Background: The epidermis is a stratified epithelium whose differentiation program is triggered in part by calcium. Dysregulation of keratinocyte differentiation may lead to non-melanoma skin cancers, including cutaneous squamous cell carcinoma (cSCC). The compound 2-aminoethoxydiphenyl borate (2-APB) modulates calcium signaling by altering activity of calcium-permeable channels of the transient receptor potential (TRP) and ORAI families, and is therefore poised to govern signaling pathways that control the balance of keratinocyte proliferation and differentiation. Objective: We sought to determine whether 2-APB alters differentiation of normal human keratinocytes and progression of human cSCCs models in vitro. Methods: Primary human keratinocyte cultures were treated with 2-APB and levels of proliferation (EdU incorporation) and differentiation markers [quantitative PCR (qPCR)] were assessed. Human cSCC biopsies and cell lines were analyzed for TRP and ORAI gene expression via qPCR. cSCC cell lines were cultured in organtypic cultures and analyzed for growth and invasiveness after 2-APB or vehicle treatment. Results: Culturing human keratinocytes with 2‑APB arrested cell proliferation, triggered differentiation-gene expression and altered epidermal stratification, indicating that 2-APB application is sufficient to promote differentiation. In human organotypic cSCC cultures, 2‑APB attenuated tumor growth and invasiveness. Finally, expression of a panel of 2‑APB-targeted ion channels (TRPV3, TRPV1, TRPC1, OraI1, OraI2 and OraI3) was dysregulated in high-risk cSCC biopsies. Conclusions: Collectively, these findings identify 2-APB as a potential therapeutic for high-risk cSCCs.

2-APB arrests human keratinocyte proliferation and inhibits cutaneous squamous cell carcinoma in vitro