Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup.

We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53-wild type; DAOY and UW402: TP53-mutated). MB cell lines molecular subgroup was confirmed and TP53 mutations were validated. Cell viability, clonogenicity and apoptosis were evaluated after ATO treatment at different concentrations (1-16 µM) alone or combined with irradiation doses (0.5, 1, 2 and 4 Gy). Rad51 and Ku86 proteins were evaluated by WB. ATO treatment reduced cell viability for all SHH-MB cell lines. Significant decrease of clonogenic capacity and higher apoptosis rates were also observed after ATO exposure, being cell death more pronounced (>70%) for the SHH-MB TP53-mutated. Combined treatment of ATO with irradiation also reduced colonies formation in UW402 tumor cells, which was independent of DNA damage repair proteins Rad51 and Ku86. In silico analyses suggested that a set of genes from cell cycle and p53 pathways are differentially expressed in SHH tumor subtypes, suggesting that cell lines may respond to therapies according to the gene expression profiles. Herein, we showed ATO cytotoxicity in pediatric SHH cell lines, with marked radiosensitizing effect for the MB-SHH TP53-mutated cells. These results highlight the potential of ATO, alone or in combination with radiotherapy, supporting further clinical investigations.

Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGFß2 in breast cells.

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGFß and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGFß2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. BIOLOGICAL SIGNIFICANCE: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGFß2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages.

Misshapen coordinates protrusion restriction and actomyosin contractility during collective cell migration.

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.

Expression profiles and prognostic value of miRNAs in retinoblastoma.

Current cure rates for retinoblastoma (RB) are very high in developed countries. Nonetheless, in less privileged places worldwide, delayed diagnosis and refusal to adhere to treatment still endure an obstacle to improve overall patient survival. Thus, the access to consistent biomarkers for diagnosis at an earlier stage may facilitate treatment and improve outcomes. Over recent years, much attention has been focused on miRNAs, key post-transcriptional regulators that when altered, largely contribute to carcinogenesis and tumor progression. Many of the ~ 2500 microRNAs described in humans have shown differential expression profiles in tumors. In this review, we summarize current data about the roles of miRNAs in RB along with their value as diagnostic/prognostic factors using electronic databases such as PubMed. We reviewed the importance of miRNA in RB biology and discussed their implications in clinic intervention. Several miRNAs have pointed out reliable diagnostic and prognostic molecular biomarkers. The emergence of targeted therapies has significantly improved cancer treatment. In the near future, the modulation of miRNAs will represent a good treatment strategy.

MiR-708-5p is inversely associated with EWS/FLI1 Ewing sarcoma but does not represent a prognostic predictor.

BACKGROUND: Overall survival of Ewing sarcoma (EWS) remains poor and less than 30% of patients with metastatic or recurrent disease survive despite current treatments. Thus, there is a constant search for new biomarkers for diagnosis, prognosis and prediction of therapy. Numerous studies have reported the abnormal expression of miR-708-5p in tumors of different origins. However, its role in EWS remains unclear. PROCEDURE: qRT-PCR was performed in nineteen consecutive EWS samples and twelve non-tumor bone samples from age-matched controls. Functional assays were performed in SK-ES-1 cells transfected with miR-708 lentiviral-based vectors and results analyzed in terms of clonogenicity, migration, invasion and western blot. RESULTS: We show that miR-708-5p is downregulated in EWS tissues though no associations with any prognostic features such as HUVOS grade, event or survival were found in our cohort. Nonetheless, expression levels of this micro-RNA were inversely associated with the presence of the EWS/FLI1 translocation. When miR-708-5p was transfected into the SK-ES-1 cell line, it did not affect migration or clonogenicity, but promoted a significant increase on the invasive potential of cells endorsed with high expression of MMP2. CONCLUSIONS: Taken together, our results suggest that despite downregulated in EWS samples, this miRNA might represent a secondary genetic alteration derived from the pleiotropic cellular effects of the abnormal EWS/FLI1 transcription factor that does not affect tumor growth but instead, is related with the promotion of tumor invasion, not being suitable for future therapeutic intervention.