Migration Of Hepatocellular Carcinoma Cell Lines Research Articles

DACH1 is a novel predictive and prognostic biomarker in hepatocellular carcinoma as a negative regulator of Wnt/β-catenin signaling.

The cell fate determination factor Dachshund (DACH1) functions as a novel suppressor in the progression of various neoplasms. Previous study has suggested that hypermethylation of promoter region was responsible for the reduction of DACH1 expression in hepatocellular carcinoma (HCC), and associated with the progression of HCC, but the clinical significance and the exact molecular mechanisms of DACH1 in the progression of HCC remain unclear. In this study, we employed public microarray data analysis and tissue microarrays (TMAs) technologies and showed that DACH1 expression was reduced in HCC even at early stage and associated with the tumor progression. Notably, Kaplan-Meier analysis further indicated DACH1 could be an independent prognostic factor for the overall survival of HCC. Further, mechanistic studies revealed that overexpression of DACH1 inhibited the growth and migration of HCC cell line, which were dependent in part on the inactivation of Wnt pathway via phosphorylation of GSK3β to suppress β-catenin. In agreement, the abundance of DACH1 was inversely correlated with several Wnt target genes. Collectively, our study indicated β-catenin is a novel target of DACH1 in HCC.

Abstract 2046: TGF-β drives tumor-progression via JAK/STAT3-signaling in advanced HCC.

Abstract Background: More than 80% of Hepatocellular carcinoma (HCC) patients present at advanced stages not amenable to curative therapies. Currently existing palliative therapies achieve a minimal survival benefit of 3 months. Hence, there is an urgent need for novel therapeutic strategies. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its Smad2/3 signaling axis. Inactivation of this axis has been implicated in hepatocarcinogenesis. In patients with advanced HCC, TGF- β serum concentrations are elevated and correlated to disease stage, metastases and worse prognosis. Our aims were to evaluate the status and function of the TGF-β/Smad2 signaling axis in advanced HCC. Material and Methods: Immunohistochemical analysis for pSer465/467-Smad2 in human HCC samples was performed using Tissue Micro Arrays. In vitro studies were performed using established human HCC cell lines (HepG2, Hep3B, PLC/PRF/5, HUH-7, SNU-398, SNU-449). Pathway activation following TGF-β stimulation was evaluated by immunoblot analysis and immunofluorescence microscopy. Cell proliferation was assessed by cell quantification and MTS-assay. Apoptosis was quantified morphologically by DAPI stain and fluorescence microscopy, and biochemically by caspase-3/7 assay. Migration was assessed by gap closure assay, and invasion by Matrigel invasion assay. Results: Intranuclear pSer465/467-Smad2 was identified in up to 51% of advanced HCC (n=73) and in 89% of HCC metastases (n=9). In 50% of human HCC cell lines, TGF-β treatment (10 ng/mL) resulted in Ser465/467-phosphorylation and nuclear translocation of the transcription factor Smad2. Exclusively in HCC cells with an intact TGF-β/Smad2-signaling cascade, TGF-β stimulation induced Tyr705-phosporylation of the transcription factor STAT3. TGF-β induced STAT3 Tyr705-phosporylation is inhibited by Smad2-directed gene silencing. Further, pyridine 6 mediated Janus Kinase-1/2 (JAK1/2) inhibition prevented TGF-β induced Tyr705-phosporylation of STAT3. Cell proliferation of HCC cells was inhibited by TGF-β. However, TGF-β treatment significantly induced invasion and migration in HCC cell lines with intact TGF-β/Smad2 signaling. Further, TGF-β induced resistance to TRAIL-induced apoptosis in these cells. However, co-treatment with the STAT3-inhibitor Stattic (10 nM) completely abrogates TGF-β mediated HCC cell migration and invasion, and sensitizes these HCC cell lines to TRAIL-mediated apoptosis. Conclusion: In advanced HCC, the TGF-β/Smad signaling axis is constitutively activated in the majority of primary HCC tumors and HCC metastases. The function of TGF-β/Smad2 in human HCC cell lines has switched from tumor inhibitor to a driver of tumor progression via JAK/STAT3 signaling. STAT3-inhibition reverses the TGF-β tumor-promoting effects. Hence, JAK/STAT3 targeting might be a promising new strategy to restore TGF-β tumor suppressor function in advanced HCC. Citation Format: Andres Rojas, Jessica Cromheecke, Mong-Hong Lee, Mien-Chie Hung, Boris Blechacz. TGF-β drives tumor-progression via JAK/STAT3-signaling in advanced HCC. [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 2046. doi:10.1158/1538-7445.AM2013-2046

Upregulation of microRNA-372 associates with tumor progression and prognosis in hepatocellular carcinoma

MicroRNA-372 (miR-372) has been demonstrated to play a crucial role in cellular proliferation and apoptosis of cancer cells. However, its effects in hepatocellular carcinoma (HCC) have not been explored. The aim of this study was to investigate the clinical significance of miR-372 in human HCC. Quantitative RT-PCR was performed to detect miR-372 expression in HCC clinical samples and cell lines. Then, Kaplan-Meier and Cox proportional regression analyses were performed to determine the association of miR-372 expression with survival of HCC patients. Moreover, the effects of miR-372 on tumorigenicity of HCC cell lines were evaluated by in vitro assays. miR-372 expression in HCC tissues was significantly higher than in the corresponding normal adjacent liver tissues (P < 0.001). There was a correlation between miR-372 upregulation and advanced TNM stage of HCC patients (P = 0.02). In addition, HCC patients with higher miR-372 expression had significantly poorer recurrence-free survival (P = 0.006) and overall survival (P = 0.001). Multivariate analysis revealed that high miR-372 expression was an independent predictor of poor prognosis (for recurrence-free survival: Hazard Ratio [HR] 6.826, P = 0.01; for overall survival: HR 9.533, P = 0.008). Moreover, in vitro assays demonstrated that the ectopic expression of miR-372 may significantly promote the cellular proliferation, invasion, and migration of HCC cell lines. Our findings showed that miR-372 may serve as a potent prognostic marker for tumor recurrence and survival of HCC patients. Furthermore, miR-372 has been identified as a promoter for tumorigenicity of HCC cells, suggesting that it might be a prospective therapeutic target for HCC.

Abstract C42: p16-enhanced the migration phenotypes in hepatocellular carcinoma cells is Rb-independent.

Abstract The occurrence of tumor metastasis to the extra-hepatic region contributes to a poor prognosis of hepatocellular carcinoma (HCC). However, the molecular mechanisms of metastasis in HCC remain unclear. Our previous studies showed that concomitant loss of Ink4a/Arf locus accelerated tumor formation and metastasis in mice with liver specific Trp53 deletion, indicating roles of Ink4a/Arf locus in tumorigenesis and metastasis. In this study, we focus on the p16 (Ink4a), a well-known tumor suppressor in regulation of cell cycle. When re-introduction of p16 into Trp53 and Ink4a/Arf-null liver cancer cells, we unexpectedly observed that p16 could enhance cell migration in HCC cell lines, including murine and human orgins by transwell assay and ectopic p16 expression also increased colonization in the lungs via tail vein injection into immune-deficient mice. Interestingly, there was no significant change in expression of epithelial mesenchymal transition (EMT)-related proteins. We further demonstrated that activity of Cdc42 and Rac1 was increased in MM189 cell with p16 over-expression but activity of RhoA was partially decreased. By knockdown of small GTPase, Cdc42 GTPase was required for p16-enhanced cell migration, whereas the related RhoA GTPase inhibited p16-mediated migration. Analysis of p16 mutants indicated that the Cdk4 interacting domain was required for stimulation of HCC cell migration, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. We therefore hypothesized that p16-mediated migration is not involved in pRb-dependent pathways. To validate our hypothesis, knockdown of pRb did not impair p16-enhanced migration in HepG2 cells with ectopic p16 expression. Our study suggested that p16-enhanced migration is not mediated by pRb-dependent pathways and more studies needs to be performed to elucidate this. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C42.

Cimetidine inhibits epidermal growth factor‐induced cell signaling

Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)-induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines. HCC cell lines (Hep3B, HLF, SK-Hep-1, JHH-2, PLC/PRF/5 and HLE) were used and cell proliferation was assessed by [3H]-thymidine incorporation assay. Cell migration was measured by in vitro cell migration assay. Biological effects of cimetidine were assessed with human EGF receptor (EGFR)-expressing mouse fibroblast cells (NR6-WT). The autophosphorylation of EGFR and the activation of other downstream effectors were analyzed by immunoprecipitation and immunoblotting. The concentration of intracellular cyclic AMP (cAMP) was measured by competitive enzyme immunoassay. Cimetidine inhibited both EGF-induced cell proliferation and migration in Hep3B, HLF, SK-Hep-1 and JHH-2, while cimetidine did not affect EGF-induced cell proliferation and migration in PLC/PRF/5 and HLE. Cimetidine was revealed to disrupt the EGF-induced autophosphorylation of EGFR and its downstream effectors, mitogen activated protein kinases and phospholipase C-gamma. To define the molecular basis of this negative regulation, we identified that cimetidine significantly decreased intracellular cAMP levels and that decrement of cAMP inhibited autophosphorylation of EGFR. The cell permeable cAMP analog, CPT-cAMPS reversed the cimetidine-induced inhibition of EGF-induced cell proliferation and cell migration by restoring autophosphorylation of EGFR. Cimetidine inhibited EGF-induced cell proliferation and migration in HCC cell lines by decreasing the concentration of intracellular cAMP levels. Cimetidine may be a candidate chemopreventive agent for HCC.