RNF43 inhibits PD-L1 expression via β-catenin in melanoma cells and promotes CD8+ T cell-mediated anti-tumor immune reaction

doi:10.3760/cma.j.cn371439-20230404-00079

Abstract

Abstract:

Objective To investigate the regulatory effects of ring finger protein 43 （RNF43） on CD8⁺ T cell-mediated anti-tumor immune reaction in melanoma. Methods RNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection； In vivo proliferation of mouse melanoma cells line B16-OVA in the Lv-Ctrl-OE，Lv-RNF43-OE，Lv-Ctrl-KD and Lv-RNF43-KD groups was detected by subcutaneous tumorigenesis assay in mice，and the expression levels of CD8⁺ T cells perforin and interferon γ （IFN-γ） in tumor immune microenvironment of melanoma were detected by flow cytometry； The expression levels of β-catenin and programmed death-ligand 1 （PD-L1） mRNA in cells were detected by quantitative real-time PCR assay； The effect of RNF43 on the transcriptional regulation of PD-L1 was detected by dual-luciferase reporter gene assay. Results Stable RNF43 over-expressing and RNF43 knockdown mouse melanoma cells lines Lv-RNF43-OE and Lv-RNF43-KD were successfully constructed. The results of subcutaneous tumorigenesis experiment in mice showed that the tumor mass of the Lv-RNF43-OE group was （0.08±0.06） g，which was significantly smaller than that of the Lv-Ctrl-OE group [（1.04±0.52） g]，with a statistically significant difference （t=3.71，P=0.032）； The tumor mass of Lv-RNF43-KD group was （1.94±0.29） g，with no statistically significant difference （t=-1.70，P=0.164） compared with that of the Lv-Ctrl-KD group （1.15±0.74） g. The flow cytometry results showed that the fluorescence intensity of CD8⁺ T cell perforin in the Lv-RNF43-OE group was 9 034 ± 2 628，which was significantly higher than that in the Lv-Ctrl-OE group （3 847 ±1 637），with a statistically significant difference （t=-3.35，P=0.015）； The fluorescence intensity of CD8⁺ T cell perforin in the Lv-RNF43-KD group was 966±247，which was significantly lower than that in the Lv-Ctrl-KD group （2 226±646），with a statistically significant difference （t=3.16，P=0.034）； The fluorescence intensity of IFN-γ of CD8⁺ T cell in the Lv-RNF43-OE group was 2 422±429，which was significantly higher than that of 1 688±324 in the Lv-Ctrl-OE group，with a statistically significant difference （t=-2.73，P=0.034）； The fluorescence intensity of IFN-γ of CD8⁺ T cell in the Lv-RNF43-KD group was 614 （454，863），with a statistically significant difference （Z=-1.96，P=0.050） compared with 1 159 （1 152，2 068） in the Lv-Ctrl-KD group. The results of quantitative real-time PCR showed that the relative expression level of β-catenin mRNA in the Lv-RNF43-OE group was 0.67±0.16，which was significantly lower than that of 1.00±0.11 in the Lv-Ctrl-OE group，with a statistically significant difference （t=2.98，P=0.041）； The relative expression level of PD-L1 mRNA in the Lv-RNF43-OE group was 0.32±0.09，which was significantly lower than that of 1.00±0.09 in the Lv-Ctrl-OE group，with a statistically significant difference （t=9.13，P=0.001）. The results of the dual-luciferase reporter gene assay showed that the PD-L1 promoter luciferase activity in the pCMV6-NC，RNF43，RNF43+β-catenin and β-catenin groups were 1.00±0.00，0.84±0.00，1.49±0.00 and 1.57±0.03 （F=2 218.33，P<0.001）. Further pairwise comparison showed that compared with the pCMV6-NC group，PD-L1 promoter luciferase activity was significantly lower in the RNF43 group （P<0.001） and significantly higher in the RNF43+β-catenin and β-catenin groups （P<0.001； P=0.003）； compared with the RNF43 group，PD-L1 promoter luciferase activity was significantly higher in the RNF43+β-catenin group （P<0.001）. Conclusion RNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression of β-catenin and promote CD8⁺ T cell-mediated anti-tumor immune reaction.

Key words: Melanoma, Beta catenin, Ring finger protein 43

Wu Minhang, Sun Wenzheng, Yu Qingzhuo, Guo Rong, Ye Hui, Du Ying, Qiu Jin, An Huazhang, Cao Lili. RNF43 inhibits PD-L1 expression via β-catenin in melanoma cells and promotes CD8⁺ T cell-mediated anti-tumor immune reaction[J]. Journal of International Oncology, 2023, 50(7): 407-412.

Figures/Tables 5

References 12

[1]	National Health Commission of the People’s Republic of China. Chinese guidelines for diagnosis and treatment of melanoma 2018 (English version)[J]. Chin J Cancer Res, 2019, 31(4): 578-585. DOI: 10.21147/j.issn.1000-9604.2019.04.02. doi: 10.21147/j.issn.1000-9604.2019.04.02
[2]	Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients[J]. Nature, 2014, 515(7528): 563-567. DOI: 10.1038/nature14011. doi: 10.1038/nature14011
[3]	Tsukiyama T, Zou J, Kim J, et al. A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis[J]. Nat Commun, 2020, 11(1): 4586. DOI: 10.1038/s41467-020-18257-3. doi: 10.1038/s41467-020-18257-3 pmid: 32934222
[4]	Tsukiyama T, Fukui A, Terai S, et al. Molecular role of RNF43 in canonical and noncanonical Wnt signaling[J]. Mol Cell Biol, 2015, 35(11): 2007-2023. DOI: 10.1128/MCB.00159-15. doi: 10.1128/MCB.00159-15 pmid: 25825523
[5]	Neumeyer V, Grandl M, Dietl A, et al. Loss of endogenous RNF43 function enhances proliferation and tumour growth of intestinal and gastric cells[J]. Carcinogenesis, 2019, 40(4): 551-559. DOI: 10.1093/carcin/bgy152. doi: 10.1093/carcin/bgy152 pmid: 30380024
[6]	Sakamoto H, Kuboki Y, Hatori T, et al. Clinicopathological significance of somatic RNF43 mutation and aberrant expression of ring finger protein 43 in intraductal papillary mucinous neoplasms of the pancreas[J]. Mod Pathol, 2015, 28(2): 261-267. DOI: 10.1038/modpathol.2014.98. doi: 10.1038/modpathol.2014.98
[7]	Luo H, Ma C. Identification of prognostic genes in uveal melanoma microenvironment[J]. PLoS One, 2020, 15(11): e0242263. DOI: 10.1371/journal.pone.0242263. doi: 10.1371/journal.pone.0242263
[8]	Radaszkiewicz T, Nosková M, Gömöryová K, et al. RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy[J]. Elife, 2021, 10: e65759. DOI: 10.7554/eLife.65759. doi: 10.7554/eLife.65759
[9]	Gajos-Michniewicz A, Czyz M. WNT signaling in melanoma[J]. Int J Mol Sci, 2020, 21(14): 4852. DOI: 10.3390/ijms21144852. doi: 10.3390/ijms21144852
[10]	Martínez-Lostao L, Anel A, Pardo J. How do cytotoxic lymphocytes kill cancer cells?[J]. Clin Cancer Res, 2015, 21(22): 5047-5056. DOI: 10.1158/1078-0432.CCR-15-0685. doi: 10.1158/1078-0432.CCR-15-0685 pmid: 26567364
[11]	Alspach E, Lussier DM, Schreiber RD. Interferon γ and its important roles in promoting and inhibiting spontaneous and therapeutic cancer immunity[J]. Cold Spring Harb Perspect Biol, 2019, 11(3): a028480. DOI: 10.1101/cshperspect.a028480. doi: 10.1101/cshperspect.a028480
[12]	Du L, Lee JH, Jiang H, et al. β-catenin induces transcriptional expression of PD-L1 to promote glioblastoma immune evasion[J]. J Exp Med, 2020, 217(11): e20191115. DOI: 10.1084/jem.2019 1115. doi: 10.1084/jem.2019

基因	引物
β-catenin	正向：5’-ATGGAGCCGGACAGAAAAGC-3’
	反向：5’-TGGGAGGTGTCAACATCTTCTT-3’
PD-L1	正向：5’-GCTCCAAAGGACTTGTACGTG-3’
	反向：5’-TGATCTGAAGGGCAGCATTTC-3’
β-actin	正向：5’-AGCCATGTACGTAGCCATCC-3’
	反向：5’-TTTGATGTCACGCACGATTT-3’

[1]	Hong Anlan, Cao Meng, Wang Yan, Fang Fang. Research progress on lncRNAs as members of ceRNA network in melanoma [J]. Journal of International Oncology, 2022, 49(1): 61-64.
[2]	Wang Qian, Tan Xiaojun, Zhang Donghui, Luo Kai. Application of β-catenin, Cyclin D1 and DKK1 in pathologically aided diagnosis of breast cancer [J]. Journal of International Oncology, 2021, 48(7): 402-408.
[3]	Zhang Ruizhi, Chen Xin, Zhang Peng, Tao Kaixiong. New progress on diagnosis and treatment of anorectal malignant melanoma [J]. Journal of International Oncology, 2020, 47(6): 377-380.
[4]	Du Mingli, Li Guixiang, Wu Wenjuan, Zhao Lei. Application of circulating tumor DNA in targeted therapy and immunotherapy of metastatic melanoma [J]. Journal of International Oncology, 2020, 47(6): 381-384.
[5]	Jin Lan, Kang Xiaojing. Drug resistance mechanism and treatment strategy for targeted therapy of melanoma [J]. Journal of International Oncology, 2018, 45(7): 444-448.
[6]	Miao Qiuju, Wang Yifei, Xu Xiulian.. Molecular targeted therapy in malignant melanoma [J]. Journal of International Oncology, 2018, 45(11): 699-702.
[7]	Xia Dechun, Kang Xiaojing. Role and mechanism of Notch signaling pathway in melanoma growth and metastasis [J]. Journal of International Oncology, 2018, 45(10): 635-638.
[8]	Wang Yanjun, Kang Xiaojing. Biomarkers for malignant melanoma [J]. Journal of International Oncology, 2018, 45(1): 59-.
[9]	Chai Li, Kang Xiaojing. Non-coding small RNA and melanoma [J]. Journal of International Oncology, 2017, 44(7): 541-543.
[10]	Sun Chengyu, Xu Yuqing. Immunotherapy in melanoma patients with autoimmune disease [J]. Journal of International Oncology, 2017, 44(4): 310-312.
[11]	He Yan, Tie Jun, Tang Jun. Role of microphthalmia-associated transcription factor in melanoma metastasis [J]. Journal of International Oncology, 2017, 44(4): 281-283.
[12]	Tang Jianqin, Hou Xiaoyang, Jiang Guan, Wei Zhiping, Liu Yanqun. Application of nanotechnology in the diagnosis and therapy of melanoma [J]. Journal of International Oncology, 2016, 43(11): 871-873.
[13]	Qiu Wangwang, Yang Zhili, Zheng Qi. Ring finger protein 43 gene and its function in digestive system cancer [J]. Journal of International Oncology, 2016, 43(1): 56-59.
[14]	Wang Shixiong, Li Yaoping. Ring finger protein 43 gene and cancer [J]. Journal of International Oncology, 2016, 43(1): 23-25.
[15]	MA Ya-Nan, WANG Bao-Hong, SU Qing-Hong, XU Xiao-Qun, WANG Jun-Fu. Effect of miR27a on cell proliferation and apoptosis in human melanoma cell line WM239 [J]. Journal of International Oncology, 2015, 42(3): 161-164.