E‑cadherin regulates proliferation of colorectal cancer stem cells through NANOG

Abstract

Cancer stem cells (CSCs) possess a self‑renewal ability and display tumorigenic potential in immunodeficient mice. Colorectal CSCs are thought to be a uniform population and no functionally distinct subpopulations have been identified. Because E‑cadherin is an essential molecule for self‑renewal of embryonic stem cells, we examined E‑cadherin expression, which may play a role in maintaining the properties of CSCs, in EpCAMhigh/CD44+ colorectal CSCs from human primary colorectal cancers. We obtained 18 surgical specimens of human primary colorectal cancer. CD44, EpCAM, and E‑cadherin expression were analyzed by fluorescence‑activated cell sorting. Sorted EpCAMhigh/CD44+ colorectal CSCs were injected into immunodeficient mice to estimate the tumorigenic potential. Genetic profiles were analyzed by cDNA microarray. Notably, colorectal CSCs could be divided into two populations based on the E‑cadherin expression status, and they exhibited different pathological characteristics. Compared to E‑cadherin‑negative colorectal CSCs, E‑cadherin‑positive (EC+) colorectal CSCs demonstrated higher tumor growth potential in vivo. EC+ colorectal CSCs revealed a higher expression of the pluripotency factor NANOG, which contributed to the higher tumor growth potential of EC+ colorectal CSCs through control of cyclin D1 expression. These findings are the first demonstration of functionally distinct subpopulations of colorectal CSCs in human clinical samples.

Figure 1.

Tumorigenic potential of EpCAM^high/CD44⁺ colorectal cancer cells in vivo. (A) Flow cytometric analysis of a human colorectal cancer. A small EpCAM^high/CD44⁺ population was detected in the human colorectal cancer. (B) Only EpCAM^high/CD44⁺ cells had tumorigenic potential in NOD/SCID mice. (C) Flow cytometric analysis of a xenograft tumor. EpCAM^high/CD44⁺ cells gave rise to EpCAM^positive/CD44⁻ cells and reconstituted the expression profiles of the primary sample. (D) H&E staining of a primary and a xenograft tumor. The histology of the xenograft tumor was the same as that of the primary tumor. (E) Serial transplantation recapitulated the histology of the primary tumors.

Figure 2.

E-cadherin expression in EpCAM^high/CD44⁺ colorectal cancer cells. (A) EpCAM^high/CD44⁺ colorectal cancer cells contained an E-cadherin⁺ and an E-cadherin⁻ population. (B) Immunohistochemical staining of a human colorectal cancer specimen. The red arrow indicates CD44 (black) and E-cadherin (green) double-positive cells. The yellow arrow indicates CD44 (black) positive but E-cadherin negative cells. (C) N-cadherin was not expressed in EpCAM^high/CD44⁺ colorectal cancer cells.

Figure 3.

Higher tumorigenic potential of E-cadherin⁺ colorectal CSCs. (A) Image of a mouse bearing a xenograft tumor from EC⁺ and EC⁻ colorectal CSCs and flow cytometric analyses of their phenotype. (B) Tumor weight of xenograft tumors. Error bars represent the 95% confidence interval. EC⁺ colorectal CSCs produced significantly larger tumors compared to EC⁻ colorectal CSCs. *P<0.01 (C) Histopathological analysis of xenograft tumors from EC⁺ CSCs and EC⁻ CSCs.

Figure 4.

Upregulation of NANOG expression in EC⁺ CSCs. cDNA microanalysis of (A) total gene expression, (B) transcription-related (including NANOG) gene expression, and (C) EMT-related gene expression in EC⁺ and EC⁻ CSCs. (A) Total gene expression differed between EC⁺ CSCs and EC⁻ CSCs. (B) NANOG expression was significantly upregulated in EC⁺ CSCs vs. EC⁻ CSCs. (C) Expression of genes related to EMT did not differ between EC⁺ CSCs and EC⁻ CSCs. (D) Immunofluorescence staining of a human colorectal cancer specimen. NANOG was expressed in the nucleus of CD44 and E-cadherin double-positive colorectal cancer cells (yellow arrow).

Figure 5.

NANOG regulates colorectal cancer cell proliferation through the control of cyclin D1 expression. (A) Growth curve of HCT116 colorectal cancer cells. NANOG siRNA-transfected cells exhibited decreased cell proliferation compared to the control. (B) Quantitative RT-PCR (*P<0.01) and (C) RT-PCR identified NANOG suppression in siRNA-transfected cells. (D) An immunofluorescence study revealed the downregulation of NANOG protein after siRNA transfection. (E) RT-PCR for cyclins. Cyclin D1 and cyclin B1 mRNA levels were decreased in siRNA-transfected cells, but the expression levels of other cyclins, p21, and p27 were not changed.

Figure 6.

The concept of this study. The CSC population is a heterogeneous population that contains EC⁺ and EC⁻ cells. Although both EC⁺ cells and EC⁻ cells have the ability to produce a tumor in immunodeficient mice, EC⁺ colorectal CSCs have higher tumor growth ability. E-cadherin and NANOG may regulate colorectal CSC proliferation through the control of cyclin D1 expression.