Smad protein expression and activation in transforming growth factor-beta refractory human squamous cell carcinoma cells.
In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-beta (TGF-beta) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-beta control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-beta signaling pathway by combining measurements of the levels and the activation state of Smad signaling intermediates with DNA-based diagnostic assays. In this report, we demonstrate the validity of this approach using a panel of seven squamous cell carcinoma (SCC) lines known to be refractory to TGF-beta-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and-3. Furthermore, treatment with TGF-beta induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-beta type II receptor. Three of the remaining SCC lines failed to express the common mediator Smad4, two on the basis of loss of transcription and one by a posttranscriptional mechanism. Thus, a mechanism for TGF-beta resistance was identified in five of the seven tumor cell lines. Interestingly, in the two remaining lines, no abnormalities of signaling intermediates were found, and TGF-beta was able to activate TGF-beta-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-beta is due to factors extraneous to the TGF-beta pathway itself. Application of our protein-based strategy to interrogate the TGF-beta signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-beta resistant in vivo.
Yan
W
W
Department of Internal Medicine and Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520-8032, USA.
Vellucci
V F
VF
Reiss
M
M
D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D015398:Signal Transduction; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta