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Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used.


Colonospheres formed in vitro exhibited higher expression of colon CSCs markers LGR5, CD44, CD166 and Musashi-1 along with putative CSC marker EpCAM, compared to the corresponding parental cancer cells and also exhibit the ability to form spheroids under extreme limiting dilution, indicating the predominance of CSCs in colonospheres. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to ≤ 1% in the corresponding parental cells. Additionally, colonospheres showed reduced membrane bound β-catenin but had increased levels of total β-catenin, cyclin-D1 and c-myc and down regulation of axin-1 and phosphorylated β-catenin. Increased expression of β-catenin was associated with a marked transcriptional activation of TCF/LEF. The latter was greatly decreased following down regulation of β-catenin by the corresponding siRNA, leading to a marked reduction in CD44 positive cells as well as colonospheres formation. In contrast, upregulation of c-myc, a down-stream effector of TCF/LEF greatly augmented the formation of colonospheres.


Our data suggest that colonospheres formed by colon cancer cell lines are highly enriched in CSCs and that Wnt/β-catenin pathway plays a critical role in growth and maintenance of colonospheres.


Biology | Oncology