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   NPC T1 Cancer Proteomics

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Theme leaders: Geert Kops (UMCU) & Jeroen Demmers (Erasmus MC)

Background

The Cancer Proteomics theme is a collaboration with the Cancer Genomics Centre of NGI and will study various aspect of cancer biology, including a comparison between tumor proteomes, the analysis of protein complexes and protein modifications

Proteomics of breast and lung cancer (Foekens, Philipsen, Grosveld and Demmers). We will focus on breast- and lung cancer, the most frequent cause of cancer deaths in the Netherlands. Evidently, identification of appropriate protein targets for these groups of patients is of vital importance. We therefore aim to find potential drug targets and markers of tumor aggressiveness by performing comparative proteomics as well as kinase activity profiling.  We will develop heavy-chain only antibodies against these targets, for use in diagnostic, prognostic and possibly therapeutic approaches. Ubiquitin modification in DNA damage repair (Hoeijmakers, Demmers). Post-translational protein modifications with ubiquitin play a key role in regulating the linked DNA damage repair process. To unravel the role of the ubiquitin-proteasome-system during DNA-repair we will determine which proteins are modified with ubiquitin during the UV-induced DNA damage response, and study their function. Chromatin dynamics and epigenetic changes (Van Leeuwen, Krijgsveld). A novel procedure, site-specific Recombination-induced Epitope Tag Alteration to mark New and Existing proteins (RETANE), allows the isolation of old and newly formed proteins. This procedure will be used to study chromatin dynamics to understand epigenetic changes. Proteomics of signal transduction and cell cycle (Bos, Clevers, Kops, Heck). Defects in signal transduction networks play an important role in cancer development. We will study these networks in various systems.  We will analyse protein complexes that associate with Tcf4, with axin and with beta-catenin in the nucleus and cytoplasm of colon cancer cells before and after removal of beta-catenin by siRNA, and of crypts and villi freshly isolated from mice to unravel the unresolved critical switch in Wnt signalling. We will isolate supramolecular complexes, like focal adhesions and junction contact points under various conditions and will analyse them by mass spectrometry. Finally, we will study Mps1 signalling to control the spindle checkpoint. Mps1 is currently a very promising target for tumor intervention.

Approach

Proteomics of breast and lung cancer. Since most, if not all, of the proteins identified will not be tumor-specific, a quantitative rather than aqualitative approach is prudent. We will apply enzymatic O18 labelling, a post-processing labelling procedure for quantitative comparison of tumour and healthy samples. Heavy chain-only antibodies will be generated in proprietary transgenic mice and characterized on BiaCore equipment (Grosveld, Demmers). Kinase profiling wil be performed on PamChip® 256 arrays (Foekens, Sieuwerts, Pamgene). Ubiquitin modification in DNA damage repair will be studied using dedicated cell labelling procedures with tagged ubiquitin (Hoeijmakers, Demmers). A wide range of relevant DNA repair mutant cell lines and animal models is available for functional analysis (Hoeijmaker, Vermeulen, Lans). Chromatin dynamics and epigenetic changes will be studied in yeast. ”old” and “new” proteins will be labelled and isolated and will be subject to MS analysis for protein modifications
Proteomics of signal transduction and cell cycle. Protein complexes will be isolated and analysed for interacting proteins and for protein modifications. For quantitatative analysis we will use either Silac, or invitro O18 labeling. Complexes will be isolated prior to and after stimulation tomonitor dynamics. Results will be evaluated by immunofluorescence for co-localization and siRNA for biological relevance for the processes under study in the matching projects.

Deliverables

  • Lung tumor membrane protein signatures that will be used to select diagnostic, prognostic and –in the longer term- possibly even therapeutic applications
  • A proteome profile of breast tumors
  • A list of modifications of repair proteins and their function
  • A differential profile of chromatin modifications from old and new protein
  • A list and functional analysis of interacting proteins and protein modifications involved in Wnt signaling
  • A list and functional analysis of interacting proteins and protein modifications involved in Rap1 signaling
  • A list and functional analysis of interacting proteins and protein modifications involved in Mps1 signaling