Integrin α6β4 signaling stimulates progression of multiple types of cancer in part by altering the transcriptome. Notably, expression of AREG and EREG positively correlates with expression of and signaling through integrin α6β4 (Fig. 2A), supporting our previous work13. To determine if AREG and EREG expression is regulated by DNA methylation, Panc1-2G6 cells were treated with the DNA methyltransferase inhibitor 5-aza-CdR at indicated concentrations, harvested at indicated time points, and RNA analyzed by QPCR. We found that both AREG and EREG mRNA expression increased in a time and dose dependent manner (Fig. 2B) demonstrating the susceptibility of AREG and EREG to DNA methylation. Furthermore, integrin α6β4 was required for induction of AREG and EREG mediated by 5-aza-CdR, as knocking down the integrin β4 in Panc1-3D7 cells hindered epigenetic induction of AREG and EREG expression (Fig. 2C). Considering that epigenetic changes are reversible, AsPC1 and Suit2 cells, high expressers of integrin α6β4, AREG, and EREG, were treated with the methyl donor S-adenosylmethionine (SAM) and assessed for AREG and EREG expression by QPCR. These data revealed a 50% decrease in expression of AREG and EREG as seen in Fig. 2D. Taken together these data indicate that signaling from integrin α6β4 and DNA demethylation are required to drive AREG and EREG expression.Figure 2AREG and EREG expression is mediated by DNA demethylation in response to signaling from the integrin α6β4. (A) Expression of AREG and EREG was compared in Panc1-2G6 (low α6β4) and cells expressing a dominant negative α6β4 (Panc1-β4ΔCyt), Panc1-3D7, Suit2, and AsPC1 (high α6β4; in order of increasing expression) cell lines. (B) Panc1-2G6 cells (low α6β4) were treated with vehicle only (control) or with 1 μM or 5 μM 5-aza-2′-deoxycitine (5-aza-CdR) in fresh medium daily for 3 or 5 days. (C) Panc1-3D7 stably expressing an shRNA targeting the β4 subunit or a non-targeting (NT) shRNA control vector were treated with 2 μM 5-aza-CdR for 3 days and then assessed for AREG and EREG expression. (D) AsPC1 and Suit2 (high α6β4) were treated with vehicle only (control) or 80 μM S-adenosylmethione (SAM) in fresh medium daily for 5 days (E,F). Panc1-2G6 (E) and Panc1-3D7 (F) cells were treated with 2 μM 5-aza-CdR for 24 or 72 hours, 5-aza-CdR was removed and cells were either collected immediately or maintained in culture for 10 days. (G–I) Cells with high integrin α6β4 were treated with vehicle only (control) or 0.5 μM JQ1 overnight and harvested for analysis by QPCR. For all experiments RT-PCR was used to convert RNA to cDNA and QPCR was used to assess AREG and EREG expression. Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls, unless otherwise indicated.