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. 2023 Oct 2;133(19):e166295.
doi: 10.1172/JCI166295.

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

Shahanshah Khan  1 , Youn-Tae Kwak  1 , Lan Peng  1 , Shuiqing Hu  1 , Brandi L Cantarel  2 , Cheryl M Lewis  3 , Yunpeng Gao  1 , Ram S Mani  1 , Thirumala-Devi Kanneganti  4 , Hasan Zaki  1
Affiliations

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

Shahanshah Khan et al. J Clin Invest. .

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.

Keywords: Colorectal cancer; Gastroenterology; Tumor suppressors.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. NLRP12 suppresses invasive adenocarcinoma and downregulates genes involved in proliferation, epithelial-mesenchymal transition (EMT), and invasion.
Colorectal tumorigenesis was induced in WT and Nlrp12–/– mice (n = 16/group) using the AOM plus DSS regimen. (A and B) Mice were sacrificed 12 weeks after AOM, and tumor burden was counted. (C and D) Tumor-bearing colon sections (n = 7) were stained with H&E and examined histopathologically. (C) Representative images of H&E-stained colons. The arrow indicates the invasion of the tumor into the stroma. Scale bars: 100 μm. (D) Semiquantitative scoring for low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive adenocarcinoma. Data represent mean ± SEM. Experiments were repeated at least 3 times, and data from a representative experiment are presented. (E) RNA isolated from tumors was used for RNA-seq analysis. The heatmap shows the expression of selected genes. (F–H) The expression of MMPs (F), EMT markers (G), and Wnt target genes (H) in the tumor was measured by real-time RT-PCR. Data represent mean ± SEM. 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 unpaired, 2-tailed Student’s t test.
Figure 2
Figure 2. CRC susceptibility of Nlrp12–/– mice is independent of gut microbiota composition.
WT and Nlrp12–/– mice (n = 8–9/group) were cohoused for 4 weeks. (A–C) Fecal DNA was isolated from separately housed (SH) or cohoused (CH) WT and Nlrp12–/– mice and sequenced for the 16S rRNA gene. (A and B) α-Diversity showing species richness was analyzed by nonparametric Shannon (npShannon) index (A) and Inverse Simpson index (B). Data represent mean ± SEM. (C) Stack bar plot showing the relative distribution of the microbial community at family levels. (D–H) Colorectal tumorigenesis was induced in cohoused WT and Nlrp12–/– mice with AOM plus DSS. (D and E) Mice were sacrificed 12 weeks after AOM, and tumor burden was counted. (F) Representative images of H&E-stained colons. The arrow indicates the invasion of colorectal tumor into the stroma. Scale bars: 100 μm. (G and H) Semiquantitative histopathological scorings for low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasiveness. (I) RNA isolated from tumors was used to measure the expression of the indicated genes by real-time RT-PCR. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney test (A and B) or unpaired, 2-tailed Student’s t test (E and G–I). Experiments were repeated 2 times, and data from a representative experiment are presented.
Figure 3
Figure 3. Increased tumorigenesis of Nlrp12–/– mice is associated with higher β-catenin activation in the tumor.
WT and Nlrp12–/– mice were separately housed (SH) (n = 6/group) or cohoused (CH) (n = 7/group). Colorectal tumorigenesis was induced by AOM plus DSS treatment. Mice were sacrificed 12 weeks after AOM injection. (A–D) Tumors were analyzed for the activation of indicated signaling pathways by Western blotting. Band intensities of p-P65, p-IκBa, and p-ERK were measured densitometrically. Data represent mean ± SEM. (E–H) The expression of β-catenin, cMyc, and cyclin D1 in tumors was analyzed by Western blotting. Band intensities of β-catenin, cMyc, and cyclin D1 were measured densitometrically. Data represent mean ± SEM. (I and J) Colon sections were stained for Ki67 and positive cells (red) were counted under 20× objective (n = 3 mice/group). Scale bars: 200 μm. Data represent mean ± SEM. (K) Tumor-bearing colon sections were immunostained for β-catenin (red). The nuclei were stained with DAPI (blue). Scale bars: 200 μm. All experiments were repeated at least 2 times, and data from a representative experiment are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired, 2-tailed Student’s t test.
Figure 4
Figure 4. Nlrp12 deficiency promotes colorectal tumorigenesis in Apcmin/+ mice.
Apcmin/+ and Apcmin/+ Nlrp12–/– mice (n = 15/group) were sacrificed 5 months after birth, and the number of tumors was counted in the small (A) and large intestine (colon) (B). (C) Representative images of H&E-stained small intestine and colons at low (2×) magnifications. Scale bars: 100 μm. (D–G) Homogenates from tumor-bearing small intestines and colons were analyzed for the activation of β-catenin, p-P65, p-IκBα, IκBα, p-ERK, ERK, p-JNK, p-AKT, and β-actin by Western blotting. Band intensities of p-P65, p-ERK, and β-catenin relative to β-actin were measured. Data represent mean ± SEM. (H and I) RNA isolated from small intestine and tumor-bearing colons was measured for the expression of indicated genes by real-time RT-PCR. Data represent mean ± SEM. Experiments were repeated at least 3 times, and data from a representative experiment are presented. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, 2-tailed Student’s t test.
Figure 5
Figure 5. Intestinal epithelial cell–specific NLRP12 suppresses β-catenin activation and colorectal tumorigenesis.
(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).
Figure 6
Figure 6. NLRP12 regulates the Wnt/β-catenin pathway and controls proliferation and migration of cancer cells.
(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).
Figure 7
Figure 7. NLRP12 suppresses β-catenin activation via inhibition of GSK3β phosphorylation.
(A) GFP- or NLRP12-expressing HEK293T, HCT116, and HT29 cells were stimulated with Wnt3a. Cell lysates were analyzed for NLRP12, β-catenin, p-β-catenin, GSK3β, p-GSK3β, and β-actin by Western blotting. (B) Scramble or NLRP12-KO HEK293T, HCT116, and HT29 cells were stimulated with Wnt3a. At indicated time points, cells lysates were used to measure NLRP12, β-catenin, GSK3β, p-GSK3β, and β-actin by Western blotting. (C) Colon tissues were collected from healthy and tumor-bearing WT and Nlrp12–/– mice and analyzed for GSK3β and p-GSK3β by Western blotting. (D) Colorectal tumors were induced in Vil-Cre, Nlrp12fl/fl, and Nlrp12fl/fl;Vil-Cre mice with AOM plus DSS. Tumors collected 12 weeks after AOM/DSS treatment were analyzed for GSK3β, p-GSK3β, and β-actin. (E and F) Colorectal tumors and adjacent nontumor tissues from WT mice following AOM/DSS-mediated tumor induction were used to measure NLRP12, β-catenin, GSK3β, p-GSK3β, and β-actin by Western blotting. (F) Band intensities of NLRP12, β-catenin, GSK3β, and p-GSK3β as shown in E were measured by densitometry. Data represent mean ± SEM. (G and H) Human colorectal tumors and adjacent nontumor tissues were analyzed for NLRP12, β-catenin, GSK3β, p-GSK3β and α-tubulin by Western blotting. Band intensities of NLRP12, β-catenin, GSK3β, and p-GSK3β were measured by densitometry. Data represent mean ± SEM. *P < 0.05, **P < 0.01 by unpaired, 2-tailed Student’s t test. Experiments represented in A and B were repeated 3 times, and those in C–H were repeated 2 times.
Figure 8
Figure 8. NLRP12 inhibits the phosphorylation of GSK3β via interaction with STK38.
(A and B) HEK293T and HCT116 cells overexpressing FLAG-tagged NLRP12 or GFP were stimulated with Wnt3a. NLRP12 was pulled down with an anti-FLAG antibody, and the immunoprecipitation (IP) product and the input were immunoblotted for NLRP12, STK38, GSK3β, and β-catenin. (C–E) GFP-FLAG or STK38-FLAG was overexpressed in HEK293T cells and stimulated with Wnt3a. (C) Following IP of STK38, the IP product and the input were immunoblotted for STK38, GSK3β, p-GSK3β, NLRP12, and β-catenin. (D) Cell lysates were analyzed for STK38, β-catenin, p-β-catenin, GSK3β, and p-GSK3β. (E) The expression of indicated genes in HEK293T cells following stimulation with Wnt3a was measured by real-time RT-PCR. Data represent mean ± SD of triplicate wells. **P < 0.001, ***P < 0.0001 by unpaired, 2-tailed Student’s t test. (F) HEK293T cells expressing GFP or NLRP12 were transfected with shRNA for STK38 or control shRNA. Cells were then stimulated with Wnt3a and measured for STK38, β-catenin, p-GSK3β, and GSK3β by Western blotting. All experiments were repeated 3 times, and data of a representative experiment are presented. (G) Proposed mechanism of NLRP12-mediated inhibition of the Wnt/β-catenin pathway.
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