Integration of Transcriptomic and CRISPR-Cas9 Technologies Reveal FOXA2 as a Tumor Suppressor Gene in Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) has a median survival of six months and a five-year survival of <5%, making it one of the most lethal human cancers. This poor prognosis is due to the uniformly advanced disease stage at the time of diagnosis and to its profound resistance to existing therapies. Ductal adenocarcinomas makes up between 75 to 92% of pancreatic neoplasms. Premalignant lesions, known as pancreatic intraepithelial neoplasms (PanINs) are of ductal origin and found in close physical proximity with advanced malignant tumors. PanINs are thought to be precursors of ductal adenocarcinoma, as they progress toward increasingly atypical histological stages. Previous studies have implicated the role of different signaling pathways in the pathogenesis of pancreatic cancer, however the role of transcriptomic alterations have not been well characterized. The aim of this dissertation was to characterize the role and function of a new transcription factor gene, called FOXA2, in the pathogenesis of pancreatic cancer. Our preliminary analysis revealed that FOXA2 gene was highly down-regulated in pancreatic cancer tissues relative to control tissue samples. Interestingly, FOXA2 gene has not been implicated previously in pancreatic oncogenesis. FOXA2 is a transcription factor that was initially identified in hepatocytes, where it binds in the promoter areas of important liver-enriched genes transthyretin, alpha 1-antitrypsin and albumin. Our data revealed that FOXA2 is significantly down-regulated in pancreatic cancer, according to qPCR and immunohistochemical analysis in human pancreatic tissues. In order to study the role of FOXA2 deletion in vivo, we utilized the CRISPR/Cas9 system to delete FOXA2 from the PANC-1 cell line (FOXA2 deletion). Subcutaneous injections in immunodeficient mice demonstrated FOXA2 deletion had a significantly higher weight and volume than the control tumors. Moreover, we identified a negative regulator of FOXA2 expression, microRNA-199a, which targets directly and reduces FOXA2 mRNA and protein expression levels in pancreatic cancer. Overexpression of microRNA-199a was observed in human pancreatic cancer tissue by qPCR and in-situ hybridization methods. In an effort to identify downstream targets of FOXA2, we transiently knocked-down FOXA2 with an siRNA and performed a gene expression array in a human pancreatic cell line. The expression data revealed plasminogen urokinase activator receptor (PLAUR) was significantly up-regulated upon FOXA2 inhibition. PLAUR is a marker of invasiveness and is responsible for the invasive phenotype we observed upon FOXA2 knockdown.