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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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Tumorigenicity assay in nude mice. A: The growth rates of tumors formed from untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or shFOXA1 (MFE-296/shFOXA1). After injection, tumor volumes were calculated every seven days. B and C: Six weeks after injection of MFE-296, MFE-296-NC, and MFE-296-shFOXA1 cells, tumors were removed, and the tumor weights and volumes were determined. Arithmetic means and SD are shown. D: Staining with hematoxylin and eosin (H&E) or immunohistochemical staining for FOXA1, AR, Notch1, Hes1, Ki67, and PCNA in mouse tumor tissues (immunohistochemical staining, 200×). *p < 0.05 compared with the NC group.
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Figure 7: Tumorigenicity assay in nude mice. A: The growth rates of tumors formed from untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or shFOXA1 (MFE-296/shFOXA1). After injection, tumor volumes were calculated every seven days. B and C: Six weeks after injection of MFE-296, MFE-296-NC, and MFE-296-shFOXA1 cells, tumors were removed, and the tumor weights and volumes were determined. Arithmetic means and SD are shown. D: Staining with hematoxylin and eosin (H&E) or immunohistochemical staining for FOXA1, AR, Notch1, Hes1, Ki67, and PCNA in mouse tumor tissues (immunohistochemical staining, 200×). *p < 0.05 compared with the NC group.

Mentions: Tumors generated by subcutaneous implantation of MFE-296 cells were used to evaluate the effect of FOXA1 on proliferation in a mouse tumor xenograft model. We measured tumor volumes in xenografted mice over a 6-week period following injection of untransfected MFE-296 (MFE-296), stably transfected with shFOXA1 (MFE-296/shFOXA1) or NC (MFE-296/NC). These measurements indicated that tumors in the MFE-296/shFOXA1 group grew significantly slower than those in the MFE-296/NC group and the MFE-296 group (Figure 7A). Six weeks after injection, tumors were removed from the mice (Figure 7C). The final mean weight and volume of tumors in the MFE-296/shFOXA1 group were significantly lower than those in the MFE-296/NC group (p < 0.05, Figure 7B and 7C). Tumor tissues were then embedded in paraffin, stained with hematoxylin and eosin (H&E), and immunohistochemically stained with antibodies against FOXA1, AR, Notch1, Hes1, Ki67, or PCNA. Lower FOXA1 expression in the MFE-296/shFOXA1 group also led to reduced staining for AR, indicating that FOXA1 also affected AR expression in vivo, in accordance with the results in vitro. As expected, the MFE-296/shFOXA1 group had significantly lower levels of Notch1 and Hes1 (Figure 7D), thus verifying the role of FOXA1 as a positive regulator of the Notch pathway in vivo. Furthermore, to determine the proliferative ability of MFE-296 cells, we performed immunohistochemical staining of Ki67 and PCNA, which are expressed as proliferation indices. The observed lower expression of Ki67 and PCNA in the MFE-296/shFOXA1 group was consistent with the smaller tumor volumes in the mouse tumor xenograft model (Figure 7D).


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)

Tumorigenicity assay in nude mice. A: The growth rates of tumors formed from untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or shFOXA1 (MFE-296/shFOXA1). After injection, tumor volumes were calculated every seven days. B and C: Six weeks after injection of MFE-296, MFE-296-NC, and MFE-296-shFOXA1 cells, tumors were removed, and the tumor weights and volumes were determined. Arithmetic means and SD are shown. D: Staining with hematoxylin and eosin (H&E) or immunohistochemical staining for FOXA1, AR, Notch1, Hes1, Ki67, and PCNA in mouse tumor tissues (immunohistochemical staining, 200×). *p < 0.05 compared with the NC group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Tumorigenicity assay in nude mice. A: The growth rates of tumors formed from untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or shFOXA1 (MFE-296/shFOXA1). After injection, tumor volumes were calculated every seven days. B and C: Six weeks after injection of MFE-296, MFE-296-NC, and MFE-296-shFOXA1 cells, tumors were removed, and the tumor weights and volumes were determined. Arithmetic means and SD are shown. D: Staining with hematoxylin and eosin (H&E) or immunohistochemical staining for FOXA1, AR, Notch1, Hes1, Ki67, and PCNA in mouse tumor tissues (immunohistochemical staining, 200×). *p < 0.05 compared with the NC group.
Mentions: Tumors generated by subcutaneous implantation of MFE-296 cells were used to evaluate the effect of FOXA1 on proliferation in a mouse tumor xenograft model. We measured tumor volumes in xenografted mice over a 6-week period following injection of untransfected MFE-296 (MFE-296), stably transfected with shFOXA1 (MFE-296/shFOXA1) or NC (MFE-296/NC). These measurements indicated that tumors in the MFE-296/shFOXA1 group grew significantly slower than those in the MFE-296/NC group and the MFE-296 group (Figure 7A). Six weeks after injection, tumors were removed from the mice (Figure 7C). The final mean weight and volume of tumors in the MFE-296/shFOXA1 group were significantly lower than those in the MFE-296/NC group (p < 0.05, Figure 7B and 7C). Tumor tissues were then embedded in paraffin, stained with hematoxylin and eosin (H&E), and immunohistochemically stained with antibodies against FOXA1, AR, Notch1, Hes1, Ki67, or PCNA. Lower FOXA1 expression in the MFE-296/shFOXA1 group also led to reduced staining for AR, indicating that FOXA1 also affected AR expression in vivo, in accordance with the results in vitro. As expected, the MFE-296/shFOXA1 group had significantly lower levels of Notch1 and Hes1 (Figure 7D), thus verifying the role of FOXA1 as a positive regulator of the Notch pathway in vivo. Furthermore, to determine the proliferative ability of MFE-296 cells, we performed immunohistochemical staining of Ki67 and PCNA, which are expressed as proliferation indices. The observed lower expression of Ki67 and PCNA in the MFE-296/shFOXA1 group was consistent with the smaller tumor volumes in the mouse tumor xenograft model (Figure 7D).

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