Project description:Non-invasive prognostic markers are needed to improve survival of colorectal cancer (CRC) patients. Towards this goal, we here apply integrative systems glycobiology approaches to tumour tissues and PBMCs from CRC patients and matching controls as well as a CRC patient-derived cell line. The untargeted -omics-driven approaches revealed that non-canonical paucimannosidic proteins from monocytic and cancer cell origins are prominent signatures in CRC tumour tissues, and that their expression associates with CRC progression. Guided by these novel relationships, we then show in vitro that N-acetyl-β-D-hexosaminidase (Hex) drives paucimannosidic protein biosynthesis in CRC cells, and is intimately involved in processes underpinning CRC metastasis (adhesion, migration, invasion). Importantly, Hex activity was elevated in PBMCs and plasma from patients with advanced CRC relative to those with early-stage disease. Notably, we show that plasma Hex activity accurately informs on CRC patient survival. Our study opens new avenues for effective prognostication and therapeutic intervention in CRC.
Project description:The PI3Kalpha-specific inhibitor Alpelisib (BYL719) has been approved for the treatment of metastatic ER+/HER2- breast cancer patients in combination with Fulvestrant. After initial response, patients develop drug resistance and disease relapses. In order to identify signalling pathways contributing to the acquired resistance to BYL719 in breast cancer, we generated BYL719-resistant T47D cells and used them together with the parental cells to perform label-free quantitative phosphoproteomics.
Project description:Oncocytic thyroid cancer is characterized by the aberrant accumulation of abnormal mitochondria in the cytoplasm and a defect in oxidative phosphorylation. We performed metabolomics analysis to compare metabolic reprogramming among the oncocytic and non-oncocytic thyroid cancer cell lines XTC.UC1 and TPC1, respectively, and a normal thyroid cell line Nthy-ori 3-1. We found that although XTC.UC1 cells exhibit higher glucose uptake than TPC1 cells, the glycolytic intermediates are not only utilized to generate end-products of glycolysis, but also diverted to branching pathways such as lipid metabolism and the serine synthesis pathway. Glutamine is preferentially used to produce glutathione to reduce oxidative stress in XTC.UC1 cells, rather than to generate α-ketoglutarate for anaplerotic flux into the TCA cycle. Thus, growth, survival and redox homeostasis of XTC.UC1 cells rely more on both glucose and glutamine than do TPC1 cells. Furthermore, XTC.UC1 cells contained higher amounts of intracellular amino acids which is due to higher expression of the amino acid transporter ASCT2 and enhanced autophagy, thus providing the building blocks for macromolecules and energy production. These metabolic alterations are required for oncocytic cancer cells to compensate their defective mitochondrial function and to alleviate excess oxidative stress.
Project description:Introducing a clinical-practical, alternative splicing activity-based proteogenomic method that identifies, in their oncogenically active states, biomarker genes bearing patient-specific GE or copy-number alterations of prognostic significance. This integrated multi-omics method uses intronic splicing enhancers (ISEs) probes to sort in situ ISE-interacting trans-acting protein factors (trans-interactome) directly from a heterogeneous tumor for subsequent mass spectrometry (MS) characterization.
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:HAX1 is a 32-kDa protein of unknown structure, involved in the regulation of apoptosis, cell migration and calcium homeostasis. It was also shown to bind mRNA. Scarcity of structural elements and the presence of a disordered region, inferred from HAX1 sequence, suggests that HAX1 is intrinsically disordered, and may have many protein-protein interactions. So far about 40 different proteins were characterized as HAX1 protein partners. In the present work, applying immunoaffinity chromatography coupled with mass spectrometry, we identified new candidates for HAX1 binding partners in cervical carcinoma cells. More than a half of these newly identified proteins represent RNA-binding proteins. There is also a big group of proteins, mostly mitochondrial, involved in cellular metabolism. These results imply that HAX1 has more protein partners than hitherto described. Subsequent analysis of these interactions may shed some light into molecular mechanisms of HAX1 functions, especially its involvement in mRNA processing and its role in protection against oxidative stress.
Project description:TTF-1/NKX2-1 was expressed by adenoviral vector and changes in gene expression were determined by RNA-sequencing. A549 cells were infected with Ad-TTF-1 or Ad-LacZ vectors and stimulated with TGF-beta for 24 hours or left untreated. Expression of polyA RNA was determined.
Project description:The MCF7 cell line represents a typical epithelial cell line and corresponds to luminal A breast cancer (estrogen-responsive). Overexpression of HAX1 was demonstrated in MCF7 cell line as well as in breast cancer samples, suggesting a role of HAX1 in breast cancer progression. HAX1 is a 32-kDa protein of unknown structure, involved in the regulation of apoptosis, cell migration and calcium homeostasis. It was also shown to bind mRNA. Scarcity of structural elements and the presence of a disordered region, inferred from HAX1 sequence, suggests that HAX1 is intrinsically disordered, and may have many protein-protein interactions. So far about 40 different proteins were characterized as HAX1 protein partners. In the present work, applying immunoaffinity chromatography coupled with mass spectrometry, we identified new candidates for HAX1 binding partners in breast cancer cells. Newly identified proteins may be divided into three, partially overlapping groups: cytoskeleton-associated proteins, GTP-ase associated proteins and RNA-binding proteins. These results imply that HAX1 has more protein partners than hitherto described. Subsequent analysis of these interactions may shed some light into molecular mechanisms of HAX1 functions.
Project description:This ArrayExpress record contains meta-data and results of quantitative analysis of cell lines from the NCI-60 panel using pressure cycling technology (PCT) and SWATH-mass spectrometry. Each cell line was analyzed in duplicate. Raw data files are available at the EMBL-EBI protemics data archive (PRIDE) at accession PXD003539 (http://www.ebi.ac.uk/pride/archive/projects/PXD003539). Since the record here does not include the raw data files and hence there is no need to explicitly link individual replicate to a raw file, each sample is only listed once in the ArrayExpress samples table for clarity.
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette. Cells were selected by G418 and used to prepare ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of G418-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016).