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Abstract

Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer and belongs to the four most lethal malignancies worldwide. HCC constitutes a major health concern as it is frequently diagnosed at late-stage of the disease and the presence of concomitant cirrhosis makes curative surgery inapplicable for most of the patients and concurrently reduces the options for systemic therapy. In fact, therapy is most effective during early stages of the disease. Hence, early diagnosis and treatment are of prime importance. Human hepatocarcinogenesis represents a step-wise process, in which accumulation of somatic mutations allows for clonal selection of driver gene mutations, which finally results in malignant transformation of premalignant Dysplastic Nodule into HCC. In the presented study, we aimed: (i) to identify and validate driver mutations of hepatocarcinogenesis; and (ii) to investigate the molecular mechanism through which they promote malignant transformation. The present thesis describes the identification of clonally expanded driver mutations of hepatocarcinogenesis via integration of whole exome sequencing and in vivo RNAi screening. In vivo shRNA screening of the identified clonally expanded variants using a mosaic mouse model of HCC revealed the tumor suppressive role of PRSS23 in the context of activated MYC and AKT1 signaling. Subsequent in vivo validation experiments using the individual variants confirmed the oncogenic potential of the identified PRSS23 variant (P230A) in MYC overexpressing Trp53 heterozygous mice. 3D computational modelling of PRSS23 located the amino acid substitution P230A in close proximity to the catalytic site of the protein suggesting a potential interfering effect on the enzymatic activity. Using a primary murine HCC cell line derived from Trp53 heterozygous mouse expressing MYC and activated AKT1 together with the wildtype or P230A variant of PRSS23, I demonstrated that overexpression of PRSS23P230A elevates the proliferation rate and cell viability of HCC cells compared to PRSS23WT overexpressing control cells, and promotes the invasiveness of these cells. The results of this thesis propose a previously unrecognized role for PRSS23 in mitochondria of HCC cells. Real-time analysis of the ATP production rate and morphological analysis of mitochondria in HCC cells demonstrated that overexpression of PRSS23P230A contributes to a metabolic switch towards enhanced oxidative phosphorylation likely via a process involving remodeling of mitochondrial cristae. Remarkably, transcriptomic analysis of HCC cells expressing wildtype or the P230A variant of PRSS23 unveiled differential regulation of several signaling pathways. In particular, MYC activation was elevated in PRSS23P230A expressing cells. In addition, enrichment of PRSS23 interacting proteins in these cellular processes further corroborated the latter finding. Strikingly, evaluating the activation level of MYC in HCC cells validated that PRSS23P230A augments MYC activation. All in all, the results of this thesis demonstrate that the P230A mutation of PRSS23 promotes malignant transformation during hepatocarcinogenesis by promoting MYC activity in HCC cells involving structural and functional remodeling of mitochondria.