The Role of Cannabinoids and their Receptors in Breast 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 Mohamad Mostafa Abdelhamid Elbaz Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2016 Dissertation Committee: Dr. Ramesh Ganju, Ph.D., Advisor Dr. Sujit Basu, M.D., Ph.D. Dr. Kalpana Ghoshal, Ph.D. Dr. Xue-Feng Bai, M.D., Ph.D. Copyright by Mohamad Mostafa Abdelhamid Elbaz 2016 Abstract Breast cancer is a heterogeneous disease that has different biological and clinical behaviors. It represents the second cause of cancer deaths among US women. Cannabinoids can be classified into endocannabinoids, synthetic cannabinoids and phytocannabinoids. These cannabinoids act on different receptors such as Cannabinoid receptor-1 (CB1R), Cannabinoid receptor-2 (CB2R), Transient receptor potential vanilloid type-2 (TRPV2), etc. In this thesis, we have examined the anti-tumor role of an important phytocannabinoid, Cannabidiol (CBD), and a specific CB2R agonist synthetic cannabinoid, JWH- 015. We also studied the role of CB2R activation in cancer cells as well as immune cells and finally we analyzed the role of TRPV2 in improving the efficacy of chemotherapeutic drugs. CBD is a non-psychotropic cannabinoid compound. Its anti-tumor role and mechanism are not well studied especially in triple-negative breast cancer (TNBC). In the present study, we analyzed CBD’s anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time- that CBD significantly inhibits epidermal growth ii factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP-2 and MMP-9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor-associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction of the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of GM-CSF and CCL3 cytokines, which are important for macrophage recruitment and activation. In summary, our study shows -for the first time- that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibition EGF/EGFR signaling and modulating the tumor microenvironment (TME). These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates. TRPV2 is a cation channel that is triggered by agonists like CBD. Chemotherapy still the first line for the treatment of triple negative breast cancer (TNBC) patients, however, TNBC cells usually gain rapid resistance and unresponsiveness against chemotherapeutic drugs. In this study, we analyzed iii TRPV2 role in enhancing chemotherapeutic efficacy in TNBC. First, we found that TRPV2 is significantly up-regulated in primary and metastatic breast cancer compared to normal breast tissues. Second, we observed that TNBC patients with higher TRPV2 expression and committed to chemotherapy have significantly higher overall and recurrence free survival compared to patients with lower TRPV2 expression. We also showed that TRPV2 overexpression significantly increased doxorubicin (DOX) uptake and efficacy. We showed that TNBC cells that are subjected to combination treatment (CBD+DOX) have less viability compared to DOX-treated cells. Analysis of molecular mechanisms showed higher levels of cleaved PARP and caspase-3 in combination treatment compared to DOX alone. Further studies, revealed that CBD enhanced the uptake of DOX into TNBC cells. Importantly, we show that CBD effects are TRPV2-mediated and TRPV2 downregulation or interference by its dominant negative form inhibits DOX-mediated cytotoxicity. In vivo studies showed that (CBD+DOX) mice group has significantly reduced tumor weight and enhanced cleaved (caspase-3/PARP) levels compared to DOX group. This study suggests TRPV2 agonists as adjuvant therapy to improve the anti-tumor chemotherapeutic efficacy and suggest TRPV2 as a prognostic marker for TNBC patients who receive chemotherapy for their treatment plan. Cannabinoid receptor-2 (CB2R) is an integral part of the endocannabinoid system. It is upregulated in the primary breast cancer lesions and highly iv expressed in different types of immune cells. However, its functional role in breast tumorigenesis is not well understood. The present study was aimed at evaluating the mechanistic anti-tumor role of CB2R activation on breast cancer cells and immune cells within the breast TME. First, we analyzed the anti- tumorigenic mechanisms of CB2R activation in ERα- and ERα+ breast cancer cells. Our studies showed that CB2R specific agonist (JWH-015) inhibited EGF and IGF-I-induced migration and invasion of ERα+ and ERα- breast cancer cells. At the molecular level, JWH-015 inhibited EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and MMP-9/MMP-2. Interestingly, we found that JWH-015 significantly reduced breast cancer growth in vivo and the tumors that were derived from CB2R agonist treated mice showed reduced activation of EGFR and IGF-IR and their downstream targets compared to control group. Since CB2R is highly expressed in immune cells, we assessed the role of CB2R activation on modulation of immune cells present in tumor stroma. We observed increased tumor weight, more myeloid derived suppressor cells (MDSCs) (CD11b+/Gr-1+) and less CD3+/CD8+ cells in orthotopically injected CB2R knock out mice compared to wild type mice. Furthermore, we found that JWH-015-treated wild type mice had reduced tumor growth and metastasis, more CD3+/CD8+ cells and less MDSCs within the tumor stroma. For the first time, we show that CB2R activation might suppress breast tumor growth and metastasis through novel mechanisms of inhibiting EGF/EGFR and IGF-I/IGF-IR signaling v axes on tumor cells and modulating the immune cells’ compositions within the breast TME. Overall, our studies showed that cannabinoids such as CBD and JWH-015 exert strong anti-tumor activities against breast cancer. They both inhibit hallmark signaling pathways such as EGF/EGFR and IGF-I/IGF-IR pathways. In addition, these drugs have TME modulating potential through different mechanisms which eventually enhance the anti-tumor immunity against breast cancer cells. Finally, we show that CBD, upon activating TRPV2 channel, increases the uptake capacity of breast cancer cells and makes these cancer cells more susceptible to the anti-tumor chemotherapeutic drugs. vi Dedication This document is dedicated to my mother Thanaa Hussein, father Mostafa Elbaz, my sisters Reham, Shereen and Nariman. vii Acknowledgment I would like to thank my family who sacrifice a lot to help me through all of my life. Special thanks to my mother who passed away and I really miss her and to my father for his continuous sacrifice and support. I am very grateful and I would like to thank my advisor Dr. Ramesh Ganju. I thank him for his continuous strong guidance and encouragement during all Ph.D years. His guidance helped me a lot in all the time of research and writing of this thesis. He always encourages me and makes me believe in myself. I also thank him for giving me a great opportunity to work as a member of his amazing team. I greatly appreciate every valuable advice and every righteous direction he gives till now. I have really learnt from him how to be strong if you face any problem whether in the research or in general life. I would like to greatly thank my committee members, Dr. Sujit Basu for his insightful comments and encouragement, remarkable advises, sincere valuable guidance and unstoppable support, Dr. Kalpana Ghoshal for her aspiring guidance, immense knowledge and for continuous help and support in every aspect scientifically and personally, and Dr. Xue-Feng Bai, for priceless advises, stimulating scientific discussions, great support and brilliant suggestions during viii my years of Ph.D. I am also grateful to all of my mentors and MCDB program directors, Dr. David Bisaro and Dawn Chandler for their continuous supervision and valuable advises. I want to send special thanks to all the past and present Ganju lab members for helping me learning new approaches and techniques and for their assistance and their valuable scientific discussions. I thank them all for all the fun we have had during our collaborative work. I wish to all of them great success in their academic life. ix Vita May 2001-2006..................................Pharmacy School-Helwan University-Egypt. June 2006-2011............................…Master degree- Pharmacology Department, Helwan University-Egypt. Dec. 2011-Now..................................Graduate Research Associate, Department of Pathology, The Ohio State University. Publications 1. Elbaz M, Nasser MW, Ravi J et al. Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway; Novel anti-tumor mechanisms of Cannabidiol in breast cancer. Molecular Oncology, 2015; 9(4): 906-19. 2. Nasser MW*, Wani NA*, Ahirwar DK, Powell CA, Ravi J, Elbaz M et al. Receptor for Advanced Glycation End products (RAGE) mediates S100A7- induced breast cancer growth and metastasis via modulating tumor microenvironment. Cancer Research, 2015; 15; 75(6):974-85. 3. Elbaz M*, Mohd W. Nasser*, Ramesh K. Ganju. Conditioning the solid tumor microenvironment through inflammatory chemokines and S100 family proteins. Cancer letters 2015; 365 (1): 11-22. * Equal first author. x 4.Elbaz M, Grace Amponsah, Ramesh K. Ganju, Mohd W. Nasser. S-100 proteins. Encyclopedia of cancer. Invited book chapter 5. Ravi J, Elbaz M, Nissar A.