).

We observed a drastic reduction in lung colonization in mice treated with JWH-015 with respect to control (Fig. 32B). This suggests that JWH-015 possesses anti-metastatic property apart from its tumor suppressive effect.

4.3.6 JWH-015 inhibits NSCLC tumor growth in vivo in a subcutaneous mouse model

103

To evaluate the tumor suppressive effects of JWH-015, we used ED1 cells, syngenic with

FVB mice. To test whether JWH-015 exerts tumor suppressive effects in this cell line, we initially performed EGF induced proliferation which was significantly inhibited by JWH-

015. Then, we injected ED1 cells into the right flank of 7 week old male syngenic FVB mice to induce tumor formation. When the tumors reached palpable size, we treated the tumors with either ethanol control or JWH-015 (7.5 mg/kg) (n=5), thrice a week for 3 weeks. Tumor volume was monitored and measured every week.

Cannabinoid administration blocked the subcutaneous growth of tumor cells very significantly, as verified by tumor volume (Fig. 33A) and weight (Fig. 33B, C).

To correlate the reduction in tumor growth with important cellular parameters, we performed IHC staining of tumors for Ki67 (proliferation marker) and CD31

(vascularization marker) to dissect the effect of JWH-015 on proliferation and angiogenesis.

JWH-015 treated tumors expressed lower Ki67 and CD31 levels compared to control

(Fig. 33D).

104

A ) B 3 1600 Control JWH-015 1400 Control JWH 1200 1000 800 600 ** 400 200 * Tumor volume (mm volume Tumor 0 WK3 WK4 WK5 Week post injection C D Control JWH 2

1.5 Ki67

1 **

0.5 CD31

Tumor weight (g) weight Tumor 0 Control JWH

Figure 33: JWH-015 inhibits NSCLC growth in subcutaneous mouse model. ED1 cells were subcutaneously injected in FVB mice and palpable tumors were treated with ethanol control or JWH-015 (7.5mg/kg) every third day for 3 weeks. Tumor volume (A) was calculated using the formula= length x (width)2/2. (B) Representative tumors and (C) tumor weight measured from various experimental groups. Representative (contd.)

(Fig. 33 contd.) photomicrographs of Ki67 and CD31 staining (D) of tumors extracted from various experimental groups. P<0.05 (*) and P<0.01 (**) as calculated by Student’s t test.

105

4.3.7 JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of tumor cells by downregulation of EGFR signaling

To further confirm our in vitro findings, we performed flow analysis of digested tumor cells harvested from the subcutaneous ED1 tumors.

We observed that there was significant reduction in CD11b/F4/80/CD206 M2 macrophage population in JWH-015 treated tumors compared to control (Fig. 34A).

There was no significant difference in other immune populations like CD3, CD4, CD8 T cells and CD11b/Gr1 (myeloid derived suppressor cells) (not shown).

This was further confirmed by IHC, where there was reduced expression of Arginase-I

(M2 marker) and F4/80 (macrophage marker) in tumors treated with JWH-015 compared to control (Fig. 34B).

To investigate whether reduced macrophage recruitment inhibits EMT of tumor cells, we checked for EMT markers and found that there was downregulation of mesenchymal markers in JWH-015 treated tumors with respect to control (Fig. 35A).

106

A Control Control 14.1% PE- F4/80 F4/80+ cells gating 20

15 * 10 JWH F4/80+ JWH 8.1% PE- F4/80 cells 5

gating macrophages M2 % % 0

CD11b+ Control JWH

CD206+ B ARGINASE-I F4/80

Control JWH Control JWH

Figure 34: JWH-015 decreases macrophage recruitment to tumor site. (A) Xenograft tumors were subjected to flow analysis. F4/80 cells isolated were gated to check for

CD11b+CD206+ cells to determine the population of M2 macrophages. (B)

Representative photomicrographs of F4/80, Arginase-1 staining of tumors extracted from experimental groups. P<0.05 (*) as calculated by Student’s t test.

107

A 200 SNAIL 200 SLUG

150 150 * *

100 100 Relative Relative

Relative Relative 50 50

expression expression

0 0 Control JWH Control JWH

B Control JWH C P-EGFR 300 MMP2 250 T-EGFR 200 * P-ERK 150 100 T-ERK 50

Relative expression Relative 0 P-STAT3 Control JWH T-STAT3

Figure 35: JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of tumor cells by downregulation of EGFR signaling. (A) Xenograft tumors were subjected to Real Time PCR to determine expression of EMT markers. (B) Xenograft tumors isolated were subjected to Western blot to determine expression of EGFR signaling targets. GAPDH is loading control. (C) Xenograft tumors were subjected to

Real Time PCR to determine expression of MMP2. P<0.05 (*) as calculated by Student’s t test.

108

To prove that JWH-015 inhibits macrophage induced EMT by downregulation of EGFR pathway, we checked for EGFR targets in tumor lysates and observed marked reduction of pEGFR, pSTAT3 and pERK in tumors treated with JWH-015 with respect to control

(Fig. 35B). Also, the JWH-015 treated tumors expressed lesser MMP2 levels compared to control (Fig. 35C).

These results show that macrophages are recruited to the tumor site, which enhance EMT process by activation of EGFR pathway. This process is inhibited by treatment of tumor cells with JWH-015 leading to downregulation of EGFR signaling, thereby, reduced macrophage recruitment and attenuation of EMT.

4.4 Conclusion

Cannabinoids were originally derived from the marijuana plant Cannabis sativa.

Currently, there are more than 60 compounds isolated from this plant, apart from the synthetic and endogenous cannabinoids (249-250). Initially, they were used in patients for palliative care against emesis, pain, etc. Recently, they have been identified and studied for their potent anti-cancer properties.

Evidence from the last decade proves that these cannabinoids inhibit tumorigenesis in various cancer types like breast, lung, brain, colon, prostate, etc. These compounds affect various pathways like cell cycle, inflammation, angiogenesis, proliferation, migration,

109 invasion, metastasis, etc. Cannabinoids modulate this complex array of signaling pathways mainly through cannabinoid receptors- CB1 and CB2 (12, 251).

JWH-015 is a synthetic compound that is selective for CB2 receptor. Reports suggest that

JWH-015 inhibits growth of hepatocellular carcinoma (HCC) via activation of autophagy and apoptosis by AMPK activation and TRB3 regulation (252). Also, PPARγ plays a major role in JWH-015 induced autophagy in HCC (253). In prostate cancer cells, JWH-

015 exerted anti-proliferative effects by promoting ceramide synthesis induced cell death by regulation of JNK and AKT signaling molecules (84). JWH-015 exerts anti- tumorigenic effects in other cancer tissue types like breast (235, 254), lung (194) and lymphoblastic leukemia (255). Although the anti-tumorigenic effects of JWH-015 have been established in various cancer types, the mechanism by which JWH-015 exerts these effects is not well known, especially in lung cancer. Our data reveals a novel mechanism by which JWH-015 exibits its tumor suppressive properties in NSCLC.

In our present investigation, we identified that JWH-015 inhibits EGF induced EMT in vitro in an epithelial cell line A549 and a mesenchymal cell line CALU1. EGFR has contributed to lung cancer growth by involving in vital cellular responses like proliferation, migration, invasion, metastasis, etc. (204) Also, EMT, which is activated by receptor tyrosine signaling, oncogenes, etc. contributes to lung cancer development (226,

228, 256).

Our results confirm these data that EGF induces EMT and JWH-015 significantly inhibits this process. In epithelial cell line A549, EGF induced EMT promoted mesenchymal 110 character that is reversed by JWH-015. This was further strengthened in mesenchymal cell line- CALU1, where JWH-015 inhibits mesenchymal markers and upregulates epithelial markers. Also, JWH-015 inhibits phosphorylation of EGFR and ERK, a downstream target of EGFR pathway (257), thus proving that blockade of EMT is through downregulation of EGFR pathway.

Finally, the aggressiveness of EGF induced effects like migration and invasion were reduced by JWH-015 by decreasing the expression of invasive markers like MMP2,

VCAM-1 and FAK. This is of great importance considering the fact that EGF induced

EMT has been shown in various cancer types and expression of EMT markers like E- cadherin and vimentin may be used as predictive biomarkers in analyzing the efficacy of

EGFR inhibitors in NSCLC (227).

Our results indicate that JWH-015 inhibits TAM induced EMT in A549 cells in an indirect co-culture model. The tumor microenvironment is composed of various cellular and non-cellular elements (230, 258). Among these multiple and unique cell types, tumor associated macrophages (TAMs) are crucial components because of their diverse interactions with the cancer cells. TAMs are M2 macrophages, which are tumor promoting macrophages (231-233).

We prove that TAMs secrete EGF like ligands/factors which activate the EGFR pathway in cancer cells, thus promoting EMT. This macrophage induced EMT is significantly inhibited by treating A549 cancer cells with JWH-015, which downregulates the EGFR pathway. This data correlated with our previous result that the effects of EGF induced 111

EMT is reversed by JWH-015. Thus, blockade of tumor progression and malignancy is dependent on the interplay between cancer cells and host cells of the tumor microenvironment.

Mouse models are very important to validate the in vitro results and also to study the efficacy of the anti-tumorigenic compound in vivo. Also, the regulation of the tumor microenvironment is well understood in vivo.

We studied the effect of JWH-015 in subcutaneous xenografts of mouse ED1 cells in immunocompetent FVB mice. JWH-015 significantly reduced tumor growth which was confirmed by decrease in proliferation marker- Ki67 and angiogenic marker- CD31.

CD11b/ F4/80/ CD206 M2 macrophages which are recruited to the tumor site is effectively blocked by JWH-015.

Finally, EMT markers like N-cadherin, Snail and Slug were attenuated in JWH-015 treated tumors. This marked reduction of EMT is due to downregulation of EGFR and its targets like ERK and STAT3 by JWH-015. In a metastatic tail vein model, treatment of mice with JWH-015 reduced the number of metastatic lesions present in the lung. These experiments correlate our in vitro data that JWH-015 modulates the inflammatory microenvironment, thus inhibiting EGF mediated EMT in lung cancer cells.

112

4.5 Discussion

Overall, the malignant properties of cancer cells can only be well understood if we study the crosstalk between cancer cells and the tumor microenvironment. Thus, a novel approach is required where the anti-tumorigenic compound has to target the tumor cells as well as affect its tumor microenvironment. In our study, JWH-015 was an effective anti-tumorigenic and anti-metastatic cannabinoid agonist. Also, it targeted the inflammatory microenvironment by attenuating the recruitment of tumor associated macrophages, thus inhibiting EMT in cancer cells by downregulation of EGFR pathway.

These findings suggest a crosstalk between CB2 and EGFR, thereby exploring the possibility of CB2 agonist, JWH-015 as a novel therapeutic target for NSCLC treatment.

113

Chapter 5

Future directions

Our data reveal that the endocannabinoid Met-F-AEA (along with FAAH inhibitor

URB597) and the synthetic cannabinoid JWH-015 have anti-cancerous mechanisms in

NSCLC. They exert anti-proliferative and anti-metastatic effects in vitro and in vivo through downregulation of EGFR pathway. It would be interesting to further study these cannabinoids in detail to gain better understanding:

 Since EGFR is the crucial gene that is involved in lung carcinogenesis, additional

experiments are required to study the CB1/2-EGFR crosstalk by

immunoprecipitation assays to check for direct binding of receptors.

 We studied the effect of cannabinoids on NSCLC cells with wildtype EGFR.

Further validation is required to check for the efficacy of cannabinoids in EGFR

mutated cell lines.

 Since JWH-015 inhibited recruitment of macrophages at the tumor site, it would

be interesting to study the effect of JWH-015 on tumor growth and EMT in

macrophage depleted mouse model system.

114

 We performed preliminary experiments with gefitinib resistant A549 (A549-GR)

cells and found that JWH-015 sensitized A549-GR cells by inhibiting

proliferation and migration in these cells. This data is very crucial because, in

lung cancer, most patients become resistant to EGFR inhibitors like gefitinib.

Further evaluation with JWH-015/Met-F-AEA can lead to replacement of EGFR-

TKIs with cannabinoids.

 Since EGFR activation drives EMT, ultimately causing tumor stemness, it would

be important to determine the role of cannabinoids in inhibition of cancer

stemness by downregulation of EGFR pathway.

Cannabinoids exert a direct anti-proliferative effect on tumors of different origin. They have been shown to be anti-migratory and anti-invasive and inhibit MMPs which in turn degrade the extra-cellular matrix (ECM), thus affecting metastasis of cancer to the distant organs. Also, cannabinoids modulate other major processes in our body like energy metabolism, inflammation, etc. These data are derived not only from cell culture systems but also from more complex and clinically relevant animal models. Before cannabinoids could be used in clinical trials, there is need to explore more knowledge on several issues such as anti-tumorigenic and anti-metastatic mechanisms as well as which type of cancer patient populations would be more responsive for cannabinoid based therapies. Data presented here suggest that cannabinoids derived from different sources regulate differently signaling pathways, modulate different tumor cell types and host physiological system. It is important to understand which of the cannabinoid receptors

115 are expressed and activated in different tumors as each receptor follows a different signaling mechanism.

Furthermore, endocannabinoids- AEA and 2-AG are broken down into secondary metabolites like prostaglandin (PGE2) and epoxyeicosatetraenoic acid (EE) which enhance tumor growth and metastasis in diverse cancer types. Understanding the exact signaling by which cannabinoids function will eventually lead to targeted clinical approach. Also, the difference in cellular response to cannabinoids in different cancer types might be due to the effect of the tumor environment which involves inflammatory cells, fibroblasts, endothelial cells, macrophages, etc. Thus, there is a need for an integrative understanding of the role of cannabinoids with respect to the tumor and its microenvironment. The diversity of affecting multiple signaling pathways might pave way for developing cannabinoids that selectively obstruct a particular pathway, thus opening avenues for specific targeted treatments.

Moreover, cannabinoids are more specific to cancer cells than normal cells. The administration of single cannabinoids might produce limited relief compared to the administration of crude extract of plant containing multiple cannabinoids, terpenes and flavanoids. Thus, combination of cannabinoids with other chemotherapeutic drugs might provide a potent clinical outcome, reduce toxicity, increase specificity and overcome drug resistance complications. Additional findings in in vitro and in vivo models are needed to support studies at preclinical setting.

116

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