Expression of RBMS3 in Breast Cancer Progression

Abstract

The aim of the study was to evaluate the localization and intensity of RNA-binding motif single-stranded-interacting protein 3 (RBMS3) expression in clinical material using immunohistochemical (IHC) reactions in cases of ductal breast cancer (in vivo), and to determine the level of RBMS3 expression at both the protein and mRNA levels in breast cancer cell lines (in vitro). Moreover, the data obtained in the in vivo and in vitro studies were correlated with the clinicopathological profiles of the patients. Material for the IHC studies comprised 490 invasive ductal carcinoma (IDC) cases and 26 mastopathy tissues. Western blot and RT-qPCR were performed on four breast cancer cell lines (MCF-7, BT-474, SK-BR-3 and MDA-MB-231) and the HME1-hTERT (Me16C) normal immortalized breast epithelial cell line (control). The Kaplan-Meier plotter tool was employed to analyze the predictive value of overall survival of RBMS3 expression at the mRNA level. Cytoplasmatic RBMS3 IHC expression was observed in breast cancer cells and stromal cells. The statistical analysis revealed a significantly decreased RBMS3 expression in the cancer specimens when compared with the mastopathy tissues (p < 0.001). An increased expression of RBMS3 was corelated with HER2(+) cancer specimens (p < 0.05) and ER(-) cancer specimens (p < 0.05). In addition, a statistically significant higher expression of RBMS3 was observed in cancer stromal cells in comparison to the control and cancer cells (p < 0.0001). The statistical analysis demonstrated a significantly higher expression of RBMS3 mRNA in the SK-BR-3 cell line compared with all other cell lines (p < 0.05). A positive correlation was revealed between the expression of RBMS3, at both the mRNA and protein levels, and longer overall survival. The differences in the expression of RBMS3 in cancer cells (both in vivo and in vitro) and the stroma of breast cancer with regard to the molecular status of the tumor may indicate that RBMS3 could be a potential novel target for the development of personalized methods of treatment. RBMS3 can be an indicator of longer overall survival for potential use in breast cancer diagnostic process.

Keywords: RNA-binding protein 3 (RBMS3); cancer prevention; carcinogenesis; epithelial–mesenchymal transition (EMT); target discovery; target therapy.

Figure 1

(a) The statistical analysis revealed a significantly higher RBMS3 expression as assessed by the immunoreactive score in the control mastopathy cases compared with the cancer cells in breast cancer. (b) The breast cancer cells with positive expression of HER2 and (c) estrogen receptors presented a higher expression of RBMS3 compared with tumors lacking expression of these receptors (Mann–Whitney test * p < 0.05; *** p < 0.001) (IDC—invasive ductal carcinoma, IRS—immunoreactive score).

Figure 2

Immunohistochemical visualization of RBMS3 expression. (a) High cytoplasmic expression of RBMS3 in mastopathy cases. (b) Low expression of RBMS3 in breast cancer cells. (c) Low cytoplasmic expression of RBMS3 in the stroma of mastopathy cases and (d) high expression in the stroma cells of the breast cancer cases. Magnification ×200.

Figure 3

Analysis of RBMS3 expression in the stroma of breast cancer: (a) RBMS3 expression in the stroma of breast cancer was statistically higher than in the mastopathy cases; (b) Triple-negative (TN) cases of breast cancer displayed lower expression of RBMS3 in the stroma than the other molecular types of breast cancer combined. (c) Significant increase in RBMS3 expression in the specimens with positive expression of the progesterone receptor and (d) with positive expression of the estrogen receptor. The expression of RBMS3 was statistically lower in the cancer cells than in the stromal cells of the breast cancer specimens (e). (Mann–Whitney test ** p < 0.01, *** p < 0.001, **** p < 0.0001) (IDC—invasive ductal carcinoma).

Figure 4

In vitro analysis of RBMS3 expression in breast cancer cell lines representing the four main molecular types of breast cancer and a control cell line (Me16C). (a) The statistical analysis of RBMS3′s expression at the mRNA level showed a significantly different expression of RBMS3 in all the examined cell lines in comparison to the control Me16C cell line. (b,c) Analysis at the protein level showed a significantly higher expression of RBMS3 in the MDA-MB-231 and SK-BR-3 cell lines than in the MCF-7 and BT-474 cell lines; ((a) Bonferroni’s multiple comparison test, (b) Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5

The analysis of the clinical data regarding the survival of patients showed shorter overall survival in the group of patients without IHC expression of RBMS3 (Gehan–Breslow–Wilcoxon test p = 0.051).

Figure 6

Analysis of RBMS3 mRNA expression in 2976 cases of breast cancer, using the Kaplan–Meier estimator. The analysis revealed a significant positive correlation between the expression of RBMS3 and overall survival (p < 0.0001) [21].