Skip to main content

Advertisement

SpringerLink
Log in

ΔNp63 regulates cell proliferation, differentiation, adhesion, and migration in the BL2 subtype of basal-like breast cancer

  • Original Article
  • Published:
Tumor Biology

Abstract

Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Badve S, Dabbs DJ, Schnitt SJ, et al. Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod Pathol. 2011;24:157–67.

    Article  PubMed  Google Scholar 

  2. Lehmann BD, Pietenpol JA. Identification and use of biomarkers in treatment strategies for triple-negative breast cancer subtypes. J Pathol. 2014;232:142–50.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lehmann BD, Bauer JA, Chen X, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121:2750–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Neve RM, Chin K, Fridlyand J, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10:515–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Turner NC, Reis-Filho JS. Tackling the diversity of triple-negative breast cancer. Clin Cancer Res. 2013;19:6380–8.

    Article  CAS  PubMed  Google Scholar 

  6. Barbareschi M, Pecciarini L, Cangi MG, et al. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol. 2001;25:1054–60.

    Article  CAS  PubMed  Google Scholar 

  7. Matos I, Dufloth R, Alvarenga M, et al. p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas. Virchows Arch. 2005;447:688–94.

    Article  CAS  PubMed  Google Scholar 

  8. Nylander K, Vojtesek B, Nenutil R, et al. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol. 2002;198:417–27.

    Article  CAS  PubMed  Google Scholar 

  9. Crum CP, McKeon FD. p63 in epithelial survival, germ cell surveillance, and neoplasia. Annu Rev Pathol. 2010;5:349–71.

    Article  CAS  PubMed  Google Scholar 

  10. Nekulova M, Holcakova J, Coates P, et al. The role of p63 in cancer, stem cells and cancer stem cells. Cell Mol Biol Lett. 2011;16:296–327.

    Article  CAS  PubMed  Google Scholar 

  11. Orzol P, Holcakova J, Nekulova M, et al. The diverse oncogenic and tumour suppressor roles of p63 and p73 in cancer: a review by cancer site. Histol Histopathol. 2015;30:503–21.

    CAS  PubMed  Google Scholar 

  12. Chakrabarti R, Wei Y, Hwang J, et al. DeltaNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signalling. Nat Cell Biol. 2014;16:1004–15. 1001–1013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Memmi EM, Sanarico AG, Giacobbe A, et al. p63 sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling. Proc Natl Acad Sci U S A. 2015;112:3499–504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Buckley NE, Conlon SJ, Jirstrom K, et al. The {Delta}Np63 proteins are key allies of BRCA1 in the prevention of basal-like breast cancer. Cancer Res. 2011;71:1933–44.

    Article  CAS  PubMed  Google Scholar 

  15. Flores ER, Sengupta S, Miller JB, et al. Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell. 2005;7:363–73.

    Article  CAS  PubMed  Google Scholar 

  16. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nekulova M, Holcakova J, Nenutil R, et al. Characterization of specific p63 and p63-N-terminal isoform antibodies and their application for immunohistochemistry. Virchows Arch. 2013;463:415–25.

    Article  CAS  PubMed  Google Scholar 

  18. Senoo M, Pinto F, Crum CP, et al. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell. 2007;129:523–36.

    Article  CAS  PubMed  Google Scholar 

  19. Boldrup L, Coates PJ, Gu X, et al. DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck. J Pathol. 2007;213:384–91.

    Article  CAS  PubMed  Google Scholar 

  20. Romano RA, Ortt K, Birkaya B, et al. An active role of the delta N isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate. PLoS One. 2009;4:e5623.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Aleskandarany MA, Negm OH, Green AR, et al. Epithelial mesenchymal transition in early invasive breast cancer: an immunohistochemical and reverse phase protein array study. Breast Cancer Res Treat. 2014;145:339–48.

    Article  CAS  PubMed  Google Scholar 

  22. Carroll DK, Carroll JS, Leong CO, et al. p63 regulates an adhesion programme and cell survival in epithelial cells. Nat Cell Biol. 2006;8:551–61.

    Article  CAS  PubMed  Google Scholar 

  23. Gu X, Coates PJ, Boldrup L, et al. p63 contributes to cell invasion and migration in squamous cell carcinoma of the head and neck. Cancer Lett. 2008;263:26–34.

    Article  CAS  PubMed  Google Scholar 

  24. Barbieri CE, Tang LJ, Brown KA, et al. Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis. Cancer Res. 2006;66:7589–97.

    Article  CAS  PubMed  Google Scholar 

  25. Boldrup L, Coates PJ, Gu X, et al. DeltaNp63 isoforms differentially regulate gene expression in squamous cell carcinoma: identification of Cox-2 as a novel p63 target. J Pathol. 2009;218:428–36.

    Article  CAS  PubMed  Google Scholar 

  26. Carroll DK, Brugge JS, Attardi LD. p63, cell adhesion and survival. Cell Cycle. 2007;6:255–61.

    Article  CAS  PubMed  Google Scholar 

  27. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell. 1998;2:305–16.

    Article  CAS  PubMed  Google Scholar 

  28. Yang A, Zhu Z, Kapranov P, et al. Relationships between p63 binding, DNA sequence, transcription activity, and biological function in human cells. Mol Cell. 2006;24:593–602.

    Article  CAS  PubMed  Google Scholar 

  29. Mills AA, Zheng B, Wang XJ, et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature. 1999;398:708–13.

    Article  CAS  PubMed  Google Scholar 

  30. Yang A, Schweitzer R, Sun D, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999;398:714–8.

    Article  CAS  PubMed  Google Scholar 

  31. Li N, Singh S, Cherukuri P, et al. Reciprocal intraepithelial interactions between TP63 and hedgehog signaling regulate quiescence and activation of progenitor elaboration by mammary stem cells. Stem Cells. 2008;26:1253–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yalcin-Ozuysal O, Fiche M, Guitierrez M, et al. Antagonistic roles of Notch and p63 in controlling mammary epithelial cell fates. Cell Death Differ. 2010;17:1600–12.

    Article  CAS  PubMed  Google Scholar 

  33. Lin YL, Sengupta S, Gurdziel K, et al. p63 and p73 transcriptionally regulate genes involved in DNA repair. PLoS Genet. 2009;5:e1000680.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Lee H, Kimelman D. A dominant-negative form of p63 is required for epidermal proliferation in zebrafish. Dev Cell. 2002;2:607–16.

    Article  CAS  PubMed  Google Scholar 

  35. Wu G, Nomoto S, Hoque MO, et al. DeltaNp63alpha and TAp63alpha regulate transcription of genes with distinct biological functions in cancer and development. Cancer Res. 2003;63:2351–7.

    CAS  PubMed  Google Scholar 

  36. Mehta R, Jain RK, Sneige N, et al. Expression of high-molecular-weight cytokeratin (34betaE12) is an independent predictor of disease-free survival in patients with triple-negative tumours of the breast. J Clin Pathol. 2010;63:744–7.

    Article  CAS  PubMed  Google Scholar 

  37. Rakha EA, El-Sayed ME, Green AR, et al. Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression. Histopathology. 2007;50:434–8.

    Article  CAS  PubMed  Google Scholar 

  38. Balboni AL, Hutchinson JA, DeCastro AJ, et al. DeltaNp63alpha-mediated activation of bone morphogenetic protein signaling governs stem cell activity and plasticity in normal and malignant mammary epithelial cells. Cancer Res. 2013;73:1020–30.

    Article  CAS  PubMed  Google Scholar 

  39. Dang TT, Esparza MA, Maine EA, et al. DeltaNp63alpha promotes breast cancer cell motility through the selective activation of components of the epithelial-to-mesenchymal transition program. Cancer Res. 2015;75:3925–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tran MN, Choi W, Wszolek MF, et al. The p63 protein isoform DeltaNp63alpha inhibits epithelial-mesenchymal transition in human bladder cancer cells: role of MIR-205. J Biol Chem. 2013;288:3275–88.

    Article  CAS  PubMed  Google Scholar 

  41. Alexandrova EM, Petrenko O, Nemajerova A, et al. DeltaNp63 regulates select routes of reprogramming via multiple mechanisms. Cell Death Differ. 2013;20:1698–708.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. McDonald OG, Wu H, Timp W, et al. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat Struct Mol Biol. 2011;18:867–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Suva ML, Riggi N, Bernstein BE. Epigenetic reprogramming in cancer. Science. 2013;339:1567–70.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grant NT14602-3/2013 from IGA MZ CR, grant P206/12/G151 from GACR, and projects MEYS-NPSI-LO1413 and MH CZ – DRO (MMCI, 00209805).

Author information

Authors and Affiliations

  1. Regional Centre of Applied and Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53, Brno, Czech Republic

    Paulina Orzol, Marta Nekulova, Jitka Holcakova, Petr Muller, Borivoj Votesek & Philip J. Coates

Authors
  1. Paulina Orzol
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marta Nekulova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jitka Holcakova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Petr Muller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Borivoj Votesek
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Philip J. Coates
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Borivoj Votesek or Philip J. Coates.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

Relative levels of TP63 mRNA isoforms in HCC1806 cells. Left panel: The data are presented as percentage of the total TP63 levels for the TP63 5′ isoforms (ΔN and TA) produced by alternative promoter usage. Right panel: Percentage of the total TP63 levels for each 3′ isoform (α, β and γ) produced by alternative splicing. Note that the y axis on the left panel is logarithmic to show the very low levels of TAp63 in these cells. Error bars are S.D. (GIF 6.76 kb)

High resolution image (TIF 217 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Orzol, P., Nekulova, M., Holcakova, J. et al. ΔNp63 regulates cell proliferation, differentiation, adhesion, and migration in the BL2 subtype of basal-like breast cancer. Tumor Biol. 37, 10133–10140 (2016). https://doi.org/10.1007/s13277-016-4880-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-016-4880-x

Keywords

Search

Navigation