Functional characterization of functional role of CD133 in hepatocellular carcinoma

Abstract

CD133, also called prominin-1, has long been used as the marker for cancer stem cells in various cancers including hepatocellular carcinoma. Cell sorting of primary tumor cultures and several cancer cell lines isolated CD133-positive cells which showed an increased ability of tumor initiation, self-renew, and chemoresistance. However, the specificity of cell sorting using the anti-CD133 mouse monoclonal antibody that recognizes the glycosylation epitope of CD133 has caused concern. The attachment of the antibodies also risks triggering unwanted signaling pathways. All these limitations restricted our understanding of the downstream signaling and functionality of CD133. Here, I established the stable doxycycline inducible CD133 HLE and PLC/PRF/5 cell lines together with the intracellular domain deletion mutants of CD133 to characterize their functions. Deletion of intracellular domain 2 (C2) and 3 (C3) of CD133 lost glycosylation, which abolished the protein localization to the cell surface. Overexpression of CD133 wild type (WT) promoted cell proliferation and metastasis in HLE cells. Besides, cells with induced expression of CD133 WT and ΔC2 showed an increase in chromosome number. Using confocal immunofluorescence staining, I detected that CD133 WT expression induced centrosome amplification and multipolar mitotic spindles. CD133 WT and ΔC2 downregulated a centrosome linker protein TAX1BP2, preventing TAX1BP2 from inhibiting centrosome over-duplication. Finally, I found that the expression of CD133 WT and ΔC2 promoted cell stemness using sphere formation assay. Taken together, these findings suggest that CD133 plays a significant role in tumorigenesis and stemness in HCC, apart from being a cancer stem cell marker. This study presents evidence of CD133 C3 being the functional domain and provides insights into the association between CD133 and the centrosomal protein TAX1BP2. By targeting CD133 and its downstream targets, it can direct our chemotherapy against cancer stem cells to bring about a more promising cure.