MLKL inhibits intestinal tumorigenesis by suppressing STAT3 signaling pathway

Abstract

Mixed lineage kinase domain-like protein (MLKL) plays an important role in necroptosis, but the role and mechanism of MLKL in intestinal tumorigenesis remain unclear. Here, we found that hematopoietic- and nonhematopoietic-derived MLKL affected intestinal inflammation, but nonhematopoietic-derived MLKL primarily inhibited intestinal tumorigenesis. Loss of MLKL enhanced intestinal regeneration and the susceptibility to intestinal tumorigenesis in Apc^min/+ mice by hyperactivating the Janus kinase 2 (JAK2)/ signal transducer and activator of transcription 3 (STAT3) axis. Furthermore, MLKL deficiency increased interleukin-6 (IL-6) production in dendritic cells. Administration of anti-IL-6R antibody therapy reduced intestinal tumorigenesis in Apc^min/+Mlkl^-/- mice. Notably, low MLKL expression in human colorectal tumors, which enhanced STAT3 activation, was associated with decreased overall survival. Together, our results reveal that MLKL exhibits a suppressive effect during intestinal tumorigenesis by suppressing the IL-6/JAK2/STAT3 signals.

Keywords: Anti-IL-6R antibody therapy.; IL-6/STAT3; Intestinal tumorigenesis; MLKL.

Figure 1

MLKL in hematopoietic and nonhematopoietic cellular compartments mediates protection against intestinal tumorigenesis. (A) Schematic overview of four chimeric mice: WT → WT, WT → Mlkl^-/-, Mlkl^-/-→ WT, and Mlkl^-/- → Mlkl^-/-. (B) Weight change in the chimeric mice of the AOM/DSS model. (C) Representative images of the intestinal tumors from chimeric mice on the day 80 of the AOM/DSS model. (D) Quantification of the polyp numbers in chimeric mice in the AOM/DSS model. * p < 0.05 and *** p < 0.001 versus WT → WT groups.

Figure 2

MLKL deficiency increases intestinal tumor burden in the Apc^min/+ model. (A) Survival of Apc^min/+ and Apc^min/+Mlkl^-/- mice. (B) Polyps in representative intestines from Apc^min/+ and Apc^min/+Mlkl^-/- mice. (C and D) Quantification of polyp formation (C) and tumor load (D) in 16-week-old Apc^min/+ and Apc^min/+Mlkl^-/- intestines. (E) Distribution of tumor size in 16-week-old Apc^min/+ and Apc^min/+Mlkl^-/- mice. (F) Images of the H&E-stained intestines from Apc^min/+ and Apc^min/+Mlkl^-/- mice. (G and H) Hematocrit (G) and thymus weight (H) of 16-week-old WT, Mlkl^-/-, Apc^min/+ and Apc^min/+Mlkl^-/- mice. (I) Ki-67 staining of the representative intestines from age-matched WT, Mlkl^-/-, Apc^min/+ and Apc^min/+Mlkl^-/- mice. (J-K) Whole intestines and colonic crypts were isolated from 16-week-old Apc^min/+and Apc^min/+Mlkl^-/- mice. PCNA (J) and cleaved caspase-3 (K) were analyzed by western blotting. Scale bars: 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Apc^min/+ mice.

Figure 3

Loss of MLKL enhances the activation of STAT3. (A) Ingenuity Pathway Analysis (IPA) of the disease processes and cellular functions in 6-week-old Apc^min/+ and Apc^min/+Mlkl^-/- mice. (B) GSEA demonstrated enrichment of STAT3 target genes. (C) Heat map showing a summary of the expression of STAT3 pathway target genes in 6-week-old Apc^min/+ and Apc^min/+Mlkl^-/- mice. (D) Proteins isolated from 6-week-old Apc^min/+ and Apc^min/+Mlkl^-/- mouse intestines were analyzed by western blotting to assess STAT3 activation. (E) Images of H&E-stained intestinal tissues in 3 and 5 days after WBI. (F) Villus length in WT and Mlkl^-/- intestines were measured after WBI. (G) Proteins isolated from WT and Mlkl^-/- mice during regeneration were analyzed by western blotting to assess STAT3 activation. (H) Gene expression of intestinal tissues during regeneration (days 0 and 3) in WT and Mlkl^-/- mice. (I-J) Whole intestine tissues and colonic crypts from 16-week-old Apc^min/+and Apc^min/+Mlkl^-/- mice were lysed and measured the levels of Cyclin D1 and C-myc. Scale bars: 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Apc^min/+ mice.

Figure 4

MLKL deficiency exacerbates IL-6/STAT3 activation by promoting JAK2 phosphorylation. (A) Quantitative analysis of IL-6 mRNA expression in Apc^min/+and Apc^min/+Mlkl^-/-intestinal tumors. (B) The protein levels of IL-6 in Apc^min/+and Apc^min/+Mlkl^-/-intestinal tumors as determined by ELISA. (C) Quantitative mRNA expression of IL-6 in isolated laminar propria lymphocytes (LPLs) and intestinal epithelial cells (IECs) of intestinal tumors from Apc^min/+and Apc^min/+Mlkl^-/- mice. (D) Macrophages, dendritic cells, and T cells isolated by flow cytometric cell sorting were analyzed to assess IL-6 mRNA expression by qRT-PCR. (E) LPS-stimulated WT and Mlkl^-/- BMDCs were analyzed for quantitative mRNA expression of IL-6 by qRT-PCR (LPS, 100 ng/ml). (F) Mlkl^-/- BMDCs were overexpressed with MLKL for 48 h, cells then were stimulated with LPS and analyzed the expression of IL-6 mRNA. (G) WT and Mlkl^-/- BMDCs were pre-treated 10 μM ERK inhibitor U0126 for 2 h, and then cells then were stimulated with LPS and analyzed the expression of IL-6 mRNA by qRT-PCR. (H-I) Primary WT and Mlkl^-/-IECs isolated from 3-week-old mice were stimulated with 20 ng/mL IL-6. Cell lysates were analyzed for pSTAT3, Cyclin D1 and C-myc. (J) WT and Mlkl^-/-IECs were stimulated with 20 ng/mL IL-6. Cell lysates were analyzed for pJAK2 (Y1007/Y1008). (K) The effect of MLKL on the interaction of JAK2 with STAT3. WT and Mlkl^-/-IECs were stimulated with 20 ng/mL IL-6. Cell lysates were subjected to immunoprecipitation and western blotting with indicated antibody. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Blocking IL-6 signaling inhibits intestinal tumorigenesis in Apc^min/+Mlkl^-/- mice. (A and B) 4-week-old Apc^min/+ and Apc^min/+Mlkl^-/- mice were administrated with anti-IL6R antibody weekly for 10 weeks. Clinical score and the percent weight change were monitored. (C) Quantification of polyp formation in PBS- or anti-IL6R-treated mice as indicated. (D) Intestinal tissues were harvested from PBS-treated Apc^min/+Mlkl^-/- and anti-IL6R-treated Apc^min/+Mlkl^-/- mice and then lysed. The levels of pSTAT3, Cyclin D1 and C-myc were determined by western blotting. ** p < 0.01 and *** p < 0.001.

Figure 6

The levels of MLKL has prognostic implications in colorectal cancer. (A-B) Expression of MLKL in colorectal cancer samples detected by IHC. Overall survival of patients based on MLKL expression levels conducted by Kaplan-Meier plot. (C) Boxed plot of MLKL expression assessed at different clinical stages. (D) Pearson correlation analysis of MLKL and Cyclin D1 (P < 0.001; R = - 0.71) and MLKL and C-myc (P < 0.001; R = - 0.72) in human colorectal cancer. (E) Model of MLKL negatively impacts IL-6/STAT3 signaling in intestinal tumorigenesis. ** p < 0.01.