Claudin-7 deficiency promotes stemness properties in colorectal cancer through Sox9-mediated Wnt/β-catenin signalling

Abstract

Background: Colorectal cancer (CRC) is a common malignant tumour of the digestive tract that is characterized by high patient morbidity and mortality rates. Claudin-7 (Cldn7), a tight junction protein, was recently reported to function as a candidate tumour suppressor gene in CRC. Our previous study demonstrated that the large intestine of C57/BL6 mice showed intestinal adenomas and abnormal Ki67 expression and distribution in the intestinal crypt when Cldn7 was knocked out. The aim of this study was to further investigate whether Cldn7 deficiency has non-tight junction functions, affects intestinal stemness properties, promotes CRC and to determine the specific mechanism.

Methods: Cell proliferation assays, migration assays, apoptosis assays, tumour sphere formation assays in vitro, and subcutaneous xenograft models in vivo were used to determine the effects of Cldn7 knockdown on the biological characteristics of CRC stem cells. Western blotting, qPCR and immunofluorescence staining were performed to identify the epithelial-mesenchymal transition and the activation of Wnt/β-catenin pathway in CRC stem cells. Cldn7 inducible conditional gene knockout mice and immunohistochemical staining further verified this hypothesis in vivo. The mechanism and target of Cldn7 were determined by performing a chromatin immunoprecipitation (ChIP) assay and coimmunoprecipitation (CoIP) assay.

Results: Cldn7 knock down in CRC stem cells promoted cell proliferation, migration, and globular growth in serum-free medium and the ability to form xenograft tumours; cell apoptosis was inhibited, while the cellular epithelial-mesenchymal transition was also observed. These changes in cell characteristics were achieved by activating the Wnt/β-catenin pathway and promoting the expression of downstream target genes after β-catenin entry into the nucleus, as observed in CRC cell lines and Cldn7 gene knockout mouse experiments. Using ChIP and CoIP experiments, we initially found that Cldn7 and Sox9 interacted at the protein level to activate the Wnt/β-catenin pathway.

Conclusions: Based on our research, Cldn7 deficiency confers stemness properties in CRC through Sox9-mediated Wnt/β-catenin signalling. This result clarifies that Cldn7 plays an inhibitory role in CRC and reveals a possible molecular mechanism, which is conducive to further research on Cldn7 and cancer stem cells.

Keywords: Cancer stem cells; Claudin-7; Colorectal cancer; Epithelial–mesenchymal transition; Wnt/β-catenin.

Fig. 1

Construction of CRC stem cells with stable Cldn7 knockdown. a Cldn7 expression in SW620, SW480, HCT116, and HT29 cell lines. b HCT116^CD133+CD44+ cells were analysed using flow cytometry after stable culture, and the CD133 and CD44 double-positive rate was 97%. c, d The efficiency of Cldn7 knockdown in HCT116^CD133+CD44+ cells was detected using qPCR and Western blotting

Fig. 2

Cldn7 knockdown promoted the TIC features and biological behavior of CRC stem cells. a HCT116^CD133+CD44+ shCldn7 cells formed tumour spheres faster than HCT116^CD133+CD44+ shcontrol cells. b Both cell spheres returned to an adherent state in complete medium, and the cells dissociated from the cell sphere and continued to grow and proliferate. c Tumour growth curves showed that the tumour formation rate and tumour volume formed by HCT116^CD133+CD44+shCldn7 cells were significantly greater than control cells (P < 0.05). d Proliferation ability of HCT116^CD133+CD44+shCldn7 cells was increased (P < 0.05). e Apoptosis ability of HCT116^CD133+CD44+shCldn7 cells was weakened (P < 0.05). f Migration ability of HCT116^CD133+CD44+shCldn7 cells was greater than control cells (P < 0.05)

Fig. 3

Cldn7 knockdown promoted cancer stem cell marker expression and EMT in CRC stem cells by activating the Wnt/β-catenin pathway. a, b Sox9, Olfm4 and Ki67 expression increased in HCT116^CD133+CD44+shCldn7 cells. EMT-related protein E-cadherin was decreased, Snail-1 and vimentin were increased. With the exception of the non obvious increase in vimentin expression, qPCR results were consistent with Western blot results (P < 0.05). c, d β-catenin protein levels did not change significantly in HCT116^CD133+CD44+shCldn7 cells, but c-Myc and cyclin D1 expression increased (P < 0.05). The mRNA levels of β-catenin, c-Myc and cyclin D1 in HCT116^CD133+CD44+shCldn7 cells were all increased (P < 0.05). e In HCT116^CD133+CD44+shCldn7 cells, β-catenin was distributed not only on the cell membrane but also in the cytoplasm and nucleus at low levels. f The expression of β-catenin in the cytoplasm of CRC stem cells did not change significantly with the downregulation of Cldn7, but the level of β-catenin in the nucleus increased

Fig. 4

Cldn7 knockout/knockdown promotes the development of colorectal adenomas and xenograft tumours by activating the Wnt/β-catenin pathway in vivo. a Cldn7 was knocked out in the small and large intestines of CreERT2 mice. b, c Extensive and severe inflammatory infiltrates and adenomas were observed in the small and large intestines of CreERT2 mice, while the intestinal tissue of CreW mice was normal (as indicated by the arrow). In the large intestine of CreERT2 mice, Sox9, β-catenin, c-Myc and cyclin D1 expression increased with Cldn7 knockout, and more β-catenin was present in the cytoplasm and nuclei. Activation of the Wnt/β-catenin pathway was not detected in the small intestine of CreERT2 mice, but an increase in Sox9 expression was still observed in the small intestine of CreERT2 mice. d In the xenograft tumour tissue formed by HCT116^CD133+CD44+ shCldn7 cells, Sox9 and β-catenin expression increased, and β-catenin was observed in more cytoplasm and nuclei

Fig. 5

Expression of Cldn7 and Sox9 in CRC cell lines and clinical tissues. a Sox9 expression was obvious in SW620 cell lines with low Cldn7 expression, was weak in the high Cldn7-expressing cell lines HCT116 and HT29, and the Sox9 band was virtually undetectable in SW480 cell lines with moderate Cldn7 expression. b Cldn7 was expressed at lower levels in CRC tissues than in adjacent tissues, and Sox9 expression was significantly increased in CRC tissues. c Cldn7 was expressed at significantly lower levels in metastatic cancer tissues than in primary tumours. Sox9 expression was increased in liver metastasis and lung metastasis tissues

Fig. 6

Cldn7 interacts with Sox9 to activate the Wnt/β-catenin pathway. a ChIP showed the Sox9 protein failed to bind directly to the Cldn7 promoter region and may be regulated by other means. b CoIP results showed Cldn7 and Sox9 interacted at the protein level, but the effect was indirect