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FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer.

Qiu M, Bao W, Wang J, Yang T, He X, Liao Y, Wan X - BMC Cancer (2014)

Bottom Line: We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues.However, AR didn't influence the promotion effect of FOXA1 on cell migration and invasion.These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Xinsongjiang Road, Shanghai, China. wanxp@sjtu.edu.cn.

ABSTRACT

Background: Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear.

Methods: FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor-formation assays.

Results: We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn't influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth.

Conclusions: These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC.

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FOXA1 affects the expression of AR in human EC cells. A: FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B: Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at >85%. Magnification, ×400. C: Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D: Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E: FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F: Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G: Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H: AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p < 0.05, **p < 0.01, NS p > 0.05 compared with NC.
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Figure 2: FOXA1 affects the expression of AR in human EC cells. A: FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B: Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at >85%. Magnification, ×400. C: Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D: Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E: FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F: Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G: Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H: AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p < 0.05, **p < 0.01, NS p > 0.05 compared with NC.

Mentions: We used western blotting to examine FOXA1 and AR expression in EC cells. FOXA1 was upregulated in MFE-296 cells compared with KLE, HEC-1B, and AN3CA cells. Furthermore, the AR level was also markedly higher in MFE-296 cells than in the other three EC cell lines (Figure 2A).


FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer.

Qiu M, Bao W, Wang J, Yang T, He X, Liao Y, Wan X - BMC Cancer (2014)

FOXA1 affects the expression of AR in human EC cells. A: FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B: Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at >85%. Magnification, ×400. C: Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D: Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E: FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F: Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G: Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H: AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p < 0.05, **p < 0.01, NS p > 0.05 compared with NC.
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Figure 2: FOXA1 affects the expression of AR in human EC cells. A: FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B: Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at >85%. Magnification, ×400. C: Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D: Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E: FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F: Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G: Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H: AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p < 0.05, **p < 0.01, NS p > 0.05 compared with NC.
Mentions: We used western blotting to examine FOXA1 and AR expression in EC cells. FOXA1 was upregulated in MFE-296 cells compared with KLE, HEC-1B, and AN3CA cells. Furthermore, the AR level was also markedly higher in MFE-296 cells than in the other three EC cell lines (Figure 2A).

Bottom Line: We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues.However, AR didn't influence the promotion effect of FOXA1 on cell migration and invasion.These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Xinsongjiang Road, Shanghai, China. wanxp@sjtu.edu.cn.

ABSTRACT

Background: Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear.

Methods: FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor-formation assays.

Results: We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn't influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth.

Conclusions: These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC.

Show MeSH
Related in: MedlinePlus