RBM15 enhances paclitaxel resistance in triple-negative breast cancer by targeting m6A methylation of TNFSF9 and inducing polarization of tumor-associated macrophages to M2 phenotype

Abstract

Background: Triple-negative breast cancer (TNBC) is one of the breast cancer subtypes with a poor prognosis, and the current main treatment modalities include surgical resection and adjuvant chemotherapy. However, the development of drug resistance in tumor cells to chemotherapeutic agents poses great challenges to anticancer treatment.

Methods: Bioinformatics analysis was used to screen the up-regulated genes in paclitaxel (PTX)-resistant TNBC cells. Cell viability was measured by a CCK-8 kit. TNFSF9 (Tumor necrosis factor receptor superfamily member 9) protein level was detected by Western blot (WB) assay. PTX-resistant TNBC cell lines (MDA-MB-231/PTX, MDA-MB-468/PTX) were constructed and their drug resistance was shown by IC50. The EdU, flow cytometry, Transwell, and other commercial kits were applied to detect the proliferation, apoptosis, migration, invasion, macrophage M2 polarization, and glycolysis of PTX-resistant TNBC cells. RBM15 (RNA binding motif protein 15) levels were measured by RT-qPCR and WB assays. The RIP, MeRIP, and actinomycin D assays were used to analyze the interaction between TNFSF9 and RBM15. The effect of RBM15/TNFSF9 on PTX sensitivity in vivo was verified by xenograft tumor experiments.

Results: TNFSF9 was highly expressed in PTX-resistant TNBC cells. Silencing of TNFSF9 enhanced the sensitivity to PTX. Silencing TNFSF9 induced polarization of macrophages from M2 to M1 phenotype and the release of IL-1β and TNF-α, but decreased the levels of IL-10 and TGF-β. RBM15 targeted the N6-adenylate methylation (m⁶A) modification of TNFSF9, and overexpression of TNFSF9 could reverse the tumor-suppressing effect of silencing RBM15 on PTX-resistant TNBC cells in vitro and transplanted tumors in vivo. Samples from PTX-sensitive and PTX-resistant TNBC patients proved that RBM15 regulated TNFSF9's high expression in PTX-resistant TNBC tissues.

Conclusion: This study demonstrated that RBM15 enhanced PTX resistance in TNBC by promoting m⁶A methylation in TNFSF9 and inducing M2 polarization of tumor-associated macrophages.

Keywords: M2 polarization; PTX-resistant; RBM15; TNFSF9; Triple-negative breast cancer.

RBM15/TNFSF9 enhances the resistance of TNBC cells to PTX. RBM15 induced m⁶A modification of TNFSF9 to stabilize its expression by binding to it. Subsequently, high levels of TNFSF9 enhanced the proliferation, migration, invasion, and glycolytic pathways of PTX-resistant TNBC cells, but blocked the occurrence of apoptosis. At the same time, the M2 phenotype polarization of macrophages was also promoted. All these effects enhanced the resistance of TNBC cells to PTX

Fig. 1

TNFSF9 expression in PTX-resistant TNBC cells. The MDA-MB-231, MDA-MB-468, MDA-MB-231/PTX, and MDA-MB-468/PTX cells were treated with PTX at 5, 10, 15, 20, 25, and 30 µM. (A) The volcano plots for DEGs identified using GSE90564 datasets. (B) The intersection between the five sets of DEGs was presented by the Venn diagram. (C-F) The CCK-8 kit was used to detect the viability of parental cells (MDA-MB-231, MDA-MB-468) and PTX-resistant cells (MDA-MB-231/PTX, MDA-MB-468/PTX) as well as the IC50 of PTX. (G-H) The expression of TNFSF9 mRNA in PTX-resistant cells was detected by RT-qPCR. (I-J) WB was used to detect the protein expression of TNFSF9 in PTX-resistant cells

Fig. 2

Effect of knockdown of TNFSF9 on the progression of PTX-resistant TNBC cells. The MDA-MB-231/PTX and MDA-MB-468/PTX cells were transfected with sh-NC and sh-TNFSF9. (A) WB was used to detect the knockdown efficiency of PTX-resistant TNBC cell lines with knockdown of TNFSF9. (B) The IC50 of PTX was measured by CCK-8 assay. (C-D) The effect of TNFSF9 knockdown on the proliferation of PTX-resistant cells was determined by EDU assay. (E) Flow cytometry was applied to detect the effect of TNFSF9 knockdown on the apoptosis of PTX-resistant cells. (F-G) Transwell assay was applied to determine the effect of TNFSF9 knockdown on the migration and invasion of PTX-resistant cells. (H-J) Commercial kits were used to detect the effects of TNFSF9 knockdown on glucose consumption, lactate production and ATP level.

Fig. 3

Effect of silencing TNFSF9 on macrophage polarization. The MDA-MB-231/PTX and MDA-MB-468/PTX cells were transfected with sh-NC and sh-TNFSF9. (A-B) The expression of M2 marker CD206 and M1 marker INOS in THP1-M0 cells were detected by flow cytometry. (C-D) RT-qPCR was used to measure the expression of M2 marker IL-10 and TGF-β, M1 markers IL-1β and TNF-α

Fig. 4

RBM15 increases the stability of TNFSF9 through m⁶A modification. The MDA-MB-231/PTX and MDA-MB-468/PTX cells were transfected with sh-NC and sh-RBM15. (A) Genes associated with TNFSF9 in BRCA screened by the linkedomics website. (B) Genes positively associated with TNFSF9 in BRCA screened by the linkedomics website. (C) Genes negatively associated with TNFSF9 in BRCA screened by the linkedomics website. (D) The correlation between TNFSF9 and RBM15 was analyzed by Pearson correlation coefficient. (E) An m⁶A site on the mRNA of TNFSF9 was predicted by SRAMP website. (F) The potential binding sites of TNFSF9 and RBM15 were predicted by RBP suite website. (G-H) WB was used to verify the expression of RBM15 in PTX-resistant cells. (I) WB was applied to detect the protein level of RBM15 in cells with RBM15 knocked down. (J-K) Protein and mRNA levels of TNFSF9 in PTX-resistant cells with knockdown of RBM15 were detected by WB and RT-qPCR assay. (L) The interaction between RBM15 and TNFSF9 was validated through RIP assay. (M-N) The MeRIP assay was used to explore the effect of RBM15 on m⁶A modification of TNFSF9. (O-P) The effect of RBM15 on TNFSF9 mRNA stability was determined by actinomycin D

Fig. 5

Overexpression of TNFSF9 reverses the effects of RBM15 knockdown. The MDA-MB-231/PTX and MDA-MB-468/PTX cells were transfected with sh-NC + oe-NC, sh-RBM15 + oe-NC, and sh-RBM15 + oe-TNFSF9. (A) The overexpression efficiency of sh-RBM15 cell lines transfected with pcDNA-TNFSF9 was determined by WB assay. (B) CCK-8 assay was used to detect the IC50 of PTX. (C) EdU assay was used to detect the effect of TNFSF9 overexpression on the proliferation of sh-RBM15 cells. (D) Flow cytometry was utilized to detect the effect of TNFSF9 overexpression on the apoptosis of sh-RBM15 cells. (E-F) The effect of TNFSF9 overexpression on the migration and invasion of sh-RBM15 cells were determined by Transwell assay. (G-I) The commercial kits were used to detect the effect of TNFSF9 overexpression on the glycolytic pathway of sh-RBM15 cells. (J-K) The expression of M2 marker CD206 and M1 marker INOS were detected by flow cytometry. (L-M) RT-qPCR was used to measure the expression of IL-10, TGF-β, IL-1β, and TNF-α

Fig. 6

Effect of RBM15/TNFSF9 on the sensitivity of TNBC to PTX in vivo. Mice were injected with MDA-MB-231/PTX cells stably transfected with sh-NC, sh-RBM15, and sh-RBM15 + oe-TNFSF9 and randomly divided into the control (PBS) group and the PTX (20 mg/kg) treatment group. (A) The growth rate of xenograft tumors in mice (n = 5). (B) The weight of tumors after 21 days (n = 5). (C) WB was used to measure RBM15 and TNFSF9 protein levels in sh-RBM15 cells overexpressing TNFSF9

Fig. 7

The expression of RBM15 and TNFSF9 in PTX-resistant TNBC patients. (A-B) RT-qPCR was used to detect the expression of RBM15 and TNFSF9 mRNA in tumor tissues of TNBC PTX-resistant (n = 35) and PTX-sensitive (n = 17) patients. (C) The correlation analysis of RBM15 and TNFSF9 expression in tumor tissues of TNBC PTX-resistant patients (n = 35). (D) The protein expression of RBM15 and TNFSF9 in tumor tissues of TNBC PTX-resistant and PTX-sensitive patients was detected by WB assay