Identification and Validation of Novel Oncogenes and Tumor Suppressors in Pancreatic Ductal Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is driven by somatic genetic alterations. Functional studies are essential to classify PDAC alterations as either drivers or passengers of this disease. In this work I hypothesized that integrative genomics and functional studies will identify and validate novel drivers of pancreatic carcinogenesis. In the first study I uncovered common and differential roles of cyclin Ds in PDAC. Results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling in PDAC cell lines. Furthermore, CCND1 plays a role in PDAC cell migration. In the second study I identified CCDC68 as a putative tumor suppressor gene in PDAC. Loss of CCDC68 resulted in increased in vitro proliferation and in vivo tumorigenicity of PDAC cell lines associated with amplified MAPK signaling. Loss of CCDC68 function in PDAC was documented through copy number loss and the expression of novel CCDC68Δ69-114 variant devoid of tumor suppressive function. Truncated in-frame protein CCDC68Δ69-114 was shown to be a result of exon skipping in 30% of patients harboring donor splice variant rs.1344011. I also report that the expressed ratios of CCDC68 wt/Δ69-114 variants fluctuates among different patients suggesting that the tumor suppressive function of CCDC68 might be regulated by a critical balance between two isoforms. The third study explored the molecular mechanism(s) of CCDC68 tumor suppression in PDAC. To this end, the biology between tumor suppressive CCDC68wt and non-tumor suppressive CCDC68Δ69-114 variant was compared in H6c7 and 293T cells. Analysis revealed that both isoforms associate with centrosomes in interphase, disappear in mitosis and interact with mother centriole protein CEP170. However, loss of 69-114 amino acids in CCDC68 variant significantly decreased its protein stability further shown to be associated with loss of the lysine acetylation site K94. These findings suggest that the CCDC68Δ69-114 variant is not a stable protein and also point to the significance of lysine acetylation regulating the tumor suppressive properties of CCDC68 protein. Overall, this dissertation reports cyclin D1, cyclin D3 and CCDC68 as genetic alterations playing significant roles in pancreatic carcinogenesis.Ph