Identification of Smad4 transcriptional target genes

Inactivating mutations of the TGF-beta signaling pathway, including SMAD4, have been identified in a wide variety of human tumors and can lead to resistance to TGF-beta induced growth arrest. SMAD4 itself has been firmly established as a tumor suppressor while despite the massive information gathered on TGF-beta signaling the contribution of SMAD4 inactivation in tumor progression remains elusive. It is largely unknown which particular genes involved in tumor progression require SMAD4 for their transcriptional regulation. Smad4-/- murine fibroblasts were shown to display normal growth inhibition in response to TGF-beta and we hypothesized that in Smad4-/- tumors there are as yet unidentified physiological targets of Smad4 involved in tumor progression. A cDNA microarray was performed on Smad4+/+ and -/- murine fibroblasts with and without TGF-beta stimulation in order to identify genes that were deregulated in the absence of Smad4. Several potential novel targets were identified and confirmed by RT-PCR and Northern analysis. Consistent with the role of Smad4 as a tumor suppressor, when Smad4 is inactivated, fibroblasts appear to present with an increase in factors that enhance angiogenesis and a decrease in factors that inhibit the degradation of the extracellular matrix. This could potentially lead to an increased vascularization of tumors and an augmentation of metastatic ability. Further, in the absence of Smad4, TGF-beta stimulation appears to amplify the deregulation of target genes, a mechanism which could potentially enhance tumor progression in Smad4-/- tumors