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.

FTO‐mediated ZNF687 accelerates tumor growth, metastasis, and angiogenesis in colorectal cancer through the Wnt/β‐catenin pathway

“…27 The Claudin-7 knockdown modulates the Sox9mediated Wnt/β-catenin pathway to enhance stemness properties in CRC. 28 RNF6 stimulates the Wnt pathway to strengthen radio resistance in CRC. 29 In addition, FUBP1 affects the Wnt/β-catenin cascade to accelerate stemness and metastasis in CRC.…”

“…For example, KCTD9 reduces β‐catenin levels to retard the Wnt/β‐catenin pathway in CRC 27 . The Claudin‐7 knockdown modulates the Sox9‐mediated Wnt/β‐catenin pathway to enhance stemness properties in CRC 28 . RNF6 stimulates the Wnt pathway to strengthen radio resistance in CRC 29 .…”

Is High Expression of Claudin-7 in Advanced Colorectal Carcinoma Associated with a Poor Survival Rate? A Comparative Statistical and Artificial Intelligence Study

“…However, in contrast to our results Philip et al, Bhat et al and Wang et al concluded that Claudin-7 low expression or downregulation induced EMT, which plays a major role in CRC invasion, progression and metastasis process [ 30 , 31 , 32 ]. Furthermore, Xu et al analyzed the effects of Claudin-7 knockdown in CRC stem cells through cell proliferation assay, migration assay, apoptosis assay and reported changes in cell characteristics such as promotion of cell proliferation, migration, and inhibition of cell apoptosis and the presence of EMT [ 35 ].…”

The long non-coding RNA T cell leukemia homeobox 1 neighbor enhances signal transducer and activator of transcription 5A phosphorylation to promote colon cancer cell invasion, migration, and metastasis

“…RT-qPCR was performed as previously described [ 14 ]. TRIzol (Thermo Fisher Scientific) was used to extract cellular RNA, after which an All-in-One First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA) was used to prepare cDNA.…”