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.

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