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Alternative Splicing of Mdm4 in Human Melanoma

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Alternative Splicing of Mdm4 in Human Melanoma ALTERNATIVE SPLICING OF MDM4 IN HUMAN MELANOMA A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By ABDULLAH SALEM S ALATAWI Bachelor., University of Tabuk, Saudi Arabia, 2015 _________________________________________________________ 2020 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL July 29, 2020 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Abdullah Salem S Alatawi ENTITLED Alternative Splicing of MDM4 in Human Melanoma BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. _____________________________ Michael Markey, Ph.D. Thesis Director _____________________________ Madhavi P. Kadakia, Ph.D. Chair, Department of Biochemistry and Molecular Biology Committee on Final Examination: ________________________________ Michael Markey, Ph.D. Research Associate Professor of Biochemistry & Molecular Biology ________________________________ Weiwen Long, Ph.D. Associate Professor of Biochemistry & Molecular Biology ________________________________ Michael Craig, Ph.D. Research Assistant Professor of Biochemistry & Molecular Biology ________________________________ Barry Milligan, Ph.D. Interim Dean of the Graduate School ABSTRACT Alatawi, Abdullah Salem S. M.S. Department of Biochemistry and Molecular Biology, Wright State University 2020. Alternative Splicing of MDM4 in Human Melanomas Melanoma is a potentially lethal type of skin cancer and regarded to be the third most common type of skin cancer. Although melanoma is not as common as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), it is more likely to metastasize than BCC and SCC. Interestingly, the incidence of melanoma continues to go up (expected 2% in 2020), but the deaths continue to decrease (-5.3% in 2020) due to improvements in detection and treatment. The treatment of melanoma depends on several aspects but most importantly the tumor's stage and the location. In the early stages, melanoma can be removed via surgical operation, but in the late stages, melanoma usually treated with radiation or targeted therapy. Many factors predict the development of melanoma like skin pigmentation, age, sun exposure (UV light), and genetic mutations. The p53 protein initiates apoptosis and cell cycle arrest in response to stresses that cause DNA damage like UV light. However, mutant p53 is frequently detected in numerous cancers, including melanoma. MDM2 and MDM4 negatively regulate p53. Notably, a study indicates that MDM4 is overexpressed in 65% of melanoma. Therefore, there is a strong rationale to target MDM4 therapeutically, but MDM4 has several spliced variants, and some variants were detected in human and murine cancers. Yet, no studies have assessed the relative levels of MDM4 splice variants in melanoma. Therefore, in this study, we aimed to identify which variants are expressed in melanoma. We collected clinical specimens and designed specific primers for each isoform by RT-PCR. We observed no expression of MDM4-211 in either malignant and nevi samples. MDM4- iii Alt1 was present only once in nevi samples. Other variants were similarly present in nevi and malignant samples, but MDM4-A was the most commonly expressed variant in melanoma samples. Data were also collected and analyzed from publicly available databases to examine MDM4 expression in normal skin and melanoma and the survival data associated with each isoform. The results also showed that MDM4-A is the most common isoform expressed in melanoma. In conclusion, our data suggest that aberrant splicing of MDM4 occurs early in carcinogenesis of melanoma, MDM4 is more frequently amplified than its family member MDM2, that MDM4 has more change in splicing than what is observed in MDM2, and supports the view that MDM4-A is an oncogenic variant that promotes carcinogenesis in melanoma. iv Table of Contents Page I. INTRODUCTION……………………………………………….……………..……….…. 1 A. Human Melanoma…………………………………………………...…………………….. 1 B. The Biology of Melanoma…………………………………………...……………………. 2 C. Current Treatment of Cutaneous Melanoma……………………...……………………….. 4 D. Role of p53 in melanoma…………………………………………...……………………... 6 E. The p53 Interaction with MDM2/MDM4 in Melanoma……………...……………...……. 7 F. MDM4 as a Therapeutic Target in Melanoma………………………………………..…… 11 G. Alternative Splicing in Melanoma…………………………………………………..…….. 12 H. MDM4 Splice Variants …………………………………………………………………… 12 II. MATERIALS AND METHODS……………………………………………...……………17 1. Collecting FFPE Samples…………………………………………………………………..17 2. Primer Design for MDM4 Splice Variants…………………………………………………18 3. RNA Extraction & Purification from FFPE tissues………………………………………...19 4. cDNA Synthesis and RT-PCR……………………………………………………………...20 5. MDM4 Isoforms Analysis……………………………………..………..………………….21 6. Statistical Analysis………………………………………………………………………….22 III. RESULTS…………………………………………………………………………………..23 i. Mutational Gene Analysis Pointed Out that Most Melanomas have Intact p53……………23 ii. MDM4 is More Frequently Amplified in Melanoma than MDM2………….……..…….....24 iii. The expression of MDM4 is Linked to Poor Survival in Melanoma……………….……....25 v iv. The Expression of MDM4 Splice Variants in Sun exposed Skin Compare to Non-Sun Exposed skin……………………………………………………..………………………….26 v. Comparing Castle Class to Isoforms Presence Statistically Showed MDM4-S is Significant in Melanoma Samples………………………………………………………………………….27 vi. MDM4-A is The Most Common Alternative Transcript expressed In Melanoma Samples……………………………………………………………,,……………………….28 vii. Isoform Specific Expression Analysis Showed Consistent Expression of The Alternative Transcript MDM4-A in Melanoma Patients.…………………… ……………………,,,…..30 viii. MDM4-A Isoform Correlates with Poor Survival in Melanoma…………………,………...32 IV. DISCUSSION………………………………..……………………….…………….……….33 V. FUTURE DIRECTION……………………………………………………………………...37 VI. REFERENCES……………………………….…………………………………..………….38 vi List of Figures Page Figure 1: Schematic illustration of the domain structures of p53, MDM2, and MDM4…………………………………………………………………………………...9 Figure 2: Two Proposed Models of MDM2 and MDM4 interaction with p53………………11 Figure 3: Schematic representation of MDM4 mRNA organization in exons (according to the GeneBank NM 002393)…………………………………………………………………16 Figure 4: Most melanomas have an intact p53 gene ……………….……………..…….24 Figure 5: MDM4 is frequently amplified in melanoma, in contrast with MDM2……....25 Figure 6: MDM4 expression does not significantly correlate with patient survival …..….26 Figure 7: MDM4 isoform expression in normal skin ………………………..…………………..…….…27 Figure 8: Statistical significance analysis of MDM4 isoforms in melanoma specimens..28 Figure 9: MDM4 isoform expression in melanocytic lesions is different from normal skin ………………………………………………………………………………………………………………………………..……..…31 Figure 10: MDM4-A isoform correlates with poor survival ………………………………………..…32 Figure 11: Model of Carcinogenesis of melanoma with timeline of MDM2/4/p53 …...…36 vii List of Tables Page Table 1: Melanoma Samples…………………………………………………………………………………………….17 Table 2: Nevi Samples………………………………………………………………..…………………………………….18 Table 3: The amount of RNA in each sample…………………………………………………………………….19 Table 4: Summary of RT-PCR Results in Melanoma and Nevi samples………………..……….……29 viii ACKNOWLEDGMENTS I wish to express my sincere thankfulness and appreciation to my advisers Dr. Michael P Markey, for believing in me. Thank you, Dr. Markey, for your patience, guidance, and support. Thank you, Dr. Markey, for training me on many laboratory techniques and the knowledge that I gained during my time under your supervision. I'd also like to acknowledge Dr. Nicholas Shamma, the man who made this project possible by sharing his clinical specimens with us, thank you, Dr. Shamma. I would also like to thank Dr. Weiwen Long and Dr. Michael Craig for taking the time to serve as committee members. Thank you, Dr. Long and Dr. Craig, for suggestions and everything. I was fortunate to be in the lab with someone like Stacy Simmons; she helped me many times, thank you, Stacy. I met lots of friendly and great people at the BMB Department; thank you, BMB community. Special thank goes to SACM and University of Tabuk for the scholarship and financial support. I am grateful to my mother Munirah, my father Salem, Najwd, Omar, Jamilah, Mohammed, and Amal, who have provided me with moral and emotional support. I am also grateful to my other family members and friends who have supported me along the way, especially Melanie Schloemer. Thank you all. The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. ix I. INTRODUCTION: A. Human Melanomas Melanoma is considered to be the third most widespread sort of skin carcinoma and is responsible for many of the skin cancers fatalities in the United States (Jemal, A et al., 2011) (Jemal, A et al., 2013) (Gruber SB et al., 2006) (The US Department of Health and Human Services, 2014). Worldwide, the rate of melanoma occurrence has been growing dramatically more than any other malignancy. (Ali, Z et al., 2013). Melanoma is the fifth most common cancer among males and the seventh most common cancer among females (Reed, K. B et al., 2012). A study performed on evaluating the gender role in the survival of melanoma patients showed that female patients exhibit notably more prolonged survival than male patients (Morgese, F et al., 2015). According to the U.S. Department of Health and Human Services, melanoma incidence rates have been rising over the past 40
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