Melanoma NOS1 expression promotes dysfunctional IFN signaling

Abstract

In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells.

Figure 1. Modulation of IFN-α-p-STAT1 in PBMCs by melanoma cell lines.

(A) Top left: Histograms of p-STAT1 levels in 25 melanoma cell lines. Isotype, basal, and IFN-α-p-STAT1 are displayed in the top, middle, and bottom panels to exemplify IFN-α-p-STAT1 variability. Bottom left: Transwell coculture of melanoma cells and PBMCs. Right: IFN-α-p-STAT1 (top) and basal p-STAT1 (bottom) in CD4⁺, CD8⁺, and monocyte subsets of PBMCs from 4 donors in triplicate experiments after a 7-day coculture with 12 melanoma cell lines (blue bar) or alone (Mono; red bar). (B) Top: Average IFN-α-p-STAT1 levels in CD4⁺, CD8⁺, and monocyte subsets from 4 donors cocultured with 12 melanoma cell lines or alone, as shown in A. Cocultured results were ranked according to IFN-α-p-STAT1 levels (*P < 0.05, **P < 0.005, and ***P < 0.0005, Wilcoxon test): 5 cell lines with strong inhibitory effects (reduction of IFN-α-p-STAT1 by 50% compared with PBMCs cultured alone) were determined to be L-mels, and the rest H-mels. Bottom: IFN-α-p-STAT1 in PBMC subsets cocultured with L-mels or H-mels (P = 0.0005, Wilcoxon test). (C) Top: Average IFN-α-p-STAT1 levels in L-mels before and after coculture were lower than those in H-mel cell lines (Mann-Whitney U test, P = 0.048 and 0.018) before and after coculture with PBMCs (shown for individual cell lines at the bottom). IFN-α-p-STAT1 was enhanced significantly after coculture with PBMCs only in H-mels (P = 0.047, Wilcoxon test). (D) IFN-α-p-STAT1 in melanoma cells correlated with the IFN-α-p-STAT1 in respective cocultures of CD4⁺, CD8⁺, and monocyte subsets (Spearman’s correlation).

Figure 2. Transcriptional signatures specific for L-mels.

(A) PCA based on global gene expression classified 12 melanoma cell lines into three groups according to immune phenotypes. (B) Unsupervised hierarchical clustering based on global gene expression by average linkage. (C) Heatmap based on 6,771 genes differentially (Student’s t test cutoff of P < 0.05) expressed between L-mels and H-mels.

Figure 3. Functional validation of the L-mel signature on independent cell lines.

(A) Heatmap based on the same genes, but including 41 additional melanoma cell lines (red) in addition to the original 12 (L-mels, green; H-mels including S-mels, blue). The 3 red and 3 blue arrows indicate 6 cell lines (3 classified as H-mels and 3 as L-mels) randomly selected for validation of the predicted modulation on PBMCs according to the 6,771 gene signatures. (B) The 3 lines predicted to be L-mels (blue arrows) and a previously tested L-mel (888) significantly inhibited IFN-α-p-STAT1 (Wilcoxon test, P = 0.0039 for all four L-mels; *P < 0.05; **P < 0.005; ***P < 0.0005), while the 3 new (red arrows) and the previously tested H-mels did not.

Figure 4. Signal pathways affected by the L-mel signature.

(A) IFN signaling pathway according to IPA, which highlighted JAK1, JAK2, IFNA1, and IFNA2 downregulation in L-mels (green). (B) G-coupled receptor pathway according to IPA with receptor families. Gi-coupled receptors were upregulated in L-mels (red).

Figure 5. 12q22-24 amplification is a genomic marker for L-mel and targets the NOS1 gene.

(A) Top: Chromosomal amplifications (red) and deletions (blue) in L-mels (above) and H-mels (below). Differences between L-mels and H-mels focused on chr8 and chr12 (blue squares). Bottom panel: Ten segments (red triangles) identified by ANOVA comparing L-mels with H-mels (cutoff of P < 0.01, fold change of less than –2 or greater than 2). The segments are located in chr8, chr10, and chr12 (4, 1, and 5 segments, respectively). (B) Top: Copy number values in chr12q22-24 including the most significant segment and 2 other flanking segments in which the NOS1 gene is located (cutoff of P < 0.00001). Bottom: Venn diagram assembling 168 genes from 3 overlapping segments in chr12q22-24 with the 6,771 differentially expressed genes between L-mels and H-mels; 19 were in common between the two platforms, and their symbols are displayed. (C) Left: NOS1 mRNA values in L-mels (blue) were higher than those in H-mels (red) (P = 0.001, Mann-Whitney U test). Right: NOS1 protein and RNA correlation in 8 melanoma lines tested by intracellular FACS analysis. (D) Left: Correlation between NOS1 mRNA values in melanoma cells (x axis) and IFN-α-p-STAT1 levels (y axis) in corresponding cocultured CD4⁺ and CD8⁺ T cells and monocytes. Right: Correlation between NOS1 mRNA values in melanoma cells (x axis) and their IFN-α-p-STAT1 levels (y axis). All correlation analyses in this figure are based on Spearman’s correlation test.

Figure 6. Functional validation of NOS1 as a factor inducing immune suppression.

(A) Left: IFN-α-p-STAT1 in CD4⁺ and CD8⁺ cells and monocytes following inhibition with NONOate (NO donor). Middle and right: CarPITO (scavenger of NO) and L-NAME (nonselective inhibitor of NOS family) induced recovery of CD4⁺ T cell suppression by 3 L-mels. (B) Left and middle: Selective inhibitors of NOS1 (NPLA) and NOS2 (1400W) reversed the suppression of IFN-α-p-STAT1 in cocultured PBMC subsets. IFN-α-pSTAT inhibition of PBMC subsets by L-mels was reversed by both NPLA (left panel) and 1400W (right panel). (C) Left: NPLA reversed IFN-α-p-STAT1 suppression in purified CD3⁺ T cell subsets. Right: 1400W had no effect in the same conditions. (D) Left: Nitration in PBMC subsets cocultured with 3 L-mels and one with an H-mel. Middle: Nitration of PBMC subsets increased according to the NONOate concentration and was inversely correlated with IFN-α-p-STAT1. Right: Reduction of nitration in PBMC subsets by L-NAME. (E) NOS1 genetic knockdown cell line 3107s significantly restored IFN-α-p-STAT1 inhibition in CD4⁺, CD8⁺, and monocyte subsets in a coculture system compared with that in parent cell line 3107 or control cell line 3107c (paired Student’s t test, P = 0.003 for both 3107 and 3107c). In all experiments, the 3 L-mel cell lines used were 888, 3107, and A375, while the H-mel cell line was 1858. Data for L-mels are presented cumulatively for simplicity, although comparable results were obtained with each individual cell line. All experiments were repeated at least six times. *P < 0.05, **P < 0.05, and ***P < 0.005 were derived from the Wilcoxon test or Spearman’s correlation.

Figure 7. NOS1 expression by melanoma metastases.

(A) mRNA levels of IFN-α–related signaling genes in 9 melanoma metastases (left) and simultaneously collected autologous PBMCs (middle) presented as fold change compared with healthy donors’ PBMCs. Right: Scatter plot displaying correlative values for transcripts shown in the previous two panels comparing melanoma metastases (y axis) and PBMCs (x axis); n = 63. (B) Scatter plots correlating ex vivo IFN-α-p-STAT1 (left), IFN-α-p-STAT3/p-STAT1 (middle), and IFN-α-pIRF7 (right) with NOS1 expression in 8 available PBMCs (y axis). (C) Left: lack of normal distribution of NOS1 expression in melanoma metastases (P < 0.01, Kolmogorov-Smirnov normality test) and ranking of the 113 cases into high- and low-expression groups according to the mean NOS1 expression value (4.40). Right: NOS1 expression in melanoma was inversely correlated with therapeutic outcome (Spearman’s rank correlation coefficient). The high-expression NOS1 group was significantly enriched with NR cases (P = 0.011, χ² test); (D) Left: NOS1 expression in 113 melanoma metastases from patients receiving adoptive TIL therapy segregated according to response to therapy (CR, PR, and NR; P values refer to one-way ANOVA). Right: NOS1 expression in overall response (CR + PR) compared with NR cases (unpaired Student’s t test). (E) Hierarchical clustering of the 113 metastases according to NOS1, NOS2, and NOS3 displaying three groups, one with concordantly high expression of the 3 NOS genes and enriched in NRs, the other with low expression, and the third with discordant expression. All correlative analyses are based on Pearson’s correlation test.