Kenji Fukasawa - US grants

APPLICANT'S DESCRIPTION: Chromosome instability (and resulting aneuploidy) has been recognized as a hallmark of cancer and contributes to multi-step carcinogenesis by facilitating the accumulation of genetic lesions required for the acquisition of various malignant phenotypes. However, much remains to be learned regarding the causes and mechanisms of chromosome instability in cancer. We have recently found that p53 tumor suppressor protein, the product of the most commonly mutated genes in human cancers, is involved in the control of the centrosome duplication cycle. Loss or mutational inactivation of p53 results in uncontrolled amplification of centrosomes. A deleterious consequence of centrosome hyperamplification is most prominently featured during mitosis by the formation of aberrant spindles organized by multiple centrosomes (spindle poles), leading to an increased frequency of chromosome segregation errors. Moreover, centrosome hyperamplification is commonly observed in human cancers, and that the occurrence of centrosome hyperamplification in those tumors is strongly correlated with loss or mutational inactivation of p53. This suggests that centrosome hyperamplification induced by mutation of p53 is one major factor that contributes to chromosome instability in human cancers. The goal of this research proposal is to elucidate the molecular mechanisms that control initiation of centrosome duplication, coordination of the centrosome and DNA duplication cycles, and suppression of centrosome reduplication within a single cell cycle. Elucidation of this unexplored function of p53 at a molecular level will provide vital information for fully understanding the tumor suppressor activity of p53, the role of p53 mutation in carcinogenesis, and also for designing effective cancer intervention protocols targeting p53 to block centrosome duplication. Such an approach may prove effective in cancer intervention, since centrosome duplication, like DNA replication, is restricted to proliferating cells. Moreover, blocking the centrosome duplication process results in suppression of chromosome instability as well as cell division.

DESCRIPTION (provided by applicant): Chromosome instability is a formidable force in the multi-step carcinogenesis by facilitating the accumulation of genetic lesions required for the acquisition of malignant phenotypes. During last several years, there is an accumulation of evidence that abnormal amplification of centrosomes due to the deregulated duplication of centrosomes is common in human tumors. A deleterious consequence of centrosome hyperamplification is featured during mitosis by the formation of aberrant spindles organized by multiple spindle poles, leading to an increased frequency of chromosome segregation errors. Thus, centrosome hyperamplification is one major factor that contributes to chromosome instability in human cancers. Centrosome duplication is triggered by cyclin-dependent kinase (CDK)2/cyclin E (and/or cyclin A) in a kinase activity-dependent manner. Because CDK2/cyclin E also drives cells to initiate DNA synthesis, the temporal activation of CDK2/cyclin E occurring in the mid-late G1 is believed to coordinate centrosome duplication and other cell cycle events, including DNA replication. We have recently found that nucleophosmin (NPM)/B23 is a key centrosomal target of CDK2 in the initiation of centrosome duplication. NPM/B23 is directly phosphorylated by CDK2/cyclin E, and dissociates from the centrosomes upon CDK2/cyclin E-mediated phosphorylation. Microinjection of anti-NPM/B23 antibody as well as expression of a dominant negative NPM/B23 inhibits centrosome duplication. These results suggest that dissociation of centrosomal NPM/B23 induced by CDK2-mediated phosphorylation is a critical event for the initiation of centrosome duplication, constituting a licensing system for centrosome duplication, ensuring the coordination of centrosome and DNA duplication as well as restricting centrosome duplication to occur once within a single cell cycle. Elucidation of the functional role of NPM/B23 in centrosome duplication, especially in association with CDK2/cyclin E (and cyclin A), at a molecular level will provide crucial information for further understanding of the regulation of centrosome duplication, which leads to the potential of designing effective cancer intervention protocols targeting centrosome duplication. Such an approach may prove effective, since centrosome duplication, like DNA replication, is restricted to proliferating cells. Moreover, blocking the centrosome duplication process results in suppression of chromosome instability as well as cell division.

[unreadable] DESCRIPTION (provided by applicant): Chromosome instability (and resulting aneuploidy) has been recognized as a hallmark of cancer cells, and contribute to multi-step carcinogenesis by facilitating the accumulation of genetic lesions required for acquisition of various malignant phenotypes. However, much remains to be learned in respect to the causes and mechanisms of chromosome instability in cancer. A number of studies have shown that abnormal amplification of centrosomes (generation of more than two centrosomes) occurs frequently in almost all types of human cancers, and there is a strong association between occurrence of centrosome amplification and aneuploidy. The detrimental consequence of centrosome amplification is prominently featured during mitosis by the formation of aberrant spindles organized by multiple centrosomes (spindle poles), leading to an increased frequency of chromosome segregation errors. Indeed, centrosome amplification is now widely accepted as one of the major factors that contribute to chromosome instability in cancer. Centrosome amplification occurs by several mechanisms, among which uncontrolled duplication of centrosomes is perhaps the leading cause. We and others have previously shown that loss or inactivating mutation of p53 tumor suppressor protein results in dysregulation of centrosome duplication, resulting in a high frequency of centrosome amplification, which undoubtedly contributes to the overall tumor suceptibility phenotype associated with loss or mutation of p53. During the current grant period, we have focused on how p53 is involved in the regulation of centrosome duplication, and we have made many critical findings for further understanding of the molecular mechanisms underlying this important cellular event. In this application, in addition to our continuing efforts to elucidate the molecular mechanism of how p53 itself regulates centrosome duplication and how loss of p53 leads to centrosome amplification, we will extend our studies to the upstream molecules/events of p53. This is critical to fully understand the p53-dependent regulation of duplication and numeral homeostasis of centrosomes as a cellular phenomenon rather than a "single-out" biological event. The studies proposed here will not only provide vital information for fully grasping the tumor suppressor activities of p53 and the role of p53 mutation in carcinogenesis, but also lead to effective cancer intervention protocols targeting centrosome duplication. Such an approach may prove effective in cancer intervention, since centrosome duplication, like DNA synthesis, is restricted to proliferating cells. Moreover, targeted inhibition of centrosome duplication will not only block cell division, but also suppress chromosome instability. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chromosome instability (and resulting aneuploidy) has been recognized as a hallmark of cancer cells, and contributes to tumor progression by facilitating the genetic alterations required for acquisition of malignant phenotypes. Numerous studies have shown the frequent occurrence of abnormal amplification of centrosomes (generation of more than two centrosomes) in almost all types of solid tumors and certain types of lymphoma and leukemia, and the strong association between occurrence of centrosome amplification and aneuploidy. The detrimental consequence of centrosome amplification is prominently featured during mitosis: the presence of multiple centrosomes (spindle poles) results in mitotic defects, leading to an increased frequency of chromosome segregation errors. Indeed, centrosome amplification is now widely accepted as one of the major factors that contribute to chromosome instability in cancer. Centrosome amplification is caused primarily by uncontrolled duplication of centrosomes in a single cell cycle. We have recently found that ROCK II (Rho-associated kinase) localizes to centrosomes, and acts as a key regulatory protein for the initiation of centrosome duplication in association with nucleophosmin (NPM/B23). NPM/B23 is one of the major targets of CDK2-cyclin E, which plays a key role in initiating centrosome duplication. CDK2-cyclin E-mediated phosphorylation renders NPM/B23 a higher binding affinity to ROCK II, and ROCK II becomes super- activated by NPM/B23 binding. ROCK II then promotes the initiation of centrosome duplication in a centrosome localization- and kinase activity-dependent manner. ROCK II activation is also controlled by binding to an activated Rho protein (Rho-GTP), a major signal transducer downstream of many growth factor receptors (receptor tyrosine kinases/RTKs). These observations put forward to a model, in which two pathways (CDK2-NPM/B23 pathway and RTK-Rho-ROCK II pathway) converge by the physical interaction between ROCK II and NPM/B23 and super-activation of ROCK II to initiate centrosome duplication. In this research proposal, we will molecularly dissect the how ROCK II is controlled by these cancer-associated proteins to drive centrosome duplication, and how ROCK II controls centrosome duplication through focusing of the protein called LASP1, which we have recently identified as a potential centrosomal target of ROCK II for the initiation of centrosome duplication. Based on the facts that centrosome duplication occurs only in proliferating cells, and centrosome amplification is the major cause of chromosome instability in cancer, centrosome duplication and centrosome amplification can be effective targets for the cancer intervention protocols. Our research proposal that aim to understand the molecular mechanisms of the regulation of centrosome duplication and induction of centrosome amplification will provide vital information on this regard. PUBLIC HEALTH RELEVANCE: Cancer is the result of accumulation of many genetic alterations. Gain or loss of chromosomes (chromosome instability), which occurs frequently in cancer, introduces many genetic alterations simultaneously, hence accelerating tumors to acquire further malignant characteristics. In this research grant application, we will examine the mechanisms of how various oncoproteins and tumor suppressors, which are frequently mutated in cancer, maintain the integrity of chromosomes via controlling the duplication of centrosomes. The centrosome plays a critical role in the accurate segregation of chromosomes to two daughter cells during mitosis. Mutations of those oncoproteins and tumor suppressor proteins result in numeral abnormality of centrosomes, leading to mitotic defects and consequentially destabilization of chromosomes. As centrosome duplication occurs in actively proliferating cells such as cancer cells, centrosome duplication can be an effective target of cancer intervention, and the findings from our proposed studies will provide critical information on the development of superior cancer intervention protocols targeting centrosomes and cancer-associated proteins that control centrosome duplication.