Abstract 2204: Integrin α6β4 regulates expression of Areg and Ereg through DNA repair-dependent mechanisms

The integrin α6β4 is overexpressed in and contributes to the invasive potential of pancreatic carcinoma cells. We have previously shown this property is due to the ability of integrin α6β4 to alter the transcriptome, in part, through the active DNA demethylation of pro-metastatic genes such as S100A4/metastasin. In this study, we find that amphiregulin (Areg) and epiregulin (Ereg), which are ligands for the epidermal growth factor receptor (EGFR), are dramatically upregulated by integrin α6β4 in pancreatic carcinomas. EGFR and associated ligands contribute to cancer cell proliferation, invasion, angiogenesis, self-sufficiency in growth signals and apoptosis resistance. To determine if Areg and Ereg transcription is regulated by DNA methylation, we treated pancreatic cancer cells that have low integrin α6β4 expression with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine (DAC) resulting in stable overexpression of Areg and Ereg, as measured by QPCR. Furthermore, suppression of integrin α6β4 by RNAi, hindered induction by DAC. Pancreatic cancer cells with high integrin α6β4 were treated with the methyl donor S-adenosylmethionine, resulting in inhibition of Areg and Ereg expression. Currently, the most accepted mechanism for transcriptional upregulation by active DNA demethylation is through DNA repair. To determine if DNA repair regulates Areg and Ereg expression, we modulated DNA repair by treating pancreatic cancer cells with Gemcitabine, altering expression of Gadd45A and thymine DNA glycosylase (TDG), or inhibition of Parp-1. Gemcitabine inhibits multiple components of DNA repair including Gadd45A-mediated DNA demethylation, while Gadd45A, TDG and Parp-1 are involved in DNA demethylation by DNA repair. Gemcitabine treatment, RNAi-mediated knockdown of Gadd45A or TDG, and Parp-1 inhibition resulted in decreased expression of Areg and Ereg when integrin α6β4 is overexpressed. Conversely, cDNA overexpression of Gadd45A increased expression of Areg and Ereg. Unexpectedly, bisulfite conversion and sequencing of CpG islands in the Areg and Ereg promoters revealed that integrin α6β4 does not control demethylation of the proximal promoters, as these were not methylated. However, a super enhancer associated with Areg and Ereg, has been identified, and may be the target of integrin α6β4 signaling. To block super enhancer function, cells were treated with the bromodomain inhibitor JQ1, which resulted in a dramatic decrease in expression of Areg and Ereg, indicating regulation via a super enhancer. Taken together these data indicate that the integrin α6β4 controls expression of Areg and Ereg by targeting the DNA repair machinery to regulatory regions of Areg and Ereg; this regulatory region is most likely located in the Areg and Ereg associated super-enhancer. Citation Format: Brittany L. Carpenter, Kathleen L. O9Connor. Integrin α6β4 regulates expression of Areg and Ereg through DNA repair-dependent mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2204. doi:10.1158/1538-7445.AM2015-2204