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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 induces migration and invasion in EC cells. A: Cell migration of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. The abbreviation “HPF” on the y axis means one high power field. B: Cell migration of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells were subjected to transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. C: Cell invasion of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was shown in the right panel. D: Cell invasion of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells by three independent experiments were subjected to transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was showed in the right panel. (Magnification, 200×). *p < 0.05, **p < 0.01, ***p < 0.001, and NS p > 0.05.
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Figure 6: FOXA1 induces migration and invasion in EC cells. A: Cell migration of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. The abbreviation “HPF” on the y axis means one high power field. B: Cell migration of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells were subjected to transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. C: Cell invasion of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was shown in the right panel. D: Cell invasion of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells by three independent experiments were subjected to transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was showed in the right panel. (Magnification, 200×). *p < 0.05, **p < 0.01, ***p < 0.001, and NS p > 0.05.

Mentions: Our immunohistochemistry results revealed that patients with myometrial invasion displayed higher FOXA1 expression. With this observation in mind, we hypothesized that functional expression of FOXA1 might induce tumor metastasis in EC. To explore the role of FOXA1 in the regulation of metastatic function and to determine whether AR is involved in FOXA1-mediated regulation of metastatic function, we examined the migration and invasion ability of MFE-296/shFOXA1 and AN3CA/exFOXA1 cells after exAR or siAR cotransfection using transwell migration and invasion assays. MFE-296/shFOXA1 cells displayed a decreased rate of migration compared to MFE-296/NC cells (Figure 6A). However, cotransfection of MFE-296/shFOXA1 cells with exAR (MFE-296/shFOXA1 + exAR) did not rescue the migration to the levels observed in MFE-296/NC or untransfected cells (MFE-296) (Figure 6A). Furthermore, AN3CA/exFOXA1 cells exhibited a high migration rate as compared with AN3CA/NC cells, but cotransfection with siAR (AN3CA/exFOXA1 + siAR) did not significantly attenuate the migration rate (Figure 6B).


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 induces migration and invasion in EC cells. A: Cell migration of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. The abbreviation “HPF” on the y axis means one high power field. B: Cell migration of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells were subjected to transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. C: Cell invasion of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was shown in the right panel. D: Cell invasion of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells by three independent experiments were subjected to transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was showed in the right panel. (Magnification, 200×). *p < 0.05, **p < 0.01, ***p < 0.001, and NS p > 0.05.
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Figure 6: FOXA1 induces migration and invasion in EC cells. A: Cell migration of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. The abbreviation “HPF” on the y axis means one high power field. B: Cell migration of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells were subjected to transwell migration analysis (Left). The mean ± SD number of migrated cells of three independent experiments was showed in the right panel. C: Cell invasion of MFE-296, MFE-296/NC, MFE-296/shFOXA1 and MFE-296/shFOXA1 + exAR cells was assessed by the transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was shown in the right panel. D: Cell invasion of AN3CA, AN3CA/NC, AN3CA/exFOXA1 and AN3CA/exFOXA1 + siAR cells by three independent experiments were subjected to transwell invasion analysis (Left). The mean ± SD number of invased cells of three independent experiments was showed in the right panel. (Magnification, 200×). *p < 0.05, **p < 0.01, ***p < 0.001, and NS p > 0.05.
Mentions: Our immunohistochemistry results revealed that patients with myometrial invasion displayed higher FOXA1 expression. With this observation in mind, we hypothesized that functional expression of FOXA1 might induce tumor metastasis in EC. To explore the role of FOXA1 in the regulation of metastatic function and to determine whether AR is involved in FOXA1-mediated regulation of metastatic function, we examined the migration and invasion ability of MFE-296/shFOXA1 and AN3CA/exFOXA1 cells after exAR or siAR cotransfection using transwell migration and invasion assays. MFE-296/shFOXA1 cells displayed a decreased rate of migration compared to MFE-296/NC cells (Figure 6A). However, cotransfection of MFE-296/shFOXA1 cells with exAR (MFE-296/shFOXA1 + exAR) did not rescue the migration to the levels observed in MFE-296/NC or untransfected cells (MFE-296) (Figure 6A). Furthermore, AN3CA/exFOXA1 cells exhibited a high migration rate as compared with AN3CA/NC cells, but cotransfection with siAR (AN3CA/exFOXA1 + siAR) did not significantly attenuate the migration rate (Figure 6B).

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