Crosstalk Between Cannabinoid Receptors and Epidermal
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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 By 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 Copyright by Janani Ravi 2015 Abstract (i) The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti- cancer 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. iv Dedication This document is dedicated to my mother Rajeswari, father Ravi, sister Pavithra and grandparents. v 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. vi 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 ix 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