Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for >90% of diagnosed cases. This is an aggressive malignancy, with the majority of patients dying within a year of diagnosis. A small proportion of cells within HCC possess stem cell characteristics and are termed liver cancer stem cells (LCSCs). These LCSCs are highly resistant to traditional therapies. As a result, there is a need to directly target LCSCs to improve patient survival. Our study establishes the role IL-6 plays in mediating LCSC tumorigenesis.
Western blots on DEN-induced HCC tumours demonstrated a strong association between IL-6 and stem cells, with increased expression of IL-6 and stem cell markers in DEN-induced HCC. Western blots and functional assays on primary cell lines derived from DEN-induced HCC showed tumour aggressiveness corresponded with increased IL-6 signalling. IL-6 signalling within these cells was also linked to the percentage of LCSCs, with higher percentages of tumour sphere formation.
To determine the direct effect of IL-6 on stem cell viability, HuH7 cells stably overexpressing IL-6 were grown as xenografts. Xenografts overexpressing IL-6 appear to have an increased percentage of stem cells, with western blots showing significantly higher expression of the stem cell markers Epcam, CD44 and keratin. There was a significant reduction in the expression of the mature hepatocytes marker albumin. To evaluate the effect of IL-6 on LCSC tumorigenesis, cell lines of purified liver cancer stem cells (Celprogen, Cat. No. 36116-43) were transfected with an IL-6 overexpression plasmid. Promoter luciferase vector assays showed that IL-6 overexpression in these LCSCs induced the expression of the key protumorigenic pathways Notch, NFκβ, Myc/Max, HIF-1, MAPK/ERK and MAPK/JNK.
Our study suggests that IL-6 regulates the presence of stem cells in HCC, and that LCSCs are a major regulator of IL-6 induced HCC tumorigenesis.