National Center Institute, National Institutes of Health, USA
Proteogenomics: New Era of Opportunities in Cancer Biology and Precision Medicine
Despite significant progress in understanding cancer through massively parallel sequencing genome programs, the complexity that comprises its diseases remains a daunting barrier. Today we know that molecular drivers of cancer are derived not just from DNA alterations alone, but from protein expression and activity at the cellular pathway level - proteomics. To predict the downstream effects of gene alterations, orthogonal technologies such as next-generation proteomics are needed. This proteogenomics approach (interplay between proteome and genome) is anticipated to transform oncology care from one that relies mainly on trial-and-error treatment strategies based on the anatomy of the tumor, to one that is more precisely based on the tumor’s molecular profile. This seminar will discuss how genomics, transcriptomics, and proteomics must all be brought together in the quest to understand the etiology of cancer, in addition to highlighting efforts by the U.S. National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) program in this area of biomedical research. CPTAC began with the purpose of developing standardized (rigor & reproducibility) proteomic assays and workflows, in order to complement genomic and transcriptomic analyses. CPTAC’s proteogenomics approach was recently successful in demonstrating the scientific benefits of integrating proteomics with genomics to produce a more unified understanding of cancer biology and possibly therapeutic interventions for patients, while creating open community resources that are widely used by the global cancer community. This seminar will also highlight the recently announced APOLLO (Applied Proteogenomics OrganizationaL Learning and Outcomes) program and the efforts of the International Proteogenomic Consortium. APOLLO brings together the U.S. National Cancer Institute, U.S. Department of Defense, and the U.S. Department of Veterans Affairs to create the nation’s first healthcare system in which cancer patients will be routinely screened for genomic abnormalities and proteomic information with the goal of matching their tumor type to a specific targeted therapy.
A novel data-independent acquisition (DIA) mass spectrometry approach integrated with RNA-seq for deep proteogenomic profiling
We have recently initiated integrated analysis of mRNAs with high resolution mass spectrometry for proteogenomic analysis of individual cell types. The availability of corresponding RNA-seq data provides a unique opportunity to search a database of peptides encoded only by mRNAs expressed in the individual cell types – i.e. intra-exonic as well as junctional (encoded by exon-exon junctions) peptides. This strategy also allowed us to directly interrogate the non-synonymous SNPs that were translated into proteins. However, one problem of current data-dependent approaches is that the more abundant peptides are readily identified while those that are less abundant (e.g. regulatory proteins such as transcription factors, certain alternative protein isoforms, proteins with alternative start sites, peptides from translation of microORFs) are not sampled. Data-independent acquisition (DIA) strategies provide an opportunity to sample peptides that would otherwise not be selected for fragmentation. However, the current implementation of such DIA approaches relies on very large m/z windows, which precludes identification of low abundance peptides. We have developed a novel pipeline to carry out DIA analysis on an Orbitrap Fusion Lumos mass spectrometer using very small m/z windows for fragmentation. We analyzed primary human umbilical vein endothelial cell (HUVEC) lysates by scanning sequentially through 150 Th windows in each LC-MS/MS run using the advanced quadrupole mass filter followed by fragmentation of peptide ions in the HCD cell in small m/z windows and ultimate detection of fragment ions in the Orbitrap mass analyzer. Several runs were carried out to cover the entire (350-1,450 m/z) mass range on an Orbitrap Fusion Lumos mass spectrometer. Our strategy allowed us to “enrich” several low abundance proteins and peptides, including post-translationally identified peptides, which would otherwise be missed by conventional strategies. The expression of these low abundance proteins was corroborated with the expression of transcripts in RNA-seq data. Overall, this approach can be coupled to any upstream fractionation/enrichment method for a more comprehensive characterization of the proteome.
Ulsan National Institute of Science and Technology, Korea
Current identities of phosphoinositide-specific phospholipase C isozymes
Phosphoinositide-specific phospholipase C play a critical role on cellular function. PLC hydrolysis phosphatidylinosito 4, 5-bisphosphate (PIP2) to produce inositol-1,4,5-triphosphate(IP3) and diacylglycerol(DAG) in ligand-mediated signal transduction. Currently, thirteen mammal PLC isozymes have been identified and are divided into six subtypes ; : PLC (1-4), (1-2), 1,3,4), , , and (1-2).
PLC isozymes are ubiquitously distributed and regulates cellular functions in diverse tissues. PLCγ is activated in response to many growth factors that bind to receptor tyrosine kinase(RTK). PLCis participated in GPCR-mediated signaling. Although the results of many studies suggest that PLCγ1 has important functions.
From PLCγ1-deleted mice in cell or tissue, we observed that PLC-γ1 is involved in brain development, neuropsychiatry disorder, colorectal tumorigenesis and so on.
Forebrain-PLCγ1 knockout mice display abnormal behaviors including hyper-locomotor activity, reduced social interaction, and decreased social communication. We observed that imbalance between excitation and inhibition in PLCγ1-deleted hippocampus contributes to abnormal behaviors. Molecular studies revealed that BDNF/TrkB signaling is impaired in PLCγ1 deleted hippocampus. Taken together, our findings demonstrate a critical roles of PLCγ1 for BDNF/TrkB signaling activation and neuropsychiatric functions.
PLCγ1 is highly expressed in cancers as compared to normal tissues, and plays a crucial role in tumorigenesis. Using a colitis-associated cancer model, we show that although deletion of PLCγ1 in intestinal epithelial cells decrease inflammation, it leads to a dramatic decrease in tumor load without affecting tumor size. PLCγ1 f/f mice enhance colitis-induced epithelial damage and increase expression of pro-inflammatory cytokines that may serve as tumor growth factors. This is linked to resisted inflammation during tumor promotion. PLCγ1 may link inflammation-associated epithelial damage and tumor development.
We believe that studies on PLCγ1 conditional knockout mice will contribute to the understanding of not only the cell-specific functions of PLCγ1, but also its pathophysiological roles.
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