NLRP12 downregulates the Wnt/β-catenin pathway via interaction with STK38 to suppress colorectal cancer
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
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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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 6

NLRP12 regulates the Wnt/β-catenin pathway and controls proliferation and migration of cancer cells.

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NLRP12 regulates the Wnt/β-catenin pathway and controls proliferation an...
(A–D) Crypts from the small intestine of WT and Nlrp12–/– mice were cultured in a 3D organoid culture system. (A and B) Organoid growth was monitored, and organoid sizes were measured. (C) Organoids were stimulated with Wnt3a, and the activation of β-catenin was measured by Western blotting. (D) RNA isolated from Wnt3a-stimulated organoids was measured for the expression of Ctnnb1, Ccnd1, cMyc, Axin2, Yap1, and MKi67. Data represent mean ± SD. Experiments were repeated 3 times, and data of a representative experiment are presented. (E–J) Nlrp12-knockout (Nlrp12-KO) MC38 cells were generated with CRISPR/Cas9. (E and F) Control and Nlrp12-KO MC38 cells were cultured, and cell proliferation was monitored by IncuCyte. (G and H) MC38 and its Nlrp12-KO clones were cultured, and colony formation was measured by clonogenic assay. (I and J) Wound healing of MC38 and Nlrp12-KO MC38 cells was monitored by IncuCyte. (I) Representative images showing wound on cultured cells (left panel), and migration and cell confluence in wound area (right panel). Data represent mean ± SD of 8 wells (F) and 24 wells (J). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, 2-tailed Student’s t test (B, D, and H) or multiple t test (F and J). Experiments were repeated 3 times, and data of a representative experiment are presented. Scale bars: 100 μm (A), 400 μm (E), and 600 μm (I).