NLRP12 downregulates colorectal tumorigenesis via suppression of the Wnt/b-catenin pathway
Introduction
Advanced stages of colorectal cancer (CRC) are rarely curable, underscoring the importance of a better understanding of the molecular mechanisms of CRC progression, invasion, and metastasis. We previously demonstrated that mice deficient in Nlrp12, a cytosolic innate immune sensor, are susceptible to CRC. NLRP12 has been characterized as a negative regulator of NF-kB and MAPK pathways. However, the precise mechanism of NLRP12-mediated protection against colorectal cancer (CRC) was unknown.
Methods
We used azoxymethane (AOM) plus dextran sodium sulfate (DSS) induced colorectal tumorigenesis in mice. Data were validated in APCmin model of colorectal tumorigenesis. Tumors were analyzed histopathologically, and measured for gene expression and signaling pathway activation. NLRP12 was overexpressed or knocked down in colorectal cancer cell lines to examine the role of NLRP12 in the regualtion of the Wnt/b-catenin pathway. Using in vitro assays and biochemical studies, we explore the molecular mechanism of NLRP12-mediated regulation of the Wnt/b-catenin pathway.
Results
Nlrp12-/- mice developed increased tumor burden and showed higher incidence of invasive adenocarcinoma following treatment with AOM/DSS regimen. RNA-seq analysis revealed that tumors of Nlrp12-deficient mice express higher levels of protooncogenes (cMyc, Ccnd1), Wnt target genes (Ctnnb1, Axin2, Yap1, Tcf3, Lgr5), matrix metalloproteinases, and epithelial to mesenchymal transition markers (Vim, Fn1, Foxc2, Zeb2, Ezh2) compared to wild-type tumor. Interestingly, higher expression of these genes in Nlrp12-deficient tumors was not linked to any inflammatory signaling pathways as there was no difference in the activation of NF-kB, ERK, JNK, AKT, and STAT3 signaling pathways between Nlrp12−/− and wild-type tumors. However, we observed higher activation of β-catenin in Nlrp12-deficient tumors. Using intestinal epithelial cell specific Nlrp12-conditional knockout mice we showed that Nlrp12 deficiency in intestinal epithelial cells leads to increased tumorigenesis, metastasis, and β-catenin activation. To further understand the role of NLRP12 in the regulation of the Wnt/β-catenin, we overexpressed NLRP12 in HEK293T cells and HCT116 cells followed by stimulation with Wnt ligand Wnt3A. NLRP12 overexpressed cells showed significantly reduced activation of β-catenin and expression of Wnt target genes. Consistently, knocking down of NLRP12 in HEK293T and HCT116 cells caused higher activation of the Wnt/β-catenin pathway. Further, Nlrp12-deficient epithelial and CRC cells showed increased proliferation and migration along with higher activation of Wnt/β-catenin in vitro. Biochemical analysis revealed that NLRP12-mediated suppression of β-catenin activation is associated with reduced phosphorylation of GSK3β. Finally, human and mouse colorectal tumors showed reduced expression of NLRP12 along with increased activation of β-catenin and GSK3β phosphorylation, underscoring the clinical relevance of NLRP12 in the regulation of the Wnt/β-catenin pathway and colorectal tumorigenesis.
Conclusion
We discovered a novel function of NLRP12 involved in the negative regulation of the Wnt/β-catenin pathway in the intestinal epithelium. With this regulatory function, NLRP12 serves as a molecular checkpoint for proliferation, EMT, and invasion of CRC. The NLRP12-GSK3β-β-catenin signaling axis could be a potential therapeutic target for CRC treatment.