Breast cancer quantitative proteome and proteogenomic landscape.

In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.

SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization.

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.

Publisher Correction: Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow.

In the original version of this Article, extraneous text not belonging to the Article was accidentally appended to the results section. This error has now been corrected in both the PDF and HTML versions of the Article.

Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow.

Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3-10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.

Correction: Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification.

[This corrects the article DOI: 10.1371/journal.pone.0166067.].

Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification.

BACKGROUND & AIMS: Adenocarcinomas of the pancreatobiliary system are currently classified by their primary anatomical location. In particular, the pathological diagnosis of intrahepatic cholangiocarcinoma is still considered as a diagnosis of exclusion of metastatic adenocarcinoma. Periampullary cancers have been previously classified according to the histological type of differentiation (pancreatobiliary, intestinal), but overlapping morphological features hinder their differential diagnosis. We performed an integrative immunohistochemical analysis of pancreato-biliary tumors to improve their diagnosis and prediction of outcome. METHODS: This was a retrospective observational cohort study on patients with adenocarcinoma of the pancreatobiliary system who underwent diagnostic core needle biopsy or surgical resection at a tertiary referral center. 409 tumor samples were analyzed with up to 27 conventional antibodies used in diagnostic pathology. Immunohistochemical scoring system was the percentage of stained tumor cells. Bioinformatic analysis, internal validation, and survival analysis were performed. RESULTS: Hierarchical clustering and differential expression analysis identified three immunohistochemical tumor types (extrahepatic pancreatobiliary, intestinal, and intrahepatic cholangiocarcinoma) and the discriminant markers between them. Among patients who underwent surgical resection of their primary tumor with curative intent, the intestinal type showed an adjusted hazard ratio of 0.19 for overall survival (95% confidence interval 0.05-0.72; p value = 0.014) compared to the extrahepatic pancreatobiliary type. CONCLUSIONS: Integrative immunohistochemical classification of adenocarcinomas of the pancreatobiliary system results in a characteristic immunohistochemical profile for intrahepatic cholangiocarcinoma and intestinal type adenocarcinoma, which helps in distinguishing them from metastatic and pancreatobiliary type adenocarcinoma, respectively. A diagnostic immunohistochemical panel and additional extended panels of discriminant markers are proposed as guidance for their pathological diagnosis.

TAp73 suppresses tumor angiogenesis through repression of proangiogenic cytokines and HIF-1α activity.

The p53-family member TAp73 is known to function as a tumor suppressor and regulates genomic integrity, cellular proliferation, and apoptosis; however, its role in tumor angiogenesis is poorly understood. Here we demonstrate that TAp73 regulates tumor angiogenesis through repression of proangiogenic and proinflammatory cytokines. Importantly, loss of TAp73 results in highly vascularized tumors, as well as an increase in vessel permeability resulting from disruption of vascular endothelial-cadherin junctions between endothelial cells. In contrast, loss of the oncogenic p73 isoform ΔNp73 leads to reduced blood vessel formation in tumors. Furthermore, we show that up-regulated ΔNp73 levels are associated with increased angiogenesis in human breast cancer and that inhibition of TAp73 results in an accumulation of HIF-1α and up-regulation of HIF-1α target genes. Taken together, our data demonstrate that loss of TAp73 or ΔNp73 up-regulation activates the angiogenic switch that stimulates tumor growth and progression.

Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution.

Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ~170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.