Browsing by Subject "Carcinoma, hepatocellular."

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Open Access
Heterogeneity of hepatocellular malignant phenotype
(2006) Öztürk, Nuri
Hepatocellular carcinoma (HCC) is one of the most wide-spread carcinomas throughout the world - responsible for more than 600,000 deaths annually - and is strongly associated with several etiological factors; including aflatoxin B1, alcohol, and Hepatitis virus B and C. In HCC, many genes undergo somatic aberrations with a tendency to cluster at genes involved in cell cycle regulation, in the p53 and canonical Wnt signaling pathways, and in the TGF-β/IGF axis. Almost a third of HCCs display mutations affecting canonical Wnt signalling. However, the role of canonical Wnt signaling aberrations in HCC is not known in detail, since transgenic mice which express mutant β-catenin (an integral component of canonical Wnt signaling) do not develop liver tumours. To study the heterogeneity of hepatocellular malignancies, we have concentrated on canonical Wnt signaling in HCC cell lines. We have found that canonical Wnt signaling was active in 80% of well-differentiated, and 14% of poorly-differentiated cell lines respectively. Furthermore, ectopic expression of a mutant β-catenin resulted in strong canonical Wnt activity in well-differentiated, but not in poorly-differentiated HCC cells. Our findings suggested that heterogeneity in HCC exists even in the same pathway as exemplified by differential canonical Wnt signaling activity in well- and poorlydifferentiated HCC cell lines. During this study, we produced monoclonal antibodies against β-catenin to distinguish between the pools of nuclear/cytoplasmic and membraneassociated β-catenin in cells, since it is believed that the nuclear β-catenin pool is more potent in tumorigenesis. Monoclonal antibody (MAb), 4C9 recognised β-catenin out of adherens junctions, while another MAb, 9E10 recognised all β-catenin forms even though their epitopes were adjacent. Differential Wnt signaling activity in HCC cell lines prompted us to investigate the interactions between β-catenin and other molecules, which have important functions in hepatocytes and may affect β-catenin/TCF transcriptional activity. C/EBPα is a potent inhibitor of cell proliferation in HCC cell lines and is involved in liver-specific gene expression, and some somatic alterations of it have been observed in AML and HCC. We investigated the effect of C/EBPα on β-catenin signalling. We have found that C/EBPα inhibits mutant β-catenin-TCF transcriptional activity, and physically interacts with β- catenin in HCC cells. While we were analyzing some stably mock-transfected Huh7 clones to use as controls, we observed heterogeneity in their proliferation rates. Further analysis of these clones revealed that some clones ceased to proliferate when passaged extensively. One of these clones (C3) was not tumorigenic in immunodeficient mice. Based on these observations, we hypothesized that some cancer cells could produce senescencent progeny in cell culture. Indeed, we showed that breast- and liver-cancer-derived cells display senescencent phenotypes at variable ratios. By using our experimental system, we also showed that replicative senescence program may work independently of functional p53 and p16 pathways, and the SIP1 gene is partially responsible for replicative senescent phenotypes in our Huh7-derived senescent clone C3. Overexpression of mouse SIP1 in p53- and Rb-deficient Hep3B cells induced partial senescent phenotypes at early passages. However, stable Hep3B cells repressed mouse SIP1 expression by an unknown mechanism and escaped senescent arrest in late passages. Our results suggest that Wnt pathway may have a dual role in hepatocellular malignancy, as it is active/easily inducible in well-differentiated HCC cells and inactive/repressed in poorly-differentiated ones. The further study of β-catenin in tumor samples by using our monoclonal antibodies may reveal new aspects in β-catenin signaling. However, the mechanism of these phenomena and the inhibition of β-cateninTCF signaling by C/EBPα require more study to reach a more comprehensive conclusion. The study of reprogramming of replicative senescence in HCC-derived cells indicated that senescence program may work independent of p53 and p16 pathways and heterogeneity of hepatocellular malignancy exists even within the established HCC derived cell lines.
Open Access
Systems biology approach for targeted therapy of liver cancer P13K/AKT/MTOR pathway inhibitors : an ally or rival for sorafenib'e
(2014) Erşahin, Tülin
Hepatocellular carcinoma (HCC) is one of the leading causes of cancerrelated mortality worldwide. It is the second most frequent cause of cancer death in men, and the sixth in women due to its aggressive behavior and resistance to conventional therapies. Sorafenib (Nexavar, BAY43-9006), a multi-kinase inhibitor with anti-angiogenic functions, is the only FDA-approved molecular-targeted agent for the treatment of patients with advanced HCC. Yet, Sorafenib shows limited overall survival benefit associated with resistance and tumor recurrence. Current mono-target- or single pathway-centric drug designs are not sufficient for effective therapy of advanced HCC. Negative results of clinical trials on targeted therapies for advanced HCC are due to clinical heterogeneity, complexity of cirrhotic background and interconnected regulation of cancer hallmarks through compensatory signaling pathways with redundant functions. Secretion of growth factors, pro-inflammatory and immune-suppressive cytokines and chemokines in the tumor microenvironment and consequent activation of tumor-promoting signaling cascades confer resistance to Sorafenib treatment. RAF/MEK/ERK and PI3K/AKT/mTOR are the major tumorpromoting signaling pathways with regulatory functions in all hallmarks of HCC. They have redundant functions and inhibition of one pathway can stimulate compensatory signaling from the other pathway. Since Sorafenib targets angiogenic VEGFR and PDGFR kinases and RAF/MEK/ERK signaling, the primary mechanism of resistance to Sorafenib and tumor recurrence in HCC patients emerges to be the compensatory signaling from the PI3K/AKT pathway. Therefore, we anticipated that combined treatment with Sorafenib and PI3K/AKT inhibitors could reverse drug resistance in HCC. In this study, we analyzed the synergistic effects of Sorafenib and PI3K/AKT inhibitors on HCC cell growth and migration, determined possible mechanisms underlying synergistic mechanism of action by transcriptome analysis, and further showed that combination therapy leads to tumor regression in PTEN-deficient HCC xenografts in vivo. We showed that PTEN-deficient HCC cells with constitutively active PI3K/AKT signaling depend on the alpha isoform of PI3K (p110-α) for survival and co-targeting these cells with the isoform specific PI3K inhibitor (PI3Ki- α, PIK-75) overcomes resistance to Sorafenib. Indeed, while dual-targeting of PTENdeficient HCC cells with Sorafenib and PI3Ki-α results in synergistic growth inhibition, dual-targeting these cells with Sorafenib and a beta isoform specific inhibitor of PI3K (PI3Ki-α, TGX-221) leads to an antagonistic increase in tumor growth compared to single treatment with Sorafenib, since inhibiting p110-! promotes compensatory signaling from p110-α. We also investigated the cytotoxic effects of inhibiting Akt kinase by using an Akt2 isoform-specific inhibitor (Akti-2) and a general Akt inhibitor targeting both isoforms (Akti-1,2). Akti-1,2 and Akti-2 did not induce anti-growth or pro-apoptotic mechanisms, but they were highly effective in reducing migration. Akt2 isoform is specifically overexpressed in HCC, and is correlated with its progression. Moreover, the Akt2 isoform-specific role of Akt kinase on migration has been demonstrated in breast cancer, and depletion of AKT2, but not AKT1, was shown to promote regression of PTEN-deficient prostate cancer xenografts. Based on these findings, we predicted a prominent role of Akt2 in PTEN-deficient HCC cells and examined the therapeutic efficacy of combined treatment of Sorafenib and Akti-2 in vivo in athymic mice bearing Mahlavu tumor xenografts. After 3 weeks of treatment, tumor growth was reduced significantly in all tested groups (Sorafenib, Akti-2 and Combination) compared to the control group. Substantial intra-tumoral necrosis produced a temporary increase in tumor size but resulted in significant reduction in tumor weight (p<0.001) in combination-treated mice compared to Sorafenib-treated mice and further produced tumor regressions. In this study, we showed cytotoxic activity of a new PI3K α soform specific kinase inhibitor (PI3Ki-α / PIK-75) at 0.1 µM that acts synergistically with Sorafenib in vitro, determined the predominant role of PI3K isoform p110α in PTEN-deficient HCC cells, and revealed synergistic anti-tumor effect of combining Sorafenib with a new Akt isoform 2 specific kinase inhibitor (Akti-2) in vitro and in vivo.