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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 promotes proliferation of human EC cells by affecting AR-mediated transcription. A: Proliferation in MFE-296 cells transfected with NC or shFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). B: Proliferation in AN3CA cells transfected with NC or exFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). C: Assessment of proliferation by the MTT assay in MFE-296 cells transfected with NC or shFOXA1. D: Assessment of proliferation by the MTT assay in AN3CA cells transfected with NC or exFOXA1. E: Left: Colony-formation assay of untransfected MFE-296 cells (WT) and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR. Right: Graphical representation of the fold change in the number of colonies 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). F: Proliferation of MFE-296, MFE-296/NC, MFE-296/shFOXA1, or MFE-296/shFOXA1 + exAR cells was assessed by MTT assay. The right panel reiterates the data in the left panel at 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 and NS p > 0.05.
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Figure 5: FOXA1 promotes proliferation of human EC cells by affecting AR-mediated transcription. A: Proliferation in MFE-296 cells transfected with NC or shFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). B: Proliferation in AN3CA cells transfected with NC or exFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). C: Assessment of proliferation by the MTT assay in MFE-296 cells transfected with NC or shFOXA1. D: Assessment of proliferation by the MTT assay in AN3CA cells transfected with NC or exFOXA1. E: Left: Colony-formation assay of untransfected MFE-296 cells (WT) and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR. Right: Graphical representation of the fold change in the number of colonies 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). F: Proliferation of MFE-296, MFE-296/NC, MFE-296/shFOXA1, or MFE-296/shFOXA1 + exAR cells was assessed by MTT assay. The right panel reiterates the data in the left panel at 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 and NS p > 0.05.

Mentions: To examine the role of FOXA1 in EC cell proliferation, we assessed the effect of FOXA1 in colony-forming and MTT assays. In the colony-forming assay, MFE-296 cell transfected with shFOXA1 showed significantly decreased colony-forming ability when compared with MFE-296 cells transfected with NC (Figure 5A). Moreover, upregualtion of FOXA1 in AN3CA cells showed increased colony-forming ability compared with NC cells (Figure 5B). In the MTT assay, downregulation of FOXA1 in MFE-296 cells resulted in poor cell viability (Figure 5C), and upregulation of FOXA1 in AN3CA cells caused increased cell viability (Figure 5D). These data indicated that FOXA1 promoted cell proliferation.


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 promotes proliferation of human EC cells by affecting AR-mediated transcription. A: Proliferation in MFE-296 cells transfected with NC or shFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). B: Proliferation in AN3CA cells transfected with NC or exFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). C: Assessment of proliferation by the MTT assay in MFE-296 cells transfected with NC or shFOXA1. D: Assessment of proliferation by the MTT assay in AN3CA cells transfected with NC or exFOXA1. E: Left: Colony-formation assay of untransfected MFE-296 cells (WT) and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR. Right: Graphical representation of the fold change in the number of colonies 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). F: Proliferation of MFE-296, MFE-296/NC, MFE-296/shFOXA1, or MFE-296/shFOXA1 + exAR cells was assessed by MTT assay. The right panel reiterates the data in the left panel at 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 and NS p > 0.05.
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Figure 5: FOXA1 promotes proliferation of human EC cells by affecting AR-mediated transcription. A: Proliferation in MFE-296 cells transfected with NC or shFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). B: Proliferation in AN3CA cells transfected with NC or exFOXA1 was assessed by the colony-forming assay (Left) and further quantified in the number of colonies of triplicate experiments (Right). C: Assessment of proliferation by the MTT assay in MFE-296 cells transfected with NC or shFOXA1. D: Assessment of proliferation by the MTT assay in AN3CA cells transfected with NC or exFOXA1. E: Left: Colony-formation assay of untransfected MFE-296 cells (WT) and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR. Right: Graphical representation of the fold change in the number of colonies 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). F: Proliferation of MFE-296, MFE-296/NC, MFE-296/shFOXA1, or MFE-296/shFOXA1 + exAR cells was assessed by MTT assay. The right panel reiterates the data in the left panel at 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 and NS p > 0.05.
Mentions: To examine the role of FOXA1 in EC cell proliferation, we assessed the effect of FOXA1 in colony-forming and MTT assays. In the colony-forming assay, MFE-296 cell transfected with shFOXA1 showed significantly decreased colony-forming ability when compared with MFE-296 cells transfected with NC (Figure 5A). Moreover, upregualtion of FOXA1 in AN3CA cells showed increased colony-forming ability compared with NC cells (Figure 5B). In the MTT assay, downregulation of FOXA1 in MFE-296 cells resulted in poor cell viability (Figure 5C), and upregulation of FOXA1 in AN3CA cells caused increased cell viability (Figure 5D). These data indicated that FOXA1 promoted cell proliferation.

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