ASAPAutomated Sonication-Free Acid-Assisted Proteomesfrom Cells and FFPE Tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens

ASAPAutomated Sonication-Free Acid-Assisted Proteomesfrom Cells and FFPE Tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens

ASAPAutomated Sonication-Free Acid-Assisted Proteomesfrom Cells and FFPE Tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens

ASAPAutomated Sonication-Free Acid-Assisted Proteomesfrom Cells and FFPE Tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens

Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition.

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors

Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break repair

The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSB-inducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNA-end resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDRWork in Y.S. laboratory is funded by research grants from the Dr Miriam and Sheldon G. Adelson Medical Research Foundation; The A-T Children's Project; The Israel Science Foundation (Joint ISF-NSFC Program with the National Natural Science Foundation of China); The Israel Cancer Research Fund; Work in P.H. lab was supported by R+D+I grant from the Spanish Ministry of Economy and Competitivity [SAF2013-43255-P]; ERC Starting Grant [DSBRECA]; PhD fellowship from the Spanish Ministry of Education (FPU to R.P.-C.); The R.A. lab is supported by NWO grant [NGI 93512001 to R.A.]; The Human Frontier Science Program [LT000640/2013 to A.P.U.]; The work in T.G. lab was supported by The I-CORE Program of the Planning and Budgeting Committee of the Israel Ministry of Education; Y.S. is a Research Professor of the Israel Cancer Research Fund. Funding for open access charge: research grant money.Peer reviewe

<i>EXO1</i> modular genes are tightly co-expressed and associated with poor prognosis in breast cancer patients

<p>: (A) Identification of <i>EXO1</i> modular genes using meta-analysis of correlation coefficients from multiple breast tumor datasets. Schematic <i>EXO1</i> centred network shows that <i>EXO1</i> co-expressed genes are involved in cell cycle and DNA repair. (B) and (C) Elevated expression of <i>EXO1</i> modular genes was associated with poor clinical outcome in breast tumor cohorts. (D) Heatmap showing the expression pattern of <i>EXO1</i> modular genes in 51 breast cancer cell lines. <i>EXO1</i> modular genes are highly expressed in aggressive and basal cell lines. I-Invasive, N-Non-invasive, B-Basal, L-Luminal and ER status of the cell lines are shown on the top.</p

Specific inhibition of splicing factor activity by decoy RNA oligonucleotides

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.ISSN:2041-172

Identification of genes associated with poor clinical outcome in 1q amplicon of breast tumors.

<p>(A) High frequency amplification of 1q region in multiple cohorts of breast tumors. Graphs I – VI represent inferred copy-number of probes at chromosome 1 from the aCGH studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B61" target="_blank">61</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B66" target="_blank">66</a>]. These graphical representations were the outcome of data visualized through Progenetix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B67" target="_blank">67</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077553#B68" target="_blank">68</a>]. Copy number gain and loss are represented with green and red colors respectively. In all these five cohorts, 1q is amplified in 40 - 60 % of tumors. (B) Meta analysis – workflow for the identification 1q genes associated with patient survival. (C) Cox regression coefficient, hazard ratio and p-value of the shortlisted 1q genes, (D) Chromosomal map showing the locations of clinically significant 1q genes. (E) The identified 1q candidate genes showing an elevated expression in breast tumors when compared to the non-cancerous breast tissues in 3 different cohorts. </p