Periostin drives extracellular matrix degradation, stemness, and chemoresistance by activating the MAPK/ERK signaling pathway in triple–negative breast cancer cells

Figures

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  Fig. 1. ACM and PA increase the expression of MMPs and stemness–related molecules in TNBCs. a–b. qPCR analysis of the expression of MMPs (MMP2, MMP9, and MMP13) (a) and stemness–related molecules (MYC, SOX2, and NANOG) (b) in TNBCs (MDA–MB–231 and SUM159PT cell lines) treated with NC or ACM for 24 h. c–d. qPCR analysis of the expression of MMPs (c) and stemness–related molecules (d) in MDA–MB–231 and SUM159PT cells treated with vehicle or PA for 24 h. NC: negative control, ACM: adipogenic conditioned medium. *P < 0.05

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  Fig. 2. ACM and PA induce the expression and secretion of periostin in TNBCs. a–b. MDA–MB–231 and SUM159PT cells were treated with NC or ACM for 24 h, and then the POSTN mRNA and protein levels were determined by qPCR (a) and Western blotting (b) analysis. c–d. MDA–MB–231 and SUM159PT cells were treated with vehicle or PA for 24 h, and then the POSTN mRNA and protein levels were determined by qPCR (c) and Western blotting (d) analysis. e–f. The concentration of periostin in the cell culture medium was determined by ELISA. NC: negative control; ACM blank: ACM alone; ACM with: ACM cocultured with TNBCs. ns: no significance; *P < 0.05

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  Fig. 3. Inhibition of periostin restores the cellular processes in TNBCs. a–b. The results of the qPCR (a) and Western blotting (b) analysis of the specificity of shRNA against POSTN in MDA–MB–231 and SUM159PT cells, where β–actin was used as the control. c–d. The results of the qPCR analysis of the MMPs (MMP2, MMP9, and MMP13) (c) and stemness–related molecules (MYC, SOX2, and NANOG) (d) in MDA–MB–231 and SUM159PT cells transfected with shPOSTN or shCtrl for 48 h. e–f. The results of the qPCR analysis of the MMPs (e) and stemness–related molecules (f) in MDA–MB–231 and SUM159PT cells treated with rhPeriostin or IgG for 24 h. *P < 0.05

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  Fig. 4. ACM and PA function in TNBCs by regulating the expression and secretion of periostin. a–c. MDA–MB–231 and SUM159PT cells were treated with vehicle or PA for 24 h after transfection with shPOSTN, and then the related gene expression was detected by qPCR. d–f. MDA–MB–231 and SUM159PT cells were treated with vehicle or PA and/or periostin NeuAbs for 24 h, and then the related gene expression was detected by qPCR. *P < 0.05

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  Fig. 5. Periostin promotes the tolerance of TNBCs toward paclitaxel chemotherapy. a–b. A CCK–8 assay was used to determine cell proliferation in MDA–MB–231 and SUM159PT cells under the indicated conditions. c–d. A CCK–8 assay was used to determine the cellular viability when MDA–MB–231 and SUM159PT cells were treated with 3 µg/ml PTX under the indicated conditions. *P < 0.05

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  Fig. 6. Periostin activates the MAPK signaling pathway to promote extracellular matrix degradation, stemness, and chemoresistance in TNBCs. (a) Four samples representative of the transcriptome heatmap. (b) Volcano plot of the RNA–seq results. c–d. GO term enrichment analysis in the BP category (c) and KEGG pathway enrichment analysis (d) of the upregulated genes in the rhPeriostin group compared with the IgG group. The bars marked in red were the ones we focused on. e–f. Western blotting analysis of the indicated protein levels in MDA–MB–231 cells treated with IgG or rhPeriostin and/or U0126–EtOH for 24 h. The densitometry results of p–ERK1/2 (upper and bottom strips) were expressed as fold change in the protein levels when compared with IgG–treated MDA–MB–231 cells after being normalized to β–actin (e). g–h. qPCR analysis of the indicated gene expression in MDA–MB–231 cells treated with IgG or rhperiostin and/or U0126–EtOH for 24 h. i. CCK–8 analysis of the cellular viability when MDA–MB–231 cells were treated with 3 µg/ml PTX in the indicated conditions. *P < 0.05

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  Fig. 7. High POSTN expression correlates with poor survival in patients with TNBC. Kaplan–Meier curves of survival in TNBC patients, as calculated using TCGA data. The median expression value was used to separate the POSTN–low and POSTN–high groups based on the Kaplan–Meier curve. a–b. POSTN expression was assessed for its association with OS, RFS, and DMFS in TNBC patients. The expression groups were divided into high (red) and low (black). The survival times are shown in months, and HR and confidence intervals (in brackets) are shown for the gene