1. ¹Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Trieste, Italy
2. ²Cancer Research-United Kingdom Laboratories, Department of Oncology, Imperial College London, Hammersmith Hospital, London, UK
3. ³Dipartimento di Scienze e Tecnologie Biomediche, Universita' degli Studi di Udine, Udine, Italy

Correspondence: Dr F Demarchi or Professor C Schneider, Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), AREA Science Park, Padriciano 99, 34012 Trieste, Italy. E-mails: francesca.demarchi@lncib.it or schneide@lncib.it

Received 30 April 2008; Revised 16 October 2008; Accepted 24 October 2008; Published online 24 November 2008.

Abstract

Here, we show that FoxO3A transcription factor is upregulated upon calpain small-1 (CAPNS1) depletion both in mouse embryonic fibroblasts (MEFs) and in the human mammary carcinoma cell line MCF-7. On starvation, CAPNS1 depletion is associated with a higher rate of FoxO3A dephosphorylation and translocation to the nucleus and to a sharper increase in the levels of p27Kip1 and Bim, the products of two FoxO target genes. Notably, FoxO3A depletion in CAPNS1-/- MEFs reduces both the induction of Bim and apoptosis. Both okadaic acid treatment and silencing of the protein phosphatase 2A (PP2A) catalytic subunit can partially reduce starvation-induced FoxO3A activation and apoptosis in CAPNS1-/- fibroblasts. PP2A associates more tightly with Akt in CAPNS1 knockout cells, indicating that PP2A is involved in calpain-mediated FoxO regulation. Finally, we show that PP2A regulatory subunits B56 alpha and gamma are in vitro substrates of calpain, and calpain regulates B56 alpha stability in vivo, suggesting a direct role of calpain in the regulation of PP2A function. In conclusion, for the first time we report that CAPNS1 interferes with PP2A-Akt interaction consequently affecting FoxO3A-dependent cell death. Calpain inhibition might therefore be exploited as a tool to induce apoptosis in tumors sensitive to FoxO activation.