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    Research Grants Awarded

    Cyclin D1-Cdk4 Kinase In Human Breast Cancers

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    D-type cyclins (D1, D2 and D3) are components of the core cell cycle machinery. These proteins represent the ultimate cell cycle recipients of the mitogenic and oncogenic pathways. Once induced, D-cyclins bind and activate cyclin-dependent kinases, CDK4 and CDK6. These cyclin D-CDK4 and D-CDK6 complexes phosphorylate cellular proteins, thereby allowing cell cycle progression. Consistent with their growth-promoting functions, amplification of cyclin or CDK genes, and overexpression of the proteins is seen in a large number of human cancers. The best documented is involvement of cyclin D1 overexpression in the majority of human mammary carcinomas. Importantly, overexpression of cyclin D1 is usually accompanied by the hyperactivation of cyclin D1-CDK4 kinase activity. Moreover, mice engineered to overexpress CDK4, or cyclin D1, are prone to mammary carcinomas. Collectively, these results indicate that hyperactivated kinase activity of cyclin D1-CDK4 complexes is a driving force of mammary neoplasia. In the past, we found that knockout mice lacking cyclin D1, or lacking CDK4, were completely resistant to mammary carcinomas triggered by the ErbB2 oncogene. We also engineered a knock-in strain of mice expressing kinase-dead cyclin D1-CDK4 complexes. We found that these mice developed normally, but were resistant to ErbB2-driven mammary carcinomas, revealing a differential requirement for cyclin D1-CDK4 kinase in breast neoplasia, but not in normal development. We also found that knock down of cyclin D1, or knock down of CDK4, or replacement of the wild-type CDK4 with a kinase-dead CDK4 point mutant in mouse breast cancer cells blocked the ability of these cells to form tumors upon injection into mammary glands of the recipient animals. Collectively, these results indicate that cyclin D1-CDK4 kinase is not only required for breast cancer initiation, but the continued presence of this kinase is critically required to maintain the tumorigenic potential of cancer cells. Currently, the most fundamental question in the field is: what are the critical targets of cyclin D1-CDK4 kinase in breast cancer cells? Cyclin D1-CDK4 was previously shown to phosphorylate, and functionally inactivate three cell cycle inhibitory proteins: the retinoblastoma, p107 and p130 proteins, and Smad3 transcription factor. However, inactivation of these proteins (through mutations or deletions) occurs relatively rarely in breast cancers, whereas cyclin D1-CDK4 hyperactivation is seen in the majority of tumors, suggesting that other critical proteins are being targeted by D1-CDK4 kinase in breast cancer cells. Moreover, human breast cancers preferentially ?choose? to overexpress cyclin D1, but not D2 or D3, and CDK4, but not CDK6, suggesting the presence of D1-CDK4-specific targets in breast cancer cells. We hypothesize that the function of cyclin D1-CDK4 kinase in cancer cells goes well beyond cell cycle progression. We hypothesize that this kinase, when hyperactive, contributes to cancer formation by affecting several other processes such as cell death and DNA repair. The goal of this application is to identify the full set of phosphorylation targets of cyclin D1-CDK4 in human breast cancer cells. Annotation of the entire human proteome, and development of novel proteomic approaches offer an unprecedented opportunity to resolve this critical issue. Aim 1. To search for cyclin D1-CDK4 substrates among cyclin D1- and CDK4- associated proteins in human breast cancer cells. We will take advantage of the observation that cyclins and CDKs form stable complexes with their phosphorylation targets. We will use immunoaffinity purification coupled to novel high-sensitivity shot-gun mass spec sequencing, to define the whole set of cyclin D1- and CDK4- interacting proteins in breast cancer cells. We will next test these interactors for their ability to be phosphorylated by cyclin D1-CDK4 in vitro and in vivo. In our preliminary analyses we determined that this approach is feasible, and we defined over 40 interactors of cyclin D1 in MCF7 breast cancer cells. Aim 2. To perform a proteome-wide search for cyclin D1-CDK4 targets. Annotation of all open reading frames that constitute the human proteome open the possibility to perform proteome-wide screens for cyclin D1-CDK4 targets. We performed such proteome-wide computational screen, and obtained a list of 445 ?most likely? cyclin D1-CDK4 targets in the human proteome. In a high-throughput approach, we tested over 200 of these proteins for their ability to be phosphorylated by cyclin D1-CDK4 kinase, and we identified several potential novel substrates. In the proposed work, we will extend this screen to all 445 proteins. In our follow-up, mechanistic studies, we will first focus on proteins that were identified both in our proteome-wide screen and were identified by us as cyclin D1-CDK4 interactors in breast cancer cells (Aim 1). This study will provide unprecedented insights into the molecular function of cyclin D1-CDK4 kinase in driving breast neoplasia, by defining the full repertoire of D1-CDK4 substrates in breast cancer cells. We will be able to design rational anti-cancer strategies, based on interfering with D1-CDK4 targets identified in this study. We predict that cyclin D1-CDK4 substrates represent breast cancer oncogenes or tumor suppressor genes that are activated (or inactivated) in a fraction of human breast cancers. Therefore, in the near future we will initiate screens to search for mutations in cyclin D1-CDK4 targets - identified in this work - in human breast cancers.

    Lay Abstract:
    Cyclin D1 protein is a key component of the ?cell cycle machinery? which controls cell proliferation. Cyclin D1 also serves as a recipient of oncogenic (cancer-causing) signals. Once turned on, cyclin D1 pairs with a protein called ?cyclin-dependent kinase CDK4?. Cyclin D1-CDK4 pairs put phosphate groups on target proteins (i.e. ?phosphorylate? them), thereby turning these targeted proteins on, or off in cancer cells. Abnormally high levels of cyclin D1 are seen in over 50% of human breast cancers. Importantly, these cancers show greatly elevated activity of cyclin D1-CDK4. Moreover, mice engineered to display elevated levels of cyclin D1, or CDK4, develop breast cancers. These results reveal that hyperactivated cyclin D1-CDK4 activity is a driving force of mammary neoplasia. In the past, we found that mice without cyclin D1, or without CDK4, were completely resistant to mammary carcinomas triggered by the oncogene (cancer-causing gene) ErbB2 (i.e. these mice never developed any tumors). We also found that replacing active CDK4 with an inactive variant in mouse breast cancer cells completely blocked the ability of these cells to form tumors. These results indicate that cyclin D1-CDK4 function is not only required for breast cancer initiation, but the continued activity of cyclin D1-CDK4 is critically required to maintain the tumorigenic potential of cancer cells. Currently, the most fundamental question in the field is: what are the critical targets of cyclin D1-CDK4 in breast cancer cells? (i.e. which proteins are phosphorylated in breast cancer cells by cyclin D1-CDK4?) Despite over a decade of research, the identity of these targets is unknown. However, sequencing of the entire human genome, and development of novel ?proteomic? technologies offer us an unprecedented opportunity to resolve this critical issue. We hypothesize that cyclin D1-CDK4, when hyperactive in cancer cells, contributes to cancer formation not only by affecting the proliferation of cells, but also by affecting several other processes such as cell death and repair of damaged DNA. The goal of this application is to identify the full set of cyclin D1-CDK4 targets in human breast cancer cells. We will use two complementary approaches. In the first approach, we will take advantage of the observation that cyclin-CDK molecules often physically bind their targets. Therefore, we will employ novel, recently developed techniques (the so-called ?high-sensitivity mass spectrometry?) to define the whole set of cyclin D1- and CDK4-binding proteins in breast cancer cells. We will next test these proteins for their ability to be phosphorylated, thus activated or inactivated by cyclin D1-CDK4. The sequencing of the entire human genome allows us to predict the identity of all human proteins. We used a computer program to screen all human proteins for a possibility of them being phosphorylation targets of cyclin D1-CDK4. We obtained a list of 445 ?most likely? cyclin D1-CDK4 targets. In a high-throughput approach, we tested over 200 of these proteins for their ability to be phosphorylated by cyclin D1-CDK4, and we identified several potential novel targets. In the proposed work, we will extend this screen to all 445 proteins. We will then investigate in detail proteins that were identified by us in our computer screen and were identified by us as cyclin D1-CDK4- binding proteins in breast cancer cells. This study will provide unprecedented insights into the molecular function of cyclin D1-CDK4 in driving breast neoplasia, by defining the full repertoire of D1-CDK4 targets in breast cancer cells. We will be able to design rational anti-cancer strategies, based on interfering with cyclin D1-CDK4 targets identified in this study. We predict that cyclin D1-CDK4 phosphorylation targets represent breast cancer genes that are activated (or inactivated) in a fraction of human breast cancers. Therefore, in the near future we will initiate screens to search for mutations in cyclin D1-CDK4 targets - identified in this work - in human breast cancers. We believe that this study is important to breast cancer patients because it may offer a new treatment, based on interfering with cyclin D1-CDK4 targets identified in this study.