De Menna, Marta (2010) Hierarchy of early molecular events induced by oncogenic Ras in thyroid epithelial cells. [Tesi di dottorato] (Unpublished)

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Using an inducible thyroid transformation system (FRTL-5/ERTM-H-RasV12cells), the gene expression profile changes at early times after oncogene activation were analyzed. I observed that Ras activation is able to rapidly reorganize gene expression, by inducing profound changes in the expression of several genes in few hours. Among these, one of the most strongly repressed is Wnt4. Wnt4 is a secreted glycoprotein that through its binding to cell surface receptors, is able to activate canonical beta catenin-dependent signaling as well as non-canonical pathways, depending on the cell and receptor context. Although its role in cancer has not been deeply investigated, wnt4 has been found either overexpressed or downregulated in different types of cancer. In the in vitro transformation system used Wnt4 repression is associated with the acquisition of the neoplastic phenotype. Ras in fact is able to repress its expression between one and six hours after its activation, and to chronically repress wnt4 expression in stably transformed cells. To study the role of wnt4 repression in thyroid transformation, I generated an FRTL-5/ERTM-H-RasV12 cell line constitutively expressing Wnt4 in order to ectopically maintain its expression when Ras abolishes the expression of the endogenous Wnt4. Forced expression of Wnt4 does not interfere with the ability of Ras to induce loss of differentiation and TSH-independent cell proliferation. However, I observed that, in the presence of forced expression of Wnt4, the ability of Ras to promote cell migration in FRTL-5 cells is severely impaired. Measurement, by real-time RT-PCR, of Wnt4 mRNA in a panel of human thyroid tumors of different histotypes, show that Wnt4 expression is significantly lower in cancer respect to normal thyroid tissue. Signaling experiments using either chemical inhibitors of Ras downstream pathways or Ras effector domain mutants suggest the involvement of MAPK pathway in the repression of Wnt4. From a molecular point of view the restore of Wnt4 expression in presence of oncogenic Ras interferes with the downregulation of Rho activity managed by oncogenic Ras. Moreover I observed a switch in cytoskeleton reorganization from membrane ruffles formation to stress fiber formation when both oncogenic Ras and Wnt4 are expressed. Taken together these results suggest that Wnt4 repression could be involved in the promotion of the aggressive character of thyroid carcinomas bearing oncogenic Ras.

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