The N-terminal polypeptide derived from viral macrophage inflammatory protein II reverses breast cancer epithelial-to-mesenchymal transition via a PDGFRα-dependent mechanism

Abstract

NT21MP, a 21-residue peptide derived from the viral macrophage inflammatory protein II, competed effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in breast cancer. Its role in tumor epithelial-to-mesenchymal transition (EMT) regulation remains unknown. In this study, we evaluated the reversal of EMT upon NT21MP treatment and examined its role in the inhibition of EMT in breast cancer. The parental cells of breast cancer (SKBR-3 and MCF-7) and paclitaxel-resistant (SKBR-3 PR and MCF-7 PR) cells were studied in vitro and in combined immunodeficient mice. The mice injected with SKBR-3 PR cells were treated with NT21MP through the tail vein or intraperitoneally with paclitaxel or saline. Sections from tumors were evaluated for tumor weight and EMT markers based on Western blot. In vitro, the effects of NT21MP, CXCR4 and PDGFRα on tumor EMT were assessed by relative quantitative real-time reverse transcription-polymerase chain reaction, western blot and biological activity in breast cancer cell lines expressing high or low levels of CXCR4. Our results illustrated that NT21MP could reverse the phenotype of EMT in paclitaxel-resistant cells. Furthermore, we found that NT21MP governed PR-mediated EMT partly due to controlling platelet-derived growth factors A and B (PDGFA and PDGFB) and their receptor (PDGFRα). More importantly, NT21MP down-regulated AKT and ERK1/2 activity, which were activated by PDGFRα, and eventually reversed the EMT. Together, these results indicated that CXCR4 overexpression drives acquired paclitaxel resistance, partly by activating the PDGFA and PDGFB/PDGFRα autocrine signaling loops that activate AKT and ERK1/2. Inhibition of the oncogenic EMT process by targeting CXCR4/PDGFRα-mediated pathways using NT21MP may provide a novel therapeutic approach towards breast cancer.

Keywords: PDGFRα; breast cancer; epithelial-mesenchymal transition; paclitaxel; viral macrophage inflammatory protein II.

Figure 1. Platelet-derived growth factor and receptor levels in paclitaxel-resistant breast cancer cells

(A), Quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis was used to detect the expressions of platelet-derived growth factor (A), (B), and receptor α (PDGFA, PDGFB, and PDGFRα, respectively) in MCF-7 and MCF-7/PR cells. (B), Quantitative RT-PCR analysis was used to detect the expressions of PDGFA, PDGFB, and PDGFRα in SKBR-3 and SKBR-3/PR cells. *P < 0.05 PR vs control.

Figure 2. Expression of stromal cell–derived factor 1α (SDF-1α) and CXCR4 in breast cancer cells

(A), SDF-1α expression was detected by quantitative reverse transcription–polymerase chain reaction in SKBR-3, MCF-7, and MDA-MB-231 cells. (B), ELISA was used to measure the levels of SDF-1α secreted by the SKBR-3, MCF-7, and MDA-MB-231 cells. (C), ELISA was used to measure the levels of SDF-1α secreted in parental and resistant cells. (D), The CXCR4 level in MCF-7, MCF-7 PR, SKBR-3, and SKBR-3 PR cells analyzed by qRT-PCR. (E), The CXCR4 level in these cells analyzed by flow cytometry using antibody for CXCR4. (F), The mean fluorescence intensity of CXCR4 level analyzed by flow cytometry. *P < 0.05 vs SKBR-3 cells, ^#P < 0.05 PR vs control.

Figure 3. Effect of NT21MP on MCF-7 and SKBR-3 cell drug resistance

(A), Quantitative RT-PCR assays were conducted to detect the expression of resistant markers in parental and PR cells and the NT21MP treatment group. (B), Quantitative PCR assays were conducted to detect the expression of PDGFA, PDGFB, and PDGFRα in parental and PR cells and the NT21MP treatment group. (C), Western blotting results for the expression of resistant markers in MCF-7 and MCF-7 PR cells and the NT21MP treatment group. (D), Western blotting results for the expression of resistant markers in SKBR-3 and SKBR-3 PR cells and the NT21MP treatment group. *P < 0.05 and **P < 0.01 PR vs control and #P < 0.05 NT21MP treatment vs control in PR cells.

Figure 4. Effect of NT21MP on PR cell biological activity

(A), Sulforhodamine B assays were performed to measure the proliferation in PR cells treated with NT21MP. (B), Wound healing assays were used to detect the motility in PR cells treated with NT21MP. (C), Invasion assays were conducted in PR cells treated with NT21MP. (D), Flow cytometry was used to evaluate the cell cycles of PR cells after treatment with NT21MP. (E), Apoptosis was detected in PR cells after treatment with 1 μg/mL NT21MP. *P < 0.05, PR vs control; ^#P < 0.05, NT21MP treatment vs control in PR cells.

Figure 5. Construction of CXCR4 over- and underexpressing breast cancer cell lines

(A) and (B), Quantitative reverse transcription–polymerase chain reaction (RT-PCR) and western blotting were conducted to measure CXCR4 expression in SKBR-3, MCF-7, and MDA-MB-231 cells, respectively. *P < 0.05 vs SKBR-3 cells. (C), Western blotting analysis was performed to detect the expression of CXCR4 in MDA-MB-231 cells treated with CXCR4-overexpressing (pcDNA-CXCR4). CP: control pcDNA-CXCR4; PC1-5: pcDNA-CXCR4 1-5. (D), Quantitative results are illustrated for panel C. *P < 0.05 vs control. (E), Western blotting analysis was performed to detect the expression of CXCR4 in SKBR-3 cells treated with CXCR4 siRNA. BC: blank control siRNA; NC: negative siRNA; CS1-3: CXCR4 siRNA 1-3. (F), Quantitative results are illustrated for panel E. *P < 0.05 vs control.

Figure 6. NT21MP regulates drug resistance via CXCR4

(A), Quantitative RT-PCR assays were conducted to detect the expression of EMT markers in MDA-MB-231 and PC-MDA-MB-231 cells or in SKBR-3 and CS-SKBR-3 cells with CXCR4 siRNA after 100 ng/mL SDF-1α treatment alone or combined with 1 μg/mL NT21MP. (B), Quantitative RT-PCR assays were conducted to detect the expressions of PDGFA, PDGFB, and PDGFRα for panel A. *P < 0.05, SDF-1α, CXCR4-overexpressing, and si-CXCR4 group vs control; ^#P < 0.05, NT21MP treatment vs SDF-1α group. (C), Western blotting results for panel B.

Figure 7. Effect of CXCR4 on cell proliferation and migration abilities

(A), Sulforhodamine B (SRB) assays were conducted to detect the proliferation capacities in MDA-MB-231 and PC-MDA-MB-231 cells or SKBR-3 and CS-SKBR-3 cells after treatment with 100 ng/mL SDF-1α alone or combined with 1 μg/mL NT21MP. (B), Invasion assays were conducted in MDA-MB-231 and PC-MDA-MB-231 cells or SKBR-3 and CS-SKBR-3 cells after SDF-1α treatment alone or combined with NT21MP. PC MDA-MB-231: CXCR4 over-expression in MDA-MB-231 cells and CS-SKBR-3: CXCR4 siRNA in SKBR-3 cells. *P < 0.05, SDF-1α, CXCR4-overexpressing, and si-CXCR4 group vs control; ^#P < 0.05, NT21MP treatment vs SDF-1α group.

Figure 8. Inhibition of PDGFRα enhances the effect of NT21MP-reversed EMT and invasion in SKBR-3 PR cells

(A), Western blot assays were used to detect signal pathway molecule expression. In each group, the cells were treated after SDF-1α treatment alone or combined with NT21MP or PDGFRα inhibitor or activator in SKBR-3 PR cells. (B), Western blotting analysis was performed to detect the expressions of EMT markers in SKBR-3 PR cells treated with PDGFRα inhibitor alone or combined with NT21MP. (C), Invasion assays were conducted in SKBR-3 PR cells treated with PDGFRα inhibitor alone or combined with NT21MP. (D), Western blotting analysis was performed to detect the expression of PDGFRα in SKBR-3 PR cells treated with PDGFRα siRNA. BC: blank control siRNA; NC: negative siRNA; PS1-3: PDGFRα siRNA 1-3. (E), Western blotting analysis was performed to detect the expression of EMT markers in SKBR-3 PR cells treated with PDGFRα siRNA alone or combined with NT21MP.

Figure 9. NT21MP enhances PR cells to paclitaxel sensitivity in vivo

(A), Photographs of tumor size at the time of euthanization in which NT21MP retarded the growth of SKBR-3 PR cells in mice. (B), Total tumor weights in mice were measured at the time of euthanization. *P < 0.05, NT21MP group vs control. (C), Western blotting analysis was performed to detect the expression of PDGFRα and EMT markers.