Ubiquitin-specific protease 1 facilitates tumor immune escape from natural killer cells and predicts the prognosis in small cell lung cancer

Abstract

Objective: Small cell lung cancer (SCLC) is commonly recognized as the most fatal lung cancer type. Despite substantial advances in immune checkpoint blockade therapies for treating solid cancers, their benefits are limited to a minority of patients with SCLC. In the present study, novel indicators for predicting the outcomes and molecular targets for SCLC treatment were elucidated.

Methods: We conducted bioinformatics analysis to identify the key genes associated with tumor-infiltrating lymphocytes in SCLC. The functional role of the key gene identified in SCLC was determined both in vitro and in vivo.

Results: A significant correlation was observed between patient survival and CD56dim natural killer (NK) cell proportion. Furthermore, we noted that the hub gene ubiquitin-specific protease 1 (USP1) is closely correlated with both CD56dim NK cells and overall survival in SCLC. Bioinformatics analysis revealed that USP1 is upregulated in SCLC. In addition, gene set enrichment analysis revealed that USP1 overexpression hinders NK cell-mediated immune responses. By co-cultivating NK-92 cells with SCLC cells, we demonstrated that NK cell cytotoxicity against SCLC could be improved either via USP1 knock-down or pharmacological inhibition. Furthermore, using a nude-mice xenograft tumor model, we noted that USP1 inhibition effectively suppressed tumor proliferation and increased the expression of NK cell-associated markers.

Conclusions: Our study findings highlight the importance of NK cells in regulating SCLC. USP1 overexpression can inhibit NK cell-mediated immunity; therefore, USP1 may serve not only as a prognostic biomarker but also as a potential molecular target of SCLC therapy.

Keywords: Immune escape; Natural killer (NK) cell; Prognosis; Small cell lung cancer (SCLC); Ubiquitin-specific protease 1 (USP1).

Figure 1. CD56^dim NK cell abundance was an independent prognostic factor for patients with SCLC. (A) Kaplan-Meier curves depicting the overall survival in the George’s cohort (Log-rank test). (B) Kaplan-Meier curves depicting the overall survival in the GSE60052 cohort (Log-rank test). (C) Multivariate Cox regression analysis conducted on the George’s cohort to analyze the impact of CD56^dim NK cell abundance on overall survival. (D) Multivariate Cox regression analysis conducted on the GSE60052 cohort to analyze the impact of CD56^dim NK cell abundance on overall survival.

Figure 2. USP1 was identified as the hub gene correlated with CD56^dim NK cells. (A) The flowchart for hub gene identification. (B) Venn diagram of hub genes identification. (C) USP1 expression was notably correlated to overall survival (Log-rank test). (D) Multivariate Cox regression analysis revealed that USP1 gene expression was an independent prognostic factor for overall survival.

Figure 3. USP1 overexpression inhibited NK cell-mediated immunity. (A) USP1 expression was compared between tumor and normal samples in the GSE149507 cohort. The statistical significance was determined using the Mann-Whitney U test. ****p ≤ 0.0001. (B) USP1 expression was compared between SCLC, NSCLC, and normal lung samples in Oncomine database. The statistical significance was determined using the Mann-Whitney U test. **p ≤ 0.01; ****p ≤ 0.0001. (C) The expression of USP1 was evaluated in various types of cancer cell lines from the CCLE database. (D) GSEA analysis was performed to analyze NK cell-related pathways.

Figure 4. USP1 inhibition enhanced SCLC sensitivity to NK cell cytotoxicity. (A) Western blotting was performed to assess USP1 expression in NCI-H446 cells following siRNA transfection. (B) The cytotoxicity of NK-92 cells against NCI-H446 cells was measured using LDH assay at different effector–target (E:T) ratios. The unpaired t-test was used to perform statistical analysis (**p < 0.01). (C) Flow cytometry was employed to detect apoptosis in NCI-H446 cells co-cultured with NK-92 cells after siRNA transfection. (D) The apoptosis rate was calculated using the mean and standard deviation. The unpaired t-test was used to perform statistical analysis (ns, not significant; **p < 0.01). (E) Immunofluorescence assay was conducted on NCI-H446 cells after exposure to different doses of ML323 (Scale bar, 10 μm). (F) Western blotting was performed to assess the expression of USP1. (G) Flow cytometry was utilized for apoptosis detection in NCI-H446 cells co-cultured with NK-92 cells after treatment with ML323. (H) The apoptosis rate was calculated using the mean and standard deviation. The unpaired t-test was used to perform statistical analysis (ns, not significant; **p < 0.01).

Figure 5. USP1 inhibition suppressed SCLC growth in vivo. (A) NCI-H446 cells (1 × 10⁷) were subcutaneously injected into athymic nude mice. Nine days after tumor transplantation, mice were intratumorally injected with either ML323 or PBS daily for 11 days. (B) Representative pictures depicting the excised subcutaneous tumors on day 28. (C) Subcutaneous tumor size was measured every 3 days using calipers. Tumor volume was calculated using the following formula: (length × width²)/2. The calculation was performed using mean and standard deviation. The unpaired t-test was used to perform statistical analysis (***p < 0.001). (D) Tumor weight was measured during harvest; the results were presented as mean ± standard deviation (unpaired t-test, ***p < 0.001). (E) Representative pictures of immunohistochemical staining of NCR1 in SCLC (Scale bar, 100 μm).

Figure S1. GSEA revealed that USP1 was negatively associated with NK cell related pathways, including KEGG natural killer cell mediated cytotoxicity (A), Go natural killer cell activation (B) and Go natural killer cell mediated immunity (C).