Regulation of Calpain Activity by c-Myc through Calpastatin and Promotion of Transformation in c-Myc-negative Cells by Calpastatin Suppression

Abstract

The c-Myc transcription factor is commonly dysregulated in cancer. c-Myc also sensitizes cells to apoptosis induced by a variety of toxic events. c-Myc turnover is rapid and mediated by the proteasome and intracellular calpains. Therefore, c-Myc accumulation could contribute to cell death associated with protease inhibitors. We investigated the response of c-Myc-positive and c-Myc-negative rat fibroblast cells to proteasome and calpain inhibitors. Apoptosis induced by the proteasome inhibitor, epoxomycin, was c-Myc-independent, whereas apoptosis induced by the calpain inhibitor, PD150606, or by knockdown of calpain small subunit 1 (CPNS1) was strongly dependent on c-Myc. HL60 cells knocked down for c-Myc expression exhibited reduced calpain activity and decreased sensitivity to PD150606 but not epoxomycin. Calpain inhibitor- or CPNS1 knockdown-induced apoptosis in c-Myc-positive fibroblasts was associated with cell detachment and could be prevented by plating cells on fibronectin, suggesting an anoikis phenomenon. c-Myc stimulated calpain activity by suppressing calpastatin expression, the endogenous calpain inhibitor. Knockdown of calpastatin in c-Myc-negative cells led to a restoration of calpain activity, enhanced cell growth, cell cycle redistribution, anchorage independence, and tumorigenicity in immunodeficient mice. Taken together, these results indicate that c-Myc regulates calpain activity through calpastatin; apoptosis induced by calpain inhibition is dependent on c-Myc, and calpastatin knockdown promotes transformation in c-Myc-negative cells.