NLRP12 downregulates the Wnt/β-catenin pathway via interaction with STK38 to suppress colorectal cancer
Shahanshah Khan, … , Thirumala-Devi Kanneganti, Hasan Zaki
Shahanshah Khan, … , Thirumala-Devi Kanneganti, Hasan Zaki
Published August 15, 2023
Citation Information: J Clin Invest. 2023;133(19):e166295. https://doi.org/10.1172/JCI166295.
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Research Article Gastroenterology

NLRP12 downregulates the Wnt/β-catenin pathway via interaction with STK38 to suppress colorectal cancer

Abstract

Colorectal cancer (CRC) at advanced stages is rarely curable, underscoring the importance of exploring the mechanism of CRC progression and invasion. NOD-like receptor family member NLRP12 was shown to suppress colorectal tumorigenesis, but the precise mechanism was unknown. Here, we demonstrate that invasive adenocarcinoma development in Nlrp12-deficient mice is associated with elevated expression of genes involved in proliferation, matrix degradation, and epithelial-mesenchymal transition. Signaling pathway analysis revealed higher activation of the Wnt/β-catenin pathway, but not NF-κB and MAPK pathways, in the Nlrp12-deficient tumors. Using Nlrp12–conditional knockout mice, we revealed that NLRP12 downregulates β-catenin activation in intestinal epithelial cells, thereby suppressing colorectal tumorigenesis. Consistent with this, Nlrp12-deficient intestinal organoids and CRC cells showed increased proliferation, accompanied by higher activation of β-catenin in vitro. With proteomic studies, we identified STK38 as an interacting partner of NLRP12 involved in the inhibition of phosphorylation of GSK3β, leading to the degradation of β-catenin. Consistently, the expression of NLRP12 was significantly reduced, while p-GSK3β and β-catenin were upregulated in mouse and human colorectal tumor tissues. In summary, NLRP12 is a potent negative regulator of the Wnt/β-catenin pathway, and the NLRP12/STK38/GSK3β signaling axis could be a promising therapeutic target for CRC.

Authors

Shahanshah Khan, Youn-Tae Kwak, Lan Peng, Shuiqing Hu, Brandi L. Cantarel, Cheryl M. Lewis, Yunpeng Gao, Ram S. Mani, Thirumala-Devi Kanneganti, Hasan Zaki

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Figure 5

Intestinal epithelial cell–specific NLRP12 suppresses β-catenin activation and colorectal tumorigenesis.

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Intestinal epithelial cell–specific NLRP12 suppresses β-catenin activati...
(A–G) Vil-Cre (n = 10), Nlrp12fl/fl (n = 12), and Nlrp12fl/fl;Vil-Cre (n = 13) mice were treated with AOM plus DSS to induce colorectal tumorigenesis. (A and B) Tumor burden at 12 weeks after AOM/DSS treatment was counted. (C) Representative H&E-stained images of colorectal tumors. Scale bars: 100 μm. The arrow indicates tumor invasion into the stroma. (D) Percentage of mice showing invasive colorectal adenocarcinoma. (E) Tumor homogenates were analyzed for p-P65, p-ERK, ERK, β-catenin, cyclin D1, cMyc, and β-actin by Western blotting. (F) Densitometric analysis of band intensity of β-catenin, cyclin D1, cMyc, p-P65, and p-ERK. (G) The expression of indicated genes was measured by real-time RT-PCR. Data represent mean ± SEM. Experiments were repeated at least 2 times, and data from a representative experiment are presented. (H–K) HEK293T cells overexpressing GFP or NLRP12 were stimulated with Wnt3a. (H) Cell lysates were analyzed for NLRP12, β-catenin, cMyc, cyclin D1, and β-actin. (I) The localization of β-catenin was analyzed by Western blotting of β-catenin in cytoplasmic and nuclear fractions. (J) Nuclear localization of β-catenin (red) was visualized microscopically. Scale bars: 100 μm. (K) The expression of indicated Wnt target genes was measured by real-time RT-PCR. Data represent mean ± SD of triplicate wells. (L and M) Mouse embryonic fibroblasts (MEFs) from WT and Nlrp12–/– mice were cultured and stimulated with Wnt3a. (L) The activation of β-catenin was measured by Western blotting. (M) The expression of Wnt target genes, including Ctnnb1, Ccnd1, cMyc, Axin2, and MKi67, was measured by real-time RT-PCR. Data represent mean ± SD of triplicate wells. Experiments were repeated 2 times, and data from a representative experiment are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA (B, D, F, and G) or unpaired, 2-tailed Student’s t test (K and M).