RBMS2 Chemosensitizes Breast Cancer Cells to Doxorubicin by Regulating BMF Expression

Abstract

Chemoresistance is closely related to the therapeutic effect and prognosis in breast cancer patients. Increasing evidences demonstrated that RNA binding proteins (RBPs) have notable roles in regulating cancer cell proliferation, metastasis and chemotherapeutic sensitivity. RNA binding motif single stranded interacting protein 2 (RBMS2), an RBP, has been considered to be a tumor suppressor in several cancers. However, its role of doxorubicin sensitivity in breast cancer patients has not yet been fully revealed. Here, we performed doxorubicin cytotoxicity assay, flow cytometry and mouse xenograft model to examine the influence of RBMS2 on doxorubicin sensitization in vitro and in vivo. RIP assay and dual-luciferase reporter assay were performed to explore the relationship between RBMS2 and BMF. Our data demonstrated that upregulation of RBMS2 in breast cancer cells could enhance sensitivity to doxorubicin and promote apoptosis in the presence of doxorubicin, while inhibition of RBMS2 showed an opposite trend. Moreover, this chemosensitizing effect of RBMS2 could be reversed by the inhibition of Bcl-2 modifying factor (BMF). RBMS2 positively regulated BMF expression and increased BMF-induced expression of (cleaved) caspase 3, (cleaved) caspase 9 and poly (ADP-Ribose) polymerase (PARP). These results uncovered a novel mechanism for RBMS2 in the sensibilization of doxorubicin, suggesting that RBMS2 may act as a potential therapeutic target for drug-resistant breast cancer.

Keywords: BMF; RBMS2; apoptosis; breast cancer; chemosensitization; doxorubicin.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1

RBMS2 could sensitize breast cancer cells to DOX in vitro. RBMS2 overexpression and knockdown cell lines were treated with different dosages of DOX for 24 h, respectively. CCK-8 assay was used to examine the cell viability (A and D for MCF-7, B and E for SUM 1315) and IC50 value (C and F) of DOX. Transfected MCF-7 (G, H) and SUM 1315 (I, J) cells were treated with DOX (0 or 1 µg/ml) for 2 weeks. Representative images (left panel) and quantification (right panel) of colonies in the colony formation assay. Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 2

Overexpression of RBMS2 could enhance the therapeutic effect of DOX in vivo. Tumor volumes were measured in different treatment groups (A. B). The excised tumor lumps were weighed (C) and photographed (D). Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 3

RBMS2 could induce apoptosis and apoptosis related proteins in breast cancer cell lines. Flow cytometry analysis of apoptosis in RBMS2 overexpression and knockdown MCF-7 and SUM 1315 cells with or without DOX treatment (A, B). The statistical calculation of apoptotic rates was shown (C). The levels of (cleaved) caspase 3, (cleaved) caspase 9 and PARP in MCF-7 and SUM 1315 cells transfected with RBMS2 overexpression and knockdown were detected by Western blot (D). The top band of RBMS2 is the exogenously band in western blot. Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 4

RBMS2 regulated the expression of pro-apoptotic protein BMF. Volcano plot represented the distribution of mapped transcripts (A). The size and color of the dots meant the number of enriched genes and the adjusted p values, respectively. BMF was found to be positively correlated with RBMS2 in breast cancer both from TIMER (Tumor Immune Estimation Resource, https://cistrome.shinyapps.io/timer) database (B), the patients' samples from our hospital (C). Correlation heatmap were used to analyze the correlation between RBMS2 and BMF (D). R1, R2, R3 and V5-1, V5-2 and V5-3 represented RBMS2-overexpressed group and the control group, respectively. The red arrow indicated that BMF was positively correlated with RBMS2. Expression of BMF in breast cancer tissues and normal tissues (E, F). Overexpression of RBMS2 significantly increased the expression of BMF at both mRNA and protein (G, I) levels. BMF was significantly down regulated after RBMS2 knock down at both mRNA and protein levels (H, J). The top band of RBMS2 is the exogenously band in western blot. Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 5

RBMS2 could increase BMF mRNA stability. In MCF-7 and SUM 1315 cell lines, RBMS2 overexpression prolonged the half-life of BMF mRNA after treated with Act D at a concentration of 5 µg/ml (A, B), while knockdown of RBMS2 shortened the half-life of BMF mRNA (C, D). MCF-7 and SUM 1315 cells lysates were immunoprecipitated with RBMS2 or IgG antibody and analyzed using PCR (E, G) and RT-qPCR (F, H) to detect transcript levels of BMF. Schematic diagram containing BMF 3'-UTR (upper) and AREs mutant region (lower) (I). The reporter containing BMF 3'-UTR was increased after overexpression of RBMS2 in MCF-7 (J) and SUM 1315 (K) cell lines. Firefly luciferase activity was detected and normalized to Renilla luciferase activity. Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 6

BMF could reverse the sensitization to DOX induced by RBMS2 both in vivo and in vitro. Small interfering RNA of BMF was transfected into RBMS2 overexpression MCF- 7 and SUM 1315 cells. Transfection efficiency was confirmed via RT-qPCR and western blot (A, B). The top band of RBMS2 is the exogenously band in western blot. The sensitivity to DOX of MCF-7 and SUM 1315 cells mentioned above was examined using CCK-8 and colony formation. CCK-8 assay was used to examine cell viability (C for MCF-7, D for SUM 1315) and IC50 value (E) of DOX. Representative photographs (F, I), quantification (G, J) and cell survival after DOX treatment (H, K) were shown by colony formation assay. Tumor volumes were measured in different treatment groups (L). The excised tumor lumps were weighed (M) and photographed (N). Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.

Figure 7

BMF could rescue the apoptosis and apoptosis related proteins induced by RBMS2 in breast cancer cell lines. Apoptosis and apoptosis related proteins were detected by flow cytometry analysis (A, B, C) and western blot (D, E). Data were shown as mean ± SD. *p <0.05, **p <0.01, ***p<0.001.