FOXA1 promotes prostate cancer angiogenesis by inducing multiple pro-angiogenic factors expression

Abstract

Purpose

FOXA1, as a pioneering transcription factor, has been shown to drive prostate cancer progression. Previous studies showed that FOXA1 expression in prostate cancer was positively associated with cancer angiolymphatic invasion and metastasis. However, the mechanism underlying the correlation between FOXA1 and prostate cancer angiolymphatic invasion and metastasis remains largely unclear.

Methods

Herein, we set out to investigate the role of FOXA1 in the interactions between prostate cancer cells and endothelial cells. Endothelial cells' phenotypes were assessed through CCK‐8 assay, Transwell migration assay, and tube formation assay. The angiogenic factors acting on endothelial cells mediated by FOXA1were characterized by RNA-seq, qPCR array, angiogenesis cytokines array, and ELISA assay. The impact of FOXA1 on tumor angiogenesis was examined in a xenograft model in nude mice. The effect of FOXA1 on prostate cancer angiogenesis was validated on a primary prostate cancer tissue microarray.

Results

FOXA1 expression in prostate cancer cells promoted endothelial cell proliferation, migration, and tube formation in vitro. Mechanistically, FOXA1 increased pro-angiogenic factors production, including EGF, Endothelin-1, and Endoglin. Moreover, in vivo study showed that FOXA1 facilitated tumor angiogenesis. Furthermore, clinical samples investigation indicated that FOXA1 enhanced prostate cancer angiogenesis.

Conclusion

Overall, these findings illustrated a tumor angiogenesis-promoting role of FOXA1 in prostate cancer.

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Acknowledgements

We thank Prof. Deshui Jia for his helpful suggestions, Dr. Chenyi Jiang, for his precious technical support and coaching. This work was supported by the Natural Science Foundation of Shanghai, China (Grant no: 19ZR1441200).

Funding

This work was sponsored by the Natural Science Foundation of Shanghai, China (Grant no: 19ZR1441200).

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Authors

Contributions

Conception and design: XW, BH. Development of methodology: YS, XW. Acquisition of data: YS, YZ. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): YS, YZ. Drafting the article or revising it critically for important intellectual content: YS, XW. Study supervision: BH, XW.

Corresponding authors

Correspondence to Bangmin Han or Xiaohai Wang.

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Supplementary Information

Below is the link to the electronic supplementary material.

432_2021_3730_MOESM1_ESM.pdf

Supplementary Fig. S1 Identification of primary HUVEC cells by immunofluorescence.a. Bright field of primary HUVEC cells. b. Immunofluorescence microscopy of primary HUVEC cells stained with DAPI. c. Immunofluorescence microscopy of primary HUVEC cells stained with vWF. d. Immunofluorescence microscopy of primary HUVEC cells stained with CD31. Scale bars, 50 μm (PDF 1272 KB)

432_2021_3730_MOESM2_ESM.pdf

Supplementary Fig. S2 Functional enrichment analysis of differentially expressed genes between oeNC-DU145 and oeFOXA1-DU145 cells. a. The top 10 upregulated pathways between oeNC-DU145 and oeFOXA1-DU145 cells identified by the GO enrichment analysis. b. The top 20 upregulated pathways in oeFOXA1-DU145 cells were identified by the KEGG enrichment analysis (PDF 264 KB)

432_2021_3730_MOESM3_ESM.pdf

Supplementary Fig. S3 Quantitative RT-PCR analyses of the mRNA extracted from the excised xenograft tumors containing oeNC-DU145 (n=5) or oeFOXA1-DU145 cells (n=6). a. Quantitative PCR determination of human FOXA1, mouse CD31, mouse VE-Cadherin. b. Quantitative PCR determination of human EGF, Endothelin-1, Endoglin. mCD31, mouse CD31; mVE-Cad, mouse VE-Cadherin. **P<0.01; ***P<0.001 (PDF 17593 KB)

432_2021_3730_MOESM4_ESM.tif

Supplementary Fig. S4 Functional assays of HUVEC cells cultured with different LNCaP cells or LNCaP conditioned media. a. Quantitative RT-PCR assays of FOXA1 expression in LNCaP cells with FOXA1 knockdown using two different shRNAs. b. Western blotting assays of FOXA1 expression in LNCaP cells with FOXA1 knockdown using two different shRNAs. c. Growth curves of HUVECs co-cultured with different LNCaP cells. d. Growth curves of HUVECs cultured with conditioned media from various LNCaP cells. e. Transwell migration assays of HUVECs co-cultured with different LNCaP cells. Scale bars, 50 μm. f. Transwell migration assays of HUVECs cultured with conditioned media from various LNCaP cells. Scale bars, 50 μm. g. Tube formation assays of HUVECs co-cultured with different LNCaP cells. Scale bars, 250 μm. h. Tube formation assays of HUVECs cultured with conditioned media from various LNCaP cells. Scale bars, 250 μm. *P<0.05. **P<0.01. ***P<0.001 (TIF 11205 KB)

432_2021_3730_MOESM5_ESM.tif

Supplementary Fig. S5 FOXA1 knockdown in prostate cancer cells suppressed the secretion of pro-angiogenic cytokines. Quantitative PCR determination of HIF-1α, VEGFA, FGF2, HGF, TGF-β, Endoglin, Endothelin-1, EGF, THBS1, and PLG gene expression in shNC-LNCaP and shFOXA1-LNCaP cells. ns: not significant. **P<0.01. ***P<0.001 (TIF 901 KB)

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Supplementary Fig. S6 The pro-migration of endothelial cells by FOXA1 overexpression in prostate cancer cells was reversed by antibodies to EGF, Endoglin, and Endothelin-1. (oeFOXA1-DU145-CM as the control group, the other groups as the case groups. **P<0.01; ***P<0.001; ****P<0.0001) (TIF 8824 KB)

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Su, Y., Zhang, Y., Zhao, J. et al. FOXA1 promotes prostate cancer angiogenesis by inducing multiple pro-angiogenic factors expression. J Cancer Res Clin Oncol (2021). https://doi.org/10.1007/s00432-021-03730-3

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Keywords

  • FOXA1
  • Prostate cancer
  • Tumor angiogenesis
  • Endothelial cells