Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Feb 17;17(3):869-881.
doi: 10.7150/ijbs.56152. eCollection 2021.

MLKL inhibits intestinal tumorigenesis by suppressing STAT3 signaling pathway

Qun Zhao  1   2 , Xinran Cheng  1 , Jian Guo  1 , Yun Bi  1 , Li Kuang  3 , Jianhua Ren  3 , Jing Zhong  1 , Longrui Pan  1 , Xudong Zhang  1 , Yang Guo  1 , Yongqiang Liu  4 , Shu Jin  5 , Yan Tan  1 , Xianjun Yu  1
Affiliations

MLKL inhibits intestinal tumorigenesis by suppressing STAT3 signaling pathway

Qun Zhao et al. Int J Biol Sci. .

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 Apcmin/+ 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 Apcmin/+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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
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
Figure 2
MLKL deficiency increases intestinal tumor burden in the Apcmin/+ model. (A) Survival of Apcmin/+ and Apcmin/+Mlkl-/- mice. (B) Polyps in representative intestines from Apcmin/+ and Apcmin/+Mlkl-/- mice. (C and D) Quantification of polyp formation (C) and tumor load (D) in 16-week-old Apcmin/+ and Apcmin/+Mlkl-/- intestines. (E) Distribution of tumor size in 16-week-old Apcmin/+ and Apcmin/+Mlkl-/- mice. (F) Images of the H&E-stained intestines from Apcmin/+ and Apcmin/+Mlkl-/- mice. (G and H) Hematocrit (G) and thymus weight (H) of 16-week-old WT, Mlkl-/-, Apcmin/+ and Apcmin/+Mlkl-/- mice. (I) Ki-67 staining of the representative intestines from age-matched WT, Mlkl-/-, Apcmin/+ and Apcmin/+Mlkl-/- mice. (J-K) Whole intestines and colonic crypts were isolated from 16-week-old Apcmin/+and Apcmin/+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 Apcmin/+ mice.
Figure 3
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 Apcmin/+ and Apcmin/+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 Apcmin/+ and Apcmin/+Mlkl-/- mice. (D) Proteins isolated from 6-week-old Apcmin/+ and Apcmin/+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 Apcmin/+and Apcmin/+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 Apcmin/+ mice.
Figure 4
Figure 4
MLKL deficiency exacerbates IL-6/STAT3 activation by promoting JAK2 phosphorylation. (A) Quantitative analysis of IL-6 mRNA expression in Apcmin/+and Apcmin/+Mlkl-/-intestinal tumors. (B) The protein levels of IL-6 in Apcmin/+and Apcmin/+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 Apcmin/+and Apcmin/+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
Figure 5
Blocking IL-6 signaling inhibits intestinal tumorigenesis in Apcmin/+Mlkl-/- mice. (A and B) 4-week-old Apcmin/+ and Apcmin/+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 Apcmin/+Mlkl-/- and anti-IL6R-treated Apcmin/+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
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Goding Sauer A, Fedewa SA, Butterly LF, Anderson JC. et al. Colorectal cancer statistics, 2020. CA Cancer J Clin. 2020;70:145–64. - PubMed
    1. Islami F, Goding Sauer A, Miller KD, Siegel RL, Fedewa SA, Jacobs EJ. et al. Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States. CA Cancer J Clin. 2018;68:31–54. - PubMed
    1. Woolf SH. The best screening test for colorectal cancer-a personal choice. N Engl J Med. 2000;343:1641–3. - PubMed
    1. Walsh JM, Terdiman JP. Colorectal cancer screening: scientific review. JAMA. 2003;289:1288–96. - PubMed
    1. Sun L, Wang H, Wang Z, He S, Chen S, Liao D. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012;148:213–27. - PubMed

Publication types