Research Grants Awarded
Molecular Targeting Of DNA Repair: Determining The Functional Status Of The FA/BRCA Pathway In Non-Familial Breast Cancers
Tumor Cell Biology V
The main goal of this prospective translational research proposal is to analyze sporadic breast cancers for DNA repair defects resulting from an impaired function of the BRCA1 and BRCA2 genes. In contrast to familial cancers, somatic mutations in BRCA1/2 are not commonly found. Similarly, there have been no reports of sporadic mutations in any of the many Fanconi Anemia (FA) genes, which interact with BRCA1/2 in a common “FA/BRCA” pathway to promote HR. However, genetic or epigenetic inactivation of FA/BRCA pathway components may be frequently present in sporadic cancers. Cells deficient in the FA/BRCA pathway are impaired in the DNA repair process of homologous recombination (HR) and display a pronounced hypersensitivity to crosslinking agents, such as cisplatin, but also to radiation. Additionally, these cells may be sensitive to other classes of DNA damaging drugs. These findings provide a rationale basis for molecularly targeted therapies and individualized chemotherapies. However, considering the complex network-like nature of the FA/BRCA pathway with many yet undiscovered components, genotyping of single genes, such as BRCA1 or FancF , is likely insufficient to detect patients who could benefit from individualized treatments. We propose to employ an innovative biomarker assay to determine the functional status of the FA/BRCA pathway in sporadic breast cancers. We seek to take advantage of the observation that components of the FA/BRCA pathway, namely BRCA1, FancD2 and Rad51, form subnuclear protein foci at sites of DNA repair following exposure to radiation or cisplatin. Using immunofluorescent techniques, we will detect the foci response for each of these proteins as a surrogate marker of the function of the FA/BRCA pathway. We will test the hypothesis that a functional impairment of the pathway, as evident by abrogated damage-induced foci formation, can be detected in approximately 25% of cancers. We will enroll 30 patients undergoing mastectomies. Three samples from each tumor specimen will be exposed to radiation, cisplatin, or mock treatment ex-vivo . The foci response will be classified as normal, abrogated, or partially impaired. The methylation status of the BRCA1 and FancF genes will be determined to detect underlying epigenetic silencing of these genes. We will also determine the physiological relevance of a reduced foci response. Using an in-vitro approach, variable levels of functional BRCA1 or FancD2 protein will be generated by RNA interference or by using an inducible expression system. We seek to determine a threshold in the proficiency of foci formation, above which the observed cytotoxicity from cisplatin is equal to the cell kill seen in cells with fully functional BRCA1 or FancD2. Finally, we will test the hypothesis that FA/BRCA-deficient cells are also hypersensitive to other drugs that produce DNA damage, which requires proficient HR for cell survival.
Breast cancer is the most common type of cancer among American women. About 5 to 10% of breast cancers are inherited (i.e., familial). Of these, approximately half are caused by mutations in the BRCA1 or BRCA2 genes. These genes protect cells from damage to their DNA, which, if left unrepaired, can lead to cancer development. Recent discoveries have linked the function of BRCA1/2 to a rare disorder called Fanconi Anemia (FA). FA is associated with various abnormalities including a cancer predisposition and can result from mutations in any of several FA genes. The FA and BRCA1/2 genes are regarded as forming a common “FA/BRCA” pathway, which promotes the repair of damaged DNA, specifically by a process called homologous recombination (HR). Importantly, cells with defects in HR become hypersensitive to certain chemotherapeutic agents, such as cisplatin, but also to radiation. Recent research suggests that the genes that form the FA/BRCA pathway may also be dysfunctional in non-familial breast cancers, affecting at least one in four women. Thus, if we could determine at the time of cancer diagnosis if a woman’s tumor has a dysfunctional FA/BRCA pathway, we could offer her a potentially highly effective, individualized treatment with cisplatin (or a related type of drug). However, currently there is no easy way to test individual breast cancers for the function of the FA/BRCA pathway, which consists of a complex network of multiple genes interacting with each other. We propose to develop a novel biomarker test to determine the function of the FA/BRCA pathway in non-familial breast cancers. We seek to take advantage of the observation that three proteins that are central to the FA/BRCA pathway accumulate at sites of DNA damage caused by cisplatin or radiation. These proteins interact with each other to remove the damage via HR and can be visualized in a cell as dots or ‘foci’ using a fluorescence technique. Inactivation of any of the genes that are part of the FA/BRCA pathway may impair the formation of foci. We will analyze the tumor tissues from patients undergoing breast surgeries for their cancers. Each of the tumors will be exposed to radiation or cisplatin in the laboratory, placed in nutrient medium and incubated at body temperature for several hours so that the repair proteins can form foci. We will count the cells that exhibit foci and determine for each tumor whether the formation of foci is normal or abnormal. We will also determine whether an abnormal formation of foci is due to the most commonly observed alteration of the BRCA1 and FancF genes in non-familial cancers. In addition, we will ask by how much the foci formation needs to be reduced to be considered abnormal and to be causing hypersensitivity to cisplatin. We will also investigate if FA/BRCA-deficient cells are hypersensitive not only to cisplatin but also other drugs that produce DNA damage and that are commonly used in breast cancer therapy.