PMID- 31088527 OWN - NLM STAT- MEDLINE DCOM- 20190910 LR - 20231213 IS - 1471-2407 (Electronic) IS - 1471-2407 (Linking) VI - 19 IP - 1 DP - 2019 May 14 TI - Induction of tolerogenic dendritic cells by activated TGF-beta/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells. PG - 439 LID - 10.1186/s12885-019-5670-9 [doi] LID - 439 AB - BACKGROUND: Dendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function. METHODS: Short hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay. RESULTS: RIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-beta1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-beta1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-beta1 antibody. TGF-beta1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-beta1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-beta stimulation. CONCLUSIONS: Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-beta1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-beta1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance. FAU - Zhong, Ming AU - Zhong M AUID- ORCID: 0000-0002-1869-2901 AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. zhongming0410@163.com. FAU - Zhong, Cheng AU - Zhong C AD - Division of Stem Cell Dynamics, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan. FAU - Cui, Wen AU - Cui W AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. cuiwenmd@163.com. FAU - Wang, Guanghui AU - Wang G AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Zheng, Gongpu AU - Zheng G AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Li, Li AU - Li L AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Zhang, Jing AU - Zhang J AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Ren, Rujing AU - Ren R AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Gao, Huijei AU - Gao H AD - Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao, 276826, China. FAU - Wang, Tingting AU - Wang T AD - People's Hospital of Rizhao, Rizhao, China. FAU - Li, Xin AU - Li X AD - People's Hospital of Rizhao, Rizhao, China. FAU - Che, Jiantu AU - Che J AD - S&V Biological Science and Technology Co., Ltd., Beijing, China. FAU - Gohda, Eiichi AU - Gohda E AD - Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. LA - eng GR - ZR2014HM038/Natural Science Foundation of Shandong Province/ PT - Journal Article DEP - 20190514 PL - England TA - BMC Cancer JT - BMC cancer JID - 100967800 RN - 0 (Receptors, Immunologic) RN - 0 (SMAD2 protein, human) RN - 0 (Smad2 Protein) RN - 0 (TGFB1 protein, human) RN - 0 (Transforming Growth Factor beta1) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 3.6.1.- (RIGI protein, human) RN - EC 3.6.4.13 (DEAD Box Protein 58) SB - IM MH - Animals MH - Carcinoma, Hepatocellular/genetics/metabolism/*pathology MH - Cell Line, Tumor MH - Cells, Cultured MH - Coculture Techniques MH - DEAD Box Protein 58/*deficiency MH - Dendritic Cells/*cytology/metabolism MH - Gene Expression Regulation, Neoplastic MH - Humans MH - Liver Neoplasms/genetics/metabolism/*pathology MH - Male MH - Mice MH - Neoplasm Transplantation MH - Neoplastic Stem Cells/*metabolism MH - Proto-Oncogene Proteins c-akt/metabolism MH - Receptors, Immunologic MH - *Signal Transduction MH - Smad2 Protein/metabolism MH - Transforming Growth Factor beta1/metabolism MH - Tumor Microenvironment MH - Up-Regulation PMC - PMC6515680 OTO - NOTNLM OT - Hepatocellular carcinoma OT - Immune tolerance OT - Mixed lymphocyte reaction OT - Tumor microenvironment OT - Tumor-infiltrating dendritic cell COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The cell lines used in this study don't require ethical approval. The studies employing CD14(+) monocytes and CD3(+) T cells from PBMCs of healthy donors were approved by the Medical Ethics Committee of Jining Medical College and the Medical Ethics Committee of People's Hospital of Rizhao, and written informed consent was obtained from all donors. All animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institute of Health and approved by the Animal Care and Use Committee at Jining Medical College (Additional file 3: Checklist S1). CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EDAT- 2019/05/16 06:00 MHDA- 2019/09/11 06:00 PMCR- 2019/05/14 CRDT- 2019/05/16 06:00 PHST- 2018/06/20 00:00 [received] PHST- 2019/05/02 00:00 [accepted] PHST- 2019/05/16 06:00 [entrez] PHST- 2019/05/16 06:00 [pubmed] PHST- 2019/09/11 06:00 [medline] PHST- 2019/05/14 00:00 [pmc-release] AID - 10.1186/s12885-019-5670-9 [pii] AID - 5670 [pii] AID - 10.1186/s12885-019-5670-9 [doi] PST - epublish SO - BMC Cancer. 2019 May 14;19(1):439. doi: 10.1186/s12885-019-5670-9.