CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells

Abstract

Previously, we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. In this study, we report that cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediates the apoptotic effect of bortezomib in HCC. Silencing PP2A by small interference RNA (siRNA) abolishes bortezomib-induced down-regulation of phospho-Akt and apoptosis. Bortezomib increases PP2A activity in sensitive HCC cells, including Sk-Hep1, Hep3B and Huh-7, but not in resistant PLC5 cells. Bortezomib down-regulates CIP2A in a dose- and time-dependent manner in all sensitive HCC cells, whereas no alterations in CIP2A were found in resistant PLC5 cells. Knockdown of CIP2A by siRNA restored bortezomib's effects on apoptosis and PP2A activity in PLC5 cells. Moreover, over-expression of CIP2A up-regulated phospho-Akt and protected Sk-Hep1 cells from bortezomib-induced apoptosis. It is significant that, ectopic expression of CIP2A decreased Akt-related PP2A activity, whereas silencing CIP2A increased this activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells, furthermore, our in vivo data showed that bortezomib down-regulates CIP2A and up-regulates PP2A activity in Huh-7 tumors, but not in PLC5 tumors. In conclusion, inhibition of CIP2A determines the effects of bortezomib on apoptosis and PP2A-dependent Akt inactivation in HCC.

CIP2A signature reveals the MYC dependency of CIP2A-regulated phenotypes and its clinical association with breast cancer subtypes

“…These results suggest that MYC contributes to the CIP2A-mediated stimulation of HeLa cell colony growth. Recently, several independent studies have demon- Identification of the CIP2A-regulated transcriptome M Niemelä et al strated that CIP2A inhibits cellular PP2A activity (Junttila et al, 2007;Chen et al, 2010;Guenebeaud et al, 2010). Moreover, in HeLa cells used for this study, siRNA-mediated CIP2A inhibition induced cellular PP2A activity (data not shown).…”

Nilotinib Induces Autophagy in Hepatocellular Carcinoma through AMPK Activation

“…After 10 min of incubation at 30°C, the malachite dye was added, and free phosphate was measured by optical density at 650 nm. To avoid variability due to differences in the amounts of immunoprecipitated protein between samples, the phosphatase activities were normalized to the amount of PP2A immunoprecipitated, as detected and quantified by immunoblot analysis for each treatment group (17).…”

“…The immunoprecipitates were harvested using protein G PLUS-agarose beads (Santa Cruz Biotechnology) that were washed once with regular washing buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40), twice with high salt washing buffer (50 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40), and another time with regular washing buffer. Immunoprecipitates were then eluted by boiling the beads for 5 min in SDS/ PAGE sample buffer and characterized by Western blotting with appropriate antibodies (17).…”

“…5B). We next examined other cellular regulators of PP2A including cancerous inhibitor of PP2A (CIP2A) and SET (17). Nilotinib did not alter the expression of CIP2A and SET in our cells (Fig.…”

“…PP2A exists as a heterotrimeric enzyme composed of a 36-kDa catalytic subunit or C subunit, a 64-kDa scaffolding A subunit, and multiple regulatory B subunits that are thought to influence enzyme activity, substrate specificity, and subcellular localization (17). To fur-ther dissect the mechanism of AMPK phosphorylation induced by nilotinib, we inspected the effect of PP2A.…”