The overall goal of this proposal is to test whether targeting individual cell cycle proteins represents a highly selective therapeutic strategy in different types of human breast cancer. The proliferation of mammalian cells is driven by the core cell cycle machinery operating in cell nucleus. The key components of this machinery are proteins called cyclins, which bind, activate and provide substrate specificity to their associated cyclin-dependent kinases (CDKs). These cyclin-CDK complexes phosphorylate cellular proteins, thereby driving cell proliferation. Mouse gene knockout experiments demonstrated that individual cyclins and CDKs are dispensable for development and for normal proliferation of the majority of cell types. In contrast, these proteins are essential for the initiation and for maintenance of specific cancer types, depending on the genetic lesion they carry. Relevant for this application, our laboratory recently demonstrated that an ubiquitous, global shutdown of cyclin 01 in mice bearing MMTV-Erb82 (HER2) driven breast cancers blocked tumor cell proliferation and triggered tumor cell senescence, without having any obvious impact on animals' physiology. Importantly, administration of an inhibitor of CDK4 and CDK6 (PD 0332991) to tumor bearing animals had the same effect, namely it caused senescence of Erb82-driven breast cancer cells. These observations suggest that inhibition of CDK4/6 kinase activity may represent a very effective therapeutic strategy in women with HER2-positive (HER2+) breast cancers. In the work proposed in Aim 1, we will extend our analyses to human HER2+ breast cancers. We will take advantage of a very large collection of human breast cancer cell lines (including several HER2+) assembled by Dr. Polyak. We will also use xenografts of primary HER2+ breast cancers, to test the effect of CDK4/6 inhibition on human mammary carcinomas. Lastly, we will elucidate how human HER2+ breast cancer cells develop resistance to CDK4/6 inhibition. In Aim 2, we will extend our approach to triple-negative breast cancers, where very few therapeutic options are available. We will test our hypothesis that this specific cancer type depends of cyclin E-CDK1 and/or A-CDK1 kinase. The Specific Aims are: Aim 1. To determine the response of human Erb82-positive (HER2+) breast cancers to cyclin D-CDK4/6 inhibition; Aim 2. To study the requirement for CDK1 function in triple-negative breast cancers