Zhi-Wei Li1, Beicheng Sun2, Ting Gong1, Sheng Guo3, Jianhua Zhang4, Junlong Wang1, Atsushi Sugawara5, Meisheng Jiang6, Junjun Yan7, Alexandra Gurary8, Xin Zheng1, Bifeng Gao9, Shu-Yuan Xiao10, Wenlian Chen11, Chi Ma1, Christine Farrar12, Chenjun Zhu1, Owen T M Chan13, Can Xin1, Andrew Winnicki1, John Winnicki1, Mingxin Tang14, Ryan Park1, Mary Winnicki1, Katrina Diener9, Zhanwei Wang1, Qicai Liu15, Catherine H Chu1, Zhaohui L Arter1, Peibin Yue1, Lindsay Alpert16, George S Hui8, Peiwen Fei1, James Turkson1, Wentian Yang17, Guangyu Wu18, Ailin Tao19, Joe W Ramos1, Stefan Moisyadi5, Randall F Holcombe1, Wei Jia11, Lutz Birnbaumer20, Xiqiao Zhou21, Wen-Ming Chu22. 1. Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, Hawaii. 2. Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China. 3. Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, Hawaii; Department of Endocrine, Genetics and Metabolism, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China. 4. Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, Hawaii; Department of Pediatrics, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. 5. Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 6. Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California. 7. Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. 8. Department of Tropical Medicine, Medical Microbiology, and Pharmacology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 9. Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado. 10. Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China; Department of Pathology, University of Chicago, Chicago, Illinois. 11. Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii. 12. The Microscopy, Imaging, and Flow Cytometry Shared Resource, University of Hawaii Cancer Center, Honolulu, Hawaii. 13. Pathology Core, University of Hawaii Cancer Center, Honolulu, Hawaii. 14. Center for Cardiovascular Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 15. Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, Hawaii; Department of Cardiology and Heart Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China. 16. Department of Pathology, University of Chicago, Chicago, Illinois. 17. Department of Orthopedics, Rhode Island Hospital, Brown University Alpert Medical School, Providence, Rhode Island. 18. Department of Pharmacology and Toxicology, Augusta University, Augusta, Georgia. 19. The Second Affiliated Hospital, The State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Guangzhou Medical University, Guangzhou, Guangdong, China. 20. Neurobiology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; Institute for Biomedical Research (BIOMED), Universidad Católica Argentina, Buenos Aires, Argentina. 21. Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. Electronic address: zhouxiqiao@njmu.edu.cn. 22. Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, Hawaii; The Second Affiliated Hospital, The State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Guangzhou Medical University, Guangzhou, Guangdong, China. Electronic address: wchu@cc.hawaii.edu.
Abstract
BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.
BACKGROUND & AIMS:Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS:GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public humancolorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS:GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.
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