Wen-Chi Feng

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.

OSU-03012, a Novel Celecoxib Derivative, Induces Reactive Oxygen Species–Related Autophagy in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Systemic treatments for HCC have been largely unsuccessful. OSU-03012 is a derivative of celecoxib with anticancer activity. The mechanism of action is presumably 3-phosphoinositide-dependent kinase 1 (PDK1) inhibition. This study investigated the potential of OSU-03012 as a treatment for HCC. OSU-03012 inhibited cell growth of Huh7, Hep3B, and HepG2 cells with IC(50) below 1 mumol/L. In Huh7 cells, OSU-03012 did not suppress PDK1 or AKT activity. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and flow cytometry analysis indicated that OSU-03012 did not induce cellular apoptosis. Instead, morphologic studies by light and electron microscopy, as well as special biological staining with monodansylcadaverine, acridine orange, and microtubule-associated protein 1 light chain 3, revealed OSU-03012-induced autophagy of Huh7 cells. This OSU-03012-induced autophagy was inhibited by 3-methyladenine. Moreover, reactive oxygen species (ROS) accumulation was detected after OSU-03012 treatment. Blocking ROS accumulation with ROS scavengers inhibited autophagy formation, indicating that ROS accumulation and subsequent autophagy formation might be a major mechanism of action of OSU-03012. Daily oral treatment of BALB/c nude mice with OSU-03012 suppressed the growth of Huh7 tumor xenografts. Electron microscopic observation indicated that OSU-03012 induced autophagy in vivo. Together, our results show that OSU-03012 induces autophagic cell death but not apoptosis in HCC and that the autophagy-inducing activity is at least partially related to ROS accumulation.

Induction of DNA Damage-Inducible Gene GADD45β Contributes to Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma Cells

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45β (GADD45β), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45β expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45β mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45β induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45β induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45βor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45β promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45β induction by sorafenib. Together, our findings suggest that GADD45β induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.

Combinations of Mtorc1 Inhibitor Rad001 with Gemcitabine and Paclitaxel for Treating Non-Hodgkin Lymphoma

Single-agent mammalian target of rapamycin complex 1 (mTORC1) inhibitors have recently been reported as effective salvage treatment in non-Hodgkin lymphoma (NHL). The combined effect of mTORC1 inhibitor, RAD001, with chemotherapeutic agents used for relapsed or refractory NHL was examined. Synergistic interactions were observed for RAD001 plus gemcitabine or paclitaxel in six NHL cell lines; enhanced gemcitabine- and paclitaxel-induced caspase-dependent apoptosis associated with down-regulation of mTOR signaling was detected. Synergistic interactions were also observed with RAD001 plus gemcitabine and paclitaxel. In conclusion, synergistic cytotoxicity was observed with RAD001 plus gemcitabine and paclitaxel in NHL cells. Combination therapy with these three drugs should be examined in patients with refractory or relapsed NHL.

Inhibition of the Wnt/β-catenin signaling pathway improves the anti-tumor effects of sorafenib against hepatocellular carcinoma

Sorafenib, a multikinase inhibitor, is currently the only approved drug for advanced hepatocellular carcinoma (HCC). The current study tested the hypothesis whether inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC. ICG-001, a small molecule which blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis-induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA-mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. Our data indicate that inhibition of the Wnt/β-catenin signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo.

Abstract 5336: Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA [Background] Sorafenib is currently the only approved systemic agent for advanced hepatocellular carcinoma (HCC); however, its efficacy is limited. Aberrant activation of WNT/β-catenin signaling pathway has been shown to mediate resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway would improve the anti-tumor effect of sorafenib in HCC. [Materials and Methods] Human HCC cell lines, including Huh7, HepG2, PLC5, and Hep3B, were included. Inhibition of WNT/β-catenin pathway was achieved by ICG-001, a small molecule disrupting the interaction of cAMP-responsive element binding (CREB)-binding protein (CBP) and β-catenin and inhibiting the β-catenin-mediated transcription activities, or by RNA interference (RNAi)- downregulation of β-catenin. The efficacy of sorafenib combined with WNT/β-catenin pathway inhibition in HCC cells was determined by MTT for cell viability, and by flow cytometry and Western blotting for apoptosis. The in vivo efficacy was evaluated in a subcutaneous xenograft mode of Huh7 cells. [Results] In multiple HCC cell lines, ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently; the combination of ICG-001 and sorafenib showed a synergistic antitumor effect. Downregulation of β-catenin by RNAi increased sorafenib sensitivity, whereas overexpression of β-catenin decreased sorafenib sensitivity in Huh7 cells. Furthermore, the sorafenib-sensitization effect by short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was offset by β-catenin overexpression. Mechanistically, sorafenib in combination of ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a more prominent downregulation of Mcl-1 in HCC cells. Finally, ICG-001 in combination of sorafenib showed a more significantly growth-retarding effect in Huh7 xenograft than either drug alone. [Conclusion] Our data indicate that inhibition of WNT/β- catenin signaling pathway improves the antitumor effect of sorafenib against HCC in vitro and in vivo.(This study was supported by grants NRPB-100CAP1020-2 and NSC-101-2314-B-002-141, 100CAP1020-2). Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2015-5336

Abstract 2625: Increased sensitivity to Wnt/beta-catenin inhibitors in hepatocellular carcinoma cells harboring activating mutation of beta-catenin

[Background] The WNT/β-catenin signaling pathway is a major oncogenic pathway of human hepatocellular carcinoma (HCC). More than 50% of HCCs have aberrant activation of WNT/β-catenin signaling, which are caused by genetic alterations, ligand activation, or cross-activation by other signaling pathways. We hypothesized that activating mutation of CTNNB1, the coding gene of β-catenin, may confer sensitivity to WNT/β-catenin inhibitors in HCC cells. [Materials and Methods] SNU398 is a human HCC cell line harboring a missense somatic mutation in exon 3 of CTNNB1 gene, resulting in S37C mutation. Huh7, HepG2, PLC5, and Hep3B are HCC cells containing no somatic mutations in exon 3 of CTNNB1 gene. HCC cells were treated with various types of WNT/β-catenin pathway inhibitors, including ICG-001 which down-regulates β-catenin/TCF transcriptional activity by interfering the binding of β-catenin with its transcriptional activation complex, XAV939 which targets tankyrases and facilitates degradation of β-catenin, and LGK974 which inhibits Porcupine, an acyltransferase responsible for palmitoylation and secretion of Wnt ligands, and evaluated for in vitro viability by MTT. [Results] ICG-001 as a single agent showed significantly greater anti-proliferation effect in SNU398 than other HCC cells. The 50%-inhibitory concentrations (IC50s) of ICG-001 were 5 μM and >10 μM for SNU398 and other HCC cells, respectively. ICG-001 also induced a more potent suppression of colony- formation, and a more significant increase of subG1 fraction detected by flow cytometry in SNU398 cell than other HCC cells. XAV939 up to 20 μM induced only modest anti-proliferative effect in all HCC cells. However, SNU398 remained the one showing the greatest sensitivity to the growth suppression induced by XAV939. The anti-proliferative effect of LGK974 was modest and showed no significant difference among different HCC cells. [Conclusion] The activating mutation of β-catenin may confer sensitivity to certain WNT/β-catenin pathway inhibitors, such as ICG-001, in HCC cells. (This study was supported by grants NRPB-100CAP1020-2). Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, LI-Chun Lu, Ta-Wen Hsu, Ann-Lii Cheng, Chih-Hung Hsu. Increased sensitivity to Wnt/beta-catenin inhibitors in hepatocellular carcinoma cells harboring activating mutation of beta-catenin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2014-2625

Abstract 2052: WNT/beta-catenin signaling inhibitors improve the anti-proliferative effect of sorafenib against hepatocellular carcinoma (HCC) cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC [Background] Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only indicated therapy for patients with advanced hepatocellular carcinoma (HCC). The aberrant activation of WNT/β-catenin signaling pathway, which is associated with the carcinogenetic process of HCC, has been shown to mediate the resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway might improve the anti-tumor effect of sorafenib in HCC. [Materials and Methods] Huh7, HepG2, PLC5, and Hep3B HCC cells were treated with sorafenib with or without inhibitors of WNT/β-catenin pathway, and evaluated for viability in vitro. WNT/β-catenin signaling inhibitors included ICG-001 which down-regulates β-catenin/TCF signaling by specifically binding to cyclic AMP response element-binding protein, and XAV939 which targets tankyrases and facilitates degradation of β-catenin. [Results] Sorafenib induced an anti-proliferative effect in HCC cells, with IC50s of 3.5±0.3 μM, 3.7±0.4 μM, 7.7±0.8 μM and 14±0.3 μM for HepG2, Huh7, PLC5, and Hep3B cells, respectively. Upon treated with 10 μM sorafenib for 24 hours, the total cellular amounts of β-catenin were significantly decreased in HepG2 and Huh7 cells, moderately decreased in PLC5 cells, and unchanged in Hep3B cells. In all HCC cells, XAV939 or ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently. The median effect analysis showed a synergistic anti-proliferative effect for the combination of ICG-001 plus sorafenib in all HCC cells. [Conclusion] Inhibitors of WNT/β- catenin signaling pathway in combination with sorafenib resulted in an improved anti-tumor effect against HCC in vitro. (The study was supported by the grant NSC 100-2325-B-002-043 from National Science Council of Taiwan) Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. WNT/beta-catenin signaling inhibitors improve the anti-proliferative effect of sorafenib against hepatocellular carcinoma (HCC) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2013-2052

Abstract 3886: Activated insulin-like growth factor (IGF) signaling pathway is a potential therapeutic target for brain metastasis from lung cancer.

Background: Brain metastasis is a formidable challenge in lung cancer treatment. The incidence has been increasing recently because of the advances of treatment which leads to prolonged survival of many patients. Methods: We established xenograft model of brain metastasis. Repeated rounds of in vivo selection and ex vivo primary cultures of the brain metastases resulted in a cell line with a high propensity to metastasize to the brain. Brain-tropism was confirmed by bioluminescence study. Gene expression profiles between parental lung cancer cells (PC9) and brain-tropic cells (PC9-Br) was compared by Agilent Gene Expression Microarray followed by Ingenuity Pathway Analysis. The biologic effects and clinical implications of the candidate molecules were further investigated. Results: Significant activation of IGF and WNT signaling pathways was detected in PC9-Br, as compared with the parental cells. We selected IGF pathway as the first step to explore the potential therapeutic implication. Western blot and RT-PCR validated the results of IGF2 overexpression and activated IGF1R (pIGF1R) in PC9-Br cells. In vitro drug sensitivity tests showed that PC9-Br, in comparison to parental cells, was more resistant to erlotinib, an EGFR TKI (IC50: 30nM for PC9 and 189nM for PC9-Br). Knocking down IGF2 expression in PC9-Br reversed the drug resistance, while adding IGF2 to PC9 enhanced the resistance. In clinical samples, there was a significantly higher expression of pIGF1R in 26 brain metastases as compared to their paired primary lung tumors (mean of H score differences=40.96, 95% C.I.=10.96∼70.96, P=0.0094). Conclusions: IGF signaling pathway is activated in brain-tropic lung cancer cells and could potentially be a therapeutic target for brain metastasis. Acknowledgement: This study was supported by Research Grant 101-M2002 from the National Taiwan University Hospital. Citation Format: Pei-Fang Wu, Ching-Hung Lin, Wen-Chang Huang, Wen-Chi Feng, Yen-Shen Lu, Chih-Hsin Yang, Ann-Lii Cheng. Activated insulin-like growth factor (IGF) signaling pathway is a potential therapeutic target for brain metastasis from lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2013-3886

Abstract 1904: Transforming growth factor-beta mediated epithelial to mesenchymal transition contributes to in vivo resistance to sorafenib in hepatocellular carcinoma

Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only approved agent for hepatocellular carcinoma (HCC). However, the majority of HCC patients are inherently resistant to sorafenib treatment. To study molecular mechanisms underlying the resistance to sorafenib in vivo, we studied immunocompromised mice implanted with xenografts of human HCC cells subcutaneously, and identified tumors that showed primary resistance to sorafenib. Primary cultured cells were established from sorafenib-resistant xenografts, and verified for in vitro and in vivo sensitivity to sorafenib. The differentially expressed genes of the resistant cells versus control cells were analyzed by cDNA microarray. The expressions of candidate markers were confirmed by Western blotting, and their significances were verified by specific pathway inhibitors. A subline of Huh7 cells was established by primary culture from a sorafenib-resistant HCC-xenograft (designated as Huh7.1.SR). Compared to Huh7 cells primarily cultured from HCC-xenograft treated with vehicle alone (designated as Huh7.1.v), Huh7.1.SR had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 5 to 10 μM. Subcutaneous xenograft studies confirmed that Huh7.1.SR retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes of Huh7.1.SR versus Huh7.1.v cells revealed several pathway networks centering at transforming growth factor beta receptor (TGFBR), hepatocyte nuclear factors, NOTCH, pro-inflammatory cytokines, and nuclear factor kappa B. Huh7.1.SR had an increased expression of TGF-beta and p-Smad2, indicating that the TGF-beta pathway was activated. Huh7.1.SR cells expressed phenotypic features of epithelial to mesenchymal transition (EMT), including higher expression of vimentin, lower expression of E-cadherin, and increased cell migration. The expressions of Slug and Twist, two well-known transcriptional factors of inducing EMT, also increased in Huh7.1.SR cells. Treatment of SD-208, an inhibitor of TGF-beta receptor, partially reversed the phenotypic features of EMT in Huh7.1.SR cells. Moreover, concomitant treatment of SD-208 improved the sensitivity to sorafenib in Huh7.1.SR cells. Our data indicate that activation of TGF-beta signaling pathway is an important mechanism mediating the resistance to sorafenib in HCC in vivo. (The study was supported by the grant NSC 100-2325-B-002-043- from National Science Council of Taiwan) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1904. doi:1538-7445.AM2012-1904