Abstract 1647: Integrin α6β4 stimulates transcription and secretion of EGFR ligands.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic carcinoma has the highest death to incidence ratio of all cancer due to frequent late diagnosis, drug-resistance and high metastatic potential. The integrin α6β4 is overexpressed in pancreatic carcinoma and plays an important role in tumor invasion and metastasis, in part by altering the transcriptome toward an invasive phenotype. In this study, we find that these transcriptional changes in pancreatic carcinomas include upregulation of cancer promoting genes such as ligands of the epidermal growth factor receptor (EGFR), epiregulin (Ereg) and amphiregulin (Areg). We have found previously that integrin α6β4 alters transcription by selectively targeting DNA demethylation or transcription factors, such as NFAT1 and NFAT5, to gene promoters. Given that Areg and Ereg are upregulated by over 1000-fold, we hypothesized that integrin α6β4 may control their expression by targeting their promoters for DNA demethylation. To test this concept, pancreatic cancer cells with low expression of integrin α6β4 were treated with DNA methyltransferase inhibitor 5-aza-2’deoxycytidine (DAC), and mRNA expression measured by Q-PCR. This treatment resulted in induction of Areg and Ereg expression indicating that these genes may be controlled by DNA methylation. Furthermore, suppression of integrin α6β4 by RNAi, hindered induction by DAC, thus suggesting that integrin α6β4 plays a role in ligand expression. To support these results, pancreatic cancer cells were treated with the methyl donor S-adenosylmethionine, allowing for global hypermethylation and silencing of genes controlled by methylation. Our results show that treatment inhibits Ereg and Areg expression in cells with high expression of integrin α6β4, providing evidence for epigenetic regulation by this integrin. Non-coding RNAs processed by Dicer often play a role in DNA methylation of specific genes. Accordingly, we used siRNA to target the microRNA processing enzyme Dicer. Dicer knockdown resulted in decreased expression of Ereg and Areg, measured by Q-PCR, indicating that small non-coding RNAs are involved in their regulation. Autocrine EGFR signaling is also regulated by altered secretion of Ereg and Areg. Using an ELISA for Areg, we measured secretion associated with variable expression of integrin α6β4. We find that integrin α6β4 stimulated secretion of Areg into the media, and this secretion is enhanced by HGF signaling. Consequently, we also provide evidence that integrin α6β4 promotes HGF-stimulated chemotaxis, invasion, and invasive growth which is facilitated by autocrine EGFR signaling. This study demonstrates that overexpression of integrin α6β4 in pancreatic cancer promotes a more aggressive phenotype by activating transcriptional expression and autocrine secretion of EGFR ligands, Ereg and Areg, that promote an invasive phenotype. Citation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kathleen L. O'Connor. Integrin α6β4 stimulates transcription and secretion of EGFR ligands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2013-1647