Transforming growth factor-beta1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells

Abstract

Aim: To investigate whether rhTGF-beta1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-beta receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells.

Methods: MCF-7 breast cancer cells were treated with rhTGF-beta1 or transfected with a recombinant vector, pIRES2-EGFP-TbetaRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1alpha.

Results: CXCR4 mRNA and protein expression were upregulated in TGF-beta1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1alpha. In MCF-7 cells transiently transfected with pIRES2-EGFP-TbetaRII-Fc, a fusion protein named TbetaRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1alpha-mediated chemotactic response.

Conclusion: TGF-beta1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1alpha-induced chemotactic response. The results suggest a link between TGF-beta1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-beta1-mediated enhancement of metastatic potential in breast cancer cells.

Figure 1

Upregulation of CXCR4 expression in TGF-β1-treated MCF-7 cells. MCF-7 cells were treated with rhTGF-β1 at the concentrations of 0 (control), 1, 10, 100 ng/mL for 48 h. (A) CXCR4 mRNA expression was assessed by real-time PCR in MCF-7 cells treated with TGF-β1 or not. Results are expressed as mean±SD for three independent experiments (n=3). Statistical significance was determined by ANOVA followed by Newman-Keuls-Student's t test. ^bP<0.05, ^cP<0.01 vscontrol. (B) CXCR4 protein expression on these MCF-7 cells was determined by flow cytometry using monoclonal anti-CXCR4 conjugated with APC. CXCR4 surface expression was higher on the treated cells than on the control cells. [B(a)], the data presented here represent a typical experiment. [B(b)] represents CXCR4 surface expression, measured as mean fluorescence intensity (MFI), on these cells, and [B(c)] is the result expressed as the percentage of gated cells. In B, each bar represents the mean of 3 independent experiments.

Figure 2

The chemotactic response of TGF-β1-treated MCF-7 cells to SDF-1α. The chemotaxis assay was performed as described in materials and methods. The results are presented as the chemotaxis index, which is a ratio between the number of cells migrated toward SDF-1α and the number of cells migrated in the absence of SDF-1α. Mean±SEM. Representative data are shown from one of three experiments. The chemotaxis data were analyzed using Student's unpaired t-test. ^cP<0.01 vs MCF-7 cells.

Figure 3

Expression of TβRII:Fc fusion protein in TβRII-Fc transfected MCF-7 cells. MCF-7 cells were transfected with pIRES2-EGFP-TβRII-Fc or pIRES2-EGFP (control) for 48 h. (A) TβRII-Fc fusion gene expression was analyzed by RT-PCR and agarose gel electrophoresis. A specific 1200 bp band was observed in TβRII-Fc transfected MCF-7 cells (lane 3), but not in the vector control (lane 2) or parental cells (lane 1), and β-actin mRNA was used as internal control. (B) Total cell lysates were analyzed by Western blotting for the presence of fusion protein, TβRII:Fc. A strong 41 kDa band was detected in TβRII-Fc transfected MCF-7 cells (lane 3), but not in vector control (lane 2) or parental cells (lane 1). (C) An ELISA assay was used to measure the TβRII:Fc fusion protein in the culture supernatants of the tested cells. Each bar represents the mean of six independent experiments.

Figure 4

Downregulation of CXCR4 expression in TβRII-Fc transfected MCF-7 cells. MCF-7 cells were transfected with pIRES2-EGFP-TβRII-Fc or pIRES2-EGFP (control) for 48 h, and then these cells were harvested and examined by quantitative RT-PCR and flow cytometry, respectively. (A) The result of quantitative RT-PCR revealed that CXCR4 mRNA expression was reduced by 60% in TβRII-Fc transfected cells as compared with that in the control cells. Data are expressed as mean±SEM of three separate experiments. Statistical significance was assessed by using Student's unpaired t-test. ^bP<0.05. (B) The flow cytometric analysis showed that the level of CXCR4 expression in pIRES2-EGFP-TβRII-Fc transfected MCF-7 cells [B(b)] was lower than that in pIRES2-EGFP transfected MCF-7 cells [B(a)]. The transfected MCF-7 cells were classified into four types according to the expression levels of CXCR4 (ie the relative level of APC fluorescence) and EGFP: CXCR4⁻EGFP⁻, CXCR4⁻EGFP⁺, CXCR4⁺EGFP⁻ and CXCR4⁺EGFP⁺. In TβRII-Fc transfected cells, CXCR4 expression was decreased on both CXCR4⁺EGFP⁻ and CXCR4⁺EGFP⁺cells, compared with those of corresponding pIRES2-EGFP transfected MCF-7 cells [B(c)]. [B(d)] represents the difference of CXCR4 expression between TβRII-Fc transfected MCF-7 cells (red line) and control cells. And the bar graph [B(e)] shows the MFI of CXCR4 expression on these MCF-7 cells. The bar graph summarizes three independent experiments.

Figure 5

A reduction of the SDF-1α-induced chemotactic response in TβRII-Fc transfected MCF-7 cells. The transfected MCF-7 cells were subjected to chemotaxis assays as described above. The results are presented as the chemotaxis index. Representative data are shown from one of three experiments, and the chemotaxis data were analyzed using Student's unpaired t-test. ^cP<0.01.