TGF-beta1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest

Ian C Paterson, Maria Davies, Andrea Stone, Suzy Huntley, Emily Smith, Miranda Pring, John W Eveson, C Max Robinson, E Kenneth Parkinson, Stephen S Prime

Research output: Contribution to journalArticle (Academic Journal)

Abstract

This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.

Original languageEnglish
Pages (from-to)1616-24
Number of pages9
JournalOncogene
Volume21
Issue number10
DOIs
Publication statusPublished - 28 Feb 2002

Keywords

  • Animals
  • Carcinoma/metabolism
  • Cell Division
  • DNA-Binding Proteins/genetics
  • G1 Phase
  • Humans
  • Keratinocytes/metabolism
  • Kinetics
  • Ligands
  • Mice
  • Mice, Nude
  • Mouth Neoplasms/metabolism
  • Neoplasm Transplantation
  • RNA, Neoplasm/biosynthesis
  • Skin Neoplasms/metabolism
  • Smad4 Protein
  • Trans-Activators/genetics
  • Transcription, Genetic
  • Transfection
  • Transforming Growth Factor beta/genetics
  • Transforming Growth Factor beta1
  • Tumor Cells, Cultured
  • Tumor Suppressor Proteins/genetics

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    Paterson, I. C., Davies, M., Stone, A., Huntley, S., Smith, E., Pring, M., Eveson, J. W., Robinson, C. M., Parkinson, E. K., & Prime, S. S. (2002). TGF-beta1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest. Oncogene, 21(10), 1616-24. https://doi.org/10.1038/sj.onc.1205217