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Voditelj |
Prof.dr.sc. Hrvoje Banfić, Medicinski fakultet Sveučilišta u Zagrebu
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Institucije partneri |
Fred Hutchinson Cancer Research Center, Seattle, WA, USA
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Trajanje projekta |
3 godine
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SAŽETAK PROJEKTA
Signaling pathways involving phosphoinositide 3-kinase (PI-3K) and phosphoinositide (PI) cycle are the most frequently targeted signaling pathways in human cancer. In addition to the signaling events at the cell membrane, the PI cycle is known to occur in nuclei and to regulate important nuclear events, including transcription, mRNA export and telomere length. The major progress in understanding the functional role of the nuclear phospholipid pathway has been recently made by genetic and biochemical studies in the budding yeast Sacharomyces cerevisiae. The aim of this proposal is to develop cell-based assays using heterologous expression of kinases involved in the endonuclear phospholipid signaling in the unicellular eukaryote, S. cerevisiae. In addition, yeast model will be used to dissect the role of particular kinases, especially phospholipase C and PI-3Ks, in regulation of principal and evolutionary conserved nuclear processes. The main hypothesis is that the expression of human proteins in yeasts will induce growth interference and thus provide a yeast cell-based assay to positively screen pharmaceutical agents. The ultimate goal is to be able to identify new, selective small molecule inhibitors of the phospholipid signaling pathway that should provide new opportunities for mechanism-based anticancer therapies. This goal will be achieved by collaborative efforts of two experienced scientists; the project leader from Croatia, who is an expert in the field of the nuclear phospholipid signaling, and Croatian co-applicant from USA, who is an expert in yeast genetics with great experience in using yeast-based positive screen for anticancer drug discovery.
ZNANSTVENA PODLOGA PROJEKTA
In the past 20 years, several studies confirmed the existence of nuclear phosphoinositide (PI) cycle which occurs independently from the classical pathway at the cell membrane (1). The nuclear PtdIns(4,5)P2 serves as a substrate for PI-phospholipase C mediated hydrolysis and phosphoinositide 3-kinase (PI-3K) -mediated phosphorylation. Although an increase in the level of diacylglycerol (DAG), Ins(1,4,5)P3 or 3-phosphorylated PtdIns has been shown to occur in nuclei in response to external stimuli or during synchronous progression through the cell cycle, there is a paucity of data on how these nuclear messengers regulate nuclear processes and for some of them, like Ins(1,4,5)P3, there are no proofs that they play the same role in the regulation of the calcium level as the classical one at the cell membrane. The major progress in understanding the functional role of the pathway has been recently made by genetic and biochemical studies in the budding yeast Sacharomyces cerevisiae. Yeasts have enzymes to generate PtdIns(4,5)P2 within the nucleus and have a nuclear PI-PLC, which can generate DAG and Ins(1,4,5)P3, the later serving as a precursor for the synthesis of higher inositol phosphates that have been convincingly proved to regulate such as important nuclear events like mRNA export (2), transcription (3) and telomere length (4). However, yeasts lack some elements of the „classical“ PI signaling as they have no Ins(1,4,5)P3 receptor in their genome and do not utilize DAG to activate protein kinase C suggesting that there are many similarities between yeast and endonuclear phospholipid signaling and that the evolutionary standpoint can be informative. Several years ago, when a possible role for the nuclear PI-PLC in nuclear envelope assembly and cell cycle emerged, one of the hypotheses suggested that the nucleus may have been the site at which phosphoinositide signaling originally evolved and that the cycle was later duplicated in the plasma membrane for the signaling purpose (5). The conservation of kinases responsible for the generation of inositol phosphates from yeast to man suggests that the primordial role of the pathway may be the regulation of some important nuclear processes, thus making the components of the endonuclear pathway an attractive future candidates for drug design.
In mammals, there are several different PI-PLC isoforms that are grouped into 6 families (-?,-?,-?,-?,-? and -?); the one that is the most consistently reported to be activated in nuclei is PI-PLC-?1. Our previous work demonstrated cyclic, MAPK-mediated activation of b splice variant of PI-PLC-?? in nuclei of mammalian cells which was important for the progression through the cell cycle (6). A member of PI-PLC subfamily delta is the only one present in all eukaryotes (including Plc1p encoded by a PLC1 in yeasts), nucleocytoplasmic shuttling of PLC-? has been confirmed in mammalian cells, and our previous study show an increase in the activity of PLC-??that was localized in the chromatin fraction of the nuclei (7). In the yeasts models, colocalization of PI-PLC-?? (i.e. Plcp1) with chromatin has been described and suggested to have a role in kinetochore function and PLC deleted cells were demonstrated to exhibit a delay at the G2/M phase of the cell cycle (8). Our preliminary data in human cells show that an increase in the PLC-activity at late G1 is partially due to the presence of immunoprecipitated PLC-?.
PI-3K pathway has been recently demonstrated to be the most frequently targeted signaling pathway in human cancer (9). PI-3Ks phosphorylate phosphoinositides at the 3' position of the inositol ring and include several isoforms that are divided into three classes based on their sequence homology, substrate preference and mechanism of activation. The best studied and the most related to cancer are class I enzymes that are complex heterodimers responsible for phosphorylation of PtdIns(4,5)P2 into PtdIns(3,4,5)P3. The class I enzymes are absent in S. cerevisiae and PtdIns(3,4,5)P3 seems to have no role in the physiology of budding yeasts. The only PI-3K in yeasts is Vps 34 which regulates vesicle trafficking; the enzyme is homologous to the mammalian class III enzyme that phosphorylates PtdIns to produce PtdIns(3)P. The least is known about physiological role and mode of regulation of mammalian class II enzymes which include three isoforms (PI-3K-C2?, -ß and -?) and show preference for PtdIns and PtdIns(4)P as substrate in vitro but, similar to Vps34, produces only PtdIns(3)P in vivo. Growth factors activate class II enzymes, and one of the isoform, PI-3K-C2? has been recently reported to regulate the migration and survival of human tumor cells (10). Our previous work demonstrated the increase in the activity of the PI-3K-C2? immunoprecipitated from the nuclei of proliferating hepatocytes and synchronized leukemia cells that was due to the calpain-mediated proteolysis and nuclear translocation of the enzyme (11,12).
The investigation of the physiological role of the particular PLC ands PI-3K isoform as possible drug-target in vivo is greatly hampered by the lack of isoform-specific small molecule inhibitors. The inhibitors used to study the role of PI-PLCs, like ET-18-OCH3, neomycin or similar compounds are highly non-specific. One of two inhibitors that are widely used to inhibit PI-3Ks, wortmannin, has broad activity within the family including the members of the PI-3K-related enzymes, such as ATR, ATM, DNA-PK and mTOR. On the other hand, class III enzymes are highly resistant to more specific inhibition by LY 294002 and class II are much less sensitive than class I enzymes. Although recent biochemical screen of several compounds identified new selective inhibitors for some class I isoforms, no promising inhibitors of class II enzymes (or PI-3K-C2?) were identified (13)
Cell-based assays designed in budding yeasts (S. cerevisiae) have recently emerged as a useful model system for anticancer drug discovery (14). In addition to possibility to express mammalian proteins with yeast homologues which may functionally complementate yeast mutations, yeast-based phenotypic screens can be designed for targets that have no yeast homologues. Human PI-3K class I and some other proteins linked with accelerated growth in human cancers block growth when expressed in yeasts and the growth of strains overexpressing PI-3K can be restored through the expression of the phosphatase PTEN, which antagonizes PI-3K action (15). This provide the basis for testing putative selective inhibitors in a positive-selection screen in which the outcome of the successful inhibition is the stimulation of the proliferation which is far more specific than negative-screen in which the effect of the selective inhibition can not be easily distinguished from a non-specific toxic effect (16).
CILJEVI I ZNAČENJE PROJEKTA
This is a proposal to develop cell-based assays using heterologous expression of phospholipases and kinases involved in the endonuclear phospholipid signaling in the unicellular eukaryote, S. cerevisiae (yeast). In addition, signaling mechanisms found to operate in the nuclei of mammalian cells will be tested in yeast models synchronously progressing through the cell cycle. These assays should provide valuable tool to dissect the role of particular enzymes, especially PLC and PI-3K isoforms, in regulation of principal and evolutionary conserved nuclear processes. The working hypothesis is that the level of phosphoinositides and inositol phosphates will change in yeast during the cell cycle and that the expression of heterologous proteins will induce growth interference and thus provide a cell-based assay to positively screen pharmaceutical agents. The ultimate goal is to be able to identify small molecule inhibitors that selectively influence the pathway. The project leader has a long-term research interest in the field of the nuclear phospholipid signaling, particularly biochemistry of phosphoinositides and the nuclear PI-PLC-?1 and PI-3K-C2?. Co-applicant is an expert in yeast genetics, particularly in chromatin and telomere biology and DNA replication checkpoints, who has successfully used yeast-based positive screen to identify new selective inhibitors of Sir2p chromatin modifying enzymes and applied these compounds as anticancer drugs (14,16).
