The incidence of cutaneous squamous cell carcinoma (cSCC) is rising. Whilst the majority are cured surgically, aggressive metastatic cSCC carry a poor prognosis. Inactivating mutations in transforming growth factor beta (TGF-β) receptors have been identified amongst genetic drivers of sporadic tumours and murine models of cSCC, suggesting a tumour suppressor function for TGF-β in normal skin. However, paradoxically, TGF-β acts as a tumour promoter in some murine model systems. Few studies have analysed the role of TGF-β/activin signalling in human normal skin, hyper-proliferative skin disorders and cSCC. Antibodies recognising phospho-SMAD proteins which are activated during canonical TGF-β/activin signalling were validated for use in immunohistochemistry. A tissue microarray comprising FFPE lesional and perilesional tissue from human primary invasive cSCC (n=238), cSCC in-situ (n=2) and keratocanthoma (n=9) were analysed in comparison with tissues from normal human scalp (n=10). Phosphorylated SMAD2 and SMAD3 were detected in normal interfollicular epidermal keratinocytes and were also highly localised to inner root sheath, matrix cells and Keratin 15 positive cells. Lesional cSCC tissue had significantly reduced activated SMAD2/3 compared to perilesional tissue, consistent with a tumour suppressor role for SMAD2/3 activators in cSCC. Increased cSCC tumour thickness inversely correlated with the presence of phospho-SMADs in tumour tissue suggesting that a reduction in canonical TGF-β/activin signalling may be associated with disease progression.

Centre for Cell Biology and Cutaneous Research Barts and the London School of Medicine and Dentistry Queen Mary University of London London E1 2AT UK

Department of Dermatology Ninewells Hospital and Medical School NHS Tayside Dundee Scotland DD1 9SY UK

Department of Pathology Ninewells Hospital and Medical School NHS Tayside Dundee Scotland DD1 9SY UK

Department of Plastic and Reconstructive Surgery Ninewells Hospital and Medical School NHS Tayside Dundee Scotland DD1 9SY UK

Dermatopathology Laboratory St John's Institute of Dermatology St Thomas' Hospital London SE1 7EH UK

Division of Cancer Research School of Medicine University of Dundee Ninewells Hospital and Medical School Dundee Scotland DD1 9SY UK

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno 656 53 Czech Republic

Tayside Tissue Bank Ninewells Hospital and Medical School NHS Tayside Dundee Scotland DD1 9SY UK

See more in PubMed

Stern RS. Prevalence of a history of skin cancer in 2007: results of an incidence-based model. Arch Dermatol. 2010;146:279–282. PubMed

Karia PS, Jambusaria-Pahlajani A, Harrington DP, Murphy GF, Qureshi AA, Schmults CD. Evaluation of american joint committee on cancer, international union against cancer, and brigham and women's hospital tumor staging for cutaneous squamous cell carcinoma. J Clin Oncol. 2014;32:327–34. PubMed PMC

Cammareri P, Rose AM, Vincent DF, Wang J, Nagano A, Libertini S, Ridgway RA, Athineos D, Coates PJ, McHugh A, Pourreyron C, Dayal JHS, Larsson J, et al. Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nature communications. 2016;7:12493. PubMed PMC

Shi Y, Massague J. Mechanisms of tgf-beta signaling from cell membrane to the nucleus. Cell. 2003;113:685–700. PubMed

Massague J. Tgfbeta in cancer. Cell. 2008;134:215–230. PubMed PMC

Inman GJ. Switching TGFbeta from a tumor suppressor to a tumor promoter. Curr Opin Genet Dev. 2011;21:93–99. PubMed

Cui W, Fowlis DJ, Bryson S, Duffie E, Ireland H, Balmain A, Akhurst RJ. TGFbeta1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice. Cell. 1996;86:531–542. PubMed

Perez-Lorenzo R, Markell LM, Hogan KA, Yuspa SH, Glick AB. Transforming growth factor beta1 enhances tumor promotion in mouse skin carcinogenesis. Carcinogenesis. 2010;31:1116–1123. PubMed PMC

Han G, Lu SL, Li AG, He W, Corless CL, Kulesz-Martin M, Wang XJ. Distinct mechanisms of tgf-beta1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis. J Clin Invest. 2005;115:1714–1723. PubMed PMC

Hoot KE, Lighthall J, Han G, Lu SL, Li A, Ju W, Kulesz-Martin M, Bottinger E, Wang XJ. Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J Clin Invest. 2008;118:2722–32. PubMed PMC

Harradine KA, Ridd K, Saunier EF, Clermont FF, Perez-Losada J, Moore DH, Epstein EH, Jr, Bastian BC, Akhurst RJ. Elevated cutaneous smad activation associates with enhanced skin tumor susceptibility in organ transplant recipients. Clin Cancer Res. 2009;15:5101–5107. PubMed PMC

Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-[beta] receptor. Nature. 1994;370:341–347. PubMed

Wieser R, Wrana JL, Massagué J. GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. The EMBO Journal. 1995;14:2199–2208. PubMed PMC

Wu JW, Hu M, Chai J, Seoane J, Huse M, Li C, Rigotti DJ, Kyin S, Muir TW, Fairman R, Massagué J, Shi Y. Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-β signaling. Mol Cell. 2001;8:1277–89. PubMed

Xu L, Chen YG, Massague J. The nuclear import function of smad2 is masked by SARA and unmasked by TGFbeta-dependent phosphorylation. Nat Cell Biol. 2000;2:559–562. PubMed

Massague J. TGF[beta] signalling in context. Nat Rev Mol Cell Biol. 2012;13:616–630. PubMed PMC

South AP, Purdie KJ, Watt SA, Haldenby S, den Breems NY, Dimon M, Arron ST, Kluk MJ, Aster JC, McHugh A, Xue DJ, Dayal JH, Robinson KS, et al. NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis. J Invest Dermatol. 2014;134:2630–2638. PubMed PMC

Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. 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. PubMed

Nenutil R, Smardova J, Pavlova S, Hanzelkova Z, Muller P, Fabian P, Hrstka R, Janotova P, Radina M, Lane DP, Coates PJ, Vojtesek B. Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry. J Pathol. 2005;207:251–259. PubMed

Mesa KR, Rompolas P, Greco V. The Dynamic Duo: Niche/Stem Cell Interdependency. Stem Cell Reports. 2015;4:961–966. PubMed PMC

Purba TS, Haslam IS, Poblet E, Jimenez F, Gandarillas A, Izeta A, Paus R. Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges. Bioessays. 2014;36:513–525. PubMed

Ito M, Kizawa K, Hamada K, Cotsarelis G. Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen. Differentiation. 2004;72:548–557. PubMed

Karia PS, Jambusaria-Pahlajani A, Harrington DP, Murphy GF, Qureshi AA, Schmults CD. Evaluation of American Joint Committee on Cancer, International Union Against Cancer, and Brigham and Women's Hospital tumor staging for cutaneous squamous cell carcinoma. J Clin Oncol. 2014;32:327–334. PubMed PMC

Quan T, He T, Kang S, Voorhees JJ, Fisher GJ. Ultraviolet irradiation alters transforming growth factor beta/smad pathway in human skin in vivo. J Invest Dermatol. 2002;119:499–506. PubMed

Wang XJ, Liefer KM, Tsai S, O'Malley BW, Roop DR. Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis. Proc Natl Acad Sci USA. 1999;96:8483–8488. PubMed PMC

Liu X, Alexander V, Vijayachandra K, Bhogte E, Diamond I, Glick A. Conditional epidermal expression of TGFbeta 1 blocks neonatal lethality but causes a reversible hyperplasia and alopecia. Proc Natl Acad Sci USA. 2001;98:9139–9144. PubMed PMC

Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T, Springer TA. Latent Tgf-[bgr] structure and activation. Nature. 2011;474:343–349. PubMed PMC

Vizan P, Miller DS, Gori I, Das D, Schmierer B, Hill CS. Controlling long-term signaling: receptor dynamics determine attenuation and refractory behavior of the TGF-beta pathway. Sci Signal. 2013;6:ra106. PubMed

Xie W, Bharathy S, Kim D, Haffty BG, Rimm DL, Reiss M. Frequent alterations of smad signaling in human head and neck squamous cell carcinomas: a tissue microarray analysis. Oncol Res. 2003;14:61–73. PubMed

Schmierer B, Hill CS. Tgfbeta-smad signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol. 2007;8:970–982. PubMed

Hinck AP. Structural studies of the TGF-βs and their receptors – insights into evolution of the TGF-β superfamily. FEBS Letters. 2012;586:1860–1870. PubMed

McDowall M, Edwards NM, Jahoda CAB, Hynd PI. The role of activins and follistatins in skin and hair follicle development and function. Cytokine & Growth Factor Reviews. 2008;19:415–426. PubMed

Hubner G, Hu Q, Smola H, Werner S. Strong induction of activin expression after injury suggests an important role of activin in wound repair. Dev Biol. 1996;173:490–498. PubMed

Shimizu A, Kato M, Nakao A, Imamura T, ten Dijke P, Heldin CH, Kawabata M, Shimada S, Miyazono K. Identification of receptors and smad proteins involved in activin signalling in a human epidermal keratinocyte cell line. Genes Cells. 1998;3:125–134. PubMed

Lange D, Persson U, Wollina U, ten Dijke P, Castelli E, Heldin CH, Funa K. Expression of tgf-beta related smad proteins in human epithelial skin tumors. Int J Oncol. 1999;14:1049–1056. PubMed

Brown KA, Pietenpol JA, Moses HL. A tale of two proteins: differential roles and regulation of smad2 and smad3 in TGF-β signaling. J Cell Biochem. 2007;101:9–33. PubMed

Tsuji Y, Denda S, Soma T, Raftery L, Momoi T, Hibino T. A potential suppressor of tgf-[beta] delays catagen progression in hair follicles. J Investig Dermatol Symp Proc. 2003;8:65–68. PubMed

Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, Botchkarev V, Handjiski B, Metz M, Hibino T, Soma T, Dotto GP, Paus R. Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. FASEB J. 2000;14:752–760. PubMed

Oshimori N, Fuchs E. Paracrine tgf-beta signaling counterbalances bmp-mediated repression in hair follicle stem cell activation. Cell stem cell. 2012;10:63–75. PubMed PMC

Pickering CR, Zhou JH, Lee JJ, Drummond JA, Peng SA, Saade RE, Tsai KY, Curry JL, Tetzlaff MT, Lai SY, Yu J, Muzny DM, Doddapaneni H, et al. Mutational landscape of aggressive cutaneous squamous cell carcinoma. Clin Cancer Res. 2014;20:6582–6592. PubMed PMC

Li YY, Hanna GJ, Laga AC, Haddad RI, Lorch JH, Hammerman PS. Genomic analysis of metastatic cutaneous squamous cell carcinoma. Clin Cancer Res. 2015;21:1447–1456. PubMed PMC

Goudie DR, D'Alessandro M, Merriman B, Lee H, Szeverenyi I, Avery S, O'Connor BD, Nelson SF, Coats SE, Stewart A, Christie L, Pichert G, Friedel J, et al. Multiple self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in TGFBR1. Nat Genet. 2011;43:365–369. PubMed

Arnault JP, Mateus C, Escudier B, Tomasic G, Wechsler J, Hollville E, Soria JC, Malka D, Sarasin A, Larcher M, Andre J, Kamsu-Kom N, Boussemart L, et al. Skin tumors induced by sorafenib; paradoxic RAS-RAF pathway activation and oncogenic mutations of hras, TP53, and TGFBr1. Clin Cancer Res. 2012;18:263–272. PubMed

Morris JC, Tan AR, Olencki TE, Shapiro GI, Dezube BJ, Reiss M, Hsu FJ, Berzofsky JA, Lawrence DP. Phase i study of gc1008 (fresolimumab): a human anti-transforming growth factor-beta (TGFβ) monoclonal antibody in patients with advanced malignant melanoma or renal cell carcinoma. PLoS ONE. 2014;9:e90353. PubMed PMC

Latil M, Nassar D, Beck B, Boumahdi S, Wang L, Brisebarre A, Dubois C, Nkusi E, Lenglez S, Checinska A, Vercauteren Drubbel A, Devos M, Declercq W, et al. Cell-type-specific chromatin states differentially prime squamous cell carcinoma tumor-initiating cells for epithelial to mesenchymal transition. Cell stem cell. 2017;20:191–204. e195. PubMed PMC

Barrette K, Van Kelst S, Wouters J, Marasigan V, Fieuws S, Agostinis P, van den Oord J, Garmyn M. Epithelial-mesenchymal transition during invasion of cutaneous squamous cell carcinoma is paralleled by AKT activation. The British journal of dermatology. 2014;171:1014–1021. PubMed

Derynck R, Muthusamy BP, Saeteurn KY. Signaling pathway cooperation in tgf-beta-induced epithelial-mesenchymal transition. Curr Opin Cell Biol. 2014;31:56–66. PubMed PMC

Davies M, Prime SS, Eveson JW, Price N, Ganapathy A, D’Mello A, Paterson IC. Transforming growth factor-β enhances invasion and metastasis in Ras-transfected human malignant epidermal keratinocytes. Int J Exp Pathol. 2012;93:148–156. PubMed PMC

Brantsch KD, Meisner C, Schonfisch B, Trilling B, Wehner-Caroli J, Rocken M, Breuninger H. Analysis of risk factors determining prognosis of cutaneous squamous-cell carcinoma: a prospective study. Lancet Oncol. 2008;9:713–720. PubMed

Breuninger H, Brantsch K, Eigentler T, Hafner HM. Comparison and evaluation of the current staging of cutaneous carcinomas. J Dtsch Dermatol Ges. 2012;10:579–586. PubMed

Giampieri S, Manning C, Hooper S, Jones L, Hill CS, Sahai E. Localised and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility. Nature cell biology. 2009;11:1287–1296. PubMed PMC

Matise LA, Palmer TD, Ashby WJ, Nashabi A, Chytil A, Aakre M, Pickup MW, Gorska AE, Zijlstra A, Moses HL. Lack of transforming growth factor-beta signaling promotes collective cancer cell invasion through tumor-stromal crosstalk. Breast Cancer Res. 2012;14:R98. PubMed PMC

Le Bras GF, Taylor C, Koumangoye RB, Revetta F, Loomans HA, Andl CD. TGFbeta loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion. Exp Cell Res. 2015;330:29–42. PubMed PMC

Proby CM, Purdie KJ, Sexton CJ, Purkis P, Navsaria HA, Stables JN, Leigh IM. Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis. Exp Dermatol. 2000;9:104–117. PubMed

Hannigan A, Smith P, Kalna G, Lo Nigro C, Orange C, O'Brien DI, Shah R, Syed N, Spender LC, Herrera B, Thurlow JK, Lattanzio L, Monteverde M, et al. Epigenetic downregulation of human disabled homolog 2 switches TGF-beta from a tumor suppressor to a tumor promoter. J Clin Invest. 2010;120:2842–2857. PubMed PMC

Slater D, Walsh M. Dataset for the histological reporting of primary invasive cutaneous squamous cell carcinoma and regional lymph nodes. The Royal College of Pathologists. 2014

Complex and variable regulation of ΔNp63 and TAp63 by TGFβ has implications for the dynamics of squamous cell epithelial to mesenchymal transition