Hui-Chuan Yu

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

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.

Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation

BACKGROUND & AIMS Previously we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. Here we report that combined treatment with sorafenib and bortezomib shows synergistic effects in HCC. METHODS HCC cell lines (PLC/PRF/5, Huh-7, and Hep3B) were treated with sorafenib and/or bortezomib and analyzed in terms of apoptosis signal transduction. In vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. RESULTS Pretreatment with sorafenib enhanced bortezomib-induced apoptotic cell death by restoring bortezomibs ability to inactivate Akt in PLC/PRF/5 cells. Knocking down Akt1 by RNA-interference overcame apoptotic resistance to bortezomib in PLC/PRF/5 cells and ectopic expression of active Akt in HCC cells abolished the bortezomib sensitizing effect of sorafenib, indicating Akt inactivation plays a key role in mediating the combinational effects. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of phospho-Akt (P-Akt) expression induced by co-treatment with sorafenib and bortezomib, and 1, 9 di-deoxy-forskolin, a PP2A agonist, restored bortezomibs effect on P-Akt and apoptosis. Importantly, silencing of PP2A by RNA-interference reduced the apoptotic effect induced by sorafenib-bortezomib co-treatment, indicating that PP2A is indispensable for mediating the effects of these drugs. Notably, sorafenib with bortezomib increased PP2A activity in PLC/PRF/5 cells without altering protein levels of PP2A complex or the interaction between PP2A and Akt proteins. Finally, sorafenib plus bortezomib significantly suppressed PLC/PRF/5 xenograft tumor growth, down-regulated P-Akt expression, and up-regulated PP2A activity. CONCLUSIONS The combination of sorafenib and bortezomib shows synergy in HCC through PP2A-dependent Akt inactivation.

Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation

Background: Nilotinib, an approved drug for leukemia, has been investigated in HCC. Results: Nilotinib induced autophagy in HCC cell lines, including PLC5, Huh-7, and Hep3B. Conclusion: Nilotinib-induced AMPK activation and subsequent autophagy is a major mode of action of nilotinib in HCC. Significance: Elucidating the mechanisms by which nilotinib works on HCC is fundamental to develop the new treatment for HCC. Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.

Bortezomib Sensitizes HCC Cells to CS-1008, an Antihuman Death Receptor 5 Antibody, through the Inhibition of CIP2A

Previously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC) cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody. Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short interfering RNA enhanced CS-1008–induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC cells abolished CS-1008–induced apoptosis, indicating that CIP2A plays an important role in the sensitizing effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008 through the inhibition of CIP2A. Mol Cancer Ther; 10(5); 892–901. ©2011 AACR.

Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells.

In this study, we investigated the effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma (HCC). LCL161 showed differential effects on apoptosis in four HCC cell lines, and the endogenous level of Bcl-2 determined the sensitivity of HCC cells to LCL161. Cytotoxicity and apoptosis were observed in sensitive PLC5 and Hep3B cells that express lower levels of Bcl-2, but not in resistant Huh-7 and SK-Hep1 cells with higher Bcl-2 expression. Down regulation of Bcl-2 by small interference RNA overcame the resistance to LCL161 in Huh-7, and the apoptotic effect was rescued in Bcl-2-expressing Hep3B. To test the hypothesis that Bcl-2 determines the sensitivity of HCC cells to LCL161, we assayed the biological effect of SC-2001, a novel Bcl-2 inhibitor derived from obatoclax, in LCL161-resistant cell lines. Huh-7 cells co-treated with LCL161 and SC-2001 showed a significant dose-dependent apoptotic effect demonstrated by sub-G1 assay and cleavage of PARP. Furthermore, the combination index (CI) of LCL161 and SC-2001 showed a convincing synergism in resistant Huh-7. In addition, the combinational therapy showed significant growth inhibition in Huh-7-bearing xenograft tumors. Notably, down regulation of Bcl-2 was observed in a tumor sample treated with LCL161 and SC-2001. In conclusion, targeting Bcl-2 with SC-2001 overcomes drug resistance to LCL161 in HCC cells thus suggesting a new anti-IAP combinational therapy for HCC.

Inhibition of CIP2A determines erlotinib-induced apoptosis in hepatocellular carcinoma

Erlotinib is a small-molecular inhibitor of epidermal growth factor receptor (EGFR). Here, we identify that cancerous inhibitor of protein phosphatase 2A (CIP2A) is a major determinant mediating erlotinib-induced apoptosis in hepatocellular carcinoma (HCC). Erlotinib showed differential effects on apoptosis in 4 human HCC cell lines. Erlotinib induced significant apoptosis in Hep3B and PLC5 cell lines; however, Huh-7 and HA59T cell lines showed resistance to erlotinib-induced apoptosis at all tested doses. Down-regulation of CIP2A, a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of erlotinib in HCC. Erlotinib inhibited CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant cells. Overexpression of CIP2A upregulated phospho-Akt and protected Hep3B cells from erlotinib-induced apoptosis. In addition, silencing CIP2A by siRNA restored the effects of erlotinib in Huh-7 cells. Moreover, adding okadaic acid, a PP2A inhibitor, abolished the effects of erlotinib on apoptosis in Hep3B cells; and forskolin, a PP2A agonist enhanced the effect of erlotinib in resistant HA59T cells. Combining Akt inhibitor MK-2206 with erlotinib restored the sensitivity of HA59T cells to erlotinib. Furthermore, in vivo xenograft data showed that erlotinib inhibited the growth of PLC5 tumor but had no effect on Huh-7 tumor. Erlotinib downregulated CIP2A and upregulated PP2A activity in PLC5 tumors, but not in Huh-7 tumors. In conclusion, inhibition of CIP2A determines the effects of erlotinib on apoptosis in HCC. CIP2A may be useful as a therapeutic biomarker for predicting clinical response to erlotinib in HCC treatment.

STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma

Purpose: Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). Experimental Design: HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib (n \ge 8 mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Results: Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Conclusions: Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. Clin Cancer Res; 20(22); 5768–76. ©2014 AACR.

Cancerous Inhibitor of Protein Phosphatase 2A Mediates Bortezomib-Induced Autophagy in Hepatocellular Carcinoma Independent of Proteasome

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.

SHP-1 is a negative regulator of epithelial-mesenchymal transition in hepatocellular carcinoma.

Epithelial-to-mesenchymal transition (EMT) is well known to involve in tumor invasion and metastasis. Src homology region 2 domain-containing phosphatase 1 (SHP-1) functions as a potent tumor suppressor and also acts as a negative regulator of p-STAT3Tyr705 oncogenic signaling. However, little is known about the molecular mechanism(s) through which SHP-1 regulates EMT during hepatocellular carcinoma (HCC) progression. Here we first reported that endogenous SHP-1 protein levels were significantly downregulated in cells with mesenchymal characteristics and negatively correlated with p-STAT3Tyr705 and vimentin but positively correlated with E-cadherin. SHP-1 overexpression abolished transforming growth factor-β1 (TGF-β1)-induced p-STAT3Tyr705 and EMT, as well inhibited migration and invasion but further rescued by signal transducer and activator of transcription factor 3 (STAT3) overexpression. Depletion of SHP-1 could induce a more increase in TGF-β1-induced p-STAT3Tyr-705 and EMT characteristics, further supporting the mechanism that suppression of TGF-β1-induced EMT is dependent on SHP-1-mediated STAT3 inactivation. Constitutively overexpressed SHP-1 tyrosine phosphatase activity by D61A-mutated SHP-1 markedly reduced TGF-β1-induced p-STAT3Tyr705 and EMT features but was not altered by C453S catalytic-dead mutant SHP-1. Consequently, SHP-1 acted as a powerful suppressor in preventing EMT by exerting its tyrosine phosphatase activity that directly downregulated p-STAT3Tyr705. Most notably, we discovered a novel SHP-1 agonist SC-43 better than sorafenib to exert more potent anti-EMT effects in vitro as well as anti-metastatic growth in vivo. In conclusion, SHP-1 is a potent suppressor of HCC EMT and metastasis, thus highlighting that SC-43–SHP-1 axis may serve as a potential therapeutic target that antagonized p-STAT3Tyr705 and thereby prevented HCC EMT and metastasis.

Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a tumor suppressor, which is functionally defective in various cancers. Previously, we found that PP2A activity determined the anticancer effect of bortezomib and erlotinib in hepatocellular carcinoma (HCC) cells. Here, we tested a novel erlotinib derivative, TD52, in four HCC cell lines, PLC5, Huh-7, Hep3B and Sk-Hep1. Using MTT and flow cytometry, we showed that TD52 had more potent apoptotic effects than erlotinib in HCC cells. TD52-induced apoptosis was associated with dose- and time- dependent reactivation of PP2A and downregulation of cancerous inhibitor of protein phosphatase 2A (CIP2A) and p-Akt. Inhibition of PP2A or ectopic expression of CIP2A or Akt in PLC5 cells abolished the effects of TD52. Furthermore, we demonstrated that TD52 affected the binding of Elk-1 to the proximal promoter of the CIP2A gene, thus downregulating transcription of CIP2A. Importantly, TD52-induced tumor inhibition was associated with reactivation of PP2A and downregulation of CIP2A and p-Akt in vivo. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A determines the apoptotic effect induced by TD52. Our findings disclose the therapeutic mechanism of this novel targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.