ROPN1 Gene Expression as a Prognostic and Predictive Biomarker in Aggressive Breast Cancer: Clinical Implications and Survival Association

Abstract

Objective: The ropporin-1 (ROPN1) gene, initially linked to sperm motility, is differentially expressed in triple negative breast cancer (TNBC), suggesting a role in tumor progression and therapy resistance. To characterize ROPN1 expression in breast cancer and evaluate its association with clinicopathological features, survival, and treatment response as a translational biomarker.

Materials and methods: Data from The Cancer Genome Atlas (1,087 patients), Sweden Cancerome Analysis Network-Breast (3,273 patients), and geodatabases were analyzed. ROPN1 transcriptional levels were assessed in relation to clinical variables and survival. Chemotherapy agents and epigenetic modulators were tested in cell lines to evaluate ROPN1 regulation.

Results: Transcriptional overexpression of ROPN1 was significantly enriched in TNBC/basal-like tumors (p<0.0001) and correlated with reduced overall survival, particularly in basal cases [hazard ratio (HR) = 1.85; 95% confidence interval (CI): 1.02-3.33; p = 0.041]. Patients treated with chemotherapy and exhibiting high ROPN1 levels had unfavorable prognosis, with an even poorer profile in untreated cohorts (HR = 4.55; 95% CI: 1.33-14.29; p = 0.01). Hypomethylation at cg00101712 (HR = 0.59; p = 0.016) and cg09298623 (HR = 0.49; p = 0.0014) CpG sites were associated with worse survival at 5 years follow-up, underscoring epigenetic regulation of this pathway as a key driver of poor outcomes. Furthermore, in vitro treatment with cisplatin, doxorubicin, and paclitaxel resulted in variable responses, with a significant reduction of ROPN1 in HCC70 and HS578T cell lines, while BT549 and MDA-MB-231 cell lines showed notable increases.

Conclusion: ROPN1 overexpression in TNBC/basal-like tumors suggests a role as a prognostic biomarker and predictor of post-chemotherapy resistance. Investigation of ROPN1 expression in breast tumors may lead to alternative strategies targeting pro-metastatic pathways and improve precision treatment for aggressive breast cancer.

Keywords: ROPN1; aggressive breast cancer; basal-like; triple negative breast cancer.

Figure 1

ROPN1 expression patterns in different clinical-pathological contexts of breast cancer. The graphs show comparisons of ROPN1 expression among subgroups for (A) estrogen receptor, (B) progesterone receptor, (C) HER2, (D) menopausal status, (E) histological type, and (F) molecular subtypes according to the PAM50 classification. Dunn’s multiple comparisons test was applied, and differences between the basal and HER2, luminal A or luminal B subtypes were significant (p<0.0001), whereas only the difference between the basal and normal-like subtypes was not significant (p = 0.2751) ER: Estrogen receptor; PR: Progesterone receptor; HER2: Human epidermal growth factor receptor type II; Peri: Perimenopausal; Pre: Premenopausal; Post: Postmenopausal; IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; B: Basal; H: HER2; LA: Luminal A; LB: Luminal B; N: Normal-like

Figure 2

Methylation profile of ROPN1 in breast cancer patients. (A) Correlation between ROPN1 mRNA levels and methylation in a population of breast cancer patients. (B) Specific correlation for the basal subtype of breast cancer, where the negative correlation is more pronounced. (C) Methylation pattern of ROPN1 according to the PAM50 classification. (D) Comparison of ROPN1 methylation in the clinical profiles of basal and non-basal breast cancer patients. (E) OS analysis based on the methylation pattern of the ROPN1 body opening site cg00101712. (F) OS analysis based on the methylation profile of ROPN1 in TSS1500 N-shore cg09298623. (G) The PPI network was constructed using the STRING database, employing the top 40 genes correlated with ROPN1 from the TCGA Firehose Legacy database. Red represents genes positively correlated with ROPN1, whereas green indicates genes negatively correlated with ROPN1. (H) Pathway enrichment analysis B: Basal; H: HER2; LA: Luminal A; LB: Luminal B; N: Normal-like; PPI: Protein-protein interaction; PAM50: Prediction analysis of microarray 50; HER2: Human epidermal growth factor receptor 2

Figure 3

Associations between ROPN1 expression and survival in breast cancer patients. (A) Overall survival analysis of all breast cancer patients. (B) Stratified survival of patients with the basal subtype of breast cancer. (C) Impact of ROPN1 expression on the survival of basal breast cancer patients undergoing chemotherapy. (D) Comparative survival analysis of basal breast cancer patients who did not receive chemotherapy. Data were obtained from the GSE96058 study and analyzed via Kaplan-Meier curves. The “Low” and “High” categories refer to patient classification based on ROPN1 gene expression levels, with “Low” indicating expression below the established cut-off value, and “High” indicating expression above this threshold. Expression pattern of ROPN1 in different cohorts. The probes 224191_x_at (E), 231535_x_at (F), and 233203_at (G) were evaluated using datasets available in the GEO database, identified as GSE76275, GSE21653, GSE32646, GSE18864, and GSE43358, respectively

Figure 4

Expression of ROPN1 in breast cancer cell lines and the effects of different treatments. (A) Basal transcription levels of ROPN1 in a panel of mammary cell lines, including distinct tumor subtypes. (B-H) Variation in ROPN1 expression after treatment with cisplatin, doxorubicin, paclitaxel, or radiotherapy. (I) Modulation of ROPN1 expression in response to the epigenetic-acting drugs 5-aza and trichostatin A