gdc-0449 and Carcinoma--Hepatocellular

Abnormal expression of chondroitin sulfate has been found in many types of cancer, while its biological functions in hepatocellular carcinoma (HCC) progression remain uninvestigated. Here, we report that chondroitin sulfate synthase 1 (CHSY1), the enzyme that mediates the polymerization step of chondroitin sulfate, is a critical mediator of malignant character in HCC that acts via modulating the activity of the hedgehog signaling. CHSY1 was up-regulated frequently in HCC where these events were associated with worse histologic grade and poor survival. Enforced expression of CHSY1 was sufficient to enhance cell growth, migration, invasion, and epithelial-mesenchymal transition, whereas silencing of CHSY1 suppressed these malignant phenotypes. Mechanistic investigations revealed that the increase of cell surface chondroitin sulfate by CHSY1 promoted sonic hedgehog binding and signaling. Inhibiting hedgehog pathway with vismodegib decreased CHSY1-induced migration, invasion, and lung metastasis of HCC cells, establishing the critical role of hedgehog signaling in mediating the effects of CHSY1 expression. Together, our results indicate that CHSY1 overexpression in HCC contributes to the malignant behaviors in cancer cells, we provide novel insights into the significance of chondroitin sulfate in hedgehog signaling and HCC pathogenesis.

Topics: Anilides; Animals; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Chondroitin Sulfates; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucuronosyltransferase; Hedgehog Proteins; Hep G2 Cells; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice, Inbred C57BL; Middle Aged; Multifunctional Enzymes; N-Acetylgalactosaminyltransferases; Neoplasm Grading; Neoplasm Invasiveness; Phenotype; Pyridines; RNA Interference; Signal Transduction; Transfection; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is a leading cause of death in Asian countries. Sonic hedgehog (Shh) pathway plays a role in hepatocarcinogenesis. We investigated the treatment effect of mouse HCC with Shh inhibitor GDC-0449.. Mouse hepatoma ML-1 cells were implanted in B6 mice. Fifteen days later, GDC-0449 (vismodegib), antagonist of smoothened, was used to treat HCC-bearing mice. The tumor size and liver histopathological features were analyzed, as well as gene expression in Shh pathways.. GDC-0449 treatment effectively reduced tumor size and cell infiltration of the HCC in mice. Gene expression of Shh pathway molecules was altered, including upregulated Shh expression and downregulated smoothened expression in tumor fractions after GDC-0449 treatment.. GDC-0449 could effectively mitigate HCC growth in vivo.

Topics: Anilides; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Down-Regulation; Gene Expression; Hedgehog Proteins; Liver Neoplasms, Experimental; Male; Mice, Inbred C57BL; Patched Receptors; Patched-1 Receptor; Pyridines; Random Allocation; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Smoothened Receptor; Transcription Factors; Up-Regulation; Zinc Finger Protein GLI1

Sorafenib leads to a survival benefit in patients with advanced hepatocellular carcinoma but its use is hampered by the occurrence of drug resistance. To investigate the molecular mechanisms involved we developed five resistant human liver cell lines in which we studied morphology, gene expression and invasive potential. The cells changed their appearance, lost E-cadherin and KRT19 and showed high expression of vimentin, indicating epithelial-to-mesenchymal transition. Resistant cells showed reduced adherent growth, became more invasive and lost liver-specific gene expression. Furthermore, following withdrawal of sorafenib, the resistant cells showed rebound growth, a phenomenon also found in patients. This cell model was further used to investigate strategies for restoration of sensitivity to sorafenib.

Topics: Anilides; Antineoplastic Agents; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromones; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Keratin-19; Liver Neoplasms; Morpholines; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins c-akt; Pyridines; Sorafenib; Vimentin

Studies of the hepatitis C virus (HCV) life-cycle rely heavily on Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh-related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here we demonstrate that Huh7.5 cells, when compared with Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers, and markedly up-regulated Hh pathway activity: Shh, >100-fold, Gli1, >30-fold, Ptc, 2-fold. In Huh7.5 cells, we found that cyclopamine, an Hh pathway antagonist, reduced HCV RNA levels by 50% compared with vehicle and inactive isomer controls. Moreover, in Huh7 cells treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2-fold and 4-fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose-response manner.. We have identified a relationship between HCV and Hh signaling where up-regulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high-level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.

Topics: Anilides; Antiviral Agents; Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Hedgehog Proteins; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Liver Neoplasms; Pyridines; Replicon; Signal Transduction; Up-Regulation; Virus Replication

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50-60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2(-/-) mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.. Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2(-/-) mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.. Unlike controls, Mdr2(-/-) mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.. Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.

Topics: Anilides; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Carcinoma, Hepatocellular; Cell Count; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Hyaluronan Receptors; Liver Cirrhosis; Liver Neoplasms; Magnetic Resonance Imaging; Mice; Osteopontin; Pyridines; Recurrence; Signal Transduction; Stem Cells; Tumor Burden