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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 AR-mediated transcription via binding with AR and activates the Notch pathway. A: Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B: Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10−7 M DHT or vehicle. C: Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D: Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [19]. E: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p < 0.05, **p < 0.01 and NS p > 0.05.
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Figure 4: FOXA1 affects AR-mediated transcription via binding with AR and activates the Notch pathway. A: Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B: Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10−7 M DHT or vehicle. C: Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D: Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [19]. E: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p < 0.05, **p < 0.01 and NS p > 0.05.

Mentions: To investigate whether FOXA1 affects AR-mediated transcription through binding to AR, we performed co-immunoprecipitation experiments. We used nuclear lysates from MFE-296 cells to conduct immunoprecipitation with anti-FOXA1. FOXA1 co-immunoprecipitated with AR, whereas immunoprecipitation with the isotype IgG control did not pull down AR or FOXA1 (Figure 4A), indicating that FOXA1 interacted with AR in MFE-296 cells. We also performed the co-immunoprecipitation experiment in AN3CA cells, which has low level of AR. As shown in Figure 4B, AR could be immunoprecipitated by anti-FOXA1 in the presence of DHT but not in its absence. This result indicated that FOXA1 and AR interacted physically. It is likely that FOXA1 affects AR-mediated transcription via binding with AR.


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 AR-mediated transcription via binding with AR and activates the Notch pathway. A: Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B: Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10−7 M DHT or vehicle. C: Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D: Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [19]. E: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p < 0.05, **p < 0.01 and NS p > 0.05.
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Figure 4: FOXA1 affects AR-mediated transcription via binding with AR and activates the Notch pathway. A: Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B: Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10−7 M DHT or vehicle. C: Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D: Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [19]. E: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F: Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p < 0.05, **p < 0.01 and NS p > 0.05.
Mentions: To investigate whether FOXA1 affects AR-mediated transcription through binding to AR, we performed co-immunoprecipitation experiments. We used nuclear lysates from MFE-296 cells to conduct immunoprecipitation with anti-FOXA1. FOXA1 co-immunoprecipitated with AR, whereas immunoprecipitation with the isotype IgG control did not pull down AR or FOXA1 (Figure 4A), indicating that FOXA1 interacted with AR in MFE-296 cells. We also performed the co-immunoprecipitation experiment in AN3CA cells, which has low level of AR. As shown in Figure 4B, AR could be immunoprecipitated by anti-FOXA1 in the presence of DHT but not in its absence. This result indicated that FOXA1 and AR interacted physically. It is likely that FOXA1 affects AR-mediated transcription via binding with AR.

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