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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.
Highlights
Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide [1]
Bortezomib induces autophagy in HCC To investigate the effect of bortezomib on autophagy in HCC, we first examined the expression of microtubule-associated protein 1 light chain 3 (LC3-II), a LC3-phosphatidyl-ethanolamine conjugate, that is a hallmark of autophagy
The stimulatory effect of bortezomib on autophagy was confirmed by an increase in the GFP-LC3 dots in the cells treated with the drug, as examined by GFP-LC3 assay (Fig. 1D top), and by an increase in acridine orange staining for acidic vesicular organelles (Fig. 1D bottom)
Summary
Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide [1]. Advanced HCC is characterized by frequent resistance to conventional chemotherapeutic agents and radiation. There is clearly a need to develop new therapeutic targets and strategies for HCC therapy. The autophagy pathway has emerged as a promising new target in cancer treatment. Autophagy plays multiple roles in cancer: it may promote cancer cell death or survival depending on the complex interactions among metabolic stress, pathways of apoptosis and autophagy [3,4,5]; and perturbation of autophagy can contribute to tumorigenesis [3,6]. A better understanding of autophagy regulation may facilitate discovery of new potential therapeutic targets in HCC
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