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.

Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit.

During zika and dengue viruse (ZIKV and DENV) outbreaks, the majority of the infected individuals remain clinically asymptomatic. Such asymptomatic individuals may occasionally acquire both arboviruses, donate blood, and contaminate haemoderivatives. The aim of this study was to characterize a ZIKV/DENV-4 coinfection in asymptomatic blood donor who donated blood during a large mixed ZIKV/DENV outbreak in the Säo Paulo State, Brazil. On the basis of post-donation information, one blood donor sample was found positive for ZIKV and DENV RNA. The DENV molecular serotyping was performed by molecular testing. The sample was also titrated on VERO E6 cells in order to define the presence of infectious arboviruses. The real-time PCR testing of the blood donor sample demonstrated very high viral load for both ZIKV and DENV. Further, molecular serotyping of DENV showed that the presence of DENV-4. The viral titration in cell culture indicated a titre of 2.75x10[6] PFU/ml which was concordant with the presence of infectious viruses in the blood donation. This is an interesting report for the simultaneous presence of infectious ZIKV and DENV-4 in asymptomatic blood sample. Special attention must be paid during mixed arboviral outbreaks for the possibility of transfusion-transmission of multiple arboviral agents.