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.

A proteomics outlook towards the elucidation of epithelial-mesenchymal transition molecular events.

The main cause of death in cancer is the spread, or metastasis, of cancer cells to distant organs with consequent tumor formation. Additionally, metastasis is a process that demands special attention, as the cellular transformations make cancer at this stage very difficult or occasionally even impossible to be cured. The main process that converts epithelial tumor cells to mesenchymal-like metastatic cells is the Epithelial to Mesenchymal Transition (EMT). This process allows stationary and polarized epithelial cells, which are connected laterally to several types of junctions as well as the basement membrane, to undergo multiple biochemical changes that enable disruption of cell-cell adherence and apical-basal polarity. Moreover, the cells undergo important reprogramming to remodel the cytoskeleton and acquire mesenchymal characteristics such as enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a large increase in the production of ECM components. As expected, the alterations of the protein complement are extensive and complex, and thus exploring this by proteomic approaches is of particular interest. Here we review the overall findings of proteome modifications during EMT, mainly focusing on molecular signatures observed in multiple proteomic studies as well as coordinated pathways, cellular processes and their clinical relevance for altered proteins. As a result, an interesting set of proteins is highlighted as potential targets to be further investigated in the context of EMT, metastasis and cancer progression.

Anti-nutritional factors and digestibility of protein in Cayocar brasiliense seeds

Abstract The Brazilian Cerrado presents a wide variety of natural products, including the Pequi (Caryocar brasiliense). The important factor that determines the dietary and nutritional ideality of protein is degestitibility. This work aims to evaluate the protein digestibility of Pequi seeds and the presence of anti-nutritional factors. The protein Pequi almonds were extracted, toasted and untoasted. Evaluation digestibility was structurally characterized by SDS-PAGE 15%, which can be used to analyze simulated gastric fluid (SFG) and digestion in intestinal fluid (SFI) in the presence of pepsin and trypsin/chymotrypsin. The extract from Pequi almonds showed inhibitory activity and was not detected hemagglutination. The intensity of the bands according to 2S albumins, after heat treatment, did not decrease in comparison to the condition native, significantly. In the crisp crude extract, which was incubated under SGF and SIF, the intensity of the corresponding bands at 8 and 3 kDa was resistant to SGF and indigestible after 4 hours under SIF. The characterization of Pequi almonds demonstrated that the product of the crude extract has anti-nutritional factors, which were confirmed by evaluating digestibility.