My long-term objective is to establish my own laboratory at a research institution to perform breast cancer research with the aim of discovering the missing links between DNA damage repair pathways and breast carcinogenesis which could be exploited for identifying novel therapeutic targets. To accomplish this goal, I joined Dr. Maria Jasin's lab at MSKCC who has performed groundbreaking studies on breast cancer genes including BRCA1, BRCA2 and RAD51 paralogs. Proposed Research: Breast cancer is the most common form of cancer identified among women in the United States. Genetic studies have revealed a direct link between breast/ovarian cancer predisposition and germ-line mutations in BRCA1, BRCA2, PALB2 and paralogs of the recombinase gene RAD51 that are involved in DNA repair by the homologous recombination (HR) pathway. Although the connection between breast cancer and BRCA1 and BRCA2 has been widely investigated, the RAD51 paralogs have been identified only recently as highly penetrant cancer susceptibility genes. Mutants in RAD51 paralog genes in rodent cells are highly sensitive to DNA damaging agents, displays genomic instability and have reduced HR. Biochemically, RAD51 paralogs exists in two different complexes, BCDX2 (RAD51B, RAD51C, RAD51D and XRCC2) and CX3 (RAD51C and XRCC3). However the contributions of individual RAD51 paralogs during early and late stages of HR and the epistatic relationship of BCDX2 and CX3 complexes has not been studied systematically in human cells. The objective of this proposal is to dissect the cellular functions f the RAD51 paralogs in normal human mammary epithelial cells to provide insight into how deficiency of RAD51 paralog genes predisposes individuals to cancer.
Specific Aim 1 : Identify the functions of the individual RAD51 paralogs in human mammary epithelial cells containing an integrated HR reporter.
Specific Aim 2 : Determine the epistatic relationship of the RAD51 paralogs to understand the role of the major complexes (BCDX2 and CX3) in HR. Research Design: 1) To identify the functions of RAD51 paralogs in MCF10A DR-GFP cells, I will generate 5 isogenic RAD51 paralog knockouts (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) using Transcription Activator-Like Effector Nuclease (TALEN)-directed gene disruptions. 2) To understand the epistatic relationships of BCDX2 and CX3 complexes, I will generate several double-mutant cell lines. I will disrupt members of both complexes (RAD51B-/-XRCC3-/-), disrupt members of only the BCDX2 complex (RAD51B-/- RAD51D-/-), and disrupt members of both complexes using a member common to both complexes (RAD51C-/- ) and one member unique to the BCDX2 complex (RAD51B-/-). The single knockout cell lines and double- mutant cell lines will be assessed for their overall viability, ability to undergo DNA replication, sensitvity to DNA damaging agents, HR proficiency, and genomic instability.
Like BRCA1 and BRCA2, RAD51 paralogs have been recently identified as highly penetrant breast and ovarian cancer genes. Pathogenic mutations for both RAD51C and RAD51D have been identified in breast and ovarian cancer patients. RAD51 paralog-deficient rodent cells exhibit chromosomal instability and increased sensitivity to genotoxic agents. These phenotypic defects are likely attributable to the involvement of RAD51 paralogs in DNA double-strand break (DSB) repair through the homologous recombination (HR) pathway. How RAD51 paralogs function in normal human cells has been poorly studied. My goal is to determine the function of each RAD51 paralog and complex in human mammary epithelial cells because they represent a good genetic model system for studying the etiology of breast cancer. My proposed work on the RAD51 paralogs will form a basis for a cellular understanding of why breast cancer is associated with RAD51 paralog mutations. Specific Aims: Objectives: Breast cancer is a leading cause of death in women in the United States. Early onset of familial breast cancer is caused by inheritance of a mutated gene;most cases are caused by loss of function of the molecular pathways that respond to and repair DNA double-strand breaks (DSBs). Prime examples are the breast cancer susceptibility genes BRCA1 and BRCA2, although recent studies have also implicated the RAD51 paralogs, all of which are involved in DSB repair by homologous recombination (HR). The involvement of BRCA1 and BRCA2 in maintaining genomic integrity to suppress breast cancer has been extensively studied. However, as RAD51 paralogs have only recently been identified as breast/ovarian cancer suppressors, their role in HR is not well understood. There are 5 RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and the individual and collaborative contributions of these genes to HR have yet to be elucidated. To understand this, I will study the effect of knocking out individual RAD51 paralog genes in normal human breast cells, to determine their molecular function in DNA repair and suppression of genomic instability associated with breast cancer. Furthermore, I will investigate the complex interactions of these related genes by knocking out different combinations, which will provide a clearer picture of how RAD51 paralogs function together during HR to prevent carcinogenesis. I propose two specific aims for this research project: 1) Identify the functions of the individual RAD51 paralogs in human mammary epithelium cells containing an integrated HR reporter. 2) Determine the epistatic relationship of the RAD51 paralogs to understand the role of the major complexes (BCDX2 and CX3) in HR. Expected outcomes and impact: RAD51 paralogs have been found mutated in patients with breast and ovarian cancer. However, it is not well understood how the different RAD51 paralogs or complexes contribute to the maintenance of genomic integrity and how the deficiency in RAD51 paralogs contributes to carcinogenesis. Therefore, by generating and characterizing RAD51 paralog knockout cell lines, this research project will provide insight into how defects in RAD51 paralog genes predispose breast cells to carcinogenesis. More broadly, a more comprehensive understanding of HR mechanisms in mammalian cells will be important for therapeutic approaches that are being developed which target the HR pathway.