Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In this report the effects of CAIII expression on liver cancer cells invasiveness were explored. Overexpression of CAIII in the HCC cell line SK-Hep1 resulted in increased anchorage-independent growth and invasiveness. And siRNA-mediated silencing of CAIII expression decreased the invasive ability of SK-Hep1 cells. Furthermore, CAIII transfectants showed elevated focal adhesion kinase (FAK) and Src activity. Silencing of FAK expression in CAIII transfectants led to suppression of HCC cell invasion. More importantly, the CAIII transfectants acidified the culture medium at an accelerated speed than the control cells did. Taken together, these data suggest that the CAIII-promoted invasive ability of HCC cells may probably be mediated through, at least in part, the FAK signaling pathway via intracellular and/or extracellular acidification.

Metabolic risk factors, such as obesity, fatty liver, high lipidemia, and diabetes mellitus are associated with increased risk for nonviral hepatocellular carcinoma (HCC); however, few nonviral HCC studies have stratified patients according to underlying etiologies. From 2005 to 2011, 3,843 patients with HCC were recruited into the Taiwan Liver Cancer Network. Of these patients, 411 (10.69%) who were negative for hepatitis B virus (HBV), surface antigen, HBV DNA, and anti‐hepatitis C virus (HCV) antibody were classified as non‐HBV non‐HCV (NBNC)‐HCC. Detailed clinical analyses of these patients were compared with age‐ and sex‐matched patients with HBV‐HCC or HCV‐HCC for the associated metabolic risk factors. For this comparison, 420 patients with HBV‐HCC and 420 patients with HCV‐HCC were selected from the 3,843 patients with HCC. Multivariate analyses showed fatty liver (by echography), high triglyceride levels (>160 mg/dL), and diabetes mellitus history to be significantly associated only with NBNC‐HCC and not with the matched patients with HBV‐ or HCV‐HCC. When the patients with HCC were further divided into four groups based on history of alcoholism and cirrhotic status, the group without alcoholism and without cirrhosis exhibited the strongest association with the metabolic risk factors. Based on trend analyses, patients with NBNC‐HCC with or without alcoholism were significantly different from the matched patients with HBV‐ or HCV‐HCC, except for patients with alcoholism and cirrhosis, in having more than two of the above three risk factors. Conclusion: Metabolic risk factors are significantly associated with nonviral HCC, especially for patients without alcoholism in Taiwan. Because the prevalence of viral HCC is decreasing due to the success of universal vaccination and antiviral therapy, strategies for cancer prevention, prediction, and surveillance for HCC will require modification. (Hepatology Communications 2018;2:747‐759)

Hepatocellular carcinoma (HCC) is the leading cancer death in Taiwan. Chronic viral hepatitis infections have long been considered as the most important risk factors for HCC in Taiwan. The previously published reports were either carried out by individual investigators with small patient numbers or by large endemic studies with limited viral marker data. Through collaboration with 5 medical centers across Taiwan, Taiwan liver cancer network (TLCN) was established in 2005. All participating centers followed a standard protocol to recruit liver cancer patients along with their biosamples and clinical data. In addition, detailed viral marker analysis for hepatitis B virus (HBV) and hepatitis C virus (HCV) were also performed. This study included 3843 HCC patients with available blood samples in TLCN (recruited from November 2005 to April 2011). There were 2153 (56.02%) patients associated with HBV (HBV group); 969 (25.21%) with HCV (HCV group); 310 (8.07%) with both HBV and HCV (HBV+HCV group); and 411 (10.69%) were negative for both HBV and HCV (non-B non-C group). Two hundred two of the 2463 HBV patients (8.20%) were HBsAg(-), but HBV DNA (+). The age, gender, cirrhosis, viral titers, and viral genotypes were all significantly different between the above 4 groups of patients. The median age of the HBV group was the youngest, and the cirrhotic rate was lowest in the non-B non-C group (only 25%). This is the largest detailed viral hepatitis marker study for HCC patients in the English literatures. Our study provided novel data on the interaction of HBV and HCV in the HCC patients and also confirmed that the HCC database of TLCN is highly representative for Taiwan and an important resource for HCC research.

<p>Supplementary Information</p>

<p>Supplementary Figure 1. The mRNA expression of Nrp1 and Nrp2 in ESCC cell lines. RT-PCR was showed the expression level of Nrp1 and Nrp2 in TE1, TE12, TE3, TE7, and TE8 cells. GAPDH was used as an internal control.</p>

<p>Supplementary Figure 2. The phosphorylated VEGFR-3 in TE-1 cells with VEGF-C and VEGF-A treatment. TE-1 cells were starved with serum-free medium for 16 hours and treated with 100 and 200 ng/mL VEGF-C as well as 200 ng/mL VEGF-A for 10 minutes, following examination of phosphorylated VEGFR-3 (p-VEGFR-3, Y1063/1068) expression by Western blot. β-actin was used as an internal control.</p>

<p>Supplementary Figure 4. The mRNA expression of miR-326 in TE-8 cells with knockdown of VEGFR-3. TE-8 cells were transiently transfected shVEGFR-3#1, #2 or shCtrl for 48 hours and isolated total RNA to assay. A, qRT-PCR was used to quantify the expression level of miR-326 in those indicated cells. B, RT-PCR was showed the expression level of VEGFR-3 in those indicated cells. GAPDH was used as an internal control. The quantities values were evaluated as percentage compared with TE-8/shCtrl cells. The columns are the mean values from three independent experiments. Bars indicate means {plus minus} SE.</p>

<p>Supplementary Figure 3. The mRNA expression of CTTN in rhVEGF-C-treated TE-1 cells. TE-1 cells were treated with a various doses of rhVEGF-C (0, 25, 50, 100 and 200 ng/mL) for 24 hours and isolated total RNA to measure the expression level of CTTN by RT-PCR. GAPDH was used as an internal control.</p>