High RNF7 expression enhances PD-1 resistance of non-small cell lung cancer cells by promoting CXCL1 expression and myeloid-derived suppressor cell recruitment via activating NF-κB signaling

doi:10.12122/j.issn.1673-4254.2024.09.10

Abstract

Abstract:

Objective To investigate the mechanism of RNF7 for regulating myeloid-derived suppressor cells (MDSCs) in non-small cell lung cancer (NSCLC). Methods TIMER2.0 database and immunohistochemistry were used to analyze RNF7 expression level and its correlation with immune cell infiltration in non-small cell lung cancer. The impact of RNF7 expression levels on prognosis of lung cancer patients was analyzed using Kaplan-Meier survival analysis. CMT-167 cells with RNF7 overexpression or knockdown were inoculated subcutaneously in C57BL/6 mice, and the mice in RNF7 knockdown group were treated with anti-PD-1 or IgG isotype control 7 days after the inoculation. The tumor tissues were harvested after 30 days for tumor volume measurement, detection of S100A8+A9 and Gr-1 expressions with immunohistochemistry, and analysis of MDSC infiltration. Gene set enrichment analysis (GSEA) was performed to identify the potential pathways regulated by RNF7 in NSCLC. Western blotting and luciferase assays were used to assess the impact of RNF7 on the NF‑κB signaling pathway. ELISA and RT-qPCR were used to measure chemokine (C-X-C motif) ligand 1 (CXCL1) expression. Results RNF7 expression was significantly upregulated in NSCLC, and high RNF7 expression levels were associated with poor prognosis of the patients (P<0.001). TIMER2.0 analysis revealed a positive correlation between RNF7 expression and MDSC infiltration (P<0.001). GSEA suggested that RNF7 was enriched in the NF-κB signaling pathway. In NSCLC cells, RNF7 knockdown significantly inhibited NF-κB activation and reduced CXCL1 expression. In the tumor-bearing mice, RNF7 overexpression significantly increased MDSC infiltration in the tumor tissue, and RNF7 knockdown obviously reduced MDSC infiltration and enhanced the efficacy of anti-PD-1 therapy. Conclusion High expression of RNF7 in NSCLC cells promotes CXCL1 expression by activating the NF-κB signaling pathway, thus leading to the chemotactic recruitment of MDSCs, which contributes to tumor resistance to anti-PD-1 therapy.

Key words: RNF7, non-small cell lung cancer, nuclear factor-κB signaling pathway, myeloid-derived suppressor cells, anti-PD-1

Na ZHONG, Huijie WANG, Wenying ZHAO, Zhengui SUN, Biao GENG. High RNF7 expression enhances PD-1 resistance of non-small cell lung cancer cells by promoting CXCL1 expression and myeloid-derived suppressor cell recruitment via activating NF-κB signaling[J]. Journal of Southern Medical University, 2024, 44(9): 1704-1711.

Figures/Tables 4

Fig. 1 RNF7 promotes lung adenocarcinoma progression. A: TIMER2.0 database analysis showing increased RNF7 mRNA expressions in multiple cancer tissues compared with the adjacent tissues. B: Survival analysis showing a close correlation between high RNF7 expression level and poor prognosis of lung cancer patients. C: RNF7 expression in lung adenocarcinoma tissues and adjacent tissues determined by immunohistochemistry. D: Kaplan-Meier survival analysis showing that patients with high RNF7 expression had shorter overall survival than those with low RNF7 expression (n=90). *P<0.05, **P<0.01, ***P<0.001.

Fig.2 RNF7 expression is positively correlated with MDSC infiltration in NSCLC. A: Correlation of RNF7 expression with MDSCs infiltration in pan-cancers predicted by TIMER2.0 database. B: Correlation of RNF7 expression with MDSCs infiltration in lung adenocarcinoma and squamous lung cancer. C: Cox regression analyses using data from TCGA indicating that high MDSC infiltration was significantly associated with poorer prognosis of NSCLC patients. D: Immunofluorescence staining of RNF7 and CD33 in lung adenocarcinoma tissue (DAPI, blue; RNF7, violet; CD33, green). E: Immunohistochemical staining of immune cell markers Gr1 and S100A8+A9 in the tissues (scale bar=50 μm). F: Infiltration of MDSCs assessed by flow cytometry in the tumors. **P<0.01.

Fig.3 RNF7 induces MDSCs recruitment by promoting NF-κB signaling pathway activation. A: GSEA analysis showing that the high expression of RNF7 in NSCLC was significantly enriched in the NF-κB signaling pathway. B: Western blotting of the key effectors in the NF-κB signaling pathway in cells with RNF7 knockdown. C: Immunoprecipitation of p-IκBα and analysis of its ubiquitination. D: Detection of protein expressions of p-65 and H3 by Western blotting. E: ELISA validation of CXCL1 levels in cell culture supernatants and RT-qPCR of CXCL1 mRNA expression in CMT-167 or H1299 cells. F: Luciferase reporter-based NF-κB activity assay. **P<0.01.

Fig. 4 RNF7 is a potential therapeutic target for immunotherapy of non-small cell lung cancer. A: Dissected tumors from each group of the mice. CMT167 Ctrl or RNF7 knockdown cells were implanted in C57BL/6 (n=5). Isotype control (IgG) or anti-PD-1 were given at 200 µg/mouse on days 7 after cell injection. B: Comparison of tumor volume among the groups. C, D: Immunohistochemical staining of immune cell markers (CD8, Gr1 and S100A8+A9) in different groups (scale bar=50 μm). E: Flow cytometry gating strategy of immune cells and the proportion of MDSCs. **P<0.01.

References 27

1	Schabath MB, Cote ML. Cancer progress and priorities: lung cancer[J]. Cancer Epidemiol Biomarkers Prev, 2019, 28(10): 1563-79.
2	Cao W, Chen HD, Yu YW, et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020[J]. Chin Med J, 2021, 134(7): 783-91.
3	Miller M, Hanna N. Advances in systemic therapy for non-small cell lung cancer[J]. BMJ, 2021, 375: n2363.
4	Sun Y, Li H. Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase[J]. Protein Cell, 2013, 4(2): 103-16.
5	Sampson C, Wang QP, Otkur W, et al. The roles of E3 ubiquitin ligases in cancer progression and targeted therapy[J]. Clin Transl Med, 2023, 13(3): e1204.
6	Huang TT, Li JW, Liu XL, et al. An integrative pan-cancer analysis revealing the difference in small ring finger family of SCF E3 ubiquitin ligases[J]. Front Immunol, 2022, 13: 968777.
7	Gu QY, Bowden GT, Normolle D, et al. SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IkappaB-alpha/NF-kappaB[J]. J Cell Biol, 2007, 178(6): 1009-23.
8	Goenka A, Khan F, Verma B, et al. Tumor microenvironment signaling and therapeutics in cancer progression[J]. Cancer Commun, 2023, 43(5): 525-61.
9	Sui HS, Dongye SY, Liu XC, et al. Immunotherapy of targeting MDSCs in tumor microenvironment[J]. Front Immunol, 2022, 13: 990463.
10	Hegde S, Leader AM, Merad M. MDSC: markers, development, states, and unaddressed complexity[J]. Immunity, 2021, 54(5): 875-84.
11	Ma J, Xu HX, Wang SJ. Immunosuppressive role of myeloid-derived suppressor cells and therapeutic targeting in lung cancer[J]. J Immunol Res, 2018, 2018: 6319649.
12	Li MSC, Mok KKS, Mok TSK. Developments in targeted therapy & immunotherapy-how non-small cell lung cancer management will change in the next decade: a narrative review[J]. Ann Transl Med, 2023, 11(10): 358.
13	Tao YR, Dai LR, Liang WL, et al. Advancements and perspectives of RBX2 as a molecular hallmark in cancer[J]. Gene, 2024, 892: 147864.
14	Li K, Shi HH, Zhang BX, et al. Myeloid-derived suppressor cells as immunosuppressive regulators and therapeutic targets in cancer[J]. Signal Transduct Target Ther, 2021, 6(1): 362.
15	Li YN, Qiao KL, Zhang XY, et al. Erratum: targeting myeloid-derived suppressor cells to attenuate vasculogenic mimicry and synergistically enhance the anti-tumor effect of PD-1 inhibitor[J]. iScience, 2022, 25(10): 105281.
16	Papadaki MA, Aggouraki D, Vetsika EK, et al. Epithelial-to-mesenchymal transition heterogeneity of circulating tumor cells and their correlation with MDSCs and tregs in HER2-negative metastatic breast cancer patients[J]. Anticancer Res, 2021, 41(2): 661-70.
17	Adah D, Hussain M, Qin LM, et al. Implications of MDSCs-targeting in lung cancer chemo-immunotherapeutics[J]. Pharmacol Res, 2016, 110: 25-34.
18	Xiong XF, Mathewson ND, Li H, et al. SAG/RBX2 E3 ubiquitin ligase differentially regulates inflammatory responses of myeloid cell subsets[J]. Front Immunol, 2018, 9: 2882.
19	Su L, Zhang F, Liu MX, et al. The Tian-Men-Dong Decoction suppresses the tumour-infiltrating G-MDSCs via IL-1β-mediated signalling in lung cancer[J]. J Ethnopharmacol, 2023, 313: 116491.
20	Li H, Tan MJ, Jia LJ, et al. Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis[J]. J Clin Invest, 2014, 124(2): 835-46.
21	Inui T, Watanabe M, Nakamoto K, et al. Bronchial epithelial cells produce CXCL1 in response to LPS and TNFα: a potential role in the pathogenesis of COPD[J]. Exp Lung Res, 2018, 44(7): 323-31.
22	Hoesel B, Schmid JA. The complexity of NF‑κB signaling in inflammation and cancer[J]. Mol Cancer, 2013, 12: 86.
23	Zhang LL, Ludden CM, Cullen AJ, et al. Nuclear factor kappa B expression in non-small cell lung cancer[J]. Biomed Pharmacother, 2023, 167: 115459.
24	Tang HC, Li H, Sun ZJ. Targeting myeloid-derived suppressor cells for cancer therapy[J]. Cancer Biol Med, 2021, 18(4): 992-1009.
25	Kumar V, Patel S, Tcyganov E, et al. The nature of myeloid-derived suppressor cells in the tumor microenvironment[J]. Trends Immunol, 2016, 37(3): 208-20.
26	Highfill SL, Cui YZ, Giles AJ, et al. Disruption of CXCR2-mediated MDSC tumor trafficking enhances anti-PD1 efficacy[J]. Sci Transl Med, 2014, 6(237): 237ra67.
27	Barry ST, Gabrilovich DI, Sansom OJ, et al. Therapeutic targeting of tumour myeloid cells[J]. Nat Rev Cancer, 2023, 23(4): 216-37.

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