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De Robertis A.,Molecular Oncology Unit | De Robertis A.,Siena Biotech Medicine Research Center | Mennillo F.,Molecular Oncology Unit | Mennillo F.,Siena Biotech Medicine Research Center | And 12 more authors.
PLoS ONE | Year: 2014

Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas. © 2014 De Robertis et al.


De Robertis A.,Siena Biotech Medicine Research Center | Valensin S.,Siena Biotech Medicine Research Center | Rossi M.,Siena Biotech Medicine Research Center | Tunici P.,Siena Biotech Medicine Research Center | And 20 more authors.
Molecular Cancer Therapeutics | Year: 2013

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway inGBMcells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/ degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics. ©2013 AACR.


PubMed | Noxamet Ltd., Siena Biotech Medicine Research Center and Sant'Anna School of Advanced Studies
Type: | Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences | Year: 2016

Preclinical imaging modalities represent an essential tool to develop a modern and translational biomedical research. To date, Optical Imaging (OI) and Magnetic Resonance Imaging (MRI) are used principally in separate studies for molecular imaging studies. We decided to combine OI and MRI together through the development of a lentiviral vector to monitor the Wnt pathway response to Lithium Chloride (LiCl) treatment. The construct was stably infected in glioblastoma cells and, after intracranial transplantation in mice, serial MRI and OI imaging sessions were performed to detect human ferritin heavy chain protein (hFTH) and firefly luciferase enzyme (FLuc) respectively. The system allowed also ex vivo analysis using a constitutive fluorescence protein expression. In mice, LiCl administration has shown significantly increment of luminescence signal and a lower signal of T2 values (P<0.05), recorded noninvasively with OI and a 7 Tesla MRI scanner. This study indicates that OI and MRI can be performed in a single in vivo experiment, providing an in vivo proof-of-concept for drug discovery projects in preclinical phase.


PubMed | Sant'Anna School of Advanced Studies, Temple University, Siena Biotech Medicine Research Center and Fondazione Toscana G. Monasterio
Type: | Journal: Cardiovascular research | Year: 2016

Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/-catenin/TCF pathway activation, an important regulator of post-ischemic cardiac remodeling.We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the -catenin-responsive TCF/Lef promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n=11). By 1.5-Tesla MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, postmortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n=6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of -catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischemic remodeling process, as demonstrated by the significant preservation of cardiac function, the 421% increase of peri-infarct arteriolar density and 433% reduction in infarct scar size compared with untreated animals.The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/-catenin/TCF activation state in response to cardiac injury and therapeutic interventions.

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