Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets. Two conditions were compared and each sample has a biological replicate. Samples are hybridized in dye-swap, resulting in 4 hybridizations.

Project description:BackgroundThrombospondins (THBSs) are a family of multidomain and secreted matricellular Ca(2+)-binding glycoproteins which has at least five members encoded by independent genes. As a THBSs family member, Thrombospondin2 (THBS2) has been reported to regulate angiogenesis. Nevertheless, the functions and clinical significance of THBS2 still remains unclear in gastric cancer.MethodsThe mRNA and protein expression levels of THBS2 were assessed in 14 paired of gastric cancer specimens and corresponding normal mucosas using quantitative real-time PCR and western blot analysis. Immunohistochemistry of THBS2 and CD34 on population-based tissue microarrays consisting of 129 gastric cancer cases were used to evaluate the prognostic significance of THBS2 and microvessel density (MVD) of each sample. Survival analyses were performed by Kaplan-Meier method and Cox's proportional hazards model. Colony formation assay, endothelial cell tube formation assay, cell migration assay and apoptosis analysis in MKN-45 and SGC-7901 cell lines were carried out to evaluate the effects of THBS2 on gastric cancer in vitro.Results85.71% (12 of 14) gastric cancer tissues expressed remarkably lower THBS2 in both mRNA and protein levels than the corresponding normal controls. Consistently, tissue microarray (TMA) results showed THBS2 levels were also inhibited in gastric cancer tissues compared with the normal controls. Moreover, we observed that patients with higher levels of THBS2 were significantly correlated with more favourable prognosis while decreased THBS2 expression were associated with poorer histological grades of gastric cancer. Additionally, our in vitro experiments further demonstrated that overexpression of THBS2 could impede both the proliferation rate and the tube formation of Human umbilical vein endothelial cells (HUVECs) in MKN-45 and SGC-7901 cell lines.ConclusionOur study suggests THBS2 is aberrantly expressed in gastric cancer and plays a critical role in cancer progression, which can be a potential prognosis predictor of gastric cancer.

Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets.

Dataset Information

Down-regulation of the tumour suppressor ?-opioid receptor predicts poor prognosis in hepatocellular carcinoma patients.

Publications

Down-regulation of the tumour suppressor κ-opioid receptor predicts poor prognosis in hepatocellular carcinoma patients.

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