Wei-Tien Tai

Activation of Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Mediates Acquired Resistance to Sorafenib in Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Sorafenib, a tyrosine kinase inhibitor, was recently approved by the United States Food and Drug Administration for HCC. In this study, we established two sorafenib-resistant HCC cell lines from Huh7, a human HCC cell line, by long-term exposure of cells to sorafenib. Sorafenib induced significant apoptosis in Huh7 cells; however, Huh7-R1 and Huh7-R2 showed significant resistance to sorafenib-induced apoptosis at the clinical relevant concentrations (up to 10 μM). Thorough comparisons of the molecular changes between Huh7 and resistant cells showed that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway played a significant role in mediating acquired resistance to sorafenib in Huh7-R1 and Huh7-R2 cells. Phospho-Akt and p85 (a regulatory subunit of PI3K) were up-regulated, whereas tumor suppressor phosphatase and tensin homolog were down-regulated in these resistant cells. In addition, ectopic expression of constitutive Akt in Huh7 demonstrated similar resistance to sorafenib. The knockdown of Akt by RNA interference reversed resistance to sorafenib in Huh7-R1 cells, indicating the importance of Akt in drug sensitivity. Furthermore, the combination of 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one dihydrochloride (MK-2206), a novel allosteric Akt inhibitor, and sorafenib restored the sensitivity of resistant cells to sorafenib-induced apoptosis. In conclusion, activation of PI3K/Akt signaling pathway mediates acquired resistance to sorafenib in HCC, and the combination of sorafenib and MK-2206, an Akt inhibitor, overcomes the resistance at clinical achievable concentrations.

Sorafenib Overcomes Trail Resistance of Hepatocellular Carcinoma Cells through the Inhibition of Stat3

Purpose: Recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, many hepatocellular carcinoma (HCC) cells show resistance to TRAIL-induced apoptosis. Here, we report that sorafenib improves the antitumor effect of TRAIL-related agents in resistant HCC. Experimental Design: HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib and/or TRAIL-related agents (TRAIL or LBY135) and analyzed in terms of apoptosis and signal transduction. In vivo efficacy was determined in nude mice with PLC5 xenografts. Results: Sorafenib, the only approved drug for HCC, sensitizes resistant HCC cells to an agonistic DR5 antibody (LBY135) and TRAIL-induced apoptosis in TRAIL-resistant HCC cells. We found that STAT3 played a significant role in mediating TRAIL sensitization. Our data showed that sorafenib downregulated phospho-STAT3 (pSTAT3) and subsequently reduced the expression levels of STAT3-related proteins (Mcl-1, survivin, and cyclin D1) in a dose- and time-dependent manner in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the TRAIL-sensitizing effect of sorafenib. Moreover, SHP-1 inhibitor reversed downregulation of pSTAT3 and apoptosis induced by sorafenib, and silencing of SHP-1 by RNA interference abolished the effects of sorafenib on pSTAT3. Notably, sorafenib increased SHP-1 activity in PLC5 cells. Finally, sorafenib plus LBY135 significantly suppressed PLC5 xenograft tumor growth. Conclusions: Sorafenib sensitizes resistant HCC cells to TRAIL-induced apoptosis at clinical achievable concentrations, and this effect is mediated via the inhibition of STAT3. Clin Cancer Res; 16(21); 5189–99. ©2010 AACR.

Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma

BACKGROUND & AIMS Recently, we reported that sorafenib sensitizes hepatocellular carcinoma (HCC) cells to TRAIL through the inhibition of signal transducer and activator of transcription 3 (STAT3). Here, we report that sorafenib inhibits HCC via a kinase-independent mechanism: SHP-1 dependent STAT3 inactivation. METHODS SC-1 is a sorafenib derivative that closely resembles sorafenib structurally but with no kinase inhibition activity. HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib or SC-1 and apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with Huh-7 xenografts. RESULTS SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis in all tested HCC cell lines. SC-1 down-regulated phosphorylation of phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested HCC cells. Expression of STAT3-driven genes, including Cyclin D1 and Survivin, was also repressed by SC-1. Luciferase reporter assay confirmed the inhibition of transcriptional activity of STAT3 in both sorafenib-treated and SC-1-treated cells. Ectopic expression of STAT3 in PLC5 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 up-regulated SHP-1 activity. Knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced apoptosis induced by SC-1. Finally, SC-1 reduced Huh-7 tumor growth significantly in vivo, which was associated with down-regulation of p-STAT3 and up-regulation of SHP-1 activity. CONCLUSIONS STAT3 is a major kinase-independent target of sorafenib in HCC.

Mcl-1-dependent activation of Beclin 1 mediates autophagic cell death induced by sorafenib and SC-59 in hepatocellular carcinoma cells

We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.

Dovitinib Induces Apoptosis and Overcomes Sorafenib Resistance in Hepatocellular Carcinoma through SHP-1–Mediated Inhibition of STAT3

The multiple kinase inhibitor dovitinib is currently under clinical investigation for hepatocellular carcinoma (HCC). Here, we investigated the mechanistic basis for the effects of dovitinib in HCCs. Dovitinib showed significant antitumor activity in HCC cell lines PLC5, Hep3B, Sk-Hep1, and Huh-7. Dovitinib downregulated phospho-STAT3 (p-STAT3) at tyrosine 705 and subsequently reduced the levels of expression of STAT3-related proteins Mcl-1, survivin, and cyclin D1 in a time-dependent manner. Ectopic expression of STAT3 abolished the apoptotic effect of dovitinib, indicating that STAT3 is indispensable in mediating the effect of dovitinib in HCC. SHP-1 inhibitor reversed downregulation of p-STAT3 and apoptosis induced by dovitinib, and silencing of SHP-1 by RNA interference abolished the effects of dovitinib on p-STAT3, indicating that SHP-1, a protein tyrosine phosphatase, mediates the effects of dovitinib. Notably, dovitinib increased SHP-1 activity in HCC cells. Incubation of dovitinib with pure SHP-1 protein enhanced its phosphatase activity, indicating that dovitinib upregulates the activity of SHP-1 via direct interactions. In addition, dovitinib induced apoptosis in two sorafenib-resistant cell lines through inhibition of STAT3, and sorafenib-resistant cells showed significant activation of STAT3, suggesting that targeting STAT3 may be a useful approach to overcome drug resistance in HCC. Finally, in vivo, dovitinib significantly suppressed growth of both Huh-7 and PLC5 xenograft tumors and downregulated p-STAT3 by increasing SHP-1 activity. In conclusion, dovitinib induces significant apoptosis in HCC cells and sorafenib-resistant cells via SHP-1–mediated inhibition of STAT3. Mol Cancer Ther; 11(2); 452–63. ©2011 AACR.

Blockade of STAT3 activation by sorafenib derivatives through enhancing SHP-1 phosphatase activity.

Previously, we demonstrated that the multiple kinase inhibitor sorafenib mediates the repression of phospho-STAT3 in hepatocellular carcinoma cells. In this study, we used this kinase-independent mechanism as a molecular basis to use sorafenib as scaffold to develop a novel class of SHP-1-activating agents. The proof of principle of this premise was provided by SC-1, which on replacement of N-methylpicolinamide by a phenylcyano group showed abolished kinase activity while retaining phospho-STAT3 repressive activity. Structural optimization of SC-1 led to compound 6, which repressed phospho-STAT3 through SHP-1 activation and inhibited PLC5 cell proliferation at sub-micromolar potency. In light of the pivotal role of phospho-STAT3 in promoting tumorigenesis and drug resistance, this novel SHP-1-activating agent may have therapeutic relevance in cancer therapy.

Discovery of novel src homology region 2 domain‐containing phosphatase 1 agonists from sorafenib for the treatment of hepatocellular carcinoma

Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma (HCC). Here, we report that Src homology region 2 (SH2) domain‐containing phosphatase 1 (SHP‐1) is a major target of sorafenib and generates a series of sorafenib derivatives to search for potent SHP‐1 agonists that may act as better anti‐HCC agents than sorafenib. Sorafenib increases SHP‐1 activity by direct interaction and impairs the association between the N‐SH2 domain and the catalytic protein tyrosine phosphatase domain of SHP‐1. Deletion of the N‐SH2 domain (dN1) or point mutation (D61A) of SHP‐1 abolished the effect of sorafenib on SHP‐1, phosphorylated signal transducer and activator of transcription 3 (p‐STAT3), and apoptosis, suggesting that sorafenib may affect SHP‐1 by triggering a conformational switch relieving its autoinhibition. Molecular docking of SHP‐1/sorafenib complex confirmed our findings in HCC cells. Furthermore, novel sorafenib derivatives SC‐43 and SC‐40 displayed more potent anti‐HCC activity than sorafenib, as measured by enhanced SHP‐1 activity, inhibition of p‐STAT3, and induction of apoptosis. SC‐43 induced substantial apoptosis in sorafenib‐resistant cells and showed better survival benefits than sorafenib in orthotopic HCC tumors. Conclusion: In this study, we identified SHP‐1 as a major target of sorafenib. SC‐43 and SC‐40, potent SHP‐1 agonists, showed better anti‐HCC effects than sorafenib in vitro and in vivo. Further clinical investigation is warranted. (Hepatology 2014;58:190–201)

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.

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.

A novel obatoclax derivative, SC-2001, induces apoptosis in hepatocellular carcinoma cells through SHP-1-dependent STAT3 inactivation

We investigated the effects of a novel compound, SC-2001, on hepatocellular carcinoma (HCC). SC-2001, which is structurally related to the Mcl-1 inhibitor obatoclax, showed better antitumor effects than obatoclax in HCC cell lines, including HepG2, PLC5 and Huh-7. Like obatoclax, SC-2001 inhibited the protein-protein interactions between Mcl-1 and Bak. However, SC-2001 downregulated the protein levels of Mcl-1 by reducing its transcription whereas obatoclax had no significant effect on Mcl-1 expression. As Mcl-1 is regulated by signal transducers and activators of transcription 3 (STAT3), we found that SC-2001 downregulated the phosphorylation of STAT3 (Tyr 705) and subsequently inhibited transcriptional activities of STAT3 in a dose-dependent manner. In addition to Mcl-1, STAT3-regulated proteins, including survivin and cyclin D1, were also repressed by SC-2001. Notably, SC-2001 reduced IL-6-induced STAT3 activation in HepG2 and PLC5 cells. Ectopic expression of STAT3 abolished the prominent apoptotic death in SC-2001-treated PLC5 cells, indicating that STAT3 is indispensable in mediating the effects of SC-2001. Importantly, SC-2001 enhanced the expression of SHP1, a negative regulator of STAT3. Inhibition of SHP-1 by either specific inhibitor or small interference RNA reduced the apoptotic effects of SC-2001, indicating that SHP-1 plays a key role in mediating SC2001-induced cell death. SC-2001 enhanced the activity of SHP-1 in all tested HCC cells including HepG2, PLC5 and Huh-7. Finally, SC-2001 reduced PLC5 tumor growth, downregulated p-STAT3 and upregulated SHP-1 expression and activity in vivo. In conclusion, our results suggest that SC-2001 induces apoptosis in HCC, and that this effect is mediated through SHP-1-dependent STAT3 inactivation.