Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Myo-inositol affects the cell-commitment induced by TGF-β in MCF-10A cells and primary mammary fibroblasts

Cells are characterized by intrinsic phenotypic plasticity. Waddington was the first to introduce the concept of “epigenetic landscape” to indicate the ability of a cell to progress from one differentiated state to another. The underlying cellular commitment is due to a wide range of factors, including molecular and microenvironmental constraints. Indeed, under specific conditions, cells can undergo trans-differentiation processes, losing their typical molecular and morphological characteristics to acquire others in response to stimuli of different natures. One of these possible stimuli is TGF-β, a pleiotropic cytokine able to regulate several biological processes. In particular, TGF-β is the main inducer of two trans-differentiation phenomena: the epithelial-mesenchymal transition (EMT) and the activation of fibroblasts into myo-fibroblasts. EMT is a trans-differentiation program by which epithelial cells lose cell-cell adhesion and acquire migratory and invasive properties, specific features of mesenchymal cells. The trans-differentiation of fibroblasts into myo-fibroblasts occurs under physiological conditions, such as during tissue repair, but also under pathological conditions, and it can lead to organ fibrosis and eventually to organ failure. Both of these trans-differentiation phenomena are closely linked to tumor development and progression. In previous works, we demonstrated that myo-inositol is able to affect cell morphology by remodeling the cytoskeleton in a highly metastatic and invasive cell line. The principal aim of this work is to highlight the cell fate of MCF-10A epithelial breast cells and of primary mammary fibroblasts upon treatment with TGF-β for several days. The second objective is to evaluate whether myo-inositol, a polyol, is able to regulate the cell commitment induced by TGF-β. To achieve these purposes, we treated MCF-10A with TGF-β for up to five days. Treatment with TGF-β triggers EMT, as demonstrated by changes in functional (increased motility and invasiveness) and morphological (transition from a cuboidal to an elongated shape) parameters. These modifications corresponded to concomitant changes in the main molecular pathways responsible for the regulation of EMT, including reduction of the expression of epithelial markers, such as E-cadherin, and keratin 18, and the increase of mesenchymal ones such as Snai1, N-cadherin and vimentin. In the meanwhile, we treated the primary fibroblasts with TGF-β for 72 hours, and we highlighted the trans-differentiation into myo-fibroblasts (the cells are more flattened and scattered), remodeling of the cytoskeleton, and increase of molecular parameters, such as the synthesis and production of the extracellular matrix components. However, the trans-differentiation induced by TGF-β in MCF-10A and primary fibroblasts can be blocked and induced towards an almost complete reversion by the addition of myo-inositol. Following treatment with myo-inositol, EMT-induced MCF-10A lose most of their mesenchymal characteristics and recover a normal epithelial phenotype, both from a morphological and functional point of view. In particular, the reversion induced by inositol dramatically affects both the motility and invasiveness of the cells and almost normalizes the molecular parameters. On the other hand, myo-fibroblasts treated with myo-inositol decrease the synthesis and the production of matrix components, and recover a less invasive and migratory phenotype. Therefore, the results obtained demonstrate that: 1) treatment with TGF-β triggers a trans-differentiation program that leads the cells to assume an unstable phenotype with different morphological and functional characteristics; 2) this phenotype can be almost completely abolished by treatment with myo-inositol with the recovery of normal morphological and functional features.