"Examining TGF-beta Inhibition, via Smad7, and the Role of The Inhibitors of Differentiation (Id) in Metastatic Melanoma"

Abstract

TGF-β, Ids and tumorigenesis are interrelated. Smad7 (S7) has been shown to potently suppress melanomagenesis through downregulation of metastasis-related genes such as MMP2, MMP9, osteopontin and CXCR4. Here, we describe other contributing factors also play a role. Human skin grafts with fluorescently-tagged melanoma cells revealed Smad7-expressing cells positioned themselves proximal to the dermal-epidermal junction and failed to form tumors; control cells invaded and formed tumors within the dermis. Smad7 inhibited &beta-catenin T41/S45 phosphorylation, which is associated with degradation and induced a 4.5-fold increase in full-length N-cadherin. Cell adhesion assays confirmed a strong interaction between Smad7-expressing cells and primary dermal fibroblasts mediated via N-cadherin; while control cells were incapable of such interaction. Skin graft analysis indicated an N-cadherin-mediated homotypic interaction between Smad7-expressing cells and primary dermal fibroblasts; in contrast to control cells. Smad7 suppressed β-catenin degradation and promoted interaction with N-cadherin, stabilizing the association with neighboring dermal fibroblasts, thus mitigating invasion. We find Smad7 limits growth to a `radial growth phase' melanoma, a significantly lessened state of disease.

To better define the role of Ids in melanomagenesis, we hypothesized expressing Id2, Id3 or Id4 along with Smad7 would revert 1205Lu/S7 cells back to the tumorigenic state. Subcutaneous injection of TGF-β-dependent 1205Lu cells co-expressing Id2, Id3, or Id4, with Smad7 bypassed the tumorigenic block mediated by Smad7, generating TGF-β-independent tumors and re-upregulated metastasis-related genes. Surprisingly, 1205Lu/S7 cells expressing Id4, but not Id2 or Id3, activated robust melanin production in amelanotic 1205Lu cells, confirmed by Fontana-Masson stain, de-novo expression of MART-1 and tyrosinase proteins. Mechanistic investigation revealed M-MITF phosphorylation and MART-1 promoter activation in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanoma. In human tumors melanin corresponded with Id4 localization. Additionally, pigment-laden CD163+ mouse histiocytes, with areas of extensive necrosis were found throughout S7/Id4 tumors only. Current chemotherapeutics for the treatment of melanoma are only marginally effective. Immunotherapy provides the most promise, yet innate immunity is often overlooked. This work better defines the role of the inhibitors of differentiation, particularly Id4, in advanced melanoma and may have implications for new pathways in developing immunotherapeutics in the treatment of melanoma.