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  • 1
    Keywords: SACCHAROMYCES-CEREVISIAE ; CELL-CYCLE ; YEAST ; PLASMA-MEMBRANE ; DEGRADATION ; NUCLEAR-PORE COMPLEX ; Half-Life ; UBIQUITIN LIGASES ; MONOMERIC RED ; END RULE PATHWAY
    Abstract: The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule-mediated protein degradation.Systematic monitoring of proteome dynamics would require simultaneous measurement of protein turnover and subcellular trafficking at the single-cell and population scales. The importance of protein turnover was introduced in 1942 by Schonheimer, who noted that "all constituents of living matter, whether functional or structural, of simple or of complex constitution, are in a steady state of rapid flux". Protein homeostasis is now understood as a balance between protein synthesis, through transcription and translation, and protein degradation, through processes such as proteasomal and lysosomal degradation, tuned in response to intrinsic and extrinsic inputs. Alterations in protein turnover are observed in aging organisms and underlie various diseases. Deregulated degradation of cell cycle control proteins such as the p53 tumor suppressor plays a critical role in many forms of human cancers. Abnormal trafficking and degradation of a mutant form of a chloride ion channel causes cystic fibrosis. Moreover, accumulation of specific proteins is linked to neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Therefore, understanding protein turnover and mobility could provide new strategies for targeted clinical interference to treat such diseases.
    Type of Publication: Journal article published
    PubMed ID: 22729030
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  • 2
    Keywords: TUMOR-CELLS ; p53 ; DNA-DAMAGE ; TUMORIGENESIS ; medulloblastoma ; REARRANGEMENTS ; ANEUPLOIDY ; CONSEQUENCES ; CANCER GENOMES ; PAIRED-END
    Abstract: A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed "complex alterations after selection and transformation (CAST)," enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.
    Type of Publication: Journal article published
    PubMed ID: 26415501
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  • 3
    Abstract: Chromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with Kras(G12D) or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.
    Type of Publication: Journal article published
    PubMed ID: 27292643
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  • 4
    Abstract: Patterns of gene expression in tumors can arise as a consequence of or result in genomic instability, characterized by the accumulation of somatic copy number alterations (SCNAs) and point mutations (PMs). Expression signatures have been widely used as markers for genomic instability, and both SCNAs and PMs could be thought to associate with distinct signatures given their different formation mechanisms. Here we test this notion by systematically investigating SCNA, PM, and transcriptome data from 2660 cancer patients representing 11 tumor types. Notably, our data indicate that similar expression signatures can be derived from correlating gene expression with either SCNA or PM load. Gene sets related to cell growth and proliferation generally associated positively, and immunoregulatory gene sets negatively, with variant burden. In-depth analyses revealed several genes whose de-regulation correlates with SCNA but not with PM burden, yielding downstream effectors of TP53 and MYC signaling unique to high-SCNA tumors. We compared our findings to expression changes observed in two different cancer mouse models with persistent mitotic chromosomal instability, observing a decrease in proliferative expression signatures. Our results suggest that overexpression of cell-cycle-related genes are a characteristic of proliferation, and likely tumor evolution, rather than ongoing genomic instability.
    Type of Publication: Journal article published
    PubMed ID: 28320919
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  • 5
    Abstract: BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and mole
    Type of Publication: Journal article published
    PubMed ID: 29753700
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  • 6
    Keywords: RECEPTOR ; CANCER ; TIME ; mechanisms ; IDENTIFICATION ; LOCALIZATION ; AKT ; SPINDLE ; SMALL-MOLECULE INHIBITOR ; KINESIN EG5
    Abstract: Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.
    Type of Publication: Journal article published
    PubMed ID: 23643362
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