Dissection of the RET/ß-catenin interaction in the TPC1 thyroid cancer cell line.

The RET receptor tyrosine kinase is a member of the cadherin superfamily and plays a pivotal role in cell survival, differentiation and proliferation. Currently, 12 ret/ptc chimeric oncogenes, characterized by the fusion between the intracellular domain of RET and different activating genes, which can cause ligand-independent dimerization and constitutive activation, have been described. ß-catenin is usually involved in the maintenance of cell-to-cell adhesion and mediates the Wnt/ß-catenin pathway important during embryogenesis and in cellular malignant transformation. Recently, a novel mechanism of RET-mediated function through the ß-catenin pathway has been reported in multiple endocrine neoplasia type 2 and in sporadic thyroid carcinomas. Here, we investigated the effects of the ZD6474, a small molecule RET-inhibitor, on RET/ß-catenin interaction. We confirmed the ZD6474 mediated-inhibition of recombinant RET kinase and of growth of cells expressing RET/PTC. Interestingly, we firstly observed reduced cellular mobility and changed morphology of TPC1 treated cells suggesting that RET-inhibitor could affect ß-catenin cellular distribution as resulted in its co-immunoprecipitation with E-cadherin. We further investigated this hypothesis showing that TPC1 treated cells displayed predominantly ß-catenin cytosolic localization. Surprisingly, RET and ß-catenin co-immunoprecipitated in both ZD6474-treated and untreated TPC1 cells, suggesting that RET/ß-catenin interaction might not be affected by RET kinase inactivation. All together these results suggest that RET kinase activation is crucial for ß-catenin stabilization (pY654), localization and its signaling pathway activation but not for ß-catenin/RET physical interactions, in human papillary thyroid carcinomas. In conclusion, ZD6474, by inhibiting RET kinase, down-modulates ß-catenin pathway leading its recruitment to the membrane by E-cadherin.

Heterogeneous phenotype of human melanoma cells with in vitro and in vivo features of tumor-initiating cells.

Melanospheres, the melanoma cells that grow as nonadherent colonies and that show in vitro self-renewing capacity and multipotency, were selected from melanoma specimens or from melanoma cell lines. Melanospheres were highly tumorigenic, and intradermal injections in severe combined immunodeficient (SCID) mice of as few as 100 cells generated tumors that maintained tumorigenic potential into subsequent recipients. Primary and serially transplanted xenografts recapitulated the phenotypic features of the original melanoma of the patient. Melanoma cells cultured in the presence of fetal calf serum (FCS) were also tumorigenic in SCID mice, although with lower efficiency; these xenografts showed a homogeneous phenotype for the expression of melanoma-associated markers, Melan-A/Mart-1, HMB45, and MITF, and contained cells with features of fully differentiated cells. Melanospheres were heterogeneous for the expression of stem cell markers and showed a significantly enhanced expression of the Nanog and Oct3/4 transcription factors when compared with adherent melanoma cells. No direct and unique correlation between any of the examined stem cell markers and in vivo tumorigenicity was found. Taken together, our data provide further evidence on the heterogeneous nature of human melanomas and show that melanospheres and their corresponding tumors, which are generated in vivo in immunocompromised mice, represent a model to investigate melanoma biology.

Interleukin-17-producing T-helper cells as new potential player mediating graft-versus-host disease in patients undergoing allogeneic stem-cell transplantation.

OBJECTIVES: Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients. METHODS: Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA. RESULTS: An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17+/interferon (IFN)-gamma- and IL-17+/IFN-gamma+ subsets and expressed IL-23 receptor. Interestingly, IFN-gamma+ TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD. CONCLUSIONS: These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.