Degree

Doctor of Philosophy (PhD)

Department

Comparative Biomedical Sciences

Document Type

Dissertation

Abstract

Anti-proliferative responses such as senescence and apoptosis are often used by normal cells to combat oncogenic insults and to prevent tumorigenesis. However, oncogenic mutations are frequently found in cancers, suggesting that additional mutations may occur to facilitate the bypass of these anti-proliferative responses. It is believed that some of these additional mutations may also contribute to the chemoresistance of cancers. This dissertation focused on identifying novel genes and biological processes implicated in chemoresistance and tumorigenesis.

Cisplatin-based chemotherapeutic regimens are frequently used for treatments of solid tumors. However, tumor cells may have inherent or acquired resistance to cisplatin, and the underlying mechanisms are largely unknown. We performed genome-wide knockout screening and found that protein translation, RNA catabolic process, and mitochondrial translational elongation and termination are the top biological processes responsible for cisplatin sensitivity in A375 human melanoma cells. In contrast, ubiquitin-dependent protein catabolic process, neddylation, and negative regulations of cellular catabolic process and canonical Wnt signaling are the top biological processes responsible for cisplatin resistance. ZNRF3, a ubiquitin ligase, enhances cisplatin resistance in normal and the melanoma cells by repressing b-Catenin, a key component of canonical Wnt signaling. ARIH1, another ubiquitin ligase, also enhances cisplatin resistance in normal and the melanoma cells. By regulating ARIH1, tumor suppressor NF2 enhances cisplatin resistance in the melanoma but not normal cells. ARIH1 is also upregulated in normal and the melanoma cells by YAP, an effector of Hippo signaling. However, the regulation of ARIH1 by NF2 is not via YAP-mediated transcription. These results shed new lights on cisplatin resistance mechanisms and may be useful for development of future treatment strategies.

By utilizing CRISPR/Cas9 genome-wide screening system, we identified 9 common genes that are involved in the bypass of anti-proliferative responses in human fibroblasts and melanocytes. Exclusively, DBT, an enzyme that involved in branched chain amino acid metabolism, and GPR4, a pH-sensing G-protein coupled receptor, are essential in mediating oncogene-induced apoptosis in human melanocytes only. These data were further confirmed by the single cell analysis of targeted populations that survived oncogenic selection. We also demonstrated that DBT deficient melanocytes suppressed oncogene-induced apoptosis comparing to the wild-type. In contrast, restoring DBT expression successfully recovered the apoptosis potential. Tumor suppressor p53 plays a key role in activating oncogene-induced apoptosis pathway. Previous studies had demonstrated that p53 is positively regulated by tumor suppressor p14ARF and negatively regulated by proto-oncogene AKT, and the oncogene activation can promote p14ARF activation and AKT inhibition. Our data suggested that DBT disruption suppresses oncogene-induced apoptosis by inactivating p53 via downregulation of p14ARF and upregulation of AKT. Collectively, these findings revealed a new understanding about the early stage development of tumorigenesis.

Date

10-12-2018

Committee Chair

Li, Shisheng

Available for download on Saturday, October 12, 2019

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