Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

We investigated the role of the ubiquitously expressed calpain 2 isoform in breast tumor cell growth, migration, signaling, and tumorigenesis. RNAi-mediated knockdown of the capn2 transcript was used to manipulate expression of the catalytic subunit of calpain 2 in the AC2M2 mouse mammary carcinoma cell line. Stable knockdown of capn2 correlated with reduced in vitro proliferation rates, soft agar colony formation efficiency, and migration rates, indicating roles for calpain 2 in mitogenesis, survival, and motogenesis. Biochemical analysis showed increased levels of protein phosphatase 2A and reduced levels of activated Akt in calpain 2-deficient cells, and this correlated with increased levels of the FoxO3a target gene product p27(Kip1), a key regulator of cell proliferation. Calpain 2 deficiency in the AC2M2 cells correlated with enhanced nuclear localization of FoxO3a, consistent with it being in a derepressed state capable of regulating transcriptional targets. Orthotopically engrafted calpain 2 knockdown AC2M2 cells generated tumors with reduced growth rates and enhanced in vivo expression of p27(Kip1). In summary, calpain 2 deficiency correlated with reduced Akt activity, increased protein phosphatase 2A levels, derepression of FoxO3a, and enhanced expression of the p27(Kip1) tumor suppressor. These observations argue that calpain 2 promotes tumor cell growth both in vitro and in vivo through the PI3K-Akt-FoxO-p27(Kip1) signaling cascade. Inhibition of calpain 2 might therefore provide therapeutic benefits in the treatment of cancer.