Abstract A15: The integrin α6β4 promotes pancreatic cancer invasion by altering DNA repair-mediated epigenetics

The integrin α6β4 is upregulated in pancreatic carcinoma and signaling from this integrin promotes metastatic properties, including cancer cell invasion. We have previously shown that the integrin α6β4 promotes such properties by dramatically altering the transcriptome toward an invasive phenotype. Furthermore, we have found that transcriptome alterations can be accomplished through targeted DNA demethylation of specific promoters, as we have shown with the pro-metastatic gene S100A4. In this study, we find that signaling from the integrin α6β4 dramatically upregulates expression of amphiregulin (Areg) and epiregulin (Ereg), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if Areg and Ereg are regulated by DNA methylation, pancreatic cancer cells with low expression of the integrin α6β4 were treated with the DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (DAC), which resulted in stable over expression of Areg and Ereg, as measured by QPCR. Furthermore, expression of a dominant negative form of integrin β4 or blockade of β4 expression by RNAi suppressed induction of Areg and Ereg by DAC. Similarly, treatment of cells with high integrin α6β4 with the methyl donor S-adenosylmethionine inhibited gene expression of Areg and Ereg. In order to confirm these results, genomic DNA from pancreatic cancer cells with either high or low expression of integrin α6β4 was isolated and whole genome bisulfite sequencing was performed. We find that cells with high integrin α6β4 have lower overall methylation levels compared to cells with low integrin α6β4. As expected, we also find lower methylation levels within the first exon of both Areg and Ereg when the integrin α6β4 is high. These regions of hypomethylation correspond to known H3K27 acetylation marks found in ENCODE databases and reported in the USCS genome browser. Thus, these data implicate promoter demethylation as the driver of Areg and Ereg gene expression mediated by integrin α6β4. It is currently accepted that active DNA demethylation can occur via DNA repair. To test our hypothesis that DNA repair is responsible for DNA demethylation of Areg and Ereg in pancreatic cancer cells, we modulated several components of the DNA repair pathway including GADD45A, TDG and Parp-1. The chemotherapeutic agent Gemcitabine has been shown to inhibit multiple components of DNA repair, as well as DNA demethylation mediated by Gadd45A. Treatment of pancreatic cancer cells with Gemcitabine resulted in a marked reduction in Areg and Ereg expression when the integrin α6β4 is high, indicating DNA repair is required for gene expression. To further test the involvement of Gadd45A, we used either RNAi-mediated knockdown or cDNA overexpression to alter Gadd45A levels and examined their effect on Areg and Ereg. In both instances, Areg and Ereg expression positively correlated with Gadd45A, particularly when the integrin α6β4 is high, indicating that Gadd45A is a rate-limiting step in Areg and Ereg overexpression. Thymine DNA glycosylase (TDG) has likewise been shown to mediate active DNA demethylation. Using stable shRNA, we show that TDG is required for Areg and Ereg expression in integrin α6β4 high cells, and nuclear localization of TDG is much higher in cells with high integrin α6β4. We further used a specific inhibitor for Parp-1 and show that Areg and Ereg expression is dependent on Parp-1. Finally, we determine that integrin α6β4 signaling accelerates DNA repair rates by 2.5-fold. Taken together, these data indicated that DNA repair is required to maintain overexpression of Areg and Ereg in response to signaling from the integrin α6β4 and that integrin α6β4 promotes this overexpression by enhancing DNA repair and DNA repair-mediated DNA demethylation at specific sites within the promoter regions of Areg and Ereg. Citation Format: Brittany L. Carpenter, Jinpeng Liu, Chi Wang, Kathleen O9Connor. The integrin α6β4 promotes pancreatic cancer invasion by altering DNA repair-mediated epigenetics. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A15.