Tumor Biology

, Volume 37, Issue 12, pp 16215–16225 | Cite as

Hedgehog/Gli1 signal pathway facilitates proliferation, invasion, and migration of cutaneous SCC through regulating VEGF

Original Article


Since hedgehog (HH)/Gli1 that contributes to cancer proliferation and metastasis has been masked for decades, the signaling pathway was investigated about its exact role in proliferation and metastasis of cutaneous squamous cell carcinoma (SCC). Sonic hedgehog homolog (Shh), GLI family zinc finger 1 (Gli1), and vascular endothelial growth factor (VEGF) expressions in cutaneous SCC tissues were analyzed with immunohistochemistry, and their correlations with cutaneous SCC patients’ prognosis were conducted with Kaplan-Meier curve. Regarding in vitro studies, effects of the HH signaling pathway, and cyclopamine on patched 1 (Ptch1), smoothened/frizzled class receptor (Smo) and VEGF expressions were assessed in A431 cells based on western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Besides, Cell Counting Kit-8 (CCK-8) assay was implemented to evaluate cell proliferation, while wound-healing assay and transwell assay were performed to assess cell migration and invasion, respectively. Mice models were also established to observe effects of Gli1 on tumor diversity and incidence during a period of 20 weeks. Positively expressed VEGF, Gli1, and Shh proteins in cutaneous SCC tissues were correlated with poor survival of patients (P < 0.05). Besides, Gli1 messenger RNA (mRNA) and VEGF mRNA were observed to be significantly over-expressed in A431 cells (P < 0.05), and they were associated with incremental cell proliferation, invasiveness, and migration, which can be reversed by the interference of VEGF siRNA. Furthermore, cyclopamine treatment could induce inhibition of cell proliferation, invasiveness, and migration and suppression of Smo, Gli1, and VEGF expressions. The mice models also confirmed that Gli1 could significantly induce rise of tumor incidence and tumor diversity, while cyclopamine statistically relieved this transformation (P < 0.05). Abnormal activation of the HH signaling pathway plays critical roles in development of cutaneous SCC either in vivo or in vitro.


Hedgehog signaling VEGF Proliferation Metastasis Cutaneous squamous cell carcinoma 


Compliance with ethical standards

Conflicts of interest



  1. 1.
    Vasiljevic N, Andersson K, Bjelkenkrantz K, Kjellstrom C, Mansson H, Nilsson E, Landberg G, Dillner J, Forslund O. The Bcl-XL inhibitor of apoptosis is preferentially expressed in cutaneous squamous cell carcinoma compared with that in keratoacanthoma. Int J Cancer J Int du Cancer. 2009;124:2361–6.CrossRefGoogle Scholar
  2. 2.
    Bluth MJ, Zaba LC, Moussai D, Suarez-Farinas M, Kaporis H, Fan L, Pierson KC, White TR, Pitts-Kiefer A, Fuentes-Duculan J, Guttman-Yassky E, Krueger JG, Lowes MA, Carucci JA. Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation. J Investig Dermatol. 2009;129:2451–62.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ambothi K, Prasad NR, Balupillai A. Ferulic acid inhibits UVB-radiation induced photocarcinogenesis through modulating inflammatory and apoptotic signaling in Swiss albino mice. Food Chem Toxicol: Int J Published Br Ind Biol Res Assoc. 2015;82:72–8.CrossRefGoogle Scholar
  4. 4.
    Okumura H, Uchikado Y, Setoyama T, Matsumoto M, Owaki T, Ishigami S, Natsugoe S. Biomarkers for predicting the response of esophageal squamous cell carcinoma to neoadjuvant chemoradiation therapy. Surg Today. 2014;44:421–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Li YY, Hanna GJ, Laga AC, Haddad RI, Lorch JH, Hammerman PS. Genomic analysis of metastatic cutaneous squamous cell carcinoma. Clinl Cancer Res: Off J Am Assoc Cancer Res. 2015;21:1447–56.CrossRefGoogle Scholar
  6. 6.
    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;9:713–20.CrossRefPubMedGoogle Scholar
  7. 7.
    Moussai D, Mitsui H, Pettersen JS, Pierson KC, Shah KR, Suarez-Farinas M, Cardinale IR, Bluth MJ, Krueger JG, Carucci JA. The human cutaneous squamous cell carcinoma microenvironment is characterized by increased lymphatic density and enhanced expression of macrophage-derived VEGF-c. J Invest Dermatol. 2011;131:229–36.CrossRefPubMedGoogle Scholar
  8. 8.
    Stettler C, Wandel S, Allemann S, Kastrati A, Morice MC, Schomig A, Pfisterer ME, Stone GW, Leon MB, de Lezo JS, Goy JJ, Park SJ, Sabate M, Suttorp MJ, Kelbaek H, Spaulding C, Menichelli M, Vermeersch P, Dirksen MT, Cervinka P, Petronio AS, Nordmann AJ, Diem P, Meier B, Zwahlen M, Reichenbach S, Trelle S, Windecker S, Juni P. Outcomes associated with drug-eluting and bare-metal stents: a collaborative network meta-analysis. Lancet. 2007;370:937–48.CrossRefPubMedGoogle Scholar
  9. 9.
    Liao X, Siu MK, CW A, Wong ES, Chan HY, Ip PP, Ngan HY, Cheung AN. Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation. Carcinogenesis. 2009;30:131–40.CrossRefPubMedGoogle Scholar
  10. 10.
    Lei J, Ma J, Ma Q, Li X, Liu H, Xu Q, Duan W, Sun Q, Xu J, Wu Z, Wu E. Hedgehog signaling regulates hypoxia induced epithelial to mesenchymal transition and invasion in pancreatic cancer cells via a ligand-independent manner. Mol Cancer. 2013;12:66.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tang AQ, Cao XC, Tian L, He L, Liu F. Apigenin inhibits the self-renewal capacity of human ovarian cancer SKOV3-derived sphere-forming cells. Mol Med Rep. 2015;11:2221–6.PubMedGoogle Scholar
  12. 12.
    Zhou JX, Jia LW, Liu WM, Miao CL, Liu S, Cao YJ, Duan EK. Role of sonic hedgehog in maintaining a pool of proliferating stem cells in the human fetal epidermis. Hum Reprod. 2006;21:1698–704.CrossRefPubMedGoogle Scholar
  13. 13.
    Shahi MH, Schiapparelli P, Afzal M, Sinha S, Rey JA, Castresana JS. Expression and epigenetic modulation of sonic hedgehog-Gli1 pathway genes in neuroblastoma cell lines and tumors. Tumour Biol: J Int Soc Oncodevelopmental Biol Med. 2011;32:113–27.CrossRefGoogle Scholar
  14. 14.
    Xin Y, Shen XD, Cheng L, Hong DF, Chen B. Perifosine inhibits S6K1-Gli1 signaling and enhances gemcitabine-induced anti-pancreatic cancer efficiency. Cancer Chemother Pharmacol. 2014;73:711–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Coultas L, Nieuwenhuis E, Anderson GA, Cabezas J, Nagy A, Henkelman RM, Hui CC, Rossant J. Hedgehog regulates distinct vascular patterning events through VEGF-dependent and -independent mechanisms. Blood. 2010;116:653–60.CrossRefPubMedGoogle Scholar
  16. 16.
    Maeda A, Nakata M, Yasuda K, Yukawa T, Saisho S, Okita R, Hirami Y, Shimizu K. Influence of vascular endothelial growth factor single nucleotide polymorphisms on non-small cell lung cancer tumor angiogenesis. Oncol Rep. 2013;29:39–44.PubMedGoogle Scholar
  17. 17.
    Supic G, Jovic N, Zeljic K, Kozomara R, Magic Z. Association of VEGF—a genetic polymorphisms with cancer risk and survival in advanced-stage oral squamous cell carcinoma patients. Oral Oncol. 2012;48:1171–7.CrossRefPubMedGoogle Scholar
  18. 18.
    Pfaff D, Philippova M, Kyriakakis E, Maslova K, Rupp K, Buechner SA, Iezzi G, Spagnoli GC, Erne P, Resink TJ. Paradoxical effects of T-cadherin on squamous cell carcinoma: up- and down-regulation increase xenograft growth by distinct mechanisms. J Pathol. 2011;225:512–24.CrossRefPubMedGoogle Scholar
  19. 19.
    Kimura Y, Morohashi S, Yoshizawa T, Suzuki T, Morohashi H, Sakamoto Y, Koyama M, Murata A, Kijima H, Hakamada K Clinicopathological significance of vascular endothelial growth factor, thymidine phosphorylase and microvessel density in colorectal cancer. Mol Med Rep. 2015.Google Scholar
  20. 20.
    Sahin E, Baycu C, Koparal AT, Burukoglu Donmez D, Bektur E Resveratrol reduces IL-6 and VEGF secretion from co-cultured a549 lung cancer cells and adipose-derived mesenchymal stem cells. Tumour Biol: J Int SocOncodev Biol Med. 2015.Google Scholar
  21. 21.
    Wieczorek E, Jablonowski Z, Tomasik B, Konecki T, Jablonska E, Gromadzinska J, Fendler W, Sosnowski M, Wasowicz W, Reszka E. MMP, VEGF and TIMP as prognostic factors in recurring bladder cancer. Clin Biochem. 2015;48:1235–40.CrossRefPubMedGoogle Scholar
  22. 22.
    Kanitz A, Imig J, Dziunycz PJ, Primorac A, Galgano A, Hofbauer GF, Gerber AP, Detmar M. The expression levels of microRNA-361-5p and its target VEGFa are inversely correlated in human cutaneous squamous cell carcinoma. PLoS One. 2012;7:e49568.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mace CJ, Trimble MR. Hysteria’, ‘functional’ or ‘psychogenic’? A survey of british neurologists’ preferences. J R Soc Med. 1991;84:471–5.PubMedPubMedCentralGoogle Scholar
  24. 24.
    Aziz MH, Manoharan HT, Sand JM, Verma AK. Protein kinase cepsilon interacts with STAT3 and regulates its activation that is essential for the development of skin cancer. Mol Carcinog. 2007;46:646–53.CrossRefPubMedGoogle Scholar
  25. 25.
    Palle K, Mani C, Tripathi K, Athar M. Aberrant Gli1 activation in DNA damage response, carcinogenesis and chemoresistance. Cancers. 2015;7:2330–51.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zhu W, You Z, Li T, Yu C, Tao G, Hu M, Chen X. Correlation of hedgehog signal activation with chemoradiotherapy sensitivity and survival in esophageal squamous cell carcinomas. Jpn J Clin Oncol. 2011;41:386–93.CrossRefPubMedGoogle Scholar
  27. 27.
    Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i, Altaba A. Hedgehog-Gli1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol: CB. 2007;17:165–72.CrossRefPubMedGoogle Scholar
  28. 28.
    Dahmane N, Sanchez P, Gitton Y, Palma V, Sun T, Beyna M, Weiner H, Ruiz i Altaba A. The sonic hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development. 2001;128:5201–12.PubMedGoogle Scholar
  29. 29.
    Mahindroo N, Punchihewa C, Fujii N. Hedgehog-Gli signaling pathway inhibitors as anticancer agents. J Med Chem. 2009;52:3829–45.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chen X, Horiuchi A, Kikuchi N, Osada R, Yoshida J, Shiozawa T, Konishi I. Hedgehog signal pathway is activated in ovarian carcinomas, correlating with cell proliferation: its inhibition leads to growth suppression and apoptosis. Cancer Sci. 2007;98:68–76.CrossRefPubMedGoogle Scholar
  31. 31.
    Taipale J, Chen JK, Cooper MK, Wang B, Mann RK, Milenkovic L, Scott MP, Beachy PA. Effects of oncogenic mutations in smoothened and patched can be reversed by cyclopamine. Nature. 2000;406:1005–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Koike C, Mizutani T, Ito T, Shimizu Y, Yamamichi N, Kameda T, Michimukai E, Kitamura N, Okamoto T, Iba H. Introduction of wild-type patched gene suppresses the oncogenic potential of human squamous cell carcinoma cell lines including a431. Oncogene. 2002;21:2670–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  1. 1.Department of ObstetricsJinan Maternity and Child Care HospitalJinanChina
  2. 2.Department of Burn and Plastic SurgeryProvincial Hospital Affiliated to Shandong UniversityJinanChina

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