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The Role of E2F7 and E2F8 in Squamous Differentiation, UV- and Chemotherapy-Induced Responses in Keratinocytes

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The Role of E2F7 and E2F8 in Squamous Differentiation, UV- and Chemotherapy-Induced Responses in Keratinocytes The role of E2F7 and E2F8 in squamous differentiation, UV- and chemotherapy-induced responses in keratinocytes Mehlika Hazar Rethinam MScBiotech (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 UQ Diamantina Institute i Abstract Among keratinocyte-derived squamous cell carcinoma (SCC), cutaneous SCC (CSCC) is the second most common cutaneous cancer type and the most common of the potentially fatal skin cancers. Approximately 90% of head and neck cancers are SCC (HNSCC) and HNSCC worldwide is the sixth most common cancer type afflicting mankind. While resectable disease may be treated by surgery with radiation or chemoradiation, there are still no curative options for advanced, unresectable disease. However, chemotherapy alone may offer a hope for unresectable, disseminated SCC, where 50-60% of patients have disease recurrence within 2 years and approximately 30% of these develop metastatic disease. The mainstay of chemotherapeutic treatment in SCC is the platinum-based drug, cisplatin, 5-fluorouracil (5- FU), taxanes, and anti-EGFR monoclonal antibody such as Cetuximab. Nonetheless, despite advances in treatment techniques, the 5-year survival rate still remains at around 55%. Therefore, there remains a lack of options for recurrent or metastatic disease. The E2F family of transcription factors has emerged as key regulators of proliferation, differentiation and response to stress and apoptotic stimuli in keratinocytes. Consistent with these roles, dysregulation of E2F expression/activity is a common occurrence in cancer and SCC in particular. Thus, better understanding of the E2F family of proteins is essential to establish how these processes are disrupted during SCC genesis. The E2F network exists as a complex map of interacting pathways, and the complete understanding of the E2F family will not be possible until physiological functions of newly identified inhibitory E2F proteins, E2F7 and E2F8, are fully revealed. I provide strong evidence, from in vitro experiments, that E2F7 plays a unique and non-redundant role in modulating UV- or chemotherapy-induced cytotoxicity whereas E2F8 has a unique and non-redundant capacity to regulate squamous differentiation. In addition, I report on a unique feedback loop between E2F1, E2F7 and E2F8 that highlights a previously unreported interdependent axis. With respect to E2F7-specific functions, my work characterised two previously unidentified pathways as therapeutic targets, E2F7/Sphk1/S1P/AKT and E2F7/RacGAP1/AKT, by which the transcription factor E2F7 suppresses doxorubicin specific sensitivity in SCC cells. Targeting these pathways has the potential to expand the clinical activity of existing chemotherapeutics. I provide, in vitro, in vivo and patient data in this study that shows that E2F7-dependent repression of doxorubicin sensitivity is mediated via the induction of Sphingosine kinase 1 (Sphk1) and Rac GTPase activating protein 1 (RacGAP1). These are ii both novel findings and have significant implications for drug resistant SCC. Specifically, I showed that (i) E2F7 is overexpressed in patient SCCs and inhibits sensitivity to doxorubicin, (ii) that E2F7-dependent doxorubicin resistance is mediated via E2F-dependent induction of Sphk1 leading to overproduction of Sphingosine-1-phosphate (S1P) which in turn activates AKT, (iii) that pharmacological inhibition of Sphk1 or AKT sensitizes SCC cells to the cytotoxic actions of doxorubicin in in vivo models of SCC resulting in profound tumour regression, iv) that induction RacGAP1 in SCC cells increases their sensitivity to doxorubicin and overexpression of RacGAP1 is linked to poor outcomes in SCC patients and v) that E2F7-dependent doxorubicin resistance is mediated via induction of RacGAP1 which in turn activates AKT-dependent pathways in vitro and in vivo. Since dysregulation of the E2F family of transcription factors is one of the most frequent targets in oncogenesis, it is likely that our findings will be relevant to many other cancer types. Moreover, our findings provide proof of concept for immediate translation to initiate a clinical trial using combinations of clinically available agents such as doxorubicin + AKT inhibitor or doxorubicin + an SK1 inhibitor. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature 1. Hazar-Rethinam M, Endo-Munoz L, Gannon O and Saunders N. Int J Mol Sci. 2011; 12(12): 8947-8960. Review. 2. Hazar-Rethinam M, Merida de Long L, Gannon OM, Topkas E, Boros S, Vargas AC, Dzienis M, Mukhopadhyay P, Simpson F, Endo-Munoz L, Saunders NA. Clin Cancer Res Published Online First November 19, 2014; doi: 10.1158/1078- 0432.CCR-14-1962. 3. Hazar-Rethinam M, Merida de Long L, Gannon OM, Boros S, Vargas AC, Dzienis M, Mukhopadhyay P, Simpson F and Saunders NA. Oncogene [Submitted; December 2014] Poster Presentations Australian Society for Medical Research Postgraduate Student Conference, Brisbane, Queensland 2014 “Sphingosine kinase 1 is a novel E2F7-dependent effector that regulates chemotherapeutic sensitivity in squamous cell carcinoma” American Association for Cancer Research Annual Meeting, Washington DC, US 2013 “E2F7 regulates sensitivity to the cytotoxic actions of anthracyclines in squamous cell carcinoma” Lorne Cancer Conference, Lorne, Victoria 2013 “E2F7 regulates sensitivity to the cytotoxic actions of anthracyclines in squamous cell carcinoma” Australian Society for Medical Research Postgraduate Student Conference, Brisbane, Queensland 2012 “E2F7 regulates sensitivity to the cytotoxic actions of anthracyclines in squamous cell carcinoma” Princess Alexandra Hospital Health Symposium, Brisbane, Queensland 2012 “E2F7 regulates sensitivity to the cytotoxic actions of anthracyclines in squamous cell carcinoma” v Molecular and Experimental Pathology Society of Australasia Conference, Adelaide, South Australia 2012 “E2F7 regulates sensitivity to the cytotoxic actions of anthracyclines in squamous cell carcinoma” Publications included in this thesis 1. Hazar-Rethinam M, Endo-Munoz L, Gannon O and Saunders N. Int J Mol Sci. 2011; 12(12): 8947-8960. Review. Partially incorporated as paragraphs in Chapter 1. A PDF version of the paper is included as an appendix (Appendix II) to this thesis. The preparation of the manuscript was the work of the candidate, with helpful suggestions on the manuscript contributed by all authors. 2. Hazar-Rethinam M, Merida de Long L, Gannon OM, Topkas E, Boros S, Vargas AC, Dzienis M, Mukhopadhyay P, Simpson F, Endo-Munoz L, Saunders NA. Clin Cancer Res Published Online First November 19, 2014; doi: 10.1158/1078- 0432.CCR-14-1962. Incorporated as Chapter 5 The preparation of the manuscript was the work of the candidate (MHR). LEM, FS and NAS contributed intellectual input to the project and edited the manuscript. SB, ACV and MD assisted with acquisition of data presented in Figure 2F. LMdL performed some of the animal work resulted in Figure 4, Figure 5E and F, Figure 6F and Figure S6B. PM and OMG provided technical support with analysis of the raw microarray data. ET assisted with the analysis of Figure 6G. All other experimental work was the work of the candidature (MHR). vi 3. Hazar-Rethinam M, Merida de Long L, Gannon OM, Boros S, Vargas AC, Dzienis M, Mukhopadhyay P, Saenz-Ponce N, Simpson F and Saunders NA. Oncogene [Submitted; December 2014] Incorporated as Chapter 6 The candidate (MHR) designed all experiments, produced all figures and wrote the manuscript. LMdL performed some of the animal work resulted in Figure 7a and b. SB, ACV, MD and NSP assisted with acquisition of data presented in Figure 3a-e. PM and OMG provided technical support with analysis of the raw microarray data explained in Figure 2a. FS critically reviewed the manuscript. NAS provided intellectual input to the project and edited the manuscript. Contributions by others to the thesis This thesis is the product of work primarily performed by myself with contributions from many people listed below and in the contributions
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