Michele Pagano - US grants

DESCRIPTION: (adapted from the investigator's abstract) Cyclin-dependent kinases (Cdks) form a family of enzymes that coordinate the cell division cycle. The periodic activation of cdks requires their association with proteins called cyclins, and their dissociation from inhibitory subunits, called Ckis. Ubiquitin-proteasome-mediated proteolysis is a major mechanism by which the protein levels of cell cycle regulators are regulated in response to mitogenic and anti-mitogenic stimuli. They have previously shown that in normal diploid cells protein levels of the cki p27 are mainly regulated by degradation via the ubiquitin-proteasome system. Similarly, degradation of other G1 regulatory proteins (Cyclin E, Cyclin D1, E2F-1) is controlled by the ubiquitin-pathway. Over-activation of positive cell cycle regulators (e.g., cyclins, E2F-1, Cdc25) and inactivation of Ckis (e.g., p27, p16) play a significant role in oncogenesis. These changes inactivity can occur through mutation, or transcriptional silencing of the corresponding gene, or through changes in protein stability. Indeed, although p27 gene has never been found altered in human tumors, they have recently observed that aggressive human colorectal carcinomas have reduced expression of p27 and enhanced proteolytic activity specific for p27. To determine whether the abnormal expression of cell cycle regulators represents novel prognostic markers for human breast cancer, they will use immunohistochemistry and in situ hybridization to analyze the expression of cell cycle regulators (p27, Cyclin D1, Cyclin E, E2F-1, and Cdc25 B) and their mRNA in archival sections from approximately six hundred human breast cancers (Specific Aim 1). These tumor samples, obtained from three different tumor banks, have concordant normal tissues and a long term follow-up. Furthermore, each tumor bank has an extensive data base containing information on clinico-pathological features and standard molecular markers, allowing them to determine the prognostic value of cell cycle regulators. They will focus the study on breast carcinomas 1 cm in size, and tumors obtained from pre-menopausal women. They hypothesize that aggressive tumors have enhanced degradation of p27 and other cell cycle inhibitors and decreased degradation of cell cycle activators, thus acquiring a distinct growth advantage. To test whether deregulated proteolysis of cell cycle regulatory proteins is responsible for their abnormal expression in breast carcinomas, they will analyze tumor breast samples for the abundance of specific degradation activities and for the levels of various ubiquitin conjugating enzymes (Ubcs) involved in cell cycle regulation (Specific Aim 2).

Cyclin-dependent kinases (cdks) form a family of enzymes that coordinate the cell division cycle. The periodic activation of cdks requires their association with proteins called cyclins, and their dissociation from inhibitory subunits, called ckis. Ubiquitin-mediated proteolysis is a major mechanism by which the protein levels of both cyclins and ckis are regulated in response to mitogenic and anti-mitogenic stimuli. Alterations in cdk regulation have been shown to result in abnormal cell growth associated with cancer. Recent evidence indicate the abnormal degradation of cell cycle proteins is associated with oncogenic events. Thus, the identification of the enzymes that regulate the degradation of ckis and cyclins will have a large impact on both basic research and cancer biology. We have previously shown that the intracellular level of the cki p27 is mainly regulated by degradation and that the ubiquitin system controls p27 degradation. Similarly, degradation of other G1 regulatory proteins (Cyclin E, Cyclin D1, p21, E2F-1, E2F-4) is controlled by the ubiquitin- pathway. Yet, the specific enzymes involved in the degradation of G1 regulatory proteins have not been identified. With this study we propose to characterize the human homologs of two yeast proteins, namely SKP-1 and CDC53, which are components of the ubiquitin pathway regulating the G1 phase of the yeast cell cycle (Specific Aim 1). Under Aim 1, we will also clone and characterize three new proteins that we have found associated specifically with Skp-1 in vivo. We will then investigate the in vivo function of human Skp-1, Cdc53, and Skp-1 associated proteins (Specific Aim 2). Finally, we will test whether human Skp-1 and Cdc53 control the levels of known G1 regulators (Specific Aim 3).

DESCRIPTION: (provided by applicant) Proteolysis of many cellular regulators is controlled by ubiquitin ligases called SCFs since they are composed of three major subunits: Skp1, cull and one of many E-box proteins (Fbps). The substrate specificity of SCFs is determined by distinct Fbp subunits that act as substrate recognition factors. We have demonstrated that the cell cycle inhibitor p27 is degraded by the ubiquitin-pathway through the F-box protein Skp2. Importantly, destabilization of p27, which we and others have documented in human lymphomas and in epithelial cancers, correlates with tumor aggressivity. Our interest in the ubiquitin pathway lead us to the identification of a family of 26 human F-box proteins and the discovery that one member of this family, called 13-Trcp, regulates the stability of the proto-oncogene B-catenin. Because of their functions in regulating cell proliferation and given our preliminary results, we hypothesize that deregulation of the F-box proteins Skp2 and B-Trcp may participate in the genesis of human cancers. In Aim 1 we will carry out immunohistochemical studies on Skp2 and B-Trcp expression in cancer samples obtained from two different tumor banks. We will concentrate our study on breast cancer, lymphoma and colorectal cancer. Our collection of tumor samples has concordant normal tissue and long-term follow-ups. In addition, these two tumor banks have extensive databases containing information on clinico-pathological features and standard molecular markers. Using univariate and multivariate analyses, we will determine the value of Skp2 and B-Trcp as novel prognostic markers for human cancers. Importantly, we will investigate possible causes for increased levels of these two F-box proteins and perform in vitro assays to measure their activities. In Aim 2 we will then generate and characterize transgenic mice overexpressing Skp2, B-Trcp or corresponding dominant-negative mutants. Using this approach we will be able to investigate the effects of enforced expression of these Fbps in the mammary gland and lymphoid organs. Histopathological alterations expected in transgenic animal models will mimic those human tumors in which Skp2 and B-Trcp are overexpressed and will help us to define oncogenic pathways underlying the molecular abnormalities occurring in human tumors. Finally, in Aim 3 we will test whether the inhibition of Skp2 results in a cytostatic or cytotoxic effect in cancer cells. This will be achieved using specific membrane permeable peptides designed on the basis of our structural and biochemical information.

DESCRIPTION (provided by applicant): SCF ubiquitin ligases complexes, composed of three major subunits, Skp1, Cull, and an F-box protein (Fbp), regulate the proteolysis of many regulators of cell proliferation The substrate specificity of SCFs is determined by distinct Fbp subunits that act as substrate recognition factors. It was demonstrated by the principal investigator that the F-box protein Skp2 is a rate-limiting component of the machinery that ubiquitinylates the cell cycle inhibitor p27, and that Skp2 requires its physical association to Cksl to bind p27 at high affinity. The principal investigator's interest in the ubiquitin pathway lead to the identification of a family of 26 human F-box proteins and to the discovery that one member of this family, called beta-Trcp, regulates the stability of the proto-oncogene beta-catenin. In Aim 1 the regulation and the role of SCFskp2 and Cks1 in the control of the mammalian cell division cycle will be studied. In particular, the function of Skp2 phosphorylation on Ser-76 and the role of an alternatively spliced Skp2, recently identified by the principal investigator, will be studied. In Aim 2, a mutant mouse in which the beta-Trcp locus has been inactivated using homologous recombination will be studied. Using this approach the principal investigator will determine the function of beta-Trcp in growth and development as well as its role in specific tissues and organs. The principal investigator will study how SCF-beta-Trcp regulates processes such as cellular proliferation and the immune response. The principal investigator will be able to determine the in vivo effects of lack of beta-Trcp on its putative substrates. Finally, in Aim 3 the principal investigator will investigate the function of another member of the newly identified human Fbp family, namely Fbl3, which he has found to be localized on the centrosome. Under Aim 3, the principal investigator will also clone and characterize three proteins that he has found associated specifically with Fb13 in vivo.

Akin to a complex engineered system, biological processes operate through many simultaneous interactions within complex networks. Traditionally, biologists have constructed "models" to capture this complexity and verify their intuitions as well as communicate how a particular biological system or subsystem actually works. To build these models, biologists rely on a very general and broad array of knowledge, and also augment it with depth and expertise obtained from small number of exemplar systems. With availability of large amount of high-throughput experimental data, biologists are also faced with the task of reconstructing models from data where relevant information may be deeply buried in layers of numerical information.
Biological models are often presented pictorially as graphs and flow charts with many components, each corresponding to a certain biochemical reaction. Such diagrams have also, but not always, been associated with mathematical models, mostly in the form of differential equations. The equations are used to perform simulations of the system, when they have a well-defined set of kinetic parameters. The model is refuted or validated depending on whether the simulated traces agree with biological data.

Often one is faced with situations, where there is no mathematical model, or the model is incomplete and they lack a complete set of parameters, and yet biologists do have detailed descriptive understanding of many of the components and their interactions. For instance, current microarray data analysis techniques draw the biologist's attention to targeted sets of genes but do not otherwise present global and dynamic perspectives (e.g., invariants) inferred collectively over a dataset. When ontologically invariants are inferred from experiments (using GOALIE redescription tool), such invariants can be compared with the known descriptive information to determine if we have complete and consistent theories about certain biological processes.
This project addresses these two scenarios by providing automated reasoning tools that bridge both computational and descriptive models in biology. The results from these tools and experimental analyses hint at the construction of efficiently testable predictions. The results of wet-lab experiments are then used to refine and amend the formal model. This feedback cycle between modeling and experimentation has proven important in obtaining a process-level understanding of the underlying cellular machinery.

The further characterization of specific parts of the mammalian cell cycle behavior (e.g. how a possibly unknown factor may allow the phosphorlyzation Cdk inhibitor p27 by Cdk2 at G1/S.)
In the longer run, understanding the wider implications of the complex regulatory and metabolic architecture of the cell cycle will provide significant insights into new applications of biology and advanced computing. In addition, they will provide new perspectives on computing by exploiting biologically driven metaphors. More importantly, the approaches developed in the context of hybrid-system (HS) models and bio-ontology will find applications to swarm robotics, social-software, e-commerce, complex interactive engineered systems, computer-security, adaptive software, etc., although from our own historical perspective, we will remain engaged in proving the first successes of this approach in biomedical applications.

[unreadable] DESCRIPTION (provided by applicant): Ubiquitin-dependent proteolysis ensures that specific protein functions are turned off at the right time, in the right place and in a unidirectional fashion. The degradation of many cell cycle regulatory proteins is controlled by two classes of ubiquitin ligases: the SCF (Skp1-Cull-F-box protein) complexes and the Anaphase Promoting Complex/Cyclosome (APC/C). In humans there are sixty-eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin conjugating enzyme. Despite the large number of F-box proteins, only three human SCF ubiquitin ligases (SCFskp2, SCF?Trcp and SCFFbw7) have well- established functions and substrates, many of which are involved in cell cycle control (e.g., Cdc25A, cyclin E, Emi1, p21, p27 and Wee1). Given the crucial function of the cell cycle machinery, altered proteolysis of cell cycle regulators is clearly a contributing determinant of the unrestrained proliferation typical of cancer cells. Significantly, of the three characterized F-box proteins, Skp2 is the product of a proto-oncogene, Fbw7 is a tumor suppressor, and overexpression of ?Trcp contributes to transformation, at least in certain epithelial tissues. During the first nine years, CA76584 supported the elucidation of the molecular and cellular mechanisms by which three ubiquitin ligase complexes (SCFSkp2, SCF?Trcp and APC/CCdh1) control cell cycle progression through the degradation of cancer-relevant substrates that regulate the activity of CDKs. Furthermore, the corruption of these pathways occurring in cancer was revealed. Novel preliminary studies show that high levels of the F-box proteins Emi1 and Emi2 correlate with an increased stability of Skp2 in human cancers, and suggest that Emi1 and Emi2 function as oncoproteins. Based on these results, the new aims proposed under the third cycle of CA76584 are focused on a new tier of deregulation of the ubiquitin ligase/cell cycle network and its involvement in cancer: To determine whether the expression of Emil and Emi2 is deregulated in tumors and to investigate the mechanisms deregulating Skp2 stability in cancer cells (Aim 1); To study the contribution of Emi2 to cancer development using tissue culture systems and in vivo experiments (Aim 2); To study the cell cycle functions of Emi2 in cancer cells and to identify its biologically significant substrates (Aim 3). As the mechanisms of the ubiquitin-mediated proteolysis of cell cycle regulators are unraveled, this team is committed to the integration of its basic research results with an understanding of malignant transformation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Temporally coordinated destruction of key cell cycle regulatory proteins by the ubiquitin-proteasome system represents an important regulatory mechanism to ensure that specific protein functions are turned off at the right time, in the right compartment and in a unidirectional fashion. Proteolysis of many core components of the cell cycle machinery is controlled by two major classes of ubiquitin ligases: the SCF (Skp1-Cul1-F-box protein) complexes and the Anaphase Promoting Complex/Cyclosome (APC/C). In humans there are sixty- eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin conjugating enzyme. Despite the large number of F-box proteins, only three human SCF ubiquitin ligases (containing the F-box proteins betaTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control (e.g., Cdc25A, cyclin E, Emi1, p21, p27, Wee1). We propose a project focused on a new tier of control of the cell cycle networks and its integration with the ubiquitin system. Using a novel screen, we have identified six novel putative SCF substrates, and we will characterize the mechanism, regulation and biological function of the degradation of one of them, namely E2F3, a protein intimately involved in the control of the cell cycle (Aim 1). We will furthermore identify and characterize those biologically significant substrates that are targeted for destruction by the F-box protein Fbw5 to regulate cell cycle progression (Aim 2). Finally, we will study the role of betaTrcp in controlling the degradation of a novel substrate identified using a biochemical screen: Claspin, a protein that is part of the DMA replication surveillance machinery (Aim 3). Given the crucial function of the cell cycle machinery, altered degradation of cell cycle regulatory proteins is clearly a contributing determinant of the unrestrained proliferation typical of cancer cells. As we continue to unravel the mechanisms of how the scheduled degradation of regulatory proteins by the ubiquitin system controls cellular proliferation, we are committed to the integration of our basic research results with an understanding of malignant transformation. It is anticipated that the results of our studies will have an impact on both basic science and cancer biology.

[unreadable] DESCRIPTION (provided by applicant): The ubiquitin system is a major regulatory mechanism of cellular processes in which speed, specificity, and timing are critical. Ubiquitin-mediated proteolysis of key substrates controls cell cycle progression, signal transduction pathways, differentiation, apoptosis, DNA repair and the immune response. This process is mediated by a multimeric machine, composed of a regulatory ubiquitin-targeting component and an effector protein degradation engine. The regulatory component, which targets ubiquitin to proteins destined for degradation, is itself composed of several multimeric elements (e.g., the SCF ubiquitin ligase complexes) that contribute much of the specificity inherent in the process. In humans, there are sixty-eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin-conjugating enzyme. Notably, only three out of 68 human SCF ubiquitin ligases (containing the F-box proteins UTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control. The remaining 65 F-box proteins are considered as "orphan" since their substrates still await discovery. We have recently developed a novel immunopurification strategy that enriches for substrates of F-box proteins followed by mass spectrometry analysis. We will systematically identify biologically significant substrates of human orphan F-box proteins (Specific Aim 1). Because of our research interest, we will focus particularly on those orphan F-box proteins that our preliminary results suggest to be involved in cell cycle control and cancer. Under Aim 2, we will validate the biologically most significant substrates identified under Aim 1. Given their critical role in regulating cell proliferation, SCF ligases are often the target of cancer- related deregulation and involved in oncogenic transformation. Therefore, the information gained from the proposed studies will be of direct relevance to cancer biology and other proliferative diseases. [unreadable] [unreadable] [unreadable]

Animal Model; Animal Models and Related Studies; Anti-Oncogenes; Antioncogenes; Area; Cancers; Clinical; Commit; Development; Emerogenes; Enrollment; Exertion; Faculty; Fostering; Funding; Gene Transcription; Generalized Growth; Genes; Genes, Cancer Suppressor; Genes, Onco-Suppressor; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Goals; Grant; Growth; Human; Human, General; Institutes; Investigator-Initiated Research; Investigators; Knowledge; Malignant; Malignant - descriptor; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Molecular; Mutation; Neoplasms; Oncogenes, Recessive; Oncogenes-Tumor Suppressors; Organizations, Peer Review; Pathway interactions; Pattern; Peer Review Organizations; Phase; Programs (PT); Programs [Publication Type]; Publications; RNA Expression; Recommendation; Recruitment Activity; Research; Research Personnel; Researchers; Resource Sharing; Sampling; Scientific Publication; Scientist; Signal Transduction Pathway; Site; Strategic Planning; Structure; Therapeutic; Time; Tissue Growth; Transcription; Transcription, Genetic; Translational Research; Translational Research Enterprise; Translational Science; Tumor Cell; Tumor Suppressing Genes; Tumor Suppressor Genes; Tumors; Work; base; cohesion; design; designing; enroll; experience; genome mutation; instrument; interest; malignancy; member; model organism; neoplasia; neoplasm/cancer; neoplastic cell; neoplastic growth; novel; oncosuppressor gene; ontogeny; pathway; programs; recruit; translation research enterprise; tumor

Unidirectional progression through the cell cycle depends on the specific, rapid, and temporally controlled proteolysis of key regulators by the ubiquitin-proteasome system (UPS). E3 ubiquitin ligases confer substrate specificity to the UPS. Therefore, it is not surprising that alteration of the functions of these enzymes contribute to the development of a wide variety of diseases, including cancer. SCF (Skp1, Cul1, F-box protein) complexes (also known as Cul1-Ring-ligases or CRL1) represent a family of E3 ubiquitin ligases involved in crucial cellular pathways as gene transcription, protein synthesis, cell division, DNA-damage checkpoints, the circadian clock, and apoptosis. F-box proteins play a pivotal role in the SCF complex by functioning as receptors that directly bind to and recruit substrates. Although certain F-box proteins have been characterized, most of them have not yet been matched to their cognate substrates and, therefore, are defined orphan. FBXO11 is an orphan F-box protein involved in regulating cell fate determination, and mutations of the gene encoding this protein have been associated with the onset and progression of pathological conditions both in humans and in animal models. Using unbiased proteomic screens, we have identified Cdt2 as a novel, putative substrate of SCFFBXO11. Cdt2 belongs to the family of DCAF proteins that are the substrate receptors of multi-subunit E3 complexes known as CRL4 (Cul4-Ring- ligase). CRL4Cdt2 controls the degradation of key regulators of proliferation (Cdt1, p21, Set8, etc.) in both normal and cancer cells; however, the mechanisms controlling the degradation of Cdt2 itself have remained unknown. Under the present application, we will study the molecular mechanisms and pathways regulated by both FBXO11 and Cdt2. To this end we will address how, when (Aim1), and why (Aim2) the SCFFBXO11 control the degradation of Cdt2. Taking together, our studies will provide novel insights into the regulatory circuits that control cell proliferation and differentiation. Moreover, they may offer a platform for future studies to explore the role of FBXO11 and Cdt2 in various cellular proliferative disorders. The proposed research is an extension of the NIH grant R37-CA076584 (07/01/1-06/30/2016) and represents a collaborative effort with Dr. Mario Rossi, a Group Leader at the BioMedicine Institute of Buenos Aires-CONICET-Partner Institute of the Max Planck Society in Argentina, the low- to middle-income countries (LMIC) site associated with this application, where the largest part of the project will be carried out under the supervision of Dr. Rossi.

DESCRIPTION (provided by applicant): The ubiquitin-proteasome system is a major regulator of cellular processes in which speed, specificity, and timing are critical (e.g., cell cycle progression, gene transcription, cell differentiation, apoptosis, and signal transduction). Ubiquitin-mediated proteolysis of key substrates is mediated by multiple machines encompassing two functions: ubiquitin-tagging (i.e. ubiquitin conjugating enzymes and ubiquitin ligases) and protein degradation (i.e. the proteasome). Ubiquitin ligases, which contribute to transfer ubiquitin to proteins destined for degradation, are often composed of several subunits (e.g., the SCF ubiquitin ligase complexes) that impart a high degree of precision to the process. In humans, there are 69 SCF ligases, each characterized by invariable, structural elements (Skp1, Cul1, and Rbx1) and a variable F- box protein subunit that provides specificity by directly recruiting the substrate to the core of the ligase. Notably, only 9 of the 69 human SCF ligases (containing the F-box proteins 2TrCP1, 2TrCP2, Fbxw7, Skp2, Fbxl3, Fbxl5, Fbxo1, Fbxo4, and Fbxo6) have well-established/accepted substrates and functions. The remaining 60 F-box proteins are considered orphans, and their substrates still await discovery. Fbxo15 is an orphan F-box protein that our preliminary data and published findings suggest to be involved in the control of stem cell self-renewal and differentiation. Our laboratory has successfully utilized two techniques for unbiased identification of F-box protein substrates. Using traditional tandem affinity purifications, we have identified novel SCF substrates, but we also developed a novel immunoaffinity/enzymatic assay that enriches for ubiquitylated substrates based on the ability of SCF complexes to ubiquitylate co-purified substrates in vitro. Based on our previous success in identifying and characterizing F-box protein substrates, we propose the following two aims. We will identify biologically significant substrates of human Fbxo15 (Specific Aim 1) and validate the Fbxo15-dependent regulation of these substrates (Specific Aim 2).

DESCRIPTION (provided by applicant): Regulated intracellular proteolysis controls virtually every aspect of cell physiology. The process is highly selective, with individual protein half-live ranging from minutes to weeks. This selectivity is ensured by specific interactions between substrates and E3 ubiquitin ligase complexes, which mark proteins for degradation with a chain of ubiquitin moieties. Many E3 ligases are modular, based on a core scaffold, with interchangeable substrate-targeting subunits, which enables one piece of core machinery to ubiquitylate many different substrates. The cullin-RING ligase (CRL) family of complexes are the archetypes for these modular ubiquitin ligases, and CRL1 ligases, better known as the SKP1-CUL1-F-box protein (SCF) complexes, are the best characterized. SCFs use a family of F-box proteins (69 in humans) as substrate adaptors to mediate the ubiquitylation and consequent degradation of a large number of regulatory proteins involved in diverse processes. During the initial years of GM57587, my laboratory investigated how two SCF complexes (SCF- SKP2 and SCF-ßTrCP) and APC/C (a CRL-like ubiquitin ligase) target various regulators of cyclin dependent kinases (CDKs) for degradation. Subsequently, to broaden our knowledge of these remarkable enzymes, we asked whether any other members of the F-box protein family play important roles in controlling the cell division cycle. For example, during the last funding cycle, we found that cyclin F (FBXO1) controls genome integrity by coordinating production of deoxyribonucleotides with DNA replication; FBH1 (FBXO18) promotes DNA double strand breakage and apoptosis in response to DNA replication stress; and FBXO11 regulates cell quiescence and differentiation. We now propose a project exploring the integration of CRL- controlled cell cycle networks with DNA replication and the DNA damage response. Via proteomic techniques, we have identified putative CRL substrates involved in these processes, and using an interdisciplinary approach that incorporates biochemistry, molecular cell biology, and somatic cell genetics, we will characterize the molecular mechanisms and biological significance of the degradation of these novel substrates.

PROJECT SUMMARY/ABSTRACT Ubiquitin-mediated proteolysis regulates the degradation of numerous proteins, thereby controlling many cellular processes, including cell cycle progression, signal transduction pathways, differentiation, and the centrosome duplication cycle. Much of the specificity inherent in the ubiquitination process is mediated by the E3 ubiquitin ligases, which bind selectively to, and recruit, the chosen substrate to the ubiquitin-conjugating enzyme. Notably, the majority of ubiquitin ligases are considered ?orphan?, because their substrates have not yet been identified. Several ubiquitin ligases localize to the centrosome and control the ubiquitination and subsequent proteasomal degradation of critical centriole duplication factors, such as CP110, PLK4, and SAS6 (by SCF-Cyclin F, SCF-?TrCP, and APC/C-Cdh1, respectively). Furthermore, ubiquitin ligases also control additional centrosomal functions, such as centriole separation (through the SCF-?TrCP-mediated degradation of Cep68). Our preliminary results show that additional ?orphan? E3 ligase complexes reside at the centrosome, and that several centrosomal proteins are degraded by the ubiquitin system during specific phases of the cell cycle. We propose a project systematically exploring the regulation of the centrosome cycle by the ubiquitin-proteasome system. We will use proteomic techniques to identify novel substrates of centrosomal E3 ligases (specific AIM 1) and will validate and biochemically characterize the most biologically significant substrates identified under AIM 1 (specific AIM 2). We will make our analysis of E3 ubiquitin ligases and centrosome interactors available as a web-accessible resource. Centrosome amplification is a common feature of the large majority of cancers and can result in chromosome instability. Furthermore, centrosome abnormalities are also associated with genetic disorders of neurons and cellular cilia. Therefore, the information gained from the proposed studies is expected to be of direct relevance to our understanding of cancer biology and other human diseases such as ciliopathies and neuronal development disorders.

ABSTRACT The mission of the Cancer Cell Biology Program (CCB) is to fulfill the promise of personalized cancer therapy by elucidating the critical signaling and metabolic networks controlling cancer cell properties, employing cutting-edge chemical biology to more effectively target rate-limiting pathways in cancer, and translating these insights to the clinic, via improved therapies or better biomarkers. To address these challenges, CCB has recruited multiple new, world-class investigators who critically complement existing areas of excellence and promote high quality, collaborative research. Co-led by Alec Kimmelman MD, PhD, recently recruited as Chair of Radiation Oncology, and Michele Pagano MD, Chair of Biochemistry and Molecular Pharmacology, HHMI investigator, and Director of the former Growth Control Program at PCC, CCB is a multi-disciplinary team of 49 members and 3 associate Members from 15 departments at NYU School of Medicine (NYUSoM) and the NYU Department of Chemistry, who perform basic, translational, and clinical research. This highly restructured program retains select members from the former Growth Control and Stem Cell programs, incorporates several members from the former Breast and GU programs, and has a substantially re-focused agenda. Research is now organized around three complementary thematic aims: Aim 1) To identify regulatory mechanisms for key cancer-relevant genes that confer selective dependencies in human tumors; Aim 2) To delineate how metabolism is reprogrammed in cancer and discover new metabolic vulnerabilities; Aim 3) To use structural, chemical, protein engineering and pharmacological approaches to target cancer cell dependencies for therapeutic benefit. Program members have >$17.2M in cancer-related funding, including $6.3M in NCI grants, $8.1M in other peer-reviewed funding, and $2.8M in non-peer reviewed support. Members are highly productive and collaborative. During this funding period, we published 604 papers, many in high-impact journals, with 11% intra-programmatic, 32% inter-programmatic and 27% inter-institutional (NCI-CC) publications. CCB contributed key new insights into our basic understanding of signaling and metabolic vulnerabilities in genetically defined cancer subtypes, uncovered new molecular targets, and designed, developed and/or tested new therapies in investigator-initiated trials (IITs) and elucidated their mechanism of action or resistance. CCB promotes the PCC mission by: (1) producing innovative, high-impact science that reveals new therapeutic targets in cancer cells and their microenvironment; (2) discovering new cancer drug molecule candidates; (3) accruing patients to high-impact, high-content clinical trials; and (4) developing sophisticated technologies beyond the reach of individual investigators via the new PCC Biologics Initiative and Developing Metabolomics shared resource. There is a particular focus on cancers impacting our catchment area (lung cancer, pancreas cancer, triple negative breast cancer and prostate cancer), a strong commitment to translation and a rich portfolio of bench-bedside-bench activities.

PROJECT ABSTRACT / SUMMARY More than 30 years have passed since the discovery of the ubiquitin-proteasome system, yet we still lack comprehensive insights into its regulatory mechanisms. This limitation arises because most of the approximately 600 ubiquitin ligase enzymes in mammals remain largely uncharacterized. Our laboratory has focused on how Cullin-RING Ubiquitin Ligase (CRL) complexes control the three essential dimensions of cellular life: proliferation, survival, and differentiation. We have played a central role in elucidating how, when, where and, most importantly, why CRLs mediate the degradation of key cellular regulators. The research in my laboratory initially focused on the paradigm of the timed regulation of the mammalian cell cycle by the ubiquitin-proteasome system, which we established a number of years ago. We have since expanded our research into five fields of study: (i) cell signaling, (ii) cell cycle control, (iii) the DNA damage response, (iv) oncology, and (v) the circadian clock. This expansion of our interests has been possible thanks to our discovery-driven approach that, in contrast to the more common hypothesis-driven approach, is unbiased. Yet, the results of genetics and proteomics screens are not our end goal, but they guide us to explain the underlying biological concepts and mechanisms. Importantly, they often lead us to unexpected and unexplored new territories, opening our horizons. In summary, we use a comprehensive and interdisciplinary approach to break new grounds and make transformative discoveries that will provide new mechanistic insights into fundamental biological processes, particularly those processes that are regulated by CRLs. The current proposed research will cover all the mechanistic studies performed in our laboratory concerning areas i-iii listed above. In particular, we will leverage our recognized expertise in the ubiquitin field to discover molecular mechanisms by which CRLs control signal transduction pathways, autophagy, cell cycle progression, and DNA repair. Moreover, we will perform biochemical, cellular and in vivo analyses to illuminate how CRLs are regulated to function as switches between diverse cell fates. These findings will substantially advance our understanding of the proper execution of crucial cellular processes, potentially shedding light on the etiologies and treatments of human diseases.