Cancerous inhibitor of protein phosphatase 2A mediates bortezomib-induced autophagy in hepatocellular carcinoma independent of proteasome

Abstract

Previously, we reported that cancerous inhibitor of protein phosphatase 2A (CIP2A) mediates the apoptotic effect of bortezomib in hepatocellular carcinoma (HCC). Here, we report a proteasome-independent mechanism by which bortezomib induces autophagy in HCC. Our data indicate that bortezomib activated autophagy in a dose- and time- dependent manner in HCC cell lines including Huh-7, Sk-Hep1, and Hep3B. Bortezomib downregulated CIP2A, phospho-Akt (P-Akt) and phospho-4EBP1 (P-4EBP1) in a dose- and time-dependent manner in all tested HCC cells. Ectopic expression of CIP2A abolished the effect of bortezomib on autophagy. Co-treatment of bortezomib and calyculin A, a PP2A inhibitor, reduced the effect of bortezomib on P-Akt, P-4EBP1, and autophagy. Increased phosphorylation of either Akt or 4EBP1 by ectopic overexpression protected cells from bortezomib-induced autophagy. Furthermore, we examined the effect of ΔBtz, a bortezomib derivative that closely resembles bortezomib structurally but has no proteasome activity, in HCC. Interestingly, ΔBtz demonstrated similar effects to bortezomib on autophagy, CIP2A, P-Akt and P-4EBP1, suggesting that the effect of bortezomib on autophagy is independent of proteasome inhibition. Moreover, our in vivo data showed that both bortezomib and ΔBtz inhibited tumor growth, downregulated CIP2A, P-Akt and induced autophagy in Huh-7 tumors. In conclusion, bortezomib induces autophagy in HCC through a CIP2A-PP2A-Akt-4EBP1 pathway.

Figure 1. Bortezomib induces autophagy in HCC.

A, dose-dependent effects of bortezomib-induced autophagy. HCC cells were treated with bortezomib at the indicated concentrations for 24 hours. Cell lysates were prepared for immunoblotting of microtubule-associated protein 1 light chain 3 (LC3). B, time-dependent effects of bortezomib-induced autophagy. Cells were exposed to bortezomib at the indicated concentrations for 24 or 48 hours. C, effects of bortezomib on LC3-II. Left, analysis of LC3-II staining by flow cytometry. Cells were treated with bortezomib at 800 nM for 16 or 24 hours. Right, analysis of LC3-II immunofluorescence. Hep3B cells were treated with bortezomib at 800 nM for 24 hours. D, top, LC3-GFP staining. Bottom, acridine orange stain.

Figure 2. Inhibition of CIP2A-PP2A-Akt-4EBP1 signaling pathway is associated with bortezomib-induced autophagy in HCC.

A, dose-dependent analysis of CIP2A, Akt and 4EBP1 in HCC cells. Cells were exposed to bortezomib at the indicated concentrations for 24 hours. B, time-dependent analysis of CIP2A, Akt and 4EBP1 in HCC cells. Cells were exposed to bortezomib at the indicated concentrations for 24 or 48 hours. C, dose-dependent analysis of autophagy-related proteins. Cells were exposed to bortezomib at the indicated concentrations for 24 hours.

Figure 3. Target validation of CIP2A-Akt-4EBP1.

A, ectopic expression of CIP2A-myc abolishes effects of bortezomib on autophagy in Huh-7 and Hep3B cells. Cells were transfected with CIP2A-myc and were selected for 8 weeks by G-418. Analysis of autophagy was performed by western blot (WB) after cells were sequentially exposed to DMSO or bortezomib (200 nM or 400 nM or 800 nM) for 24 hours. B, left, co-treatment with calyculin A, a PP2A inhibitor, reverses the effect of bortezomib on CIP2A, P-Akt, P-4EBP1 and autophagy. Cells were treated with calyculin A (1 mM) or bortezomib (800 nM) for 24 hours. Middle, ectopic expression of Akt1 reduced the effects of bortezomib on autophagy in Huh-7 cells. Right, ectopic expression of 4EBP1 reduced the effects of bortezomib on autophagy in Huh-7 cells. C, left, co-treatment with rapamycin, a mTOR inhibitor, enhanced bortezomib-induced autophagy in Huh-7 cells. Cells were treated with rapamycin (100 nM) and/or bortezomib (400 nM) for 24 hours. Right, Co-treatment with autophagy inhibitor 3-Methyladenine (3-MA) reduced bortezomib-induced autophagy. Huh7 cells were treated with bortezomib (800 nM) and/or 3-MA (1 mM) for 16 hours.

Figure 4. Proteasome-independent effects of bortezomib on autophagy and CIP2A.

A, left, chemical structures of bortezomib and ΔBtz. Middle, effects of bortezomib and ΔBtz on proteasome inhibition. Right, effects of bortezomib and ΔBtz on NF-κB. B, left, dose-dependent effects of ΔBtz on CIP2A, Akt and LC-3. Middle, time-dependent effects of ΔBtz on CIP2A, Akt and LC-3. Right, Co-treatment with autophagy inhibitor 3-MA reduced ΔBtz-induced autophagy. C, left, effects of ΔBtz on LC3-II immunofluoresence. Middle, effects of ΔBtz on acridine orange staining. Right, Sk-Hep1 cells treated with bortezomib (800 nM) or ΔBtz (800 nM) or vehicle were harvested, fixed and embedded in spur resin. Seventy nanometer thin sections were cut and examined at 120 Kv with a JEM-1400 transmission electron microscope. Arrows indicate autophagic vacuoles. D, Other proteasome inhibitors did not induce autophagy in HCC. Left, effects of MG-132 and lactacystin on CIP2A and LC-3. Right, effects of bortezomib, MG-132 and lactacystin on proteasome inhibition.

Figure 5. In vivo effect of bortezomib and ΔBtz on Huh-7 xeonograft nude mice.

A, tumor growth curves of Huh-7. Points, mean (n = 6); bars, SE. *P<0.05; ** P<0.01. B, western blot analysis of CIP2A, P-Akt, Akt1 and LC-3 in Huh-7 tumors. C, immunoblots were scanned by a UVP BioSpectrum AC image system and quantitated using VisionWork LS software to determine the ratio of the level of CIP2A to actin. Columns, mean; bars, SD (n = 3, *P<0.05). D, summary of the mode of actions of bortezomib.