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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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Related in: MedlinePlus

FOXA1 affects AR-mediated transcription. A: MFE-296 cells were treated with DHT (10−9 to 10−7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B: Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10−7 M DHT for 0–48 h. C: Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10−7 M DHT, or 10−7 M DHT plus 10−6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D: MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E: AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F: Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H: Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, NS p > 0.05.
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Figure 3: FOXA1 affects AR-mediated transcription. A: MFE-296 cells were treated with DHT (10−9 to 10−7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B: Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10−7 M DHT for 0–48 h. C: Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10−7 M DHT, or 10−7 M DHT plus 10−6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D: MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E: AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F: Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H: Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, NS p > 0.05.

Mentions: We next examined whether the FOXA1 level impacted the expression of AR target genes in EC cells. MFE-296 cells were hormone deprived and treated with vehicle or the AR pathway agonist DHT [20], and then the expression of AR target genes (XBP1, MYC, ZBTB16, and UHRF1) [12] was evaluated by qRT-PCR. This analysis confirmed that the expression of these four genes increased after treatment with DHT. Furthermore, the dose-response study (0–10−7 M DHT) and time-response study (0–48 h) indicated that 10−7 M DHT and 24 h of incubation elicited the strongest expression of AR and its target genes (Figure 3A and 3B). These data confirmed that these four genes were downstream of the AR-mediated transcription in EC cells. To partially confirm the promoting effect of DHT on AR-mediated transcription at the protein level, AR expression was examined by western blotting; DHT acted as an agonist, whereas the addition of the AR antagonist flutamide [21] reduced the DHT-enhanced expression of AR in MFE-296 cells (Figure 3C).


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. A: MFE-296 cells were treated with DHT (10−9 to 10−7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B: Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10−7 M DHT for 0–48 h. C: Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10−7 M DHT, or 10−7 M DHT plus 10−6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D: MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E: AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F: Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H: Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, NS p > 0.05.
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Figure 3: FOXA1 affects AR-mediated transcription. A: MFE-296 cells were treated with DHT (10−9 to 10−7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B: Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10−7 M DHT for 0–48 h. C: Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10−7 M DHT, or 10−7 M DHT plus 10−6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D: MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E: AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10−7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F: Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H: Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I: Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001, NS p > 0.05.
Mentions: We next examined whether the FOXA1 level impacted the expression of AR target genes in EC cells. MFE-296 cells were hormone deprived and treated with vehicle or the AR pathway agonist DHT [20], and then the expression of AR target genes (XBP1, MYC, ZBTB16, and UHRF1) [12] was evaluated by qRT-PCR. This analysis confirmed that the expression of these four genes increased after treatment with DHT. Furthermore, the dose-response study (0–10−7 M DHT) and time-response study (0–48 h) indicated that 10−7 M DHT and 24 h of incubation elicited the strongest expression of AR and its target genes (Figure 3A and 3B). These data confirmed that these four genes were downstream of the AR-mediated transcription in EC cells. To partially confirm the promoting effect of DHT on AR-mediated transcription at the protein level, AR expression was examined by western blotting; DHT acted as an agonist, whereas the addition of the AR antagonist flutamide [21] reduced the DHT-enhanced expression of AR in MFE-296 cells (Figure 3C).

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