Identification of Novel Molecular Targets for Endometrial Cancer Using a Drill-Down LC-MS/MS Approach with iTRAQ

BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma

Human Adipose-Derived Stem Cells in Madelung's Disease: Morphological and Functional Characterization

Madelung Disease (MD) is a syndrome characterized by the accumulation of aberrant symmetric adipose tissue deposits. The etiology of this disease is yet to be elucidated, even though the presence of comorbidities, either genetic or environmental, has been reported. For this reason, establishing an in vitro model for MD is considered crucial to get insights into its physiopathology. We previously established a protocol for isolation and culture of stem cells from diseased tissues. Therefore, we isolated human adipose-derived stem cells (ASC) from MD patients and compared these cells with those isolated from healthy subjects in terms of surface phenotype, growth kinetic, adipogenic differentiation potential, and molecular alterations. Moreover, we evaluated the ability of the MD-ASC secretome to affect healthy ASC. The results reported a difference in the growth kinetic and surface markers of MD-ASC compared to healthy ASC but not in adipogenic differentiation. The most commonly described mitochondrial mutations were not observed. Still, MD-ASC secretome was able to shift the healthy ASC phenotype to an MD phenotype. This work provides evidence of the possibility of exploiting a patient-based in vitro model for better understanding MD pathophysiology, possibly favoring the development of novel target therapies