The Wnt/β-catenin pathway regulates growth and maintenance of colonospheres

Abstract

BackgroundRecent 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.ResultsColonospheres 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.ConclusionOur 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.

Significance of E-cadherin and CD44 expression in patients with unresectable metastatic colorectal cancer

“…The hypothesized underlying mechanism for the association between E-cadherin loss and chemotherapy resistance is as follows: Cadherin switching [the alteration from E-cadherin to neural (N)-cadherin] occurs in tumors; N-cadherin subsequently activates the PI3K/Akt pathway (12); and the PI3K/Akt pathway induces chemotherapy resistance by decreasing apoptosis and increasing proliferation (13,14). In addition, the loss of E-cadherin induces an increase in the levels of cytoplasmic β-catenin (15), which is then relocated into the nuclei, where it activates the Wnt/β-catenin pathway (15); the Wnt/β-catenin pathway is associated with chemotherapy resistance through the maintenance and proliferation of cancer stem cells (15).…”

“…Additionally, the loss of E-cadherin is associated with chemotherapy resistance via numerous pathways, including the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway and the Wingless type (Wnt)/β-catenin pathway (12)(13)(14)(15).…”

Leptin-induced adhesion and invasion in colorectal cancer cell lines

“…This sphere-formation technique was used to isolate putative CSCs from freshly isolated brain (28), breast (29) and colon tumors (30). Furthermore, spheroid culture (or colonospheres) generated from a limited number of human CRC-derived cell lines are enriched for cells that express colonic CSC markers (31,32). Increasing evidence suggests that stem cells play a decisive role in the progression and metastasis of CRC.…”

Altered expression of Krüppel-like factor 4 and β-catenin in human gastric cancer

“…β-catenin is a key downstream molecule in the Wnt signaling pathway and binds to the TCF/LEF transcription factors to regulate and activate the transcription of target genes that are involved in embryo development, tissue self-renewal and cancer (38). β-catenin is pivotal in intracellular signaling and is a key element in one of the most significant pathways in epithelial carcinogenesis (39,40). β-catenin has been identified as an oncogene in a variety of tumors in numerous previous studies (23,41).…”