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

Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP - Mol. Cancer (2010)

Bottom Line: Increased expression of beta-catenin was associated with a marked transcriptional activation of TCF/LEF.The latter was greatly decreased following down regulation of beta-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.

Affiliation: Department of Internal Medicine, School of Medicine, Wayne State University, Detroit, MI 48201, USA. sskanwar@gmail.com

ABSTRACT

Background: 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/beta-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 ; K-ras mutant) and HT-29 (p53 mutant) were used.

Results: 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

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

CSC markers in colon cancer cells and spheroids/colonospheres. A. Representative photographs showing different colon cancer cells (HT-29, HCT-116 wt & p53-) in adherent condition and colonospheres formed by the respective cell lines. B. The comparative expression of different CSC markers in parental colon cancer cells (referred to as 'P') and the cells from corresponding spheroids (referred to as 'S'). The numbers represent percent of corresponding control normalized to β-actin.
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Figure 1: CSC markers in colon cancer cells and spheroids/colonospheres. A. Representative photographs showing different colon cancer cells (HT-29, HCT-116 wt & p53-) in adherent condition and colonospheres formed by the respective cell lines. B. The comparative expression of different CSC markers in parental colon cancer cells (referred to as 'P') and the cells from corresponding spheroids (referred to as 'S'). The numbers represent percent of corresponding control normalized to β-actin.

Mentions: Recently, we reported that FOLFOX-surviving colon cancer cells that are enriched in CSCs grow in large round, unattached floating spheroid colonies (termed colonospheres) when cultured in chemically defined serum-free medium at a relatively low density [22]. In agreement with our previous observation, our current results also show that colon cancer cells, whether they are p53-positive (HCT-116 wt) or p53-negative (HCT-116p53-/- and HT-29) form spheroid colonies in a chemically defined media (Figure 1A). The spheroids formed by these cells showed higher levels of EpCAM (also known as epithelial specific antigen or ESA) along with colon CSC markers-CD44, Musashi-1 and LGR-5 than the corresponding parental cells (Figure 1B). When the proportion of the CD44 positive cells was determined by flow cytometry in parental cells and colonospheres, they were found to be ≤ 1% in parental cell lines (Figure 2A), as opposed to more than 80% observed in colonospheres (Figure 2B). The results suggest that colonospheres formed by the colon cancer cells are enriched in CSCs. Indeed, when the colonospheres formed by HCT-116 cells were subjected to immunofluorescence staining for EpCAM, we observed bright EpCAM staining on the cell surface of each cell of colonospheres indicating the presence of CSCs in colonosphere (Figure 2B). However, since EpCAM is also expressed in normal human epithelial cells as well as in gastrointestinal carcinomas, we carried out the next set of experiments to validate and quantitate the presence of functional CSCs in colonospheres (within the confines of an in vitro system). We determined the frequency of sphere-forming cells by performing an extreme limiting dilution analysis (ELDA) for parental and colonospheres-derived HCT-116 cells. The frequency to form spheroids was found to be 5.5 fold higher for cells derived from colonospheres than those from the parent cell line (Table 1).

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

Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP - Mol. Cancer (2010)

CSC markers in colon cancer cells and spheroids/colonospheres. A. Representative photographs showing different colon cancer cells (HT-29, HCT-116 wt & p53-) in adherent condition and colonospheres formed by the respective cell lines. B. The comparative expression of different CSC markers in parental colon cancer cells (referred to as 'P') and the cells from corresponding spheroids (referred to as 'S'). The numbers represent percent of corresponding control normalized to β-actin.
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Figure 1: CSC markers in colon cancer cells and spheroids/colonospheres. A. Representative photographs showing different colon cancer cells (HT-29, HCT-116 wt & p53-) in adherent condition and colonospheres formed by the respective cell lines. B. The comparative expression of different CSC markers in parental colon cancer cells (referred to as 'P') and the cells from corresponding spheroids (referred to as 'S'). The numbers represent percent of corresponding control normalized to β-actin.
Mentions: Recently, we reported that FOLFOX-surviving colon cancer cells that are enriched in CSCs grow in large round, unattached floating spheroid colonies (termed colonospheres) when cultured in chemically defined serum-free medium at a relatively low density [22]. In agreement with our previous observation, our current results also show that colon cancer cells, whether they are p53-positive (HCT-116 wt) or p53-negative (HCT-116p53-/- and HT-29) form spheroid colonies in a chemically defined media (Figure 1A). The spheroids formed by these cells showed higher levels of EpCAM (also known as epithelial specific antigen or ESA) along with colon CSC markers-CD44, Musashi-1 and LGR-5 than the corresponding parental cells (Figure 1B). When the proportion of the CD44 positive cells was determined by flow cytometry in parental cells and colonospheres, they were found to be ≤ 1% in parental cell lines (Figure 2A), as opposed to more than 80% observed in colonospheres (Figure 2B). The results suggest that colonospheres formed by the colon cancer cells are enriched in CSCs. Indeed, when the colonospheres formed by HCT-116 cells were subjected to immunofluorescence staining for EpCAM, we observed bright EpCAM staining on the cell surface of each cell of colonospheres indicating the presence of CSCs in colonosphere (Figure 2B). However, since EpCAM is also expressed in normal human epithelial cells as well as in gastrointestinal carcinomas, we carried out the next set of experiments to validate and quantitate the presence of functional CSCs in colonospheres (within the confines of an in vitro system). We determined the frequency of sphere-forming cells by performing an extreme limiting dilution analysis (ELDA) for parental and colonospheres-derived HCT-116 cells. The frequency to form spheroids was found to be 5.5 fold higher for cells derived from colonospheres than those from the parent cell line (Table 1).

Bottom Line: Increased expression of beta-catenin was associated with a marked transcriptional activation of TCF/LEF.The latter was greatly decreased following down regulation of beta-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.

Affiliation: Department of Internal Medicine, School of Medicine, Wayne State University, Detroit, MI 48201, USA. sskanwar@gmail.com

ABSTRACT

Background: 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/beta-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 ; K-ras mutant) and HT-29 (p53 mutant) were used.

Results: 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

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

View Similar Images In: Results  - Collection
View Article: MedlinePlus - PubMed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2924313&rFormat=json&query=null&req=5