Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is ␤-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of ␤-catenin that may control its transcriptional activity, we performed a mammalian twohybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between ␤-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of ␤-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of ␤-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous ␤-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorageindependent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/␤-catenin signaling pathway.

Abstract-To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of Ϸ3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

BackgroundAlthough recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data.MethodsThis prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45pos leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45neg cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively.ResultsBy employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45neg subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45neg cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment.ConclusionThis study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0687-3) contains supplementary material, which is available to authorized users.

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-theart markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma-associated stem cells (GASC) from LGG (n540) and HGG (n573). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n513) and LGG (n512) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASCbased score was the only independent predictor of overall survival and malignant progression free-survival. Conclusions: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of gliomainitiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patientbased approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.