Cell-cell adhesion is crucial for the maintenance of epithelial integrity and is often lost as a result of Epithelial-to-Mesenchymal Transition (EMT), a process implicated in the formation of invasive tumor cells from primary carcinomas. We have previously reported that the Coxsackievirus and Adenovirus Receptor (CAR), an epithelial cell-cell adhesion molecule, is negatively regulated through both Ras-Erk and TGF-β signaling. The regulation via TGF-β takes place in the context of EMT. Here we provide evidence that the corresponding mechanism involves E2 box-binding transcriptional repressors and thus resembles the regulation of E-cadherin expression through TGF-β. Overexpression of Sip-1, one such E2 box-binding transcriptional repressor, decreases CAR promoter activity in various epithelial cell lines. Inactivation of the putative binding site in the CAR promoter prevents this decrease. Conversely, repression of endogenous Sip-1 expression by RNA interference in the non-small cell lung cancer cell line H460 increases CAR mRNA and protein levels. We measured mRNA expression of the E2 box-binding transcriptional repressors Sip-1, Zeb-1, Snail and Slug in multiple mesenchymal cell lines with low CAR levels and found different repressors expressed in different cell lines, suggesting that TGF-β interferes with CAR transcription not only via Sip-1, but also through other E2 box-binding proteins. We are currently investigating whether RNAi-mediated inhibition of the TGF-β-induced expression of Snail and Slug can restore CAR levels and adenovirus infectability in the human pancreatic cancer cell line Panc-1. We have previously shown that inhibition of TGF-β signaling via a small molecule inhibitor increases CAR levels and improves adenovirus infectability. The data presented here identify E2 box-binding transcriptional repressors as likely mediators. Since E2 box-binding proteins play a pivotal role in TGF-β-mediated EMT, our results highlight the importance of designing appropriate pharmacological inhibitors. Such inhibitors could potentially improve therapies with oncolytic adenoviruses through up-regulation of CAR, but speculatively also by delaying, preventing or reversing EMT and the formation of invasive tumor cells.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]