Expression Of Twist In Hepatocellular Carcinoma And It's Effect On Angiogenesis And Metastasis

Background&ObjectiveHepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide and shows relatively poor prognosis and rapid progression. Identification of the key molecular targets involved in hepatocarcinogenesis has significant therapeutic implications. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. Twist, a basic helix-loop-helix (bHLH) transcription factor, can regulate mesodermal development and promote tumor cell metastasis. Recent studies have shown that Twist overexpression is able to induce angiogenesis, inhibit apoptosis, and trigger epithelial-mesenchymal transition that is pivotal for the transformation into an aggressive cancer phenotype. The present study was aimed to examine the expression of Twist and evaluated its effect on tumor angiogenesis and metastasis in HCC.MethodsIn the first part, we evalued the expression Twist and VEGF mRNA by real-time RT-PCR in 30 pairs of hepatocellular carcinoma and matched non-cancerous tissues. Immunohistochemistry was carried out to analyze the protein expression of Twist and VEGF in 40 hepatocellular carcinoma cases. Staining of endothelial cells for CD34 was used to evaluate the microvessel density (MVD). In the second part, we determined the mRNA expression of E-cadherin and N-cadherin by real-time RT-PCR in 30 pairs of hepatocellular carcinoma and matched non-cancerous tissues. Immunohistochemistry was carried out to analyze the protein expression, of E-cadherin and N-cadherin in 40 hepatocellular carcinoma cases.ResultsThe results of the first part:1. The expression of Twist mRNA was increased in 73% (22/30) of the samples, as compared with the non-cancerous tissues. Immumohistochemical analyses indicated that Twist was positively expressed in 55% (22/40) of the cases.2. Up-regulated VEGF mRNA was detected in 67% (20/30) of the samples and 70% (14/20) of cases correlated with overexpression of Twist.3. There was a significant positive correlation between Twist protein expression and VEGF (r=0.479, p=0.002).4. The mean MVD in cases of Twist-positive HCC specimens was higher than that in cases of Twist-negative HCC tissues although the difference failed to reach statistical significance (82.85±31.02 versus 67.20±24.45; P=0.090)The results of the second part:1. Up-regulated N-cadherin mRNA could be detected in 60% (18/30) of samples and 83% (15/18) of cases correlated with overexpression of Twist. Immunostaining for N-cadherin protein was mostly membranous and cytoplastic and it could be detected in 52(21/40) of samples2. E-cadherin mRNA was down-regulated in 43% (13/30) of samples and 62% (8/13) of cases correlated with overexpression of twist. However, up-regulated E-cadherin mRNA could be detected in 30% (9/30) of cases. Immunohistochemical analysis showed that E-cadherin protein was down-regulated in 42% (17/40) of cases. We also found E-cadherin protein increased in 30% (12/40) of the cases.3. Spearman correlation analysis indicated that there was a significant correlation between the increases in Twist protein and N-cadherin up- regulation (r=0.383, p=0.016). There was no significant correlation between the expression of Twist protein and E-cadherin (p=0.097).4. There was a tendency that positive expression of Twist is significantly associated with intrahepatic metastasis (P=0.048).Conclusion 1. We found that Twist mRNA and protein levels were both increased in HCC as compared to non-cancerous tissues. Thus Twist may play an important role in the process of hepatocellular carcinoma and high-level of twist expression may be related to the malignant potential of tumor cells.2. Twist can promote angiogenesis via up-regulation of VEGF synthesis in human HCC tissues.3. Twist can enhance hepatocellular carcinoma metastasis through induction of N-cadherin not by suppression of E-cadherin.4. As a novel oncogene, Twist may become a new target of HCC therapy.