Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNAmediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin downregulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29ΔSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekindepleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.polarity | transcription | tumorigenesis | colorectal | tight junctions
Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDH CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDH phenotype. Next-generation sequencing in ALDH cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer. Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. .
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