Human Colon Tumors Express a Dominant-Negative Form of SIGIRR That Promotes Inflammation and Colitis-Associated Colon Cancer in Mice.
OBJECTIVE
Single immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer.


METHODS
We performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses.


RESULTS
RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectal cancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed higher levels of the inflammatory cytokines IL-17A and IL-6 had activation of the transcription factors STAT3 and NFκB. SIGIRR(N86/102S) expressed in colons of mice did not localize to the epithelial cell surface.


CONCLUSIONS
Levels of SIGIRR are lower in human colorectal tumors, compared with nontumor tissues; tumors contain the dominant-negative isoform SIGIRR(ΔE8). This mutant protein blocks localization of full-length SIGIRR to the surface of colon epithelial cells and its ability to downregulate IL1R signaling. Expression of SIGIRR(N86/102S) in the colonic epithelium of mice increases expression of inflammatory cytokines and formation and size of colitis-associated tumors.
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio.
Junjie
J
Zhao
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio.
Katarzyna
K
Bulek
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Muhammet F
MF
Gulen
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio.
Jarod A
JA
Zepp
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Department of Colorectal Surgery, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio.
Georgio
G
Karagkounis
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Bradley N
BN
Martin
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Hao
H
Zhou
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Minjia
M
Yu
Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio.
Xiuli
X
Liu
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Department of Colorectal Surgery, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio.
Emina
E
Huang
Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Paul L
PL
Fox
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Department of Colorectal Surgery, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio.
Matthew F
MF
Kalady
Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio.
Sanford D
SD
Markowitz
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. Electronic address: lix@ccf.org.
Xiaoxia
X
Li
D000818:Animals; D001397:Azoxymethane; D002462:Cell Membrane / Q000378:metabolism; D003092:Colitis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D003110:Colonic Neoplasms / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003600:Cytosol / Q000378:metabolism; D016264:Dextran Sulfate; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant; D020022:Genetic Predisposition to Disease; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D046509:Tissue Culture Techniques; D014162:Transfection
D001397:Azoxymethane; D016264:Dextran Sulfate; D018836:Inflammation Mediators; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; C546891:SIGIRR protein, human; C484529:SIGIRR protein, mouse
Colorectal Cancer; TLR-IL-1R; Tumor Suppressor