Regulation of calcium influx and signaling pathway in cancer cells via TRPV6–Numb1 interaction

Abstract

Ca2+ is a critical factor in the regulation of signal transduction and Ca2+ homeostasis is altered in different human diseases. The level of Ca2+ in cells is highly regulated through a diverse class of regulators. Among them is the transient receptor potential vanilloid 6 (TRPV6), which is a Ca2+ selective channel that absorbs Ca2+ in the small intestine. TRPV6 is overexpressed in some cancers and exhibits oncogenic potential, but its exact mechanism is still poorly understood. The Numb protein is a cell fate determinant that functions in endocytosis and as a tumor suppressor via the stabilization of p53. Numb protein consisted of four isoforms. Here, we showed a novel function of Numb1, which negatively regulates TRPV6 activity. The expression of Numb1 decreased cytosolic Ca2+ concentrations in TRPV6-transfected HEK293 cells. When all the isoforms of Numb were depleted using siRNA in a TRPV6 stable cell line, the levels of cytosolic Ca2+ increased. We observed an interaction between Numb1 and TRPV6 using co-immunoprecipitation. We confirmed this interaction using Fluorescence Resolution Energy Transfer (FRET). We identified the TRPV6 and Numb1 binding site using TRPV6 C-terminal truncation mutants and Numb1 deletion mutants. The binding site in TRPV6 was an aspartic acid at amino acid residue 716, and that binding site in Numb1 was arginine at amino acid residue 434. A Numb1 mutant, lacking TRPV6 binding activity, failed to inhibit TRPV6 activity. Every isoform of Numb knockdown, using an siRNA-based approach in MCF-7 breast cancer cells, not only showed enhanced TRPV6 expression but also both the cytosolic Ca2+ concentration and cell proliferation were increased. The down-regulated expression of TRPV6 using siRNA increased Numb protein expression; however, the cytosolic influx of Ca2+ and proliferation of the cell were decreased. To examine downstream signaling during Ca2+ influx, we performed Western blotting analysis on TRPV6 upregulated cancer cells (MCF-7, PC-3). Taken together, these results demonstrated that Numb1 interacts with TRPV6 through charged residues and inhibits its activity via the regulation of protein expression. Moreover, we provided evidence for a Ca2+-regulated cancer cell signaling pathway and that the Ca2+ channel is a target of cancer cells.