Hung Huynh

Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: Its role in tumor progression and apoptosis

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies in South East Asia. Although activation of the MEK-MAPK is often associated with cellular growth, the role of MEK-MAPK in growth and survival of hepatocarcinoma cells has not been established.MethodsImmuno-histochemistry was used to localize phosphorylated MAPK and MEK1/2 in the tissues. 3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay and ELISA were used to determine cell viability and cell proliferation. Deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells. Western blots analysis was performed to determine the levels of proteins involved in the MEK-MAPK and apoptotic pathways. Transfection study was performed to assess the role of MEK-MAPK pathway in growth and survival of liver cancer cells.ResultsWe report that phosphorylation of MEK1/2 at Ser217/221 was detected by immuno-histochemistry in 100% (46 of 46) of HCCs examined. A positive signal was localized in the nuclei of hepatocarcinoma cells but not in dysplastic hepatocytes or stromal cells. Over-expression and phosphorylation of MAPK was also detected in 91% (42 of 46) and 69% (32 of 46) of HCCs examined, respectively. The percentage of cells showing positively for phosphorylated MEK1/2 increased with advancing tumor stage. In vitro, treatment of human HepG2 and Hep3B cells with MEK1/2 specific inhibitors U0126 and PD98059 led to growth inhibition and apoptosis. U0126 induced the release of cytochrome c and increased the cleavage of caspase-3, caspase-7, and poly ADP-ribose polymerase (PARP). Inhibition of phosphatidylinositol 3-kinase (PI-3K), c-Jun N-terminal kinase (JNK) and p38 kinase activities caused only a mild apoptosis in HepG2 and Hep3B cells. Activated MEK1-transfected cells were more resistant to UO126-induced apoptosis in vitro and formed larger tumors in SCID mice than mock-transfected cells.ConclusionIn conclusion, our results demonstrate that MEK-MAPK plays an important role in the growth and survival of liver cancer cells and suggest that blocking MEK-MAPK activity may represent an alternative approach for the treatment of liver cancer.

Brivanib Alaninate, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor and Fibroblast Growth Factor Receptor Tyrosine Kinases, Induces Growth Inhibition in Mouse Models of Human Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm; surgery is the only curative option but 5-year survival rates are only 25% to 50%. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are known to be involved in growth and neovascularization of HCC. Therefore, agents that target these pathways may be effective in the treatment of HCC. The aim of this study was to determine the antineoplastic activity of brivanib alaninate, a dual inhibitor of VEGF receptor (VEGFR) and FGF receptor (FGFR) signaling pathways. Experimental Design: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. Results: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib–induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr1054/1059, increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. Conclusion: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC, and that their activation was required for liver cancer cell proliferation and survival. In the present study, we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition, elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase, but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244, when given p.o. to mice bearing these xenografts, resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK, and up-regulation of activated caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs. Targeted inhibition of the MEK-ERK pathway with AZD6244 may represent an alternative approach for the treatment of this disease. [Mol Cancer Ther 2007;6(1):138–45]

RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient‐derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient‐derived HCC xenografts resulted in a dose‐dependent inhibition of tumour growth. RAD001‐induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up‐regulation of p27Kip1 and down‐regulation of p21Cip1/Waf1, Cdk‐6, Cdk‐2, Cdk‐4, cdc‐25C, cyclin B1 and c‐Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.

Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Vascular endothelial growth factor, platelet derived growth factor and the Raf/mitogen‐activated protein kinase/extracellular signal regulated kinase (Raf/MEK/ERK) signalling pathway regulates the growth, neovascularization, invasiveness and metastatic potential of HCC. In this study, we investigated the in vivo antitumour activity and mechanisms of action of sorafenib tosylate on four patient‐derived HCC xenografts. Sorafenib dosed at 50 mg/kg and 100 mg/kg inhibited tumour growth by 85% and 96%, respectively. Sorafenib‐induced growth suppression and apoptosis were associated with inhibition of angiogenesis, down‐regulation of phospho‐platelet‐derived growth factor receptor β Tyr1021, phospho‐eIF4E Ser209, phospho‐c‐Raf Ser259, c‐Raf, Mcl‐1, Bcl‐2, Bcl‐x and positive cell cycle regulators, up‐regulation of apoptosis signalling kinase‐1, p27 and p21. Expression of IGF‐1Rβ and phosphorylation of c‐Raf Ser338, MEK1/2 Ser217/221 and ERK1/2 Thr202/Tyr204 were increased by sorafenib treatment. Phosphorylation of mammalian target‐of‐rapamycin (mTOR) targets (p70S6K, S6R and 4EBP1) was reduced by sorafenib in sorafenib‐sensitive lines but activated in sorafenib‐less‐sensitive 10–0505 xenograft. Sorafenib‐induced phosphorylation of c‐met, p70S6K and 4EBP1 was significantly reduced when 10–0505 cells were co‐treated with anti‐human anti‐HGF antibody, suggesting that treatment with sorafenib leads to increased HGF secretion and activation of c‐met and mTOR targets. Treatment of 10–0505 tumours with sorafenib plus rapamycin resulted in growth inhibition, inhibition of vascular endothelial growth factor receptor‐2 phosphorylation, increased apoptosis and completely blocked sorafenib‐induced phosphorylation of mTOR targets and cyclin B1 expression. These data also provide a strong rationale for clinical investigation of sorafenib in combination with mTOR inhibitors in patients with HCC.

AZD6244 enhances the anti-tumor activity of sorafenib in ectopic and orthotopic models of human hepatocellular carcinoma (HCC).

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential. METHODS We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry. RESULTS We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation. CONCLUSION Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.

Xenografts of Human Hepatocellular Carcinoma: A Useful Model for Testing Drugs

Purpose: Our aims were to establish and characterize primary human hepatocellular carcinoma xenografts. They were used to screen new drugs and improve our current treatment regimens used in hepatocellular carcinoma. Experimental Design: Primary hepatocellular carcinomas were used to create the xenografts. Western blotting was used to determine the changes in proteins in these xenografts before and after therapies. Apoptotic and cell proliferation were analyzed by immunohistochemistry. Results: Seven lines of xenografts were established from primary human hepatocellular carcinomas. Lines 4-1318, 2-1318, 2006, and 26-1004 grew rapidly in severe combined immunodeficient (SCID) mice and doubled its volume every 48 to 72 hours. Series 5-1318 (5-1318, 30-1004, and 29-1104) grew relatively slowly in SCID mice and required ∼6 to 10 days to double its tumor volume. Western blot analysis revealed that the growth rate of these xenografts was associated with abnormal expression of proteins associated with the cell cycle, signaling pathways, and tumor suppressor genes. Although hepatocellular carcinoma xenografts expressed the receptors for androgens, estrogens, and progesterone, their growth rate was not affected by either castration or sex steroid hormone supplementation. Cisplatin, oxaliplatin, vitamin D analogue EB1089, and Iressa had no effects on the growth rate in SCID mice. Although 5-fluorouracil exerted mild growth inhibition of these xenografts, i.p. delivery of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) or doxorubicin resulted in a significant growth inhibition. Doxorubicin-induced growth suppression was associated with elevation of p53 and p21Cip1/Waf1. In addition to up-regulation of p53 and p21Cip1/Waf1, SarCNU also increased the levels of phosphorylated cdc-2 at Tyr15. Conclusion: Hepatocellular carcinoma xenografts are powerful tools for screening drugs and SarCNU may be useful in the treatment of this fatal disease.

Bevacizumab and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma

BACKGROUND/AIMS Hepatocellular carcinoma is a leading cause of global cancer mortality, with standard chemotherapy being minimally effective in prolonging survival. We investigated if combined targeting of vascular endothelial growth factor protein and expression might affect hepatocellular carcinoma growth and angiogenesis. METHODS We treated patient-derived hepatocellular carcinoma xenografts with (i) bevacizumab; (ii) rapamycin; and (iii) bevacizumab plus rapamycin. Western blotting was employed to determine changes in the proteins. Apoptosis, vascular endothelial growth factor expression, microvessel density, and cell proliferation were analyzed by immunohistochemistry. RESULTS Hepatocellular carcinoma growth was inhibited by bevacizumab plus rapamycin treatment to a significantly greater degree than bevacizumab or rapamycin monotherapy. Reductions in tumor growth by bevacizumab plus rapamycin were associated with inhibition of downstream targets of the mammalian target-of-rapamycin pathway, reductions in vascular endothelial growth factor expression, and tumor microvessel density. Potentially additive effects of bevacizumab plus rapamycin included reductions in vascular endothelial growth factor expression, cyclin D1, and cyclin B1. In an intra-peritoneal model of hepatocellular carcinoma, bevacizumab plus rapamycin potently inhibited both intra-liver and intra-abdominal tumor growth, reduced ascites levels, and significantly prolonged mouse survival. CONCLUSIONS Bevacizumab and rapamycin, which are both clinically approved drugs, may represent a novel molecularly-targeted combination treatment for hepatocellular carcinoma.

Sorafenib induces growth suppression in mouse models of gastrointestinal stromal tumor

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Current therapeutic options include surgery and targeted molecular approaches such as imatinib and sunitinib. Our aim was to establish patient-derived GIST xenografts for the use of screening new drugs and improving current treatment regimens used in GIST. In this present study, we investigate the antitumor activity of sorafenib against patient-derived GIST xenografts. Murine xenograft models were given two oral doses of sorafenib daily for 30 days and growth of established tumor xenografts was monitored at least twice weekly by vernier caliper measurements. Western blotting was then used to determine changes in proteins in these xenografts before and after sorafenib therapy. Apoptotic and cell proliferation were analyzed by immunohistochemisty. Our data found that oral administration of sorafenib to mice, bearing patient-derived GIST xenografts, resulted in dose-dependent inhibition of tumor growth. Sorafenib-induced growth inhibition was associated with decreased cell proliferation, increased apoptosis, and reduction in tumor angiogenesis. Western blot analysis revealed that sorafenib inhibited C-Raf, phospho-extracellular signal-regulated kinase 1/2, and phospho-MEK1 (Thr286) slightly as well as phospho-c-Kit (Tyr568/Tyr570), phospho- platelet-derived growth factor receptor β (Tyr1021), and phospho-Flk1 (Tyr951), suggesting that sorafenib inhibited GIST growth by blocking the Raf/MEK/extracellular signal-regulated kinase pathway and angiogenesis. Sorafenib also induced cell cycle arrest, evident through increased levels of p15 and p27 and decreased levels of p21, cyclin A, cyclin B1, and cdc-2. Our study provides a strong rationale for the clinical investigation of sorafenib in patients with GIST as well as an established platform for further drug evaluation studies using GIST xenograft models. [Mol Cancer Ther 2009;8(1):152–9]

AZD6244 and doxorubicin induce growth suppression and apoptosis in mouse models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide, with no effective treatment for most individuals who succumb to this neoplasm. We report that treatment of primary HCC cells with the mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase 1/2 inhibitor AZD6244 (ARRY-142886) plus doxorubicin led to synergistic growth inhibition and apoptosis. In vivo administration of AZD6244, doxorubicin, or the combination of AZD6244 and doxorubicin in mice bearing 5-1318 HCC xenografts resulted in approximately 52% ± 15%, 12% ± 9%, and 76% ± 7% growth inhibition, respectively. AZD6244-inhibited tumor growth was associated with increased apoptosis, inactivation of ERK1/2, inhibition of cell proliferation, and down-regulation of cell cycle regulators, including cyclin D1, cdc-2, cyclin-dependent kinases 2 and 4, cyclin B1, and c-Myc. The AZD6244-doxorubicin combined protocol not only promoted apoptosis but also induced a synergistic effect not seen in single-agent–treated tumors, including increased expression of the p130 RB tumor suppressor gene. Our study provides a strong rationale for clinical investigation of combination therapy with the mitogen-activated protein/ERK kinase 1/2 inhibitor AZD6244 and doxorubicin in patients with HCC. [Mol Cancer Ther 2007;6(9):2468–76]