Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells
Abstract 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-inducibl...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-05-01
|
| Series: | BMC Cancer |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12885-019-5670-9 |
| _version_ | 1818408928187252736 |
|---|---|
| author | Ming Zhong Cheng Zhong Wen Cui Guanghui Wang Gongpu Zheng Li Li Jing Zhang Rujing Ren Huijei Gao Tingting Wang Xin Li Jiantu Che Eiichi Gohda |
| author_facet | Ming Zhong Cheng Zhong Wen Cui Guanghui Wang Gongpu Zheng Li Li Jing Zhang Rujing Ren Huijei Gao Tingting Wang Xin Li Jiantu Che Eiichi Gohda |
| author_sort | Ming Zhong |
| collection | DOAJ |
| description | Abstract 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-β1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-β1 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-β1 antibody. TGF-β1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-β1-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-β stimulation. Conclusions Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-β1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-β1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance. |
| first_indexed | 2024-12-14T09:51:31Z |
| format | Article |
| id | doaj.art-c0aeed2ecd1e4a229992d311ed11aa64 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-12-14T09:51:31Z |
| publishDate | 2019-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-c0aeed2ecd1e4a229992d311ed11aa642022-12-21T23:07:31ZengBMCBMC Cancer1471-24072019-05-0119111510.1186/s12885-019-5670-9Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cellsMing Zhong0Cheng Zhong1Wen Cui2Guanghui Wang3Gongpu Zheng4Li Li5Jing Zhang6Rujing Ren7Huijei Gao8Tingting Wang9Xin Li10Jiantu Che11Eiichi Gohda12Institute of Tumor Pharmacology, Jining Medical CollegeDivision of Stem Cell Dynamics, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of TokyoInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegeInstitute of Tumor Pharmacology, Jining Medical CollegePeople’s Hospital of RizhaoPeople’s Hospital of RizhaoS&V Biological Science and Technology Co., Ltd.Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAbstract 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-β1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-β1 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-β1 antibody. TGF-β1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-β1-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-β stimulation. Conclusions Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-β1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-β1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.http://link.springer.com/article/10.1186/s12885-019-5670-9Tumor-infiltrating dendritic cellImmune toleranceMixed lymphocyte reactionTumor microenvironmentHepatocellular carcinoma |
| spellingShingle | Ming Zhong Cheng Zhong Wen Cui Guanghui Wang Gongpu Zheng Li Li Jing Zhang Rujing Ren Huijei Gao Tingting Wang Xin Li Jiantu Che Eiichi Gohda Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells BMC Cancer Tumor-infiltrating dendritic cell Immune tolerance Mixed lymphocyte reaction Tumor microenvironment Hepatocellular carcinoma |
| title | Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells |
| title_full | Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells |
| title_fullStr | Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells |
| title_full_unstemmed | Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells |
| title_short | Induction of tolerogenic dendritic cells by activated TGF-β/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells |
| title_sort | induction of tolerogenic dendritic cells by activated tgf β akt smad2 signaling in rig i deficient stemness high human liver cancer cells |
| topic | Tumor-infiltrating dendritic cell Immune tolerance Mixed lymphocyte reaction Tumor microenvironment Hepatocellular carcinoma |
| url | http://link.springer.com/article/10.1186/s12885-019-5670-9 |
| work_keys_str_mv | AT mingzhong inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT chengzhong inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT wencui inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT guanghuiwang inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT gongpuzheng inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT lili inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT jingzhang inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT rujingren inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT huijeigao inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT tingtingwang inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT xinli inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT jiantuche inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells AT eiichigohda inductionoftolerogenicdendriticcellsbyactivatedtgfbaktsmad2signalinginrigideficientstemnesshighhumanlivercancercells |