DNA Methylation Affects The SP1 Transcription Factor-regulated Functions Of Forkhead FOXF2 In Breast Cancer Cells

BackgroundForkhead box F2(FOXF2) is a mesenchyme-specific transcription factor that plays a critical role in embryonic development and tissue differentiation by maintaining epithelium polarity. Our previous study demonstrated that FOXF2 is specifically expressed in basal-like breast cancer(BLBC) cells and functions as an EMT suppressor. FOXF2 deficiency enhances the metastatic ability of BLBC through the activation of the EMT program but reduces cell proliferation. It has also been demonstrated that FOXF2 play a critical role in tumorigenesis and cancer progression in other studies. However, the molecular mechanism underlying regulation of FOXF2 expression in breast cancer remains largely unknow. The present study characterized the proximal promoter region of FOXF2 with a long-range Cp G island and several putative binding sites of transcription factor SP1. Thus, we suggest that the subtype-specific expression pattern and function of FOXF2 in breast cancer cells are regulated by the combined effects of DNA methylation and SP1 transcriptional regulation.PurposesThe purposes of this sdudy are to clarify the moelcular mechanism underlying DNA methylation and SP1 in regulation of the subtype-specific expression pattern and function of FOXF2 in breast cancer.Methods1、 To determine the FOXF2 expression of human breast cancer tissues(n = 20) and breast cancer cell lines, including MCF-7, MDA-MB-453, MDA-MB-231 and MCF-10 A, the m RNA and protein expression of FOXF2 in the four breast cell lines as well as the FOXF2 m RNA expression in breast cancer tissues were detected by transcription quantitative polymerase chain reaction(RT-QPCR) and Western blot.2、 To further determine the correlation of FOXF2 expression and DNA methylation,MCF-7, MDA-MB-453, MDA-MB-231 and MCF-10 A cells were treated with the DNA methyltransferase inhibitor 5-aza-d C at concentrations of 0, 0.5, 1.0, 1.5,2.0, and 2.5 μM, and the FOXF2 expression levels were then measured by RT-QPCR and Western blot.3、 To further determine which DNMTs contribute to FOXF2 promoter methylation,si RNAs targeting DNMTs, including si DNMT1, si DNMT3 A, and si DNMT3 B,were transiently transfected into MCF-7 and MDA-MB-453 cells, and the efficiency of si RNA silencing and FOXF2 expression was measured by RT-QPCR and Western blot. To further understand the role of DNMTs-induced repression of FOXF2, we used Ch IP assays to study the binding of DNMTs to the methylated promoter using primers designed to span the entire Cp G island. Ch IP reactions were performed using anti-DNMT1, DNMT3 A, DNMT3 B in MCF-7and MDA-MB-453 cells.4、 To examine whether DNA methylation directly represses FOXF2 promoter activity, the proximal promoter region –655/+128 of FOXF2 was methylated using methylase Sss I, Hha I, and Hpa II in vitro. Then, the methylated inserts were cloned into a luciferase reporter construct. The promoter activity of the differentially methylated FOXF2 proximal promoter region was assessed by transfection of the luciferase reporter constructs into MDA-MB-231 cells.5、 Chromatin immunoprecitation(Ch IP) and dual-luciferase reporter assays were performed to demonstrate the transcriptional regulation of SP1 on FOXF2. To investigate the role of SP1 in regulation of FOXF2 expression in breast cells, an SP1 expression plasmid or a si RNA against SP1(si SP1) was transiently transfected into MDA-MB-231 and MCF-10 A cells, respectively. Then the SP1 and FOXF2 expression were detected by RT-QPCR and Western blot.Meanwhile, MDA-MB-231 and MCF10 A cells were treated with varying concentrations of mithramycin A(MTM). Then m RNA and protein expression levels of FOXF2 were measured by RT-QPCR and Western blot.6、 To demonstrate the effect of FOXF2 promoter methylation on the promoter binding of SP1, MCF-7 cells with the methylated FOXF2 proximal promoterregion were treated with 5-aza-d C and then subjected to Ch IP assays using an anti-SP1 antibody. Furthermore, dual-luciferase reporter assays were carried out in MDA-MB-231 cells with co-transfection of differentially methylated FOXF2 promoter constructs and SP1 expression plasmid or vector control.7、 To illustrated FOXF2 mediates the SP1-regulated suppression of progression and promotion of proliferation in non-methylated breast cells, the SP1 expression plasmid was co-transfected with a si RNA against FOXF2(si FOXF2) or a negative control si RNA(si Control) into MDA-MB-231 cells, and the cell migration, invasion and proliferation capacities were assessed using in vitro Transwell assays and MTT assays, respectively. In addition, TWIST1 expression,a target gene of FOXF2, was measured by Western blot. Meanwhile, si SP1 was co-transfected with the SP1 expression plasmid or vector control into MCF-10 A cells, and the cell migration, invasion and proliferation capacities and TWIST1 expression were measured according to the above-mentioned method.8、 To provide further clinical evidence for SP1/FOXF2-regulated function on the progression of different breast cancer subtypes, we mined the SP1 and FOXF2 m RNA expression data from the gene expression profiling dataset of 427 breast cancer tissues(GEO No. GSE25066) and analyzed the distant metastasis free survival(DMFS) among patients with different SP1 and FOXF2 expression statues in basal-like and luminal subtypes.Results1、 The proximal promoter region of FOXF2 was highly methylated in MCF-7 and MDA-MB-453 cells but unmethylated in MDA-MB-231 and MCF-10 A cells.Consistently, FOXF2 m RNA and protein were expressed at a high level in MDA-MB-231 and MCF-10 A cells, whereas a low level in MCF-7 and MDA-MB-453 cells was found. The methylation rate of the high FOXF2 m RNA expression group(FOXF2high; n = 10; median = 6.6%) was significantly lower than the low FOXF2 m RNA expression group(FOXF2low; n = 10; median =14.0%; P = 0.027). These results indicate that the FOXF2 expression level correlates inversely to the methylation status of the Cp G island in its proximalpromoter region in breast cancer cells.2、 The demethylation effect of 5-aza-d C on the Cp G island of FOXF2 in the highly methylated MCF-7 and MDA-MB-453 cells was confirmed by bisulfite sequencing. 5-aza-d C elicited a dose-dependent induction of FOXF2 expression in the highly methylated MCF-7 and MDA-MB-453 cells but did not affect FOXF2 expression on both m RNA and protein levels in the unmethylated MDA-MB-231 and MCF-10 A cells. These results confirmed that the methylation of the FOXF2 proximal promoter region leads to the suppression of its expression in breast cancer cell lines.3、 The results of RT-QPCR and western blot showed that FOXF2 expression was induced by DNMT1 or DNMT3 A depletion but not by DNMT3 B depletion in MCF-7 cells. We also observed that FOXF2 expression was restored by DNMT1 or DNMT3B depletion but not by DNMT3 A depletion in MDA-MB-453 cells.The Ch IP experiments indicated that DNMT1 and DNMT3 A bind to the methylated promoter whereas there is no DNMT3 B binding to these regions in MCF-7 cells, and MDA-MB-453 cells showed the binding of DNMT1 and DNM3 B to these regions expect for DNMT3 A. These results demonstrated the distinct effects of speci?c DNMTs on FOXF2 expression in different breast caner cells.4、 Dual-luciferase reporter assay indicated that FOXF2 promoter activity was repressed by methylation with methylase Sss I, Hpa II, or Hha I. Sss I, which methylates all Cp G sites, showed the greatest repression of FOXF2 promoter activity, whereas both Hha I and Hpa II, which methylate a portion of Cp G sites,showed the less repression. These results demonstrated DNA methylation represses FOXF2 promoter activity.5、 ChIP assay showed that SP1 clearly bound to the FOXF2 promoter and and dual-luciferase reporter assay indicated that SP1 enhanced the transcriptional activity of FOXF2. SP1 overexpression signi?cantly increased FOXF2 m RNA and protein exptession, whereas SP1 knockdown decreased FOXF2 expression in MDA-MB-231 and MCF-10 A cells. FOXF2 m RNA and protein expression were reduced with increasing MTM concentrations. These results indicated that SP1up-regulates FOXF2 transcriptional expression in breast cancer cells with an un-methylated FOXF2 proximal promoter region.6、 Ch IP assay showed that very weak binding of SP1 to the FOXF2 promoter in MCF-7 cells without treatment, whereas markedly enhanced binding was observed in the cells treated with 5-aza-d C. the exogenous SP1 expression enhanced significantly the activity of FOXF2 promoter with Hha I, Hpa II or no methylase treatment compared with the vector control, whereas SP1 barely activated the Sss I methylated FOXF2 promoter. dual-luciferase reporter assay revealed that the exogenous SP1 expression enhanced significantly the activity of FOXF2 promoter with Hha I, Hpa II or no methylase treatment compared with the vector control, whereas SP1 barely activated the Sss I methylated FOXF2 promoter. These results indicated that DNA methylation abrogates SP1 binding to the FOXF2 promoter.7、 SP1 overexpression could attenuat cell migration and invasion abilities, and inhibit the TWIST1 expression, and enhance the cell proliferation ability in MDA-MB-231 cells. But these affection of SP1 overexpression on MDA-MB-231 cells were reversed by si FOXF2 transfection. SP1 depletion could increase cell migration and invasion abilities, and improve the TWIST1 expression, and restrain the cell proliferation ability in MCF-10 A cells. But these affection of SP1 depletion on MCF-10 A cells were restored by FOXF2 transfection. These results suggested that FOXF2 mediates the SP1-regulated suppression of progression and promotion of proliferation in non-methylated breast cancer cells.8、 The patients were grouped into SP1high/FOXF2high(n = 111), SP1low/FOXF2high(n = 65),SP1high/FOXF2low(n = 154), and SP1low/FOXF2low(n = 97) using the optimal cutoff values of SP1 and FOXF2 m RNA expression. For basal-like subtype(n = 189), the patients in SP1low/FOXF2 low group had poorest DMFS compared with those in other three groups. Whereas, for luminal subtype(n = 238), the patients had no significant different DMFS among the four groups. These results confirmed that FOXF2 deficiency result from low SP1 expression could lead to BLBC metastasis, but do not affect luminal breast caner.Conclusions1、 DNA methylation contribute to the silencing of FOXF2 expression in breast cancer.2、 DNA methylation could inhibit the transcriptional activation of FOXF2 promoter by SP1.3、 FOXF2 mediates the SP1-regulated suppression of progression and promotion of proliferation in breast cancer cells.