SERPINE1 overexpression promotes proliferation and paclitaxel resistance of triple-negative breast cancer cells by inducing M2 macrophage polarization

doi:10.12122/j.issn.1673-4254.2025.12.03

Abstract

Abstract:

Objective To investigate the regulatory effect of Serpin Family E Member 1 (SERPINE1) on immune microenvironment and paclitaxel (PTX) resistance of triple-negative breast cancer (TNBC) cells. Methods CCK-8 assay was used to determine the half-maximal inhibitory concentration of PTX in TNBC cell line MDA-MB-231. In wild-type MDA-MB-231 cells and a PTX-resistant MDA-MB-231 cell line (MDA-MB-231/PTX) established by stepwise increasing low-dose PTX treatment, the effects of Western blot-verified transfection with SERPINE1 overexpression plasmids or SERPINE1 siRNAs on cell apoptosis were evaluated using Hoechst 33258 staining and by detecting expression levels of cleaved caspase-3 using Western blotting. The changes in proliferation of the transfected cells were assessed using EdU and CCK-8 assays. The breast cancer cells with different treatments were co-cultured with macrophages, and M1 and M2 polarization of the macrophages were analyzed with flow cytometry and Western blotting. In nude mouse models bearing subcutaneous breast cancer cell xenografts, the effects of SERPINE1 overexpression and knockdown in the engrafted cells on tumor growth and PTX resistance were evaluated. Results SERPINE1 overexpression significantly inhibited apoptosis and promoted proliferation of MDA-MB-231 cells, and SERPINE1 knockdown obviously promoted apoptosis and inhibited proliferation of MDA-MB-231/PTX cells. The macrophages co-cultured with SERPINE1-overexpressing breast cancer cells showed enhanced M2 polarization and suppressed M1 polarization with a lowered M1/M2 ratio. In the tumor-bearing nude mouse models, SERPINE1 overexpression in the engrafted cells resulted in significantly accelerated tumor growth. Conclusion In MDA-MB-231 cells, SERPINE1 overexpression promotes cell proliferation, inhibits apoptosis, and enhances PTX resistance. SERPINE1 plays a regulatory role in macrophage polarization in the immune microenvironment of breast cancer, and its high expression promotes M2 polarization of the macrophages.

Key words: triple-negative breast cancer, serpin family E member 1, paclitaxel resistance, tumor immune microenvironment, M2 macrophage polarization

Qian ZHANG, Bowen LIU, Li LEI, Ye WANG, Xinyue ZHANG, Zhangkun MAO, Peng TANG, Jinmei ZHANG, Jiayi YANG, Yanxi PENG, Ze LIU. SERPINE1 overexpression promotes proliferation and paclitaxel resistance of triple-negative breast cancer cells by inducing M2 macrophage polarization[J]. Journal of Southern Medical University, 2025, 45(12): 2551-2560.

Figures/Tables 8

Fig.1 SERPINE1 overexpression in MDA-MB-231 cells and SERPINE1 knockdown in MDA-MB-231/PTX cells. A, B: Western blotting for detecting SERPINE1 protein expression in MDA-MB-231 cells transfected with pcDNA4.0-SERPINE1 and vector plasmid (**P<0.01 vs EV group, n=3); C, D: Western blotting for detecting SERPINE1 protein expression in MDA-MB-231/PTX cells transfected with SERPINE1-siRNA and NC-siRNA. GAPDH was used as the loading control (*P<0.05 vs NC group, n=3).

Fig.2 Proliferative activity of MDA-MB-231 cells with SERPINE1 overexpression and MDA-MB-231/PTX cells with SERPINE1 knockdown. A, B: Results of CCK-8 assay of MDA-MB-231 cells (A) transfected with pcDNA4.0-SERPINE1 or the vector plasmid and MDA-MB-231/PTX cells (B) transfected with SERPINE1-siRNA or NC-siRNA (**P<0.01 vs EV group or NC group). C, D: EdU staining of MDA-MB-231 cells with SERPINE1 overexpression and MDA-MB-231/PTX cells with SERPINE1 knockdown (scale bar=50 μm).

Fig.3 Changes in apoptosis of MDA-MB-231 cells with SERPINE1 overexpression and MDA-MB-231/PTX cells with SERPINE1 knockdown. A, B: DNA damage detected by Hoechst staining in MDA-MB-231 cells with SERPINE1 overexpression and in MDA-MB-231/PTX cells with SERPINE1 knockdown (scale bar=50 μm). C: Western blotting for detecting protein levels of cleaved caspase-3 in MDA-MB-231 cells transfected with pcDNA4.0-SERPINE1 versus the vector plasmid (**P<0.01 vs EV group). D: Western blotting for detecting protein levels of cleaved caspase-3 in MDA-MB-231/PTX cells transfected with SERPINE1-siRNA versus NC-siRNA (***P<0.001 vs NC group, n=3).

Fig.4 Changes in paclitaxel resistance of MDA-MB-231 cells with SERPINE1 overexpression and MDA-MB-231/PTX cells with SERPINE1 knockdown. A: CCK-8 assay for determining IC50 value of paclitaxel in MDA-MB-231 cells transfected with pcDNA4.0-SERPINE1 versus the vector plasmid (**P<0.01 vs EV group). B: CCK-8 assay for determining the IC50 value of paclitaxel in MDA-MB-231/PTX cells transfected with SERPINE1-siRNA versus NC-siRNA (**P<0.01 vs NC group, n=3).

Fig.5 Effect of SERPINE1 overexpression in MDA-MB-231 cells and SERPINE1 knockdown in MDA-MB-231/PTX cells on M2 polarization of the co-cultured macrophages. A, B: Flow cytometry for analyzing the proportion of CD163+ macrophages co-cultured with SERPINE1-overexpressing MDA-MB-231 cells (**P<0.01 vs EV group). C, D: Flow cytometry for analyzing the proportion of CD163+ macrophages co-cultured with MDA-MB-231/PTX cells with SERPINE1 knockdown (*P<0.05 vs NC group). E, F: Western blotting for detecting the protein expression of YM1 and Arg-1 in the macrophages from the microenvironment of MDA-MB-231 cells with or without SERPINE1 overexpression (**P<0.01 vs EV group). G, H: Western blotting for detecting the protein expression of YM1 and Arg-1 in macrophages from the microenvironment of MDA-MB-231/PTX cells with or without SERPINE1 knockdown (**P<0.01 vs NC group, n=3).

Fig.6 M1 polarization of the macrophages co-cultured with MDA-MB-231 cells with SERPINE1 overexpression or MDA-MB-231/PTX cells with SERPINE1 knockdown. A, B: Flow cytometry for analyzing the proportion of CD86+ macrophages co-cultured with MDA-MB-231 cells with SERPINE1 overexpression (*P<0.05 vs EV group). C, D: Flow cytometry for analyzing the proportion of CD86+ macrophages co-cultured with MDA-MB-231/PTX cells with SERPINE1 knockdown (**P<0.01 vs NC group). E, F: Western blotting for detecting the protein expression of iNOS in macrophages from the microenvironment of MDA-MB-231 cells with or without SERPINE1 overexpression (**P<0.01 vs EV group). G, H: Western blotting for detecting the protein expression of iNOS in macrophages from the microenvironment of MDA-MB-231/PTX cells with or without SERPINE1 knockdown (**P<0.01 vs NC group, n=3).

Fig.7 M1/M2 ratio of the macrophages co-cultured with MDA-MB-231 cells with SERPINE1 overexpression (A; **P<0.01 vs EV group) and MDA-MB-231/PTX cells with SERPINE1 knockdown (B; **P<0.01 vs blank group, n=3).

Fig.8 Regulatory effect of SERPINE1 expression level on growth of triple-negative breast cancer xenografts and macrophage polarization in nude mice. A: Appearance of the tumor-bearing nude mice and the dissected tumors. B: Tumor growth curves over time in each group. C: Protein expression level of SERPINE1 in the xenograft tumor tissues from each group. D: HE staining of xenograft tumor tissues. E: Immunohistochemistry for the M2 macrophage marker Arg-1 in xenograft tumor tissues. F: Immunohistochemistry for the M1 macrophage marker iNOS in xenograft tumor tissues (scale bar=100 μm). *P<0.05, **P<0.01, ***P<0.001 vs NC group. n=6.

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