As shown in Table 1, 376 cases of primary OSC with clinical and gene expression data were downloaded from the TCGA database. Among these, 178 patients (47.3%) were aged >60 years. Federation of Gynecology and Obstetrics (FIGO) stage I disease was found in 1 patient (0.3%), stage II in 22 (5.9%), stage III in 293 (78.6%), and stage IV in 57 (15.3%). The histological grades, G1, G2, G3, and G4 were found in 0.3%, 11.5%, 88.0%, and 0.3% of the patients, respectively. The cancer status included 71 without (21.3%) and 262 with (78.7%) tumors. The patients were divided into four groups according to the primary therapy outcome: progressive disease (PD), 27 (8.9%); partial response (PR), 43 (14.1%); stable disease (SD), 22 (7.2%); and complete response (CR), 213 (69.8%). Residual tumors were found in 267 of the 376 total cases (23.5%), lymphatic invasion in 100 of 148 cases (67.6%), and venous invasion in 63 of 103 cases (61.2%). Lesions occurred in the bilateral ovaries of 253 cases. Mutations of TP53 and breast cancer susceptibility genes (BRCA) were found in 248 and 25 cases, respectively. Of the patients in this cohort, 61.2% finally succumbed to OSC (Table 1).
We compared the hazard ratios (HR) with 95% confidence interval (CI) ranges among patient information parameters. Univariate analysis identified primary therapy outcome (p < 0.001), age (p = 0.032), residual tumor (p < 0.001), ATP1A3 (p < 0.001), and ATP1A4 (p = 0.03) as prognostic factors for OS (Table 2). Multivariate analysis showed that primary therapy outcome (p < 0.001), residual tumor (p = 0.004), ATP1A2 (p = 0.006), ATP1A3 (p < 0.001), and ATP1A4 (p < 0.001) were independent risk factors for OS (Table 2).
Univariate analysis identified primary therapy outcome (p < 0.001), residual tumor (p < 0.001), ATP1A3 (p = 0.002), and ATP1A4 (p = 0.023) as prognostic factors for DSS (Table 3). Multivariate analysis showed that primary therapy outcome (p < 0.001), residual tumor (p = 0.001), ATP1A3 (p = 0.006), and ATP1A4 (p = 0.017) were independent risk factors for DSS (Table 3).
Correlation between expression of ATP1A genes and the clinical characteristics
The expression of ATP1A gene family members in OSC and normal tissues were significantly different. ATP1A1 and ATP1A3 were highly expressed in tumor tissues, whereas ATP1A2 and ATP1A4 were highly expressed in normal tissues (all p < 0.001) (Figure 1A–D). Based on the OV-AU dataset, we found that ATP1A2 was highly expressed in OC tissues (p < 0.001), whereas ATP1A3 was highly expressed in normal tissues (p = 0.002). In addition, there was no difference in the expression of ATP1A1 and ATP1A4 between OC and normal tissues (Supplementary Figure 1A–D). To evaluate the expression of the ATP1A gene family members in human pan-cancers, the RNA-Seq data from the TCGA and GTEx databases were further analyzed. Differential expression between the tumor and adjacent normal tissues for ATP1A1-4 across all TCGA tumors is shown in Supplementary Figure 2. ATP1A1 expression was significantly higher in cholangiocarcinoma, skin cutaneous melanoma and thymoma (THYM) (all p < 0.001) than in the adjacent normal tissues. ATP1A2 expression was significantly lower in breast invasive carcinoma, bladder urothelial carcinoma and colon adenocarcinoma (all p < 0.001) than in the adjacent normal tissues. ATP1A3 expression was significantly higher in adrenocortical carcinoma (p < 0.001), THYM (p < 0.001) and pheochromocytoma and paraganglioma (p < 0.01) than in the adjacent normal tissues. ATP1A4 expression was significantly higher in BRCA, lymphoid neoplasm diffuse large B-cell lymphoma and lung squamous cell carcinoma (all p < 0.001) (Supplementary Figure 2). In addition, the mRNA expression of the ATP1A gene family members in pan-cancer cell lines was also analyzed based on the CCLE database (Supplementary Figure 3). As observed, ATP1A1 was highly expressed in Ewing’s-sarcoma, melanoma, and colorectal carcinoma, whereas ATP1A3 was highly expressed in neuroblastoma, Burkitt lymphoma, and T-lymphocytic leukemia. Conversely, the expression of ATP1A2 and ATP1A4 was lower in most tumor cell lines (Supplementary Figure 3).
Next, we analyzed the sensitivity and specificity of the ATP1A gene family members in predicting the diagnosis of OSC by ROC curve. The area under the curve (AUC) for ATP1A1, ATP1A2, ATP1A3, and ATP1A4 were 0.829, 0.963, 0.892 and 0.778, respectively (Figure 1E–H). The results from the OV-AU dataset are consistent with those from the TCGA database. The AUC for ATP1A1, ATP1A2, ATP1A3, and ATP1A4 in patients with OC were 0.555, 0.729, 0.704, and 0.507, respectively (Supplementary Figure 1E–H). These results suggested that the ATP1A gene family members are potential diagnostic biomarkers for both OSC and OC.
Moreover, we analyzed the correlation between the expression of the ATP1A gene family members and the clinical features, as revealed by the Kruskal–Wallis test and Wilcoxon signed-rank test. Significant correlation was noted between ATP1A3 (p = 0.019) and FIGO stage, but no significant correlations were found between ATP1A1 (p = 0.395), ATP1A2 (p = 0.492), ATP1A4 (p = 0.07), and FIGO stage (Figure 2A–D). In addition, ATP1A3 (p < 0.001) was significantly associated with histological grade, whereas ATP1A1 (p = 0.913), ATP1A2 (p = 0.716), and ATP1A4 (p = 0.727) were not (Figures 2E–H). Although no correlation was found between ATP1As and lymphatic invasion, the p value of ATP1A3 was close to 0.05, suggesting that the lack of significance may be due to a limitation of the number of patients (Figures 2I–L). By analyzing the GSE26193 dataset, no significant correlations were found between ATP1A1 (p = 0.21), ATP1A2 (p = 0.35), ATP1A3 (p = 0.9), ATP1A4 (p = 0.59), and FIGO stage (Supplementary Figure 4A–D). In addition, we found a significant correlation between ATP1A1 and histological grade (p = 0.008), whereas ATP1A2 (p = 0.87), ATP1A3 (p = 0.882), and ATP1A4 (p = 0.453) were not (Supplementary Figure 4E–H).
Based on these mRNA expression patterns of the ATP1A gene family members in OSC, we next explored the protein expression patterns of ATP1As in OSC using HPA. As shown in Figure 3, ATP1A1 was highly expressed in the OSC tissue than in the normal ovarian tissues (Figure 3A). In addition, the protein expression of ATP1A2 and ATP1A3 was low in both normal and OSC tissues (Figure 3B, C). A lack of protein expression patterns of ATP1A4 genes in the database at present precluded our analysis of this protein.
Prognostic value of mRNA expression of ATP1A gene family members in patients with OSC
We used a Kaplan–Meier plotter (http://kmplot. com/analysis/) to analyze the prognostic values of the mRNA expression of ATP1A genes in both patients with OSC and OC patients. First, we analyzed the relationship between the mRNA expressions of distinct ATP1A gene family members and the prognoses of patients with OSC. As shown in Figure 4, for OS and DSS, a higher mRNA expression of ATP1A3 (HR = 1.60, CI: 1.23-2.08, p < 0.001; and HR = 1.57, CI: 1.19-2.08, p = 0.002, respectively) (Figure 4C, D) and ATP1A4 (HR = 0.75, CI: 0.57-0.97, p =0.030; and HR = 0.72, CI: 0.54-0.96, p = 0.023, respectively) (Figure 4G, H) were significantly associated with shorter OS and DSS in patients with OSC. However, the mRNA expression of neither ATP1A1 (HR = 1.15, CI: 0.88-1.49, p =0.298; and HR = 1.13, CI: 0.86-1.50, p =0.377, respectively) (Figure 4A, B) nor ATP1A2 (HR = 1.26, CI: 0.97-1.63, p =0.086; and HR = 1.30, CI: 0.98-1.73, p =0.064, respectively) (Figure 4E, F) showed any correlation with OS or DSS in patients with OSC. Moreover, the results of survival analysis from the GSE26193 datasets are showed in Supplementary Figure 5. It was found that higher mRNA expressions of ATP1A3 (HR = 1.86, CI: 1.13-3.07, p =0.014; and HR = 1.74, CI: 1.07-2.81, p =0.023, respectively) and ATP1A4 (HR = 1.25, CI: 1.02-1.54, p =0.029; and HR = 2.17, CI: 1.25-3.77, p = 0.0047, respectively) were significantly associated with shorter OS and progression-free survival (PFS) in patients with OC. In contrast, a higher mRNA expression of ATP1A1 was associated with longer OS or PFS (HR = 0.58, CI: 0.33-1.01, p =0.053; and HR = 0.51, CI: 0.3-0.87, p =0.011, respectively) (Supplementary Figure 5). These results suggested that the mRNA expressions of ATP1A3 and ATP1A4 were closely related to the prognosis of OC. They may be used as potential biomarkers to predict the survival of patients.
Secondly, we analyzed the effects of distinct ATP1A gene family member expression on patients’ prognosis in the subgroups of FIGO stage III, histological grade G3, TP53 mutation, and age ≥60 years. The results showed that in patients with FIGO stage III, no significant correlation was found in ATP1A1 (HR = 1.16, CI: 0.87-1.56, p = 0.315), ATP1A2 (HR = 1.24, CI: 0.92-1.66, p = 0.157), and ATP1A4 (HR = 0.77, CI: 0.58-1.04, p = 0.085), whereas the high expression of ATP1A3 (HR = 1.70, CI: 1.27-2.28, p < 0.001) was significantly associated with shorter OS (Figure 5A–D). Higher mRNA expressions of ATP1A2 (HR = 1.34, CI: 1.01-1.78, p = 0.042) and ATP1A3 (HR = 0.75, CI: 1.30-2.30, p < 0.001) were significantly associated with poor OS in patients with histological grade G3, whereas no such association was noted for ATP1A1 (HR = 1.20, CI: 0.91-1.59, p = 0.202) and ATP1A4 (HR = 0.79, CI: 0.60-1.06, p = 0.113) were not (Figure 5E–H). In addition, the high expression of ATP1A3 (HR = 1.62, CI: 1.17-2.26, p = 0.004) was significantly associated with decreased OS in patients with TP53 mutation, but no correlation was observed with ATP1A1 (HR = 1.33, CI: 0.95-1.84, p = 0.094), ATP1A2 (HR = 0.98, CI: 0.70-1.36, p = 0.881) and ATP1A4 (HR = 0.68, CI: 0.49-0.95, p = 0.023) (Figure 5I–L). Furthermore, we found that the high expression of ATP1A2 (HR = 1.48, CI: 1.02-2.15, p = 0.039) and ATP1A3 (HR = 2.23, CI: 1.54-3.24, p < 0.001) was significantly associated with poor OS in patients aged ≥60 years old, whereas no such correlation was observed with ATP1A1 (HR = 1.15, CI: 0.80-1.65, p = 0.460). Conversely, we found that the high expression of ATP1A4 (HR = 0.66, CI: 0.46-0.95, p = 0.027) was correlated with better OS (Figure 5M–P). Finally, the effects of ATP1A gene family members on DSS in patients in subgroups are shown in Supplementary Figure 6.
Based on the results of multivariate analysis, a genomic–clinical nomogram, including primary tumor therapy outcome, residual tumor, ATP1A2, ATP1A3, and ATP1A4, was established to predict the 1-, 3-, and 5-year OS and DSS in patients with OC (Figure 6A, C). The calibration curves of the nomogram for predicting these survival times indicated that it performed well (Figure 6B, D).
To further validate our data, we tested the protein expression of the ATP1A gene family members (including ATP1A1, ATP1A2, and ATP1A3) in cancer and paracancerous tissue samples of six patients with OSC. Among these six patients, one had FIGO stage IIIA and the other five had stage IIIC. No patient had lymph node metastasis. Our results showed that ATP1A1 was positively expressed in three OSC samples and negatively expressed in all the paracancerous samples; ATP1A2 was negatively expressed in both the OSC and paracancerous samples; ATP1A3 was positively expressed in five OSC samples and three paracancerous samples. Images of ATP1A gene family member expression in tumor and paracancerous tissues are presented in Figure 7.
Mutation frequency of ATP1A gene family members
Based on the cBioPortal database, we explored the mutation frequency of the ATP1A gene family members. The mutation rates of ATP1A1, ATP1A2, ATP1A3, and ATP1A4 were 4%, 5%, 3%, and 4%, respectively (Figure 8).