EPOR mRNA level: A valuable prognostic indicator for patients with ER+ breast cancer

Jing Pan, Xing-Hua Han,2, Wei Wang,2, Yue-Yin Pan,

1Department of Medical Oncology, Anhui Provincial Hospital, Anhui Medical University, Hefei 230001, P.R. China

2Department of Medical Oncology, The First Affiliated Hospital of University of Science and Technology of China, Hefei 230001, P.R. China

1. Introduction

Breast cancer (BRCA) is one of the leading causes of cancer death among women worldwide and the number of newly diagnosed is gradually increasing in recent years[1,2]. To further acquire early detection and improve the treatment effect of advanced breast cancer, a great deal of efforts has been made. But unfortunately, the mortality of breast cancer patients is still maintaining higher and becoming a globally difficult question[3,4]. Recently, accumulating studies have demonstrated that exploration and validation of newly biomarkers or prognostic factors could improve clinical outcomes of breast cancer patients to some extent.

Erythropoietin receptor (EPOR) is a transmembrane protein of 484 amino acids and a calculated mass of 52.6 kDa[5], which increases to about 60 kDa due to glycosylation and phosphorylation.Recently, many studies focus their attention on this gene mainly due to the clinical use of recombinant human erythropoietin (rHuEPO)in cancer patients. In 2003, two large randomized controlled trials highlighting unexpected and deleterious effects of therapeuticallyadministered rHuEPO were published[6,7]. Starting from this, many controversies and conflicting conclusions have been gradually discovered. Some studies have found that rHuEPO can stimulate the EPOR expression level through activating JAK2-STAT5 or PI3KAKT signal pathway and high EPOR expression will induce tumor growth and metastasis in many kinds of malignances[8-11]. On the contrary, other studies have revealed that there is no influence of rHuEPO use on the EPOR expression, and even these corresponding authors have detected the low expression level or no expression of EPOR in malignancies[12-16]. They pointed out the potential reason may be partly associated with non-specificity of the antibodies used for detection of EPOR protein[17,18]. As for breast cancer, a similar paradoxical phenomenon exists as the above. Therefore, it is rather necessary to explore the expression of EPOR in breast cancer from the aspect of mRNA and whether it has a prognostic value.

Thus, in the present study, The Cancer Genome Atlas (TCGA)and GTEx data were collected in GEPIA initially to identify the dysregulated gene EPOR. Further, bc-GenExMiner 4.1 online bioinformatics tool was used to evaluate EPOR mRNA differential expression level according to different classification of clinicopathologic parameters in patients with breast cancer.Additionally, the prognostic value between EPOR mRNA level and free survival of metastatic relapse (MR) or any event (AE, namely any relapse or death) in patients with breast cancer was investigated.

2. Materials and methods

2.1. Mining of dysregulated EPOR gene in breast cancer

GEPIA (Website: http://gepia.cancer-pku.cn/) is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9 736 tumors and 8 587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline[19]. Initially,to explore whether EPOR mRNA levels have the significance of differential expression between breast cancer and normal tissues,GEPIA was used to draw the “Expression on Box Plots” under the selection of “Breast Cancer (BRCA) Datasets”. Besides, differential expression levels of EPOR mRNA in BRCA patients were analyzed by bc-GenExMiner v4.1 (breast cancer Gene-Expression Miner v4.1, website: http://bcgenex.centregauducheau.fr/BC-GEM/GEMAccueil.php?js=1), a large free and open source database containing information about 5 861 cases of BRCA patients[20,21], based on different kinds of classified parameters: Receptor statuses (ER+ vs.ER-, PR+ vs. PR-, HER2+ vs. HER2-), Nodal status (N+ vs. N-),SBR (SBR1 vs. SBR2 vs. SBR3), NPI (NPI1 vs. NPI2 vs. NPI3),Age (≤40/40-70/≥70 and ≤51/＞51), Molecular subtypes, Basal-like(PAM50) and/or TNBC.

2.2. Correlation between EPOR mRNA expression levels and survival of breast cancer patients by bioinformatic analysis

To explore the correlation between EPOR mRNA level and the risk of MR or AE, MR-free and AE-free survival in BRCA patients,meta-analysis was done further through using bc-GenExMiner 4.1. Besides, subgroup analysis was done based on ER status, or molecular subtypes, or two types of molecular subtype predictors(SSP and SCM), respectively. The prognostic significance of EPOR mRNA level in BRCA patients was assessed by using univariate Cox regression model and drawing Kaplan-Meier curve and forest plot.Additionally, NPI-, AOL- and proliferation- adjusted analyses were further performed to verify the independent prognostic significance of EPOR mRNA in breast cancer.

3. Results

3.1. EPOR mRNA was downregulated in BRCA compared to normal tissues

Through searching the TCGA and GTEx data in GEPIA, we found that EPOR mRNA was dramatically downregulated in BRCA (1 085 cases) compared to normal tissues (291 cases) (P ＜0.05, Figure 1A). Then, in order to further know whether or not significantly differential expression levels of EPOR mRNA existed in breast cancer patients based on different kinds of classified parameters, bc-GenExMiner v4.1 was employed. The results showed that there were remarkably differential expression levels of EPOR mRNA in ER+vs. ER- patients (ER+ ＞ ER-, Figure 1B), PR+ vs. PR- patients (PR+＞ PR-, Figure 1C), NPI1 vs. NPI2 vs. NPI3patients (NPI1 ＞ NPI2 ＞NPI3), SBR1 vs. SBR2 vs. SBR3patients (SBR1 ＞ SBR2 ＞ SBR3),Basal-like vs. Not basal-like patients (Not basal-like ＞Basal-like,Figure1H), Basal-like and TNBC vs. Not basal-like and not TNBC patients (Not basal-like and not TNBC ＞Basal-like and TNBC,Figure 1I), TNBC vs. Not TNBC patients (Not TNBC ＞TNBC,Figure 1J), respectively. While, no significant expression difference of EPOR mRNA was found in HER2+ vs. HER2- patients (Figure 1D) or N+ vs. N- patients (Figure 1E).

3.2. High expression of EPOR mRNA was dramatically related to decreased risk of MR and AE in ERm or ER+breast cancer patients

By exploring in bc-GenExMiner 4.1, totally 33 studies including 5 064 patients investigated the correlation between EPOR mRNA level and MR or AE (Table 1). Initially, a preliminary study was done to investigate the prognostic analysis for EPOR mRNA expression with any ER status, any nodal status and any event. As shown in Table 2, high EPOR mRNA expression was closely related to the decreased risk of MR and AE in ERm or ER+ patients. Nevertheless,the same relationship was not detected in ER- patients (Table 2).These results suggested that EPOR mRNA level might have diverse prognostic significance in different subtypes of breast cancer.

Figure 1. Differential expression levels of EPOR mRNA in breast cancer patients based on different kinds of classified parameters.

3.3. EPOR univariate Cox regression analysis and adjusted analyses of MR based on ER status

To explore whether EPOR mRNA level had diverse prognostic significance in ER+ or ER- breast cancer patients, subgroup analysis was done further. As shown in Figure 2A, a total of 23 studies including 2 822 patients were searched to evaluate the correlation between EPOR mRNA level and MR risk in ER+ BRCA patients.The univariate Cox regression analysis demonstrated that high EPOR mRNA level was dramatically related to a lower risk of MR(HR: 0.79, P＜0.000 1; Figure 2A) and also a better MR-free survival(HR: 0.81, 95%CI: 0.70-0.94, P =0.007 2; Figure 2B). Besides, NPI-,AOL- and proliferation- adjusted analyses validated that high EPOR mRNA level was significantly correlated to a lower risk of MR in ER+ patients (HR: 0.81, 0.66 and 0.76, respectively; P=0.017 4,0.000 9 and ＜0.000 1, respectively; Figure 2C). As for ER- patients,a total of 21 studies including 1 073 patients were searched to evaluate the relationship between EPOR mRNA level and MR risk(Figure 2D). The results revealed no significant correlation no matter in univariate Cox regression analysis (Figure 2E) or in NPI-, AOL-and proliferation- adjusted analysis (Figure 2F).

3.4. EPOR univariate Cox regression analysis and adjusted analyses of AE based on ER status

Prognostic significance of EPOR mRNA on AE was examined among the breast cancer patients with different ER status. As shown in Figure 3A, a total of 31 studies including 3 631 patients were searched to evaluate the relationship between EPOR mRNA level and AR risk in ER+ BRCA patients. The univariate Cox regression analysis demonstrated that high EPOR mRNA level was dramatically related to a lower risk of AE (HR: 0.87, P=0.000 7; Figure 3A)and also a better AE-free survival (HR: 0.88, 95%CI: 0.78-0.99,P=0.029 9; Figure 3B). Besides, NPI-, AOL- and proliferationadjusted analyses were further performed (Figure 3E-G), which showed that the significant impact of EPOR mRNA level on AE risk mainly in the ER+ combined N+ subgroup, but not in the ER+combined N- subgroup. As for ER- patients, a total of 27 studies including 1 394 patients were searched to evaluate the significance of EPOR mRNA level on MR risk (Figure 3C).The results revealed no significant correlation both in univariate Cox regression analysis(Figure 3D) and NPI-, AOL- and proliferation- adjusted analyses(Figure 3H).

3.5. EPOR univariate Cox regression analysis of AE based on the molecular subtypes of SSP and SCM

Next, the prognostic significance of EPOR mRNA in breast cancer patients was evaluated under different molecular subtypes. As shown in Table 3, by using the SSP classification, high EPOR mRNA level was significantly related to decreased risk of AE in Luminal B breast cancer (HR: 0.82, 95%CI: 0.71-0.94, P=0.005 9),while no relationship was detected in basal-like, HER2+, Luminal A and normal basal-like subtypes. Moreover, to investigate the reliability of the results on the basis of SSP molecular subtype, EPOR univariate Cox analysis by SCM molecular subtype was done further. Similar to previous results, high EPOR mRNA level was significantly related to decreased risk in ER+/HER2-high proliferation group (HR: 0.86,95%CI: 0.76-0.97, P=0.0152; Table 4), but not in ER-/HER2-,HER2+ or ER+/HER2- low proliferation group (Table 4).

Table 1 The basic characteristics of studies included.

Table 2 Exhaustive EPOR univariate Coxanalysis of MR and AE in breast cancer patients.

Table 3 EPOR univariate Cox analysis of AE by SSP molecular subtype.

Figure 2. EPOR univariate Cox analysis and three types of prognostic index-adjusted analysis of MR based on ER status. Forest plots displaying univariate Cox’s analysis of EPOR mRNA expression and the risk of MR in ER+ (A) and ER- (D) breast cancer patients; Kaplan-Meier curves of EPOR mRNA expression and MR-free survival in ER+ (B) and ER- (E) breast cancer patients; three types (NPI, AOL and proliferation) of prognostic index-adjusted analysis of the correlation between EPOR mRNA expression and the risk of MR in ER+ (C) and ER- (F) breast cancer patients. Data mining was done by bc-GenExMiner v4.1.

Figure 3. EPOR univariate Cox analysis and three types of prognostic index-adjusted analysis of AE based on ER status.

Table 4 EPOR univariate Cox analysis of AE by SCM molecular subtype.

4. Discussion

Treatment of cancer patients with recombinant human erythropoiesis stimulating agents (rhESA) reduces transfusion requirements and improves quality of life[55-57]. Anemia prevention is pivotal with a view to hypoxia-driven tumor progression.Nevertheless, the negative outcomes of high-dose rhESA therapy trials on patients with breast[6] or head and neck cancers[7] have raised concern that EPO may boost tumor growth. A prerequisite for effects of EPO is the existence of functional EPOR. Previous studies have provided conflicting results[56,58], which may be partly due to nonspecificity of the antibodies used for detection of EPOR protein[17,18].Therefore, to avoid the bias induced by the protein level detection of EPOR, bioinformatic mining method was performed to explore the expression level of EPOR mRNA in breast cancer and its potential prognostic significance.First, TCGA and GTEx data in GEPIA was used to found surprisingly that EPOR mRNA was dramatically downregulated in BRCA (1 085 cases) compared to normal tissues (291 cases). Then,in order to further know whether or not significantly differential expression levels of EPOR mRNA existed in breast cancer patients based on different kinds of classified parameters, subgroup analysis was employed by bc-GenExMiner v4.1. The findings revealed that there were remarkably differential expression levels of EPOR mRNA between the favorable prognostic parameter group and unfavorable prognostic parameter group. The differential expression levels of EPOR mRNA in each subgroup were listed as follows: ER+ ＞ ER-,PR+ ＞ PR-, NPI1 ＞ NPI2 ＞ NPI3, SBR1 ＞ SBR2 ＞ SBR3, Not basal-like ＞Basal-like, Not basal-like and not TNBC ＞Basal-like and TNBC and Not TNBC ＞TNBC, respectively. As we known, breast cancer patients with the above favorable prognostic parameters were to be considered commonly had a better survival time[59-62].Thus, these results suggested that high EPOR mRNA level might be served as a protective role in breast cancer patients for longer survival.Second, the subsequent validation analyses were performed to investigate whether or not differential expression levels of EPOR mRNA were associated with the risk of MR and AE or MR-free and AE-free survival of breast cancer patients. Consistent with previous hypothesis, we found that high EPOR mRNA expression in ERm or ER+ patients (any type of N status) was remarkably related to decreased the risk of MR and AE. Simultaneously, ER+ patients with high EPOR mRNA level also had much more better MR-free and AE-free survival than those with low EPOR mRNA level.However, no similar phenomenon was detected in patients with ER-.Besides, NPI-, AOL- and proliferation- adjusted analyses were further performed to verify the above findings and the results were consistent. Additionally, univariate Cox analysis of AE was done to evaluate the prognostic significance of EPOR in BRCA patients based on SSP or SCM molecular subtypes. The results showed that high EPOR mRNA level was significantly associated with decreased risk of AE in BRCA patients with Luminal B or ER+/HER2- high proliferation group under SSP and SCM classification, respectively.Taken together, the above findings suggested that high EPOR mRNA level might be a significantly favorable indicator to predict low risk of MR and AE or longer survival in ER+ breast cancer patients.In conclusion, the present bioinformatic mining findings suggested that EPOR mRNA level might be a significant indicator to predict the risk of MR and AE in ER+ breast cancer patients. In future, this interesting observation is worthy of deeper exploration and validation from the aspect of real fundamental experiments and clinical trials.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgement

This study was supported by the Science and Technology Research Project of Anhui Province (No. 1704a0802148).

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