NOS3 mRNA expression in various normal tissues and tumours.
To comprehensively analyse NOS3 expression and distribution in human normal tissues and tumour tissues, we first analysed NOS3 mRNA expression level in 30 different normal tissues from GTEx and 33 different tumour tissues from Xena (https://xenabrowser.net/). The expression of NOS3 was highly variable across different normal tissues (Figure 1A) and tumour tissues (Figure 1B). In normal tissues, the median NOS3 expression levels varied from 5.624 (blood) to 12.8 (spleen). Tissues with the highest NOS3 expression were spleen (12.8±1.391), heart (10.64±1.099), testis (10.59±0.532), adipose tissue (10.5±0.8709), and breast (10.42±0.8376). Tissues with the lowest NOS3 expression were blood (5.624±1.325), skin (7.904±1.753), pancreas (8.363±1.159), muscle (8.415±1.022), and ovary (8.453±0.9167). In tumour tissues, NOS3 expression levels varied from 9.85 (stomach adenocarcinoma, STAD) to 5.071 (acute myeloid leukeamia, LAML). Tumour tissues with the highest NOS3 expression were STAD (9.85±1.018), kidney renal clear cell carcinoma (KIRC, 9.848±0.9791), pancreatic adenocarcinoma (PAAD, 9.297±0.8167), testicular germ cell tumours (TGCT, 9.285±0.6801), and oesophageal carcinoma (ESCA, 9.275±1.208). Tumour tissues with the lowest NOS3 expression were LAML (5.071±1.591), kidney renal papillary cell carcinoma (KIRP, 7.173±1.14), uveal melanoma (UVM, 7.278±0.9764), cervical and endocervical cancers (CESC, 7.388±1.054), and thymoma (THYM, 7.439±0.987).
NOS3 is differentially expressed in various tumours and their corresponding normal tissues.
We analysed NOS3 mRNA expression levels across tumours and their corresponding normal tissues in 21 tumour types that had three or more normal tissues data (Figure 2A). NOS3 was expressed at certain level in all tumours and normal tissues. NOS3 mRNA expression in 7 of 21 tumour types, colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), KIRC, prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), STAD, and thyroid carcinoma (THCA), was much higher than that in corresponding normal tissues, with statistical significance. In 8 of 21 tumour types, bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), CESC, kidney chromophobe (KICH), KIRP, liver hepatocellular carcinoma (LIHC), PAAD, and uterine corpus endometrial carcinoma (USEC), NOS3 mRNA expression was lower than that in corresponding normal tissues. The different expression levels among tumours indicated that NOS3 might play an anti-tumour or pro-tumour role depending on the tumour type. Further analyses of fold change between tumour and corresponding normal tissues found that NOS3 was most differentially expressed in CESC, with a more than 4-fold decrease in tumour tissues (log2 FC=2.92, p<0.0001) (Figure 2B, 2C). NOS3 expression in KIRP tumour tissues was also 4-fold decreased compared with corresponding normal tissues (log2 FC=2.36, p<0.0001). In addition, our analysis found that NOS3 mRNA expression levels in CESC and KIRP were relatively low among the 33 tumour types (Figure 1B). However, it was worth noting that there were only three corresponding normal tissues for CESC, which in some content might result in a deviation in the differential expression analyses. In addition, NOS3 expression in both STAD and READ tumour tissues was more than 2-fold greater than that in the corresponding normal tissues. Surprisingly, STAD tumour tissues had the highest NOS3 expression level of the 33 tumour types.
Association between NOS3 mRNA expression and clinical phenotypes.
We analysed the association between NOS3 expression and tumour stage in 25 tumour types that had stage information in TCGA. First, we analysed NOS3 expression across four stages by means of ANOVA analyses on 25 tumours combined and found that NOS3 was differentially expressed across stages (Figure 3A). Patients in stage III expressed the highest NOS3 levels. Subsequently, stages I and II were combined as early stage, and stages III and IV were combined as advanced stage. T-test analyses showed that NOS3 expression was higher in the advanced stage (Figure 3B, p<0.0001). Further analyses of different tumour types found that BLCA, BRCA, COAD, ESCA, KIRP, and skin cutaneous melanoma (SKCM) tumours in advanced tumour stage expressed higher NOS3 mRNA levels. In contrast, KIRC and THCA tumours in the advanced tumour stage expressed lower NOS3 mRNA levels (Figure 3C).
To analyse the relationship between NOS3 expression and overall survival of tumour patients, log-rank test was performed in 33 tumour types (Figure 4A, B). We found that NOS3 mRNA expression level was related to a worse prognosis inpatients with KIRP [median survival (low versus high): not reached versus 2941, p=0.0043, hazard ratio (HR) =2.441], mesothelioma (MESO, median survival: 732 versus 456.5, p=0.0032, HR=1.945), and STAD (median survival: 1686 versus 801, p=0.0133385, HR=1.394). While it was related to a better prognosis in patients with KIRC [median survival: 2386 versus not reached, p=0.0133, HR=0.6835] and lung adenocarcinoma (LUAD, median survival: 1454 versus 1622, p=0.0284, HR=0.7228). Moreover, KIRP had the largest HR with a 95% confidence index between 1.35 and 4.414. Besides, we found that high expression level of NOS3 was associated with both advanced tumour stage and worse prognosis in KIRP patients and was associated with early tumour stage and better prognosis in KIRC patients, indicating the potential clinical significance of NOS3 in KIRP and KIRC.
NOS3 mRNA expression in tumour cell lines.
Considering that tissue-based RNA expression detection might be complicated by the non-tumour tissues that are adjacent to tumour cells, we analysed NOS3 mRNA expression in1457 cell lines derived from 26 tumour types in the CCLE database. Initially, NOS3 expression in different cell lines was checked and the results showed that cell lines from STAD and COAD were the top two cell lines expressing the highest levels of NOS3 mRNA, and cell lines from nerve system tissues (e.g., GBM and neuroblastoma) and bone tissues (e.g., chondrosarcoma and osteosarcoma) expressed relatively lower NOS3 mRNA (Figure 5). Interestingly, NOS3 in STAD was expressed at the highest level both in stomach tissues from TCGA and in stomach cell lines from CCLE. Further analysis of the association between NOS3 mRNA and promoter DNA methylation level showed a weak correlation (Spearman correlation coefficient=0.1282, p=0.0002, Figure 5B). Spearman correlation analysis between NOS3 mRNA and copy number did not show statistical significance (p=0.1193, Figure 5C). These results indicated that promoter DNA methylation and copy number variants of the NOS3 gene might not be the main determinant of NOS3 mRNA levels.
Association between NOS3 expression and drug sensitivity
To investigate the correlation between NOS3 mRNA expression and drug sensitivity, NOS3 expression in 664 cell lines and drug response to 482 drugs were analysed. Spearman correlation analysis revealed that, only the response to one drug, ‘SR8278’, was considered to be moderately correlated with NOS3 mRNA expression, with a correlation coefficient greater than 0.3 (Figure 6A). A negative correlation indicated that a better response (smaller response AUC value) was correlated with increased expression of NOS3. ‘SR8278’ is an antagonist of the transcription factor REV-ERBα, affecting its circadian and metabolic functions. Two other drugs, ‘GSK.J4’ and ‘CIL55A’ had a correlation coefficient greater than 0.2 and were also negatively correlated.
Subsequently, Spearman analysis were performed to investigate the correlation of NOS3 expression with drug response separately in cell line types with at least 15 cell lines (a total of 10 cancer cell line types) and the drug response (correlation coefficient > 0.3) ratios were calculated. In these cancer cell line types, NOS3 mRNA expression was related to at least one drug, however, the response ratios were quite different and varied depending on the different cell line types (Figure 6B). The response ratios in breast and ovary tumour cell lines were more than 10%. Both breast and ovarian tumour occurred in females and shared some common pathological mechanisms, such as those closely related to female hormones. Therefore, we further analysed the common drugs whose responses were correlated with NOS3 expression in both breast and ovarian tumour cell lines (Figure 6C). The results showed that they shared six common drugs. Among these drugs, LY-2157299, a TGF-β1 inhibitor, was strongly associated with NOS3 expression in breast and ovarian tumour cell lines (the Spearman correlation coefficients were -0.64848 and -0.9, respectively). However, ‘SR8278’ was not contained.