Alessandro Scopelliti

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution.

Colorectal cancer stem cells (CR-CSC) are responsible for the generation and maintenance of intestinal tumors and are highly resistant to conventional chemotherapeutic agents. Aurora-A, a serine-threonine kinase involved in mitosis regulation, plays multiple key functions in tumor initiation and progression. We found that Aurora-A is overexpressed in primary colorectal tumor cells, in the CR-CSC fraction, and in stem cell-derived differentiated cells, compared with normal colon tissue. Aurora-A expression was functionally linked to centrosome amplification in CR-CSC, as indicated by the decrease in cells with multiple centrosomes that followed Aurora-A silencing. Knockdown of Aurora-A resulted in growth inhibition of CR-CSC, alteration of cell cycle kinetics, and downregulation of the expression levels of antiapoptotic Bcl-2 family members, strongly sensitizing to chemotherapy-induced cell death. Moreover, Aurora-A silencing compromised the ability to form tumor xenografts in immunocompromised mice and reduced the migratory capacity of CR-CSC. Altogether, these results indicate that Aurora-A is essential for CR-CSC regeneration and resistance to cytotoxic stimuli and suggest that therapies directed against Aurora-A may effectively target the stem cell population in colorectal cancer.