MYC-DEPENDENT DNA REPLICATION STRESS AND BREAST CANCER

Training Plan: At the Gautier Laboratory, I hope to receive training and further develop skills in working with new model systems, data interpretation, and independent development of research projects. While my previous training provided extensive insight into tumor suppressors and breast cancer, my present training at the Gautier Laboratory will help understand the role of oncogenes such as Myc in breast cancer. The research proposed here is directly relevant to breast tumors with the worst outcome and prognosis, also known as triple negative breast cancer. Studies show that Myc and other proteins controlling DNA replication are indeed deregulated in such breast tumors. My career goal is to utilize the extensive training in breast cancer research from my past and present (a) to further a better understanding of the disease to enable earlier diagnosis and (b) to better existing methods of diagnosis and treatment to improve the lives of patients who have been inflicted with breast cancer.Research Plan:Background: Myc is a potent oncogene that is deregulated in about 35 percent of breast cancers. Aberrant Myc expression is frequently associated with aggressive tumors and poor prognosis. Furthermore, the triple negative (estrogen receptor, progesterone receptor, and HER2 negative) basal-like subtype of breast cancers that are refractory to current therapy show overexpression of Myc and CycE. It is therefore vital to understand the oncogenic properties of Myc. Given that high levels of Myc and CycE lead to aberrant initiation of DNA replication, we hypothesize that the deregulation of DNA replication by oncogene activation influences the development of the triple negative breast cancers. Indeed, preliminary bioinformatic studies, conducted by applying the basal-like signature to large sets of breast tumors, have revealed that genes such as Myc that are involved in the initiation of DNA replication, but not factors acting after initiation, are differentially overexpressed in basal-breast tumors. Therefore, the main goal of our project is to evaluate Myc-driven basal-like breast cancer development.Objective/Hypothesis: Recent work in our lab has established a transcription-independent role for Myc in DNA replication. We hypothesize that regulation of DNA replication by Myc is critical for its oncogenic properties. Our overall objective is to determine how Myc functions in DNA replication under physiological conditions or when deregulated and its impact in causing genomic instability that leads to tumor formation.Specific Aims: (1) To determine whether Myc aberrantly activates the origins of DNA replication by regulating S phase kinases and/or chromatin modifications. (2) To identify genes essential for cell survival in the context of aberrant Myc expression.Study Design: Aim 1: We propose to test two hypotheses that could account for Myc activity at origins of DNA replication. First, we will assess whether Myc regulates S-phase protein kinases. We will use Xenopus cell-free extracts to compare the levels of chromatin-bound CDK2/CycE kinases in normal, Myc depleted and Myc overexpressing conditions. The consequences of overexpressing CDK/CycE in a Myc-depleted context will also be evaluated. Next, we will assess how Myc influences chromatin remodeling. We will determine whether replication defects induced by Myc depletion can be rescued by increasing histone acetylation. Conversely, we will examine whether deacetylation can abrogate the consequence of Myc overexpression. Aim 2: Since Myc overexpression triggers DNA damage, we hypothesize that cells overexpressing Myc have specific gene expression requirements to cope with Myc-dependent replication stress. To identify candidate genes, we will use a synthetic lethality screen wherein genes involved in DNA replication, repair, and cell cycle will be targeted for inactivation by siRNA with normal or overexpressed Myc.Impact: Many cancers, at the time of diagnosis, are already advanced and pathologically complex. It is therefore important to identify the early steps in tumorigenesis. The mechanistic investigation of Myc functions proposed in Aim 1 will enable the characterization of early steps in tumor formation that might lead to improved diagnosis and therefore a better chance of patient survival. Our second aim is focused on the identification of candidate genes essential to survive Myc overexpression. These findings have direct therapeutic potential because the identified genes can serve as diagnostic markers and they also will significantly aid the design of targeted therapy to combat Myc-related breast tumors associated with the worst prognosis.