Uncoupled responses of Smad 4-deficient cancer cells to TNF a result in secretion of monomeric laminin-g 2

Background: Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-g2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFa on tumor cells. Results: Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFa with an increased expression of all three chains encoding laminin-332; coincubation with TGFb and TNFa leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFa can induce expression of the g2 and b3 but not the a3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of g2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble g2 is associated with increased cell migration. Conclusions: Loss of Smad4 may lead to uncoupled induction of laminin-g2 in response to TNFa and may therefore represent one of the mechanisms which underlie accumulation of laminin-g2 at the invasive margin of a tumor. The finding, that g2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor