Overexpression of ubiquitin-conjugating enzyme 2T induces radiotherapy resistance in hepatocellular carcinoma by enriching regulatory T cells in the tumor microenvironment

doi:10.12122/j.issn.1673-4254.2024.06.16

Abstract

Abstract:

Objective To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T (UBE2T) on radiosensitivity of hepatocellular carcinoma (HCC). Methods Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector, and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay. The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice, and tumor growth following irradiation were recorded. The xenografts were collected for analyzing infiltration of CD4⁺ T cells and regulatory T cells (Tregs) using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting. The correlations of UBE2T expression with immune cell infiltration, glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database, and the results were verified in a co-culture system of Hepa1-6 cells and Tregs. Results UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models (especially in C57BL/6 mice). CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells, T follicular helper cells, M2 macrophages, monocytes, lymphocytes and Tregs in HCC. The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA, and irradiation increased infiltration of CD4⁺ T cells and Tregs in the tumor microenvironment. Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration. GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC. In the cell co-culture system, UBE2T overexpression significantly enhanced lactate production, proliferation and immunosuppressive functions of Tregs. Conclusion A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.

Key words: ubiquitin-conjugating enzyme 2T, hepato-cellular carcinoma, radiotherapy resistance, tumor immune microenvironment, regulatory T cells

Xinrong HE, Sili XIONG, Zhenru ZHU, Jingyuan SUN, Chuanhui CAO, Hui WANG. Overexpression of ubiquitin-conjugating enzyme 2T induces radiotherapy resistance in hepatocellular carcinoma by enriching regulatory T cells in the tumor microenvironment[J]. Journal of Southern Medical University, 2024, 44(6): 1149-1158.

Figures/Tables 6

Tab.1 Primers used for RT-qPCR

Primer

Sequence (5'→3')

Il-10-F

Il-10-R

GTCCTTTCACTTGCCCTCATC

CAAACYGGTCACAGCTTTCGA

Fig.1 Overexpression of UBE2T results in radiotherapy resistance in HCC. A: Relative mRNA level of Ube2t adjusted to Actin. B: Western blotting of UBE2T protein in LV-Control and LV-UBE2T cells. C, D: Colony formation assays of Hepa 1-6 cells with stable UBE2T overexpression. E: Treatment schedules of ionizing radiation (IR) and endpoint for recording. F, G: Tumor growth curves of the nude mice (F, n=5 or 6) and the dissected xenografts from the mice (G). in each group. H-J: Tumor growth curves of C57BL/6 mice (H, n=5 or 6), the tumor-bearing mice (I), and the dissected xenografts (J). In (F) and (H), statistical analyses were performed using a mixed-effects model followed by Tukey's multiple comparison test. In (A), student's t-test was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ****P<0.0001.

Fig.2 Immunological microenvironmental analysis of HCC by CIBERSORT. A: Percentages of different cell types in the two groups. B-J: Estimated fractions of DCs (B), T follicular helper cells (C), M1 macrophages (D), M2 macrophages (E), monocytes (F), lymphocytes (G), CD8+ T cells (H), Tregs (I), and CD8+T cells/Tregs (J) in the two groups. Student's t-test was used for comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DCs: Dendritic cells, Tregs: Regulatory T cells.

Fig.3 The number of Tregs is increased in HCC overexpressing UBE2T. A: Treatment schedules of IR and endpoint for analysis. B, C: Representative contour plots (B) and quantitation (C) of CD4+/CD3+ ratios (n=6-8). D, E: Representative contour plots (D) and quantitation (E) of the number of TIL Tregs (CD25+ Foxp3+) per 104 cells (n=6-8). Student's t-test was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001.

Fig.4 Effect of UBE2T expression on glycolysis levels in HCC and its regulatory effect on immune microenvironment. A, B: Concentration of glucose (A) and lactic acid (B) in the two groups with or without IR. C-E: Results of GSEA are plotted to visualize the correlation between the expression of UBE2T and the gene signatures of glycolysis and Tregs infiltration in the TCGA cohort. Student's t-test was used for comparisons. F, G: Western blotting of HK1, LDHA, and UBE2T protein in siControl, siUNE2T, LV-Control and LV-UBE2T cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.5 HCC overexpressing UBE2T promoted lactate secretion and activated Tregs.A: Schematic diagram of the co-culture model. B: Concentrations of Tregs in different co-culture groups (n=5). C, D: Representative contour plots (C) and quantitation (D) of Ki67+ ratios (n=6). E: Relative mRNA level of IL-10 adjusted to Actin in different co-culture groups (n=3). F, G: Representative contour plots (F) and quantitation (G) of the TGF‑β+ ratios (n=5). One-way ANOVA was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001.

References 52

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49.
2	Llovet JM, Kelley RK, Villanueva A, et al. Hepatocellular carcinoma[J]. Nat Rev Dis Primers, 2021, 7: 6.
3	Chen LC, Lin HY, Hung SK, et al. Role of modern radiotherapy in managing patients with hepatocellular carcinoma[J]. World J Gastroenterol, 2021, 27(20): 2434-57.
4	Kim TH, Koh YH, Kim BH, et al. Proton beam radiotherapy vs. radiofrequency ablation for recurrent hepatocellular carcinoma: a randomized phase III trial[J]. J Hepatol, 2021, 74(3): 603-12.
5	Shi CY, Li Y, Geng L, et al. Adjuvant stereotactic body radiotherapy after marginal resection for hepatocellular carcinoma with microvascular invasion: a randomised controlled trial[J]. Eur J Cancer, 2022, 166: 176-84.
6	Byun HK, Kim HJ, Im YR, et al. Dose escalation by intensity modulated radiotherapy in liver-directed concurrent chemoradiotherapy for locally advanced BCLC stage C hepatocellular carcinoma[J]. Radiother Oncol, 2019, 133: 1-8.
7	Bang A, Dawson LA. Radiotherapy for HCC: ready for prime time?[J]. JHEP Rep, 2019, 1(2): 131-7.
8	Schaue D, McBride WH. Opportunities and challenges of radiotherapy for treating cancer[J]. Nat Rev Clin Oncol, 2015, 12(9): 527-40.
9	Pointer KB, Pitroda SP, Weichselbaum RR. Radiotherapy and immunotherapy: open questions and future strategies[J]. Trends Cancer, 2022, 8(1): 9-20.
10	Koukourakis MI, Giatromanolaki A. Tumor draining lymph nodes, immune response, and radiotherapy: towards a revisal of therapeutic principles[J]. Biochim Biophys Acta Rev Cancer, 2022, 1877(3): 188704.
11	McLaughlin M, Patin EC, Pedersen M, et al. Inflammatory microenvironment remodelling by tumour cells after radiotherapy[J]. Nat Rev Cancer, 2020, 20(4): 203-17.
12	Yum S, Li MH, Chen ZJ. Old dogs, new trick: classic cancer therapies activate cGAS[J]. Cell Res, 2020, 30(8): 639-48.
13	Li JY, Zhao Y, Gong S, et al. TRIM21 inhibits irradiation-induced mitochondrial DNA release and impairs antitumour immunity in nasopharyngeal carcinoma tumour models[J]. Nat Commun, 2023, 14(1): 865.
14	Jiang XY, Ma Y, Wang T, et al. Targeting UBE2T potentiates gemcitabine efficacy in PancreaticCancer by regulating pyrimidine metabolism and replication stress[J]. Gastroenterology, 2023, 164(7): 1232-47.
15	Yu ZY, Jiang XY, Qin L, et al. A novel UBE2T inhibitor suppresses Wnt/β‑catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination[J]. Oncogene, 2021, 40(5): 1027-42.
16	Zhu ZR, Cao CH, Zhang DY, et al. UBE2T-mediated Akt ubiquitination and Akt/β-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism[J]. Cell Death Dis, 2022, 13(2): 154.
17	Sun JY, Zhu ZR, Li WW, et al. UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation[J]. J Exp Clin Cancer Res, 2020, 39(1): 222.
18	Newman AM, Liu CL, Green MR, et al. Robust enumeration of cell subsets from tissue expression profiles[J]. Nat Methods, 2015, 12(5): 453-7.
19	Rumgay H, Arnold M, Ferlay J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040[J]. J Hepatol, 2022, 77(6): 1598-606.
20	Qiao L, Dong C, Ma BL. UBE2T promotes proliferation, invasion and glycolysis of breast cancer cells by regualting the PI3K/AKT signaling pathway[J]. J Recept Signal Transduct Res, 2022, 42(2): 151-9.
21	Wang Y, Leng H, Chen H, et al. Knockdown of UBE2T inhibits osteosarcoma cell proliferation, migration, and invasion by suppressing the PI3K/akt signaling pathway[J]. Oncol Res, 2016, 24(5): 361-9.
22	Cao K, Ling XD, Jiang XY, et al. Pan-canceranalysis of UBE2T with a focus on prognostic and immunological roles in lung adenocarcinoma[J]. Respir Res, 2022, 23(1): 306.
23	Huang W, Huang HY, Xiao YZ, et al. UBE2T is upregulated, predicts poor prognosis, and promotes cell proliferation and invasion by promoting epithelial-mesenchymal transition via inhibiting autophagy in an AKT/mTOR dependent manner in ovarian cancer[J]. Cell Cycle, 2022, 21(8): 780-91.
24	Allen C, Her S, Jaffray DA. Radiotherapy for cancer: present and future[J]. Adv Drug Deliv Rev, 2017, 109: 1-2.
25	Citrin DE. Recent developments in radiotherapy[J]. N Engl J Med, 2017, 377(22): 2200-1.
26	Ahmad SS, Duke S, Jena R, et al. Advances in radiotherapy[J]. BMJ, 2012, 345(dec04 1): e7765.
27	Ohri N, Dawson LA, Krishnan S, et al. Radiotherapy for hepatocellular carcinoma: new indications and directions for future study[J]. J Natl Cancer Inst, 2016, 108(9): djw133.
28	Fang Y, Zhan YZ, Xie YW, et al. Integration of glucose and cardiolipin anabolism confers radiation resistance of HCC[J]. Hepatology, 2022, 75(6): 1386-401.
29	Hong WF, Zhang Y, Wang SW, et al. RECQL4 inhibits radiation-induced tumor immune awakening via suppressing the cGAS-STING pathway in hepatocellular carcinoma[J]. Adv Sci, 2024, 11(16): e2308009.
30	Yin H, Wang XY, Zhang X, et al. UBE2T promotes radiation resistance in non-small cell lung cancer via inducing epithelial-mesenchymal transition and the ubiquitination-mediated FOXO1 degradation[J]. Cancer Lett, 2020, 494: 121-31.
31	Zhang YH, Hu JY, Ji K, et al. CD39 inhibition and VISTA blockade may overcome radiotherapy resistance by targeting exhausted CD8⁺ T cells and immunosuppressive myeloid cells[J]. Cell Rep Med, 2023, 4(8): 101151.
32	Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment[J]. Nat Immunol, 2013, 14(10): 1014-22.
33	Song M, Yeku OO, Rafiq S, et al. Tumor derived UBR5 promotes ovarian cancer growth and metastasis through inducing immunosuppressive macrophages[J]. Nat Commun, 2020, 11(1): 6298.
34	Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P, et al. β-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma[J]. Cancer Discov, 2019, 9(8): 1124-41.
35	Dikiy S, Rudensky AY. Principles of regulatory Tcell function[J]. Immunity, 2023, 56(2): 240-55.
36	Savage PA, Klawon DEJ, Miller CH. Regulatory T cell development[J]. Annu Rev Immunol, 2020, 38: 421-53.
37	Wing JB, Tanaka A, Sakaguchi S. Human FOXP3⁺ regulatory T cell heterogeneity and function in autoimmunity and cancer[J]. Immunity, 2019, 50(2): 302-16.
38	Kang JH, Zappasodi R. Modulating Treg stability to improve cancer immunotherapy[J]. Trends Cancer, 2023, 9(11): 911-27.
39	Shan F, Somasundaram A, Bruno TC, et al. Therapeutic targeting of regulatory T cells in cancer[J]. Trends Cancer, 2022, 8(11): 944-61.
40	Togashi Y, Shitara K, Nishikawa H. Regulatory T cells in cancer immunosuppression-implications for anticancer therapy[J]. Nat Rev Clin Oncol, 2019, 16(6): 356-71.
41	Persa E, Balogh A, Sáfrány G, et al. The effect of ionizing radiation on regulatory T cells in health and disease[J]. Cancer Lett, 2015, 368(2): 252-61.
42	郭康, 刘晓敏, 戴聪, 等. UBE2T基因在乳腺癌中的差异表达及其与免疫细胞浸润之间的相关性研究: 基于生物信息学分析的结果[J]. 临床医学研究与实践, 2020, 5(34): 5-7, 15. DOI: 10.19347/j.cnki.2096-1413.202034002
43	于运亮, 李婷, 王莉莉, 等. UBE2C和UBE2T与胰腺癌患者预后及免疫细胞浸润的关系[J]. 山东医药, 2021, 61(28): 13-8. DOI: 10.3969/j.issn.1002-266X.2021.28.004
44	Pouysségur J, Marchiq I, Parks SK, et al. 'Warburg effect' controls tumor growth, bacterial, viral infections and immunity-Genetic deconstruction and therapeutic perspectives[J]. Semin Cancer Biol, 2022, 86(Pt 2): 334-46.
45	Kang H, Kim B, Park J, et al. The Warburg effect on radioresistance: survival beyond growth[J]. Biochim Biophys Acta Rev Cancer, 2023, 1878(6): 188988.
46	Gogvadze V, Zhivotovsky B, Orrenius S. The Warburg effect and mitochondrial stability in cancer cells[J]. Mol Aspects Med, 2010, 31(1): 60-74.
47	Apostolova P, Pearce EL. Lactic acid and lactate: revisiting the physiological roles in the tumor microenvironment[J]. Trends Immunol, 2022, 43(12): 969-77.
48	Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment[J]. Science, 2015, 348(6230): 74-80.
49	Park J, Hsueh PC, Li ZY, et al. Microenvironment-driven metabolic adaptations guiding CD8⁺ T cell anti-tumor immunity[J]. Immunity, 2023, 56(1): 32-42.
50	Watson MJ, Vignali PDA, Mullett SJ, et al. Metabolic support of tumour-infiltrating regulatory T cells by lactic acid[J]. Nature, 2021, 591(7851): 645-51.
51	Angelin A, Gil-de-Gómez L, Dahiya S, et al. Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments[J]. Cell Metab, 2017, 25(6): 1282-93.e7.
52	Li N, Kang YQ, Wang LL, et al. ALKBH5 regulates anti-PD-1 therapy response by modulating lactate and suppressive immune cell accumulation in tumor microenvironment[J]. Proc Natl Acad Sci U S A, 2020, 117(33): 20159-70.

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