TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Research Center of Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty Kopec 7 656 53 Brno Czech Republic

State Institute for Drug Control Šrobárova 48 100 41 Praha 10 Czech Republic

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Budi EH, Schaub JR, Decaris M, Turner S, Derynck R. TGF-β as a driver of fibrosis: Physiological roles and therapeutic opportunities. J. Pathol. 2021;254:358–373. doi: 10.1002/path.5680. PubMed DOI

David CJ, Massagué J. Contextual determinants of TGFβ action in development, immunity and cancer. Nat. Rev. Mol. Cell Biol. 2018;19:419–435. doi: 10.1038/s41580-018-0007-0. PubMed DOI PMC

Seoane J, Gomis RR. TGF-β family signaling in tumor suppression and cancer progression. Cold Spring Harb. Perspect. Biol. 2017;9:a022277. doi: 10.1101/cshperspect.a022277. PubMed DOI PMC

Zhang Y, Alexander PB, Wang X-F. TGF-β Family Signaling in the Control of Cell Proliferation and Survival. Cold Spring Harb. Perspect. Biol. 2017;9:a022145. doi: 10.1101/cshperspect.a022145. PubMed DOI PMC

Mani SA, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–715. doi: 10.1016/j.cell.2008.03.027. PubMed DOI PMC

Pang X, Tang Y-L, Liang X-H. Transforming growth factor-β signaling in head and neck squamous cell carcinoma: Insights into cellular responses. Oncol. Lett. 2018;16:4799–4806. PubMed PMC

Cammareri P, et al. Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nat. Commun. 2016;7:12493. doi: 10.1038/ncomms12493. PubMed DOI PMC

Rose AM, et al. Reduced SMAD2/3 activation independently predicts increased depth of human cutaneous squamous cell carcinoma. Oncotarget. 2018;9:14552–14566. doi: 10.18632/oncotarget.24545. PubMed DOI PMC

Pokorná Z, Vysloužil J, Hrabal V, Vojtěšek B, Coates PJ. The foggy world(s) of p63 isoform regulation in normal cells and cancer. J. Pathol. 2021;254:454–473. doi: 10.1002/path.5656. PubMed DOI

Fisher ML, Balinth S, Mills AA. ΔNp63α in cancer: importance and therapeutic opportunities. Trends Cell Biol. 2023;33:280–292. doi: 10.1016/j.tcb.2022.08.003. PubMed DOI PMC

Crum CP, McKeon FD. p63 in epithelial survival, germ cell surveillance, and neoplasia. Annu. Rev. Pathol. 2010;5:349–371. doi: 10.1146/annurev-pathol-121808-102117. PubMed DOI

Orzol P, et al. The diverse oncogenic and tumour suppressor roles of p63 and p73 in cancer: A review by cancer site. Histol. Histopathol. 2015;30:503–521. PubMed

Blanpain C, Fuchs E. p63: revving up epithelial stem-cell potential. Nat. Cell Biol. 2007;9:731–733. doi: 10.1038/ncb0707-731. PubMed DOI

Melino G, Memmi EM, Pelicci PG, Bernassola F. Maintaining epithelial stemness with p63. Sci. Signal. 2015;8:9. doi: 10.1126/scisignal.aaa1033. PubMed DOI

Campbell JD, et al. Genomic, pathway network, and immunologic features distinguishing squamous carcinomas. Cell Rep. 2018;23:194–212.e6. doi: 10.1016/j.celrep.2018.03.063. PubMed DOI PMC

Dotto GP, Rustgi AK. Squamous cell cancers: A unified perspective on biology and genetics. Cancer Cell. 2016;29:622–637. doi: 10.1016/j.ccell.2016.04.004. PubMed DOI PMC

Nekulova M, Holcakova J, Coates P, Vojtesek B. The role of p63 in cancer, stem cells and cancer stem cells. Cell. Mol. Biol. Lett. 2011;16:296–327. doi: 10.2478/s11658-011-0009-9. PubMed DOI PMC

Moses MA, et al. Molecular mechanisms of p63-mediated squamous cancer pathogenesis. Int. J. Mol. Sci. 2019;20:3590. doi: 10.3390/ijms20143590. PubMed DOI PMC

Loljung L, et al. High expression of p63 is correlated to poor prognosis in squamous cell carcinoma of the tongue. J. Oral Pathol. Med. Off. Publ. Int. Assoc. Oral Pathol. Am. Acad. Oral Pathol. 2014;43:14–19. PubMed

Bretz AC, et al. ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma. Nucleic Acids Res. 2016;44:3204–3218. doi: 10.1093/nar/gkw036. PubMed DOI PMC

Lo Muzio L, et al. p63 overexpression associates with poor prognosis in head and neck squamous cell carcinoma. Hum. Pathol. 2005;36:187–194. doi: 10.1016/j.humpath.2004.12.003. PubMed DOI

Moergel M, Abt E, Stockinger M, Kunkel M. Overexpression of p63 is associated with radiation resistance and prognosis in oral squamous cell carcinoma. Oral Oncol. 2010;46:667–671. doi: 10.1016/j.oraloncology.2010.06.012. PubMed DOI

Thurfjell N, et al. Endogenous p63 acts as a survival factor for tumour cells of SCCHN origin. Int. J. Mol. Med. 2005;16:1065–1070. PubMed

Tran MN, et al. The p63 protein isoform ΔNp63α inhibits epithelial-mesenchymal transition in human bladder cancer cells: role of MIR-205. J. Biol. Chem. 2013;288:3275–3288. doi: 10.1074/jbc.M112.408104. PubMed DOI PMC

Olsen JR, et al. p63 attenuates epithelial to mesenchymal potential in an experimental prostate cell model. PloS ONE. 2013;8:e62547. doi: 10.1371/journal.pone.0062547. PubMed DOI PMC

Mehrazarin S, et al. The p63 gene is regulated by grainyhead-like 2 (GRHL2) through reciprocal feedback and determines the epithelial phenotype in human keratinocytes. J. Biol. Chem. 2015;290:19999–20008. doi: 10.1074/jbc.M115.659144. PubMed DOI PMC

Yoh KE, et al. Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells. Proc. Natl. Acad. Sci. USA. 2016;113:E6107–E6116. doi: 10.1073/pnas.1613417113. PubMed DOI PMC

Citro S, et al. Synergistic antitumour activity of HDAC inhibitor SAHA and EGFR inhibitor gefitinib in head and neck cancer: a key role for ΔNp63α. Br. J. Cancer. 2019;120:658–667. doi: 10.1038/s41416-019-0394-9. PubMed DOI PMC

Min S, et al. p63 and its target follistatin maintain salivary gland stem/progenitor cell function through TGF-β/activin signaling. Iscience. 2020 doi: 10.1016/j.isci.2020.101524. PubMed DOI PMC

Testoni B, et al. Identification of new p63 targets in human keratinocytes. Cell Cycle Georget. Tex. 2006;5:2805–2811. doi: 10.4161/cc.5.23.3525. PubMed DOI

Sundqvist A, et al. TGFβ and EGF signaling orchestrates the AP-1- and p63 transcriptional regulation of breast cancer invasiveness. Oncogene. 2020;39:4436–4449. doi: 10.1038/s41388-020-1299-z. PubMed DOI PMC

Oh J-E, Kim RH, Shin K-H, Park N-H, Kang MK. DeltaNp63α protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes. J. Biol. Chem. 2011;286:38757–38767. doi: 10.1074/jbc.M111.244939. PubMed DOI PMC

Abraham CG, et al. ΔNp63α suppresses TGFB2 expression and RHOA activity to drive cell proliferation in squamous cell carcinomas. Cell Rep. 2018;24:3224–3236. doi: 10.1016/j.celrep.2018.08.058. PubMed DOI PMC

Adorno M, et al. A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis. Cell. 2009;137:87–98. doi: 10.1016/j.cell.2009.01.039. PubMed DOI

Vasilaki E, et al. Ras and TGF-β signaling enhance cancer progression by promoting the ΔNp63 transcriptional program. Sci. Signal. 2016;9:84. doi: 10.1126/scisignal.aag3232. PubMed DOI

Fukunishi N, et al. Induction of ΔNp63 by the newly identified keratinocyte-specific transforming growth factor β Signaling Pathway with Smad2 and IκB Kinase α in squamous cell carcinoma. Neoplasia N. Y. N. 2010;12:969–979. doi: 10.1593/neo.101054. PubMed DOI PMC

Murata K, et al. p63 - Key molecule in the early phase of epithelial abnormality in idiopathic pulmonary fibrosis. Exp. Mol. Pathol. 2007;83:367–376. doi: 10.1016/j.yexmp.2007.03.006. PubMed DOI

Bui NHB, et al. Spatiotemporal Regulation of ΔNp63 by TGFβ-regulated miRNAs Is essential for cancer metastasis. Cancer Res. 2020;80:2833–2847. doi: 10.1158/0008-5472.CAN-19-2733. PubMed DOI PMC

Mou H, et al. Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells. Cell Stem Cell. 2016;19:217–231. doi: 10.1016/j.stem.2016.05.012. PubMed DOI PMC

Chalmers FE, Dusold JE, Shaik JA, Walsh HA, Glick AB. Targeted deletion of TGFβ1 in basal keratinocytes causes profound defects in stratified squamous epithelia and aberrant melanocyte migration. Dev. Biol. 2022;485:9–23. doi: 10.1016/j.ydbio.2022.02.009. PubMed DOI PMC

Walter V, et al. Molecular subtypes in head and neck cancer exhibit distinct patterns of chromosomal gain and loss of canonical cancer genes. PloS One. 2013;8:e56823. doi: 10.1371/journal.pone.0056823. PubMed DOI PMC

Pokorna Z, Hrabal V, Tichy V, Vojtesek B, Coates PJ. DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma. Front. Oncol. 2022;12:924354. doi: 10.3389/fonc.2022.924354. PubMed DOI PMC

Jennings JC, Mohan S, Linkhart TA, Widstrom R, Baylink DJ. Comparison of the biological actions of TGF beta-1 and TGF beta-2: differential activity in endothelial cells. J. Cell. Physiol. 1988;137:167–172. doi: 10.1002/jcp.1041370120. PubMed DOI

O’Sullivan MJ, et al. In well-differentiated primary human bronchial epithelial cells, TGF-β1 and TGF-β2 induce expression of furin. Am. J. Physiol. Lung Cell. Mol. Physiol. 2021;320:L246–L253. doi: 10.1152/ajplung.00423.2020. PubMed DOI PMC

Schmierer B, Hill CS. TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nat. Rev. Mol. Cell Biol. 2007;8:970–982. doi: 10.1038/nrm2297. PubMed DOI

Davies M, et al. Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways. J. Cell. Biochem. 2005;95:918–931. doi: 10.1002/jcb.20458. PubMed DOI

Pokorna Z, Vyslouzil J, Vojtesek B, Coates PJ. Identifying pathways regulating the oncogenic p53 family member ΔNp63 provides therapeutic avenues for squamous cell carcinoma. Cell. Mol. Biol. Lett. 2022;27:18. doi: 10.1186/s11658-022-00323-x. PubMed DOI PMC

Hummer BT, Bartlett C, Henry E, Weissman BE. Expression of Smad4 in the FaDu cell line partially restores TGF-beta growth inhibition but is not sufficient to regulate fibronectin expression or suppress tumorigenicity. J. Cell. Physiol. 2003;194:289–302. doi: 10.1002/jcp.10202. PubMed DOI

Dahler AL, Cavanagh LL, Saunders NA. Suppression of keratinocyte growth and differentiation by transforming growth factor beta1 involves multiple signaling pathways. J. Invest. Dermatol. 2001;116:266–274. doi: 10.1046/j.1523-1747.2001.01243.x. PubMed DOI

Ingruber J, et al. EMT-related transcription factors and protein stabilization mechanisms involvement in cadherin switch of head and neck squamous cell carcinoma. Exp. Cell Res. 2022;414:113084. doi: 10.1016/j.yexcr.2022.113084. PubMed DOI PMC

Gluck C, et al. Molecular dissection of the oncogenic role of ETS1 in the mesenchymal subtypes of head and neck squamous cell carcinoma. PLoS Genet. 2019;15:e100825. doi: 10.1371/journal.pgen.1008250. PubMed DOI PMC

Cherukuri P, et al. Phosphorylation of ΔNp63α via a novel TGFβ/ALK5 signaling mechanism mediates the anti-clonogenic effects of TGFβ. PloS One. 2012;7:e50066. doi: 10.1371/journal.pone.0050066. PubMed DOI PMC

Niu M, et al. Noncanonical TGF-β signaling leads to FBXO3-mediated degradation of ΔNp63α promoting breast cancer metastasis and poor clinical prognosis. PLoS Biol. 2021;19:e3001113. doi: 10.1371/journal.pbio.3001113. PubMed DOI PMC

Laronda MM, et al. Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium. Dev. Biol. 2013;381:5–16. doi: 10.1016/j.ydbio.2013.06.024. PubMed DOI PMC

Oida T, Weiner HL. Depletion of TGF-β from fetal bovine serum. J. Immunol. Methods. 2010;362:195–198. doi: 10.1016/j.jim.2010.09.008. PubMed DOI PMC

Inman GJ, et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 2002;62:65–74. doi: 10.1124/mol.62.1.65. PubMed DOI

Wang T, et al. TGF-β-induced miR-21 negatively regulates the antiproliferative activity but has no effect on EMT of TGF-β in HaCaT cells. Int. J. Biochem. Cell Biol. 2012;44:366–376. doi: 10.1016/j.biocel.2011.11.012. PubMed DOI

Candi E, Amelio I, Agostini M, Melino G. MicroRNAs and p63 in epithelial stemness. Cell Death Differ. 2015;22:12–21. doi: 10.1038/cdd.2014.113. PubMed DOI PMC

Brabletz S, Schuhwerk H, Brabletz T, Stemmler MP. Dynamic EMT: a multi-tool for tumor progression. EMBO J. 2021;40:e108647. doi: 10.15252/embj.2021108647. PubMed DOI PMC

Haerinck J, Goossens S, Berx G. The epithelial-mesenchymal plasticity landscape: principles of design and mechanisms of regulation. Nat. Rev. Genet. 2023;24:590–609. doi: 10.1038/s41576-023-00601-0. PubMed DOI

Lambert AW, et al. ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells. Dev. Cell. 2022;57:2714–2730.e8. doi: 10.1016/j.devcel.2022.11.015. PubMed DOI PMC

Tyler M, Tirosh I. Decoupling epithelial-mesenchymal transitions from stromal profiles by integrative expression analysis. Nat. Commun. 2021;12:2592. doi: 10.1038/s41467-021-22800-1. PubMed DOI PMC

Jung AR, Jung C-H, Noh JK, Lee YC, Eun Y-G. Epithelial-mesenchymal transition gene signature is associated with prognosis and tumor microenvironment in head and neck squamous cell carcinoma. Sci. Rep. 2020;10:3652. doi: 10.1038/s41598-020-60707-x. PubMed DOI PMC

Kisoda S, et al. The role of partial-EMT in the progression of head and neck squamous cell carcinoma. J. Oral Biosci. 2022;64:176–182. doi: 10.1016/j.job.2022.02.004. PubMed DOI

Callahan JF, et al. Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5) J. Med. Chem. 2002;45:999–1001. doi: 10.1021/jm010493y. PubMed DOI

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC