Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids.

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial-mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

Upregulation of the oestrogen target gene SIX1 is associated with higher growth speed and decreased survival in HCV-positive women with hepatocellular carcinoma.

The male/female ratio of patients with hepatocellular carcinoma (HCC) is often unbalanced towards the male sex, indicating a sex predisposition for HCC development. A possible explanation may be attributed to different hormonal statuses, including the pro-inflammatory action of androgens in men and the protective effects of oestrogen against excessive inflammation in women. Although several studies have studied gene expression in patients with HCC, very few have attempted to identify features that could be distinctive between male and female patients. The present study aimed to identify distinctive signalling mechanisms between men and women that may be associated with HCC progression. The present study analysed a detailed microarray database that was obtained from the prospective study of 78 patients with HCC to study gene expression according to sex. In addition, the present study aimed to evaluate whether the differentially expressed genes were known oestrogen targets. Moreover, RNAs from the HCC cohort were evaluated for microRNA (miRNA/miR) expression, and a relationship between miRNA and gene expression according to sex was investigated. One gene, sineoculis homeobox homolog 1 (SIX1), which is known to be an oestrogen target gene, was revealed to be highly upregulated in hepatitis virus C (HCV)-positive female patients with HCC but not in HCV-positive male patients. In addition, SIX1 upregulation had a significant relationship with tumour growth speed (assessed as tumour doubling time in two CTs performed 6 weeks apart) and survival (P=0.009 and P=0.042, respectively) in female patients only. Furthermore, SIX1 upregulation was related with miR-421 and miR-9-5p only in male patients; however, in female patients, SIX1 upregulation had a direct relationship with miR-181b, miR-503-5p and miR-125b (miRNAs with potential oncogenic capacity), and an inverse correlation with miR139-5p, miR-26b, let7c-3p and let7c-5p (putatively oncosuppressive microRNAs). These data suggested a distinctive model for liver carcinogenesis in HCV-positive women, with downregulation of protective mechanisms against tumour progression and the activation of potential oncogenes, in relation to the oestrogen target gene SIX1. (IRB10/08_CE_UniRer; ClinicalTrials ID: NCT01657695).