Smad Protein Expression and Activation in Transforming Growth Factor-β Refractory Human Squamous Cell Carcinoma Cells

Abstract

In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-β (TGF-β) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-β control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-β 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-β-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and -3. Furthermore, treatment with TGF-β induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-β 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-β 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-β was able to activate TGF-β-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-β is due to factors extraneous to the TGF-β pathway itself. Application of our protein-based strategy to interrogate the TGF-β signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-β resistant in vivo.

Bioluminescence imaging of Smad signaling in living mice shows correlation with excitotoxic neurodegeneration

“…To determine in which CNS cells the TGF-␤ signaling pathway is activated, we used two different antibodies raised against the activated, phosphor ylated form of Smad2 (Smad2P) (23,24). Interestingly, Smad2P was localized predominantly to neurons and, in agreement with the biochemical and bioluminescent data, to hippocampal pyramidal neurons ( Fig.…”

Molecular mechanisms of head and neck cancer

“…Frequent loss of heterozygosity has been reported for chromosome 18 in OSCC implicating SMAD-2 and -4 as important players in the tumorigenic process [93]. Mutations, though relatively rare, have also been identified in the SMAD genes and other regulators of the TGF-β pathway in primary OSCC tumors and cell lines [75,92,[94][95][96][97]. CDK2AP1 and Deleted in Oral Cancer -1 (DOC-1), this gene was identified by subtractive hybridization from a hamster oral cancer model [98].…”

Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype

“…Smad4 was detected using a mouse monoclonal anti-Smad4 antibody (B-8; Santa Cruz Biotechnology). Phospho-Smad2 (pSmad2) and phospho-Smad3 (pSmad3) were detected using our own rabbit polyclonal anti-pSmad2 and anti-pSmad3 antibodies as described previously (58,59). T␤R-I and T␤R-II were detected using rabbit anti-T␤R-I and T␤R-II antibodies (SC-399 and SC-229, respectively; Santa Cruz Biotechnology).…”