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

The Tetrahymena thermophila Phagosome Proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated

Role of moesin in hyaluronan induced cell migration in glioblastoma multiforme

Abstract Background A major barrier to effective treatment of glioblastoma multiforme (GBM) is the invasion of glioma cells into the brain parenchyma rendering local therapies such as surgery and radiation therapy ineffective. GBM patients with such highly invasive and infiltrative tumors have poor prognosis with a median survival time of only about a year. However, the mechanisms leading to increased cell migration, invasion and diffused behavior of glioma cells are still poorly understood. Methods In the current study, we applied quantitative proteomics for the identification of differentially expressed proteins in GBMs as compared to non-malignant brain tissues. Results Our study led to the identification of 23 proteins showing overexpression in GBM; these include membrane proteins, moesin and CD44. The results were verified using Western blotting and immunohistochemistry in independent set of GBM and non-malignant brain tissues. Both GBM tissues and glioma cell lines (U87 / U373) demonstrated membranous expression of moesin and CD44, as revealed by immunohistochemistry and immunofluorescence, respectively. Notably, glioma cells transfected with moesin siRNA displayed reduced migration and invasion on treatment with hyaluronan (HA), an important component of the extracellular matrix in GBM. CD44, a transmembrane glycoprotein, acts as a major receptor for hyaluronan (HA). Using co-immunoprecipitation assays, we further demonstrated that moesin interacts with CD44 in glioma cells only after treatment with HA; this implicates a novel role of moesin in HA-CD44 signaling in gliomas. Conclusions Our results suggest that development of inhibitors which interfere with CD44-moesin interactions may open a new avenue in the future to mitigate cellular migration in gliomas

Study of an RNA helicase implicates small RNA–noncoding RNA interactions in programmed DNA elimination in Tetrahymena

Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and ∼28-nucleotide small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p–scnRNA complexes identify micronuclear-limited sequences is not well understood. We show that a Twi1p-associated putative RNA helicase Ema1p is required for the interaction between Twi1p and chromatin. This requirement explains the phenotypes of EMA1 KO strains, including loss of selective down-regulation of scnRNAs homologous to macronuclear-destined sequences, loss of H3K9 and K27 methylation in the developing new macronucleus, and failure to eliminate DNA. We further demonstrate that Twi1p interacts with noncoding transcripts derived from parental and developing macronuclei and this interaction is greatly reduced in the absence of Ema1p. We propose that Ema1p functions in DNA elimination by stimulating base-pairing interactions between scnRNAs and noncoding transcripts in both parental and developing new macronuclei

Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry*S⃞

Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3σ); YWHAZ (14-3-3ζ); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin α (PTHA); l-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers