Gallo, G.O. (Gabriel Osvaldo)

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    Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line
    (2013) Vacas, A. (Andrés); Alonso-Roldán, M.M. (Marta María); Rey-Martinez, J.A. (Jorge Alberto); Vera-Cano, B. (Beatriz); Saez-Castresana, J. (Javier); García-López, R. (Ricardo); Rosa-Fernández-Pacheco, F.J. (Francisco Javier) de la; Gallo, G.O. (Gabriel Osvaldo)
    Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one, direct Gli1 siRNA inhibition; and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. In general, we proved that cell viability, cell migration and cell colony formation decreased after Shh inhibition, which might confer a less tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. All these experiments give an overview of the Shh pathway in medulloblastoma, together with the demonstration of the efficacy of cyclopamine and Gli1 siRNA Shh inhibition in vitro.
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    Characterization of cellular and molecular responses of human glioblastoma to Transforming Growth Factor-β signalling pathway inhibition
    (2016-06-08) Gallo, G.O. (Gabriel Osvaldo); Dotor, J. (Javier); Saez-Castresana, J. (Javier)
    Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumour accounting for 60-70% of all gliomas. Improvements in survival over the past 100 years can be measured only in weeks, and current achieved median survival ranges only 12-15 months. A hallmark of this malignancy is the intrinsic resistance to current therapies. Numerous efforts using molecularly targeted therapeutics have not significantly changed the near uniform lethality of this disease. The TGF-β signalling pathway plays a key role in GBM. It is implicated in progression, infiltration, and chemo/radioresistance as well as in the maintenance of stem-like phenotype of GBM CSC. Several inhibitors of different elements and regulators of the TGF-β pathway have entered to clinical trials. Among them, P17 and P144 inhibitory peptides of the TGF-β pathway have been tested for the treatment of different diseases including tumours. We decided to analyse the therapeutic potential of P144 for the treatment of GBM. We found that P144 impaired in vitro cellular processes as proliferation, migration, invasiveness and tumorigenicity. Apoptosis and anoikis were significantly increased by P144. Additionally, P144 blocked the TGF-β protective effect against apoptosis. The inhibition of TGF-β signalling by P144 affected the self-renewal capacity of a putative CSC subpopulation in vitro. These results were confirmed by the analysis on Brain Tumour Initiating Cells (BTIC) isolated from human GBM biopsies. P144 decreased in vitro proliferation, migration, and self-renewal capacity of this subpopulation. The effect of P144 was impaired by hypoxia. However, the precise underlying mechanism of hypoxia on P144 must be elucidated. We confirm the inhibition of TGF-β signalling by P144 through SMAD2 phosphorylation blockade, the pivotal initiation event of the pathway, which was translated to a reduction of P-SMAD2 nuclear translocation. Both results suggested an in vitro regulation on the transcriptional target genes of the TGF-β pathway in GBM cell lines. Furthermore, we confirmed in vitro and in vivo, the upregulation of SMAD7 and the downregulation of SKI by P144 at transcriptional and translational levels. This observation strongly suggests the implication of these factors in the molecular mechanism triggered by P144. The therapeutic potential of P144 was analysed in a mouse subcutaneous tumour model. Despite that P144 impaired tumour growth and leaded to an increase in survival, negative contradictory results were obtained in the in vivo intracranial model. We can conclude that the therapeutic potential of P144 as a treatment of GBM is clear. However, previous to potential clinical development, further studies are required in order to confirm P144 effect over GBM in the brain environment, as well as to explore P144 therapeutic potential in combination with current (TMZ and/or radiation) and emerging molecular based therapies.