Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer

Oncogene volume 39, pages 1302–1317 (2020)Cite this article

Subjects

Abstract

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Warrick JI, Sjödahl G, Kaag M, Raman JD, Merrill S, Shuman L, et al. Intratumoral heterogeneity of bladder cancer by molecular subtypes and histologic variants. Eur Urol. 2019;75:18–22.

    CAS  PubMed  Google Scholar 

  2. Hovelson DH, Udager AM, McDaniel AS, Grivas P, Palmbos P, Tamura S, et al. Targeted DNA and RNA sequencing of paired urothelial and squamous bladder cancers reveals discordant genomic and transcriptomic events and unique therapeutic implications. Eur Urol. 2018;74:741–53.

    CAS  PubMed  Google Scholar 

  3. Thomsen MBH, Nordentoft I, Lamy P, Vang S, Reinert L, Mapendano CK, et al. Comprehensive multiregional analysis of molecular heterogeneity in bladder cancer. Sci Rep. 2017;7:11702.

    PubMed  PubMed Central  Google Scholar 

  4. Al-Ahmadie HA, Iyer G, Lee BH, Scott SN, Mehra R, Bagrodia A, et al. Frequent somatic CDH1 loss-of-function mutations in plasmacytoid variant bladder cancer. Nat Genet. 2016;48:356–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Guo CC, Dadhania V, Zhang L, Majewski T, Bondaruk J, Sykulski M, et al. Gene expression profile of the clinically aggressive micropapillary variant of bladder cancer. Eur Urol. 2016;70:611–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Gross K, Wronski A, Skibinski A, Phillips S, Kuperwasser C. Cell fate decisions during breast cancer development. J Dev Biol. 2016;4:4.

    PubMed  PubMed Central  Google Scholar 

  7. Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell. 2017;171:540.e25.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. DeGraff D, Clark P, Cates J, Yamashita H, Robinson V, Yu X, et al. Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation. PLoS ONE. 2012;7:13.

    Google Scholar 

  9. Reddy OL, Cates JM, Gellert LL, Crist HS, Yang Z, Yamashita H, et al. Loss of FOXA1 drives sexually dimorphic changes in urothelial differentiation and is an independent predictor of poor prognosis in bladder cancer. Am J Pathol. 2015;185:1385–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Varley C, Bacon E, Holder J, Southgate J. FOXA1 and IRF-1 intermediary transcriptional regulators of PPARc-induced urothelial cytodifferentiation. Cell Death Differ. 2009;16:103–14.

    CAS  PubMed  Google Scholar 

  11. Bertucci F, Finetti P, Birnbaum D. Basal breast cancer: a complex and deadly molecular subtype. Curr Mol Med. 2012;12:96–110.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Knobbe CB, Lapin V, Suzuki A, Mak TW. The roles of PTEN in development, physiology and tumorigenesis in mouse models: a tissue-by-tissue survey. Oncogene. 2008;27:5398–415.

    CAS  PubMed  Google Scholar 

  13. Puzio-Kuter AM, Castillo-Martin M, Kinkade CW, Wang X, Shen TH, Matos T, et al. Inactivation of p53 and Pten promotes invasive bladder cancer. Genes Dev. 2009;23:675–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Tsuruta H, Kishimoto H, Sasaki T, Horie Y, Natsui M, Shibata Y, et al. Hyperplasia and carcinomas in Pten-deficient mice and reduced PTEN protein in human bladder cancer patients. Cancer Res. 2006;66:8389–96.

    CAS  PubMed  Google Scholar 

  15. Cappellen D, Gil Diez de Medina S, Chopin D, Thiery JP, Radvanyi F. Frequent loss of heterozygosity on chromosome 10q in muscle-invasive transitional cell carcinomas of the bladder. Oncogene. 1997;14:3059–66.

    CAS  PubMed  Google Scholar 

  16. Hoadley KA, Yau C, Wolf DM, Cherniack AD, Tamborero D, Ng S, et al. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin. Cell. 2014;158:929–44.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Thorat MA, Marchio C, Morimiya A, Savage K, Nakshatri H, Reis-Filho JS, et al. Forkhead box A1 expression in breast cancer is associated with luminal subtype and good prognosis. J Clin Pathol. 2008;61:327–32.

    CAS  PubMed  Google Scholar 

  18. Warrick JI, Walter V, Yamashita H, Chung E, Shuman L, Amponsa VO, et al. FOXA1, GATA3 and PPARÉ£ cooperate to drive luminal subtype in bladder cancer: a molecular analysis of established human cell lines. Sci Rep. 2016;6:38531.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Yoo LI, Liu DW, Le Vu S, Bronson RT, Wu H, Yuan J. Pten deficiency activates distinct downstream signaling pathways in a tissue-specific manner. Cancer Res. 2006;66:1929–39.

    CAS  PubMed  Google Scholar 

  20. Staack A, Hayward SW, Baskin LS, Cunha GR. Molecular, cellular and developmental biology of urothelium as a basis of bladder regeneration. Differentiation. 2005;73:121–33.

    CAS  PubMed  Google Scholar 

  21. Jost SP, Gosling JA, Dixon JS. The morphology of normal human bladder urothelium. J Anat. 1989;167:103–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Khandelwal P, Abraham SN, Apodaca G. Cell biology and physiology of the uroepithelium. Am J Physiol Ren Physiol. 2009;297:F1477–1501.

    CAS  Google Scholar 

  23. Bertram JS, Craig AW. Specific induction of bladder cancer in mice by butyl-(4-hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response. Eur J Cancer. 1972;8:587–94.

    CAS  PubMed  Google Scholar 

  24. He Z, Kosinska W, Zhao ZL, Wu XR, Guttenplan JB. Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis. Mutat Res. 2012;742:92–95.

    CAS  PubMed  Google Scholar 

  25. Miyamoto H, Yang Z, Chen YT, Ishiguro H, Uemura H, Kubota Y, et al. Promotion of bladder cancer development and progression by androgen receptor signals. J Natl Cancer Inst. 2007;99:558–68.

    CAS  PubMed  Google Scholar 

  26. Nagao M, Suzuki E, Yasuo K, Yahagi T, Seino Y. Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder carcinogen, and related compounds. Cancer Res. 1977;37:399–407.

    CAS  PubMed  Google Scholar 

  27. Li Z, Tuteja G, Schug J, Kaestner KH. Foxa1 and Foxa2 are essential for sexual dimorphism in liver cancer. Cell. 2012;148:72–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Ohtani M, Kakizoe T, Nishio Y, Sato S, Sugimura T, Fukushima S, et al. Sequential changes of mouse bladder epithelium during induction of invasive carcinomas by N-butyl-N-(4-hydroxybutyl)nitrosamine. Cancer Res. 1986;46:2001–4.

    CAS  PubMed  Google Scholar 

  29. Oliveira PA, Palmeira C, Lourenço LM, Lopes CA. Evaluation of DNA content in preneoplastic changes of mouse urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. J Exp Clin Cancer Res. 2005;24:609–16.

    CAS  PubMed  Google Scholar 

  30. Vasconcelos-Nóbrega C, Colaço A, Lopes C, Oliveira PA. Review: BBN as an urothelial carcinogen. Vivo. 2012;26:727–39.

    Google Scholar 

  31. Cairns P, Evron E, Okami K, Halachmi N, Esteller M, Herman JG, et al. Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers. Oncogene. 1998;16:3215–8.

    CAS  PubMed  Google Scholar 

  32. Aveyard JS, Skilleter A, Habuchi T, Knowles MA. Somatic mutation of PTEN in bladder carcinoma. Br J Cancer. 1999;80:904–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Karoui M, Tresallet C, Julie C, Zimmermann U, Staroz F, Brams A, et al. Loss of heterozygosity on 10q and mutational status of PTEN and BMPR1A in colorectal primary tumours and metastases. Br J Cancer. 2004;90:1230–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Drayton RM, Peter S, Myers K, Miah S, Dudziec E, Bryant HE, et al. MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer. Oncotarget. 2014;5:6375–86.

    PubMed  PubMed Central  Google Scholar 

  35. Espinal AC, Buas MF, Wang D, Cheng DT, Sucheston-Campbell L, Hu Q, et al. FOXA1 hypermethylation: link between parity and ER-negative breast cancer in African American women? Breast Cancer Res Treat. 2017;166:559–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ghosh S, Gu F, Wang CM, Lin CL, Liu J, Wang H, et al. Genome-wide DNA methylation profiling reveals parity-associated hypermethylation of FOXA1. Breast Cancer Res Treat. 2014;147:653–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Gong C, Fujino K, Monteiro LJ, Gomes AR, Drost R, Davidson-Smith H, et al. FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer. Oncogene. 2015;34:5012–24.

    CAS  PubMed  Google Scholar 

  38. Choi W, Porten S, Kim S, Willis D, Plimack ER, Hoffman-Censits J, et al. Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy. Cancer Cell. 2014;25:152–65.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Damrauer JS, Hoadley KA, Chism DD, Fan C, Tiganelli CJ, Wobker SE, et al. Intrinsic subtypes of high-grade bladder cancer reflect the hallmarks of breast cancer biology. Proc Natl Acad Sci USA. 2014;111:3110–5.

    CAS  PubMed  Google Scholar 

  40. Van Batavia J, Yamany T, Molotkov A, Dan H, Mansukhani M, Batourina E, et al. Bladder cancers arise from distinct urothelial sub-populations. Nat Cell Biol. 2014;16:982–9891.

    PubMed  Google Scholar 

  41. He F, Melamed J, Tang MS, Huang C, Wu XR. Oncogenic HRAS activates epithelial-to-mesenchymal transition and confers stemness to p53-deficient urothelial cells to drive muscle invasion of basal subtype carcinomas. Cancer Res. 2015;75:2017–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hedegaard J, Lamy P, Nordentoft I, Algaba F, Høyer S, Ulhøi BP, et al. Comprehensive transcriptional analysis of early-stage urothelial carcinoma. Cancer Cell. 2016;30:27–42.

    CAS  PubMed  Google Scholar 

  43. Warrick JI, Kaag M, Raman JD, Chan W, Tran T, Kunchala S, et al. Squamous dysplasia of the urinary bladder: a consecutive cystectomy series. Int J Surg Pathol. 2016;24:306–14.

    PubMed  Google Scholar 

  44. Mermel CH, Schumacher SE, Hill B, Meyerson ML, Beroukhim R, Getz G. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 2011;12:R41.

    PubMed  PubMed Central  Google Scholar 

  45. Walter V, Du Y, Danilova L, Hayward MC, Hayes DN. MVisAGe identifies concordant and discordant genomic alterations of driver genes in squamous tumors. Cancer Res. 2018;78:337533–85.

    Google Scholar 

  46. Gao N, LeLay J, Vatamaniuk MZ, Rieck S, Friedman JR, Kaestner KH. Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development. Genes Dev. 2008;22:3435–48.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Lesche R, Groszer M, Gao J, Wang Y, Messing A, Sun H, et al. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis. 2002;32:148–9.

    CAS  PubMed  Google Scholar 

  48. Mo L, Cheng J, Lee EY, Sun TT, Wu XR. Gene deletion in urothelium by specific expression of Cre recombinase. Am J Physiol Ren Physiol. 2005;289:F562–568.

    CAS  Google Scholar 

  49. Network CGAR. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489:519–25.

    Google Scholar 

  50. Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xy GL, Hu YG, et al. Continuous zebularine treatmetn effectively sustanisn demethylation n human bladder cancer cells. Mol Cell Biol. 2004;24:1270–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. International Agency for Research on Cancer (IARC), Moch H. WHO classification of tumours of the urinary system and male genital organs. 4th edn. Lyon: International Agency for Research on Cancer; 2016.

  52. Team RC. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2018. http://www.R-project.org.

Download references

Funding

Supported by K99CA172122 (DJD), R00CA172122 (DJD), startup funding from the Departments of Pathology and Surgery at Pennsylvania State University College of Medicine (DJD), the Ken and Bonnie Shockey Fund for Urologic Research and the Bladder Cancer Support Group at Penn State Health. The authors wish to additionally acknowledge the expert administrative assistance of Kimberly Walker, and valuable discussions with Gordon Robertson, Mauro Castro and Clarice Groeneveld.

Author information

Author notes

  1. These authors contributed equally: Vasty Osei-Amponsa, Jenna M. Buckwalter

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, The Pennsylvania State University, College of Medicine, Hershey, PA, USA

    Vasty Osei-Amponsa, Jenna M. Buckwalter, Lauren Shuman, Zongyu Zheng, Hironobu Yamashita, Thomas Wildermuth, Justine Ellis-Mohl, Joshua I. Warrick, Jay D. Raman & David J. DeGraff

  2. Department of Surgery, Division of Urology, The Pennsylvania State University, College of Medicine, Hershey, PA, USA

    Jenna M. Buckwalter, Lauren Shuman, Hironobu Yamashita, Thomas Wildermuth, Justine Ellis-Mohl, Joshua I. Warrick, Jay D. Raman & David J. DeGraff

  3. Department of Public Health Sciences, The Pennsylvania State University, College of Medicine, Hershey, PA, USA

    Vonn Walter

  4. Department of Urology, Columbia University, New York, NY, USA

    Chang Liu & Cathy Mendelsohn

  5. Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, PA, USA

    Lisa M. Shantz & Robert P. Feehan

  6. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Hikmat Al-Ahmadie

  7. Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA

    Klaus H. Kaestner

  8. Departments of Urology and Pathology, New York University School of Medicine, New York, NY, USA

    Xue-Ru Wu

  9. Veterans Affairs New York Harbor Healthcare System, Manhattan Campus, New York, NY, USA

    Xue-Ru Wu

Authors
  1. Vasty Osei-Amponsa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jenna M. Buckwalter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lauren Shuman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zongyu Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hironobu Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vonn Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thomas Wildermuth
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Justine Ellis-Mohl
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Joshua I. Warrick
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Lisa M. Shantz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Robert P. Feehan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Hikmat Al-Ahmadie
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Cathy Mendelsohn
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jay D. Raman
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Klaus H. Kaestner
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Xue-Ru Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. David J. DeGraff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David J. DeGraff.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Osei-Amponsa, V., Buckwalter, J.M., Shuman, L. et al. Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer. Oncogene 39, 1302–1317 (2020). https://doi.org/10.1038/s41388-019-1063-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-019-1063-4

This article is cited by

Search

Advanced search

Quick links