The effect of estrogen receptor alpha on the expression of IL-6, EpCAM and K19 in hepatocellular carcinoma-cell culture and human study

Abstract

The expression of “stemness”-related markers in Hepatocellular carcinoma (HCC) was recently reported to be associated with more aggressive biological behavior and poor prognoses. Epithelial mesenchymal transition (EMT) was previously known to be involved in the generation of cancer stem cells. HCCs occur mainly in men, and estrogen and estrogen receptor alpha (ERα) were reported to play a protective role in HCC development by inhibiting secretion of IL-6, which is capable of inducing EMT. In this study, the role of ERα on the expression of cancer stem cell markers (EpCAM and keratin (K19), IL-6, EMT markers (Snail and Twist) as well as the biological behavior of HCC was investigated in both HCC cell lines and human HCC tissue samples. In PLC/PRF/5 and SNU423 cell lines, EpCAM-positive and EpCAM-negative cells were sorted by flow cytometry. ERα mRNA level was significantly higher in the EpCAM-negative cell fractions than in the EpCAM-positive cell fractions of both cell lines (P < 0.05). Snail mRNA level and Twist mRNA level were significantly higher (P < 0.05) (P = 0.06) in the EpCAM-positive cell fractions than in the EpCAM-negative cell2 fractions of both cell lines. IL-6 mRNA levels were significantly higher in the EpCAM-positive cell fractions than in the EpCAM-negative cell fractions of SNU423 cells (P = 0.03), whereas it was not detected in PLC/PRF/5 cells. Overexpression of ERα by transfection of pEGEF-C1-ERα plasmids showed a significant reduction of EpCAM and K19 expression levels in PLC/PRF/5 and SNU423 cell lines at both the mRNA and protein level (P < 0.05). Interestingly, overexpression of ERα reduced IL-6 expression at both the mRNA and protein level in SNU423 cells. However, Snail and Twist expression demonstrated no significant change after ERα overexpression in both cell lines. Overexpression of ERα significantly reduced the activities of cell proliferation, invasion and migration in both cell lines compared to the control group. We also studied 64 human HCC tissues; ERα type was evaluated by RT-PCR, and the mRNA expression levels of ERα, EpCAM, K19, IL-6, Snail and Twist were studied by real-time RT-PCR. The protein expression of EpCAM and K19 was also detected by immunohistochemical stain. Their expression levels were correlated with clinicopathologic features. HCC patients with the ERα wild type showed significantly higher mRNA levels of ERα; lower mRNA levels of EpCAM, K19 and IL-6; and lower protein expression of EpCAM and K19, compared to those with the ERα variant type (P < 0.05 in all); whereas there was no significant difference in mRNA levels of Snail and Twist between the two groups. HCC patients with the ERα variant type showed3higher incidences of portal vein invasion (P = 0.008), microvascular invasion (P = 0.024), and poor differentiation (P = 0.015), compared to those with the ERα wild type. Similarly, HCC patients with low ERα mRNA expression (lower 50%) showed higher portal vein invasion (P = 0.021) and microvascular invasion (P = 0.007) than those with high ERα mRNA expression (upper 50%). There were no significant differences in disease free survival and overall survival according ERα type and expression levels of ERα, IL-6, Snail, Twist, EpCAM and K19 at the mRNA or protein level. In conclusion, these data suggest low ERα mRNA expression and variant-ERα type are involved in the aggressive biological behavior of HCC, demonstrating high expressions of EpCAM, K19 and IL-6 in both HCC cell lines and HCC patients.