3.1 S1PR1 mRNA expression levels in different types of cancer in humans
The Oncomine database was used to analyze S1PR1 mRNA levels in tumor tissues and normal tissues of various cancer types. S1PR1 expression was lower in most tumor tissues, including sarcoma, bladder, brain, central nervous system, breast, colorectal, leukemia, lung, myeloma, and ovarian cancer tissues, than in normal tissues (Figure 1a). The mRNA-seq data from TCGA were analyzed using TIMER to verify these findings. Data from TCGA shown that the differential expression of S1PR1 between the tumor and adjacent normal tissues is shown in Figure 1b. Compared with adjacent normal tissues, S1PR1 expression was significantly reduced in BLCA, BRCA, CHOL, COAD, ESCA, HNSC, KICH, KIRP, LIHC, LUAD, LUSC, PRAD, READ, SKCM, STAD, and UCEC. However, S1PR1 expression was significantly higher in KIRC and THCA than in adjacent normal tissues (Figure 1b). These data showed that alterations in S1PR1 expression depend on the tumor type, suggesting that this gene exerts diverse functions in various tumors.
3.2 Prognostic evaluation of S1PR1 in cancers
We investigated whether S1PR1 expression is related to prognosis. The effect of S1PR1 expression on survival was evaluated by PrognoScan. Two probes (204642_at and 239401_at) matching S1PR1 were detected. Notably, S1PR1 expression was significantly related to prognosis in two types of cancer, breast cancer and lung cancer (Figure 2a–h). In three breast cancer cohorts (GSE1456-GPL96, GSE7378, and GSE12276) (30, 31), low S1PR1 expression was significantly associated with a poorer prognosis (Figure 2a–f). We used the Kaplan-Meier plotter database to further examine the prognostic value of S1PR1 in breast cancer. Poor prognosis based on recurrence-free survival in breast cancer was significantly correlated with low S1PR1 expression (HR = 0.67, P = 7.1e-13), but a significant correlation was not observed for overall survival (HR = 0.86, P = 0.17) (Figure 2g–h). Its determined that the low expression of S1PR1 is an independent risk factor for poor prognosis of breast cancer.
In addition, low S1PR1 expression was also related to the poor prognosis in two cohorts of patients with lung cancer (GSE31210 and GSE8894), as determined using two probes (204642_at and 239401_at) (Figure 2i–k). Kaplan-Meier plotter database also shows that low expression of S1PR1 is an independent risk factor for poor prognosis of lung cancer (overall survival, HR = 0.7, P = 6.9e-08; progression-free survival, HR = 0.71, P = 0.00035) (Figure 2l–n). Furthermore, we found that low S1PR1 expression is associated with a poor prognosis in patients with soft tissue, blood, and brain cancers (Figure S1a–c). In contrast, low S1PR1 expression was an independent risk factor for a good prognosis in gastric cancer (Figure S1d–g). These results confirmed the prognostic value of S1PR1 in specific types of cancer; both high and low S1PR1 expression are associated with prognosis depending on the type of cancer. Based on the consistent results for the associations between S1PR1 expression and survival in lung and breast cancer, we focus on the precise effects of S1PR1 in these two cancer types as well as the underlying mechanisms.
3.3 Correlations between clinical characteristics and S1PR1 expression in breast and lung cancer
We used the Kaplan-Meier plotter to study the relationship between S1PR1 expression and clinical characteristics in patients with breast and lung cancer. Low expression of S1PR1was associated with worse overall survival (OS) in male and female patients with lung adenocarcinoma (P < 0.05) (Table 1). In particular, low S1PR1 mRNA expression was correlated with worse OS in stage 1 (P=9.20E-13) and early-stage (AJCC stage M) (P=0.013) lung cancer (Table 1). Low S1PR1 mRNA expression was related to poor OS in patients with (P=0.023) or without (P=0.00075) smoking (Table 1). In addition, low S1PR1 mRNA expression was related to worse OS in patients whose no received chemotherapy or radiotherapy. These findings strongly suggest that low S1PR1 mRNA expression is correlated with poor OS in lung cancer (Table 1). In BRCA, low S1PR1 mRNA expression was related to poor OS in ER-positive or HER2-negative patients and in the luminal androgen receptor subtype (Table 2). Taken together, high expression of S1PR1 could be considered a good prognostic indictor for breast and lung cancer depending on the clinical characteristics.
3.4 Regulators of S1PR1 in breast and lung cancer
Use the LinkedOmics function module to detect the S1PR1 regulatory network to further understand the biological role of S1PR1 in breast and lung cancer. Figure 4a-c shows genes with significantly positive (dark red dots) and negative (dark green dots) correlations with S1PR1 (false discovery rate, FDR < 0.01). The top 50 positively and negatively related genes are shown in a heat map in Figure 3d-f. A Gene Ontology (GO)-based gene set enrichment analysis (GSEA) showed that genes that are co-expressed with S1PR1 are enriched for vasculogenesis and the purinergic receptor signaling pathway, while genes related to mitochondria and RNA transcript processing were inhibited in breast cancer (Figure 3g). Similarly, GO annotation results showed that genes co-expressed with S1PR1 are primarily associated with vasculogenesis, the purinergic receptor signaling pathway, and the phospholipase C-activating G protein coupled receptor signaling pathway, while tRNA metabolic process, RNA modification, and RNA transcript processing were inhibited in lung cancer (Figure 3h-i). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment for hematopoietic cell lineage, Staphylococcus aureus infection, and renin secretion pathways in both breast cancer and lung cancer. Spliceosome, DNA replication, and proteasome pathways were inhibited in both tumor types (Figure 3j-i). These results suggest that S1PR1 contributes to various processes in tumor development.
3.5 Genomic alterations in S1PR1 in breast cancer and lung cancer
cBioPortal database were used to determine the types and frequencies of S1PR1 alterations in BRCA, LUAD, and LUSC. S1PR1 was altered in 4% of patients with BRCA. These alterations included mRNA missense mutations, amplifications, and deletions (Figure 4a). S1PR1 was altered in 6% of patients with LUAD and 2.3% of patients with LUSC, including mRNA missense mutations, truncating mutations, amplifications, and deletions (Figure 4a). Finally, S1PR1 CNV was associated with OS in LUAD but not with OS or DFS in BRAC and LUSC (Figure 4b–d). These results suggest that mutations in S1PR1 are associated with prognosis in LUAD.
3.6 Relationship between immune and S1PR1 expression in breast cancer and lung cancer
Tumor infiltrating lymphocytes (TIL) are lymphocytes that leave the blood circulation and migrate to the vicinity of the tumor. The amount of TIL in the tumor is an important indicator to predict the prognosis of cancer patients and the response to immunotherapy [23, 24]. Tumor purity is a key factor in analyses of immune infiltration by genomic approaches . Therefore, we use TIMER to investigate whether the expression of S1PR1 in breast cancer and lung cancer is related to immune infiltration. Firstly, we found a significant negative correlation between the S1PR1 expression level and tumor purity, as determined using TIMER, in both breast cancer and lung cancer (Figure 4a-f, Left). S1PR1 is a determinant of immune infiltration in BRCA (tumor purity; r = −0.508, P = 1.76e-66), including subtypes of BRCA (BRCA-Basal: r = -0.5411, P = 1.28e-06; BRCA-Her2: r = -0.505, P = 4.44e-06 and BRCA-Luminal: r = -0.557, P = 9.15e-46). S1PR1 is related to immune infiltration in lung cancer, including LUAD (tumor purity; r = −0.353, P = 6.05e-16) and LUSC (tumor purity; r = −0.402, P = 5.20e-20).
Furthermore, the relationship between S1PR1 and specific immune infiltrates in breast cancer and lung cancer were analyzed. The S1PR1 expression level was significantly positively correlated with levels of infiltrating CD8+ T cells (r = 0.38, P = 5.97e-35), CD4+ T cells (r = 0.335, P = 1.03e-26), macrophages (r = 0.219, P = 3.67e-12), neutrophils (r = 0.168 P = 2.03e-07), and DCs (r = 0.208, P = 9.14e-11) in BRCA (Figure 5a). In BRCA-Basal, there were slight positive correlations between S1PR1 expression levels and levels of infiltrating CD8+ T cells (r = 0.279, P = 1.76e-03) and CD4+ T cells (r = 0.237, P = 8.52e-03). Similarly, there were positive correlations with infiltrating levels of CD8+ T cells (r = 0.546, P = 1.13e-05), CD4+ T cells (r = 0.529, P = 2.00e-05), neutrophils (r = 0.342, P =8.57e-03), and DCs (r =0.488, P = 1.35e-04) in BRCA-Her2. S1PR1 expression levels were positively correlated with levels of infiltrating CD8+ T cells (r = 0.147, P = 3.43e-21), CD4+ T cells (r = 0.316, P = 6.26e-14), macrophages (r = 0.151, P = 4.14e-04), neutrophils (r = 0.147, P = 6.67e-04), and DCs (r =0.213, P = 6.44e-07) in BRCA-Luminal tumors (Figure 5a). We also found that S1PR1 expression levels were positively correlated with levels of infiltrating CD8+ T cells (r = 0.308, P = 3.61e-12), macrophages (r = 0.376, P = 1.01e-17), neutrophils (r = 0.246, P = 4.15e-08), and DCs (r =0.207, P = 4.16e-06) in LUAD. In addition, there were positive correlations with levels of infiltrating B cells (r = 0.358, P = 1.27e-15), CD8+ T cells (r = 0.459, P = 3.83e-26), CD4+ T cells (r = 0.338, P = 3.98e-14), macrophages (r = 0.586, P = 2.61e-45), neutrophils (r = 0.453, P = 1.79e-25), and DCs (r =0.56, P = 2.12e-40) in LUSC. These results strongly suggest that S1PR1 plays a special role in the immune infiltration of breast cancer and lung cancer, and has a particularly strong effect on T cells, macrophages, neutrophils and DCs. Based on the observed correlations between S1PR1 and various types of immune cells in breast cancer and lung cancer indicated that S1PR1 may have high prognostic value.
3.7 Correlations between S1PR1 expression and immune marker sets
We further evaluated the correlations between S1PR1 and markers of various immune cells in breast cancer and lung cancer using TIMER and GEPIA databases (Sup Table 1). The correlations between S1PR1 expression and immune marker genes for different immune cell populations, including CD8+ T cells, T cells (general), B cells, monocytes, TAMs, M1 and M2 macrophages, neutrophils, NK cells, DCs, and various functional T cells, such as Th1 cells, Th2 cells, Tfh cells, Th17 cells, and Tregs, as well as exhausted T cells were analyzed by TIMER. After adjusting for tumor purity, S1PR1 expression levels were significantly correlated with marker sets for various immune cells, except for NK cells, Th17, and T cell exhaustion in BRAC (Table 3 and Figure 6). However, S1PR1 expression levels were highly correlated with most immune marker sets and both T cell populations and exhausted T cells in LUAD and LUSC (Table 3 and Figure 6). We further analyzed the correlation between S1PR1 expression and the markers using the GEPIA database, including data for BRAC, LUAD, and LUSC. The results for correlations between S1PR1 and markers of immune infiltrating cells were similar to those of the TIMER analysis (Sup Table 1). This further confirms that S1PR1 is significantly related to immune infiltrating cells in lung and breast cancer, suggesting that high levels of S1PR1 induce immune activity in the lung and breast cancer microenvironment.