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A NOVEL RNA APTAMER-BASED TARGETED THERAPY for OVARIAN CANCER with PRKCI GENE AMPLIFICATION by Hina Salam Rehmani July 2019 COPY

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A NOVEL RNA APTAMER-BASED TARGETED THERAPY for OVARIAN CANCER with PRKCI GENE AMPLIFICATION by Hina Salam Rehmani July 2019 COPY A NOVEL RNA APTAMER-BASED TARGETED THERAPY FOR OVARIAN CANCER WITH PRKCI GENE AMPLIFICATION By Hina Salam Rehmani Submitted to the Faculty of the Graduate School of Augusta University in partial fulfillment of the Requirements of the Degree of Doctor of Philosophy in Biochemistry and Cancer Biology July 2019 COPYRIGHT © 2019 by Hina S. Rehmani ACKNOWLEDGEMENTS Firstly, I would like to thank the many researchers in our scientific community that aided me in my research and development as a scientist. Thank you to my mentor, Dr. Shuang Huang, for opening your lab to me, motivating me, and for addressing all my questions and concerns. Thank you to all my committee members for their valuable feedback and discussions. Thank you to Drs. Hong, Morgan, Zemksov, and Islam for their patience in teaching me and sharing their technical aptitude. Thank you to Kim, Ida, Joyce and Bennie at the department for their help and thanks to the Graduate Office for their coursework guidance and support in presenting at AACR. I would also like to thank the unwavering support of my dear family, friends, and community. Overwhelming and heartfelt gratitude goes to my parents, Abdul S. Rehmani and Eram Yasmeen. It has been with your love, sacrifice and fortification that I have accomplished my goals and I hope to make you proud always. To my best friend and sister, Sadaf, thank you for all of your smart advice and kind words of encouragement at every obstacle. Thanks also to my brother Abdur Rahim, sister Abeera, and youngest brother Usman for always being sources of happiness and motivation. I am thankful to have you all as my siblings. To all my friends, especially Zainab and Rocio, thank you for all the laughs. To my dear aunt, Dr. Shafqat Rehmani, thank you for being an amazing inspiration and role model. Special thanks and lots of love goes to all my family in Pakistan, UK, Canada, and USA for rooting me on and praying for me constantly. To my supportive husband, Mohsin Saeed, thank you for adding so much happiness to my life. To our daughter, Sabrina Noor, I love you so much. Thank you to my parents-in-law, Rifat Saeed and Saeed Ullah Jan, for their kind hearts, persistent prayers and support in all my ambitions. To all of Mohsin’s lovely family, thank you for being so encouraging and generous to us. And finally, I would like to thank God Almighty for blessing my life with wonderful family and friends. I dedicate all my research efforts and energies to those affected with cancer so that there may be smarter ways to win the “battle”, with special focus on women with ovarian cancer. ABSTRACT HINA SALAM REHMANI A Novel RNA Aptamer-Based Targeted Therapy for Ovarian Cancer with PRKCI Gene Amplification (Under the direction of SHUANG HUANG) Ranked fifth in cancer death among women, ovarian cancer cure rates have remained low over the past two decades due to unsuccessful detection of early-stage disease, stagnant methodologies of treatment, and high relapse rates. Stage IV invasive epithelial ovarian cancer patients have a meager 17% relative 5-year survival rate and around 70% of patients relapse within the first two years of diagnosis. Accounting for more deaths than any other cancer of the female reproductive system, it is paramount to strategically offer ovarian cancer patients targeted therapies to improve outcome. Ovarian cancer contains a host of copy number aberrations (CNA) that can lead to the silencing or amplification of tumor suppressor genes, oncogenes, or non-coding RNAs that modify the expression of genes. Clinically, gene amplifications have prognostic and diagnostic usefulness as they can be a mechanism to promote tumorigenesis and/or attainment of drug resistance. Protein kinase C iota (PKCiota), a cytoplasmic serine-threonine protein kinase, is highly amplified and overexpressed within the 3q26 amplicon in ovarian cancer and is identified as an oncogene in multiple cancers. However, selectively targeting the iota isoform with small molecule inhibitors is challenging since it shares a 72% overall homology with another atypical PKC isoform, zeta. We found that small-molecule inhibitors currently being investigated do not offer selective inhibition nor specificity in ovarian cancer cell lines. We hypothesize that PRKCI amplification offers a unique opportunity to stratify patients into different risk categories and that specifically targeting PRKCI using a siRNA-aptamer can offer therapeutic benefits by impairing ovarian cancer cell tumorigenesis. We expect this approach to provide potent and selective anti-tumor activity compared to targeting using other pharmaceutical approaches. To test this hypothesis, we identified ovarian cancer cell lines with PRKCI gene amplification, identified a pattern of onco-addiction specificity in cell lines with amplification, and created an RNA-based aptamer that efficiently becomes internalized in ovarian cancer cells and mediates PRKCI mRNA silencing. In conclusion, we have identified a rationale to target, specifically, the PKCiota gene amplification in ovarian cancer and our RNA-based aptamer prevents ovarian tumorigenesis both in vitro and in vivo , opening a door for future therapies. KEY WORDS: Ovarian Cancer, Protein Kinase C iota, EpCAM, Aptamer Table of Contents Chapter Page 1 Introduction ........................................................................................................1 Overview of Dissertation ...............................................................................1 Ovarian Cancer ..............................................................................................2 The PKC Family ..........................................................................................22 The Atypical PKCs: PKCiota/lambda and PKCzeta ...................................24 Rationale for isotype-specific targeting within malignancies .....................27 EpCAM .......................................................................................................28 Aptamers .....................................................................................................31 2 Materials and Methods .....................................................................................34 3 Significance of PKCiota Amplification in Ovarian Cancer .............................49 Results .........................................................................................................54 4 Silencing PKCiota in Ovarian Cancer .............................................................68 Results .........................................................................................................68 5 Targeting PKCiota using Small Molecule Inhibitors.......................................80 Aurothiomalate ............................................................................................80 Auranofin .....................................................................................................81 Oncrasin-1 ...................................................................................................84 Summary .....................................................................................................85 6 Use of Small Molecule Inhibitors targeting PKCiota in Ovarian Cancer ........86 Results .........................................................................................................86 7 Aptamer Based Strategy for targeting PKCiota-Amplified Ovarian Cancer ...91 Results .........................................................................................................93 8 Discussion ......................................................................................................110 Screening Methods to Stratify Patients with PKCiota amplification ........110 Future Direction and Treatment Possibilities ............................................112 Potential Gaps and Questions Raised ........................................................113 Summary of Dissertation ...........................................................................114 References ........................................................................................................................115 Appendices .......................................................................................................................129 A Vita .................................................................................................................129 B Supplemental Material ...................................................................................129 List of Tables Table 1: Estimated New Cancer Cases & Deaths Worldwide for Ovarian Cancer & all Cancers in Women ...............................................................................................................3 Table 2: List of some FDA approved Drugs for Ovarian Cancer ................................ 19-20 Table 3: EpCAM CAR-T Cell Therapies in Active Clinical Trials ...................................30 Table 4: Cell Line Information ..........................................................................................35 Table 5: Primary and Secondary Antibodies Used ............................................................39 Table 6: Selected Genes within the Chromosome 3q26 Amplicon with over 30% amplification in patients .....................................................................................................51 Table 7: PRKCI’s Oncogenic Properties using the 10 Hallmarks of Cancer ....................53 Table 8: TCGA Copy Number Analysis
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