Barbara L. Weber - US grants

proteins; breast neoplasms; cell growth regulation; molecular cloning; mammary epithelium; neoplastic transformation; neoplasm /cancer genetics; molecular oncology; gene expression; genome; neoplastic growth; tissue /cell culture; enzyme linked immunosorbent assay; genetic library; complementary DNA;

Breast cancer is the second leading cause of cancer death in American women and family history is known to be a significant risk factor for the development of this disease., Recently, genetic linkage analysis has identified chromosome 17q2l as the locale of a gene which confers susceptibility to early-onset breast cancer. This study also adds support to previous evidence that the trait is transmitted in an autosomal dominant fashion and may involve loss or inactivation of a tumor suppressor gene. In collaboration with the group responsible for linkage analysis (Dr. M.C. King and colleagues), we plan to further localize and ultimately clone this gene. Our goal is to identify at least twenty-five families with a high incidence of breast cancer (excluding Li-Fraumeni families) with an average age at diagnosis less than 45 years. Once these families have been identified, samples will be collected for DNA preparation and linkage studies performed using mapping probes being prepared from 17q2l-q23 by Dr. King's group and the Genetic Marker Core of the Michigan Genome Center. Peripheral blood lymphocytes will be collected from living family members and we provide evidence that formalin-fixed, paraffin-embedded tissue is an excellent source of DNA for linkage analysis from deceased family members. One affected family member will also undergo karyotyping in an attempt to identify balanced translocations or other visible chromosomal abnormalities which may involve the breast cancer locus. Once the breast cancer region has been narrowed to 1-4 cM from its current estimated size of 20cM, a physical map will be constructed using YAC cloning, YAC walking and contig construction. The contigs encompassed by the most tightly linked markers will be searched for potential transcripts with several strategies including cDNA screening, CpG island identification and exon trapping. Potential candidate sequences will be analyzed for differences in expression between normal and malignant breast tissue and for the presence of mutations in affected individuals. Successful completion of this study will provide the foundation for work which promises to be pivotal in our understanding of the development of breast cancer.

DESCRIPTION: (adapted from the investigator's abstract) Breast cancer is the second leading cause of death in American women and family history is known to be a significant factor for the development of this disease. BRCA1, the breast cancer susceptibility gene located on chromosome 17 has recently been isolated. Almost simultaneously, chromosome 13q12-13 was identified as the locale of another such gene by genetic linkage analysis. In collaboration with the group responsible for isolation of BRCA1, we plan to further localize and ultimately isolate BRCA2. Our goals include identification additional families with evidence of linkage between breast cancer and genetic markers flanking BRCA2. These families will be studied for informative meiotic recombination events that will narrow the BRCA2 candidate interval and will be used for mutation analysis in testing BRCA2 candidate genes. Peripheral blood lymphocytes will be collected from living family members and paraffin-embedded tissue will be utilized as a source of DNA for linkage analysis from deceased family members. One affected family member will also undergo karyotyping in an attempt to identify balanced translocations or other visible chromosomal abnormalities which may involve the BRCA2 locus. Tumors from affected individuals in linked families will be studied for LOH in the BRCA2 candidate region as a means of further narrowing the candidate region. A physical map of the BRCA2 region is being constructed by our collaborators. The map already includes full coverage by yeast artificial chromosomes (YACs) and is approximately 50% covered with bacterial artificial chromosomes (BACs) and P1 clones. BAC and P1 genomic clones encompassed by the most tightly linked markers will be searched for potential transcripts by exon trapping and direct selection. Potential candidate cDNAs will be analyzed for the presence of germline mutations in affected individuals from BRCA2-linked families. The presence of germline mutations in families with evidence of linkage to BRCA2 will serve to identify a candidate gene as BRCA2. Once BRCA2 has been isolated (by us or others), we will expand our search for BRCA2 mutations to a bank of DNA collected from more than 300 women with a family history of breast cancer and/or a diagnosis of breast cancer before the age of 40. This panel of samples should be very useful in describing the spectrum of BRCA2 mutations in such a population, and these data will be added to an international study of genotype/phenotype correlation in families with inherited susceptibility to breast cancer as a result of germline mutations in BRCA1 or BRCA2. Perhaps of greatest significance however is that identification of BRCA2 will be pivotal in our understanding of the development of breast cancer, and will hasten the time when we can accurately define a cohort of women at very high risk of developing breast cancer and prevent the development of this devasting and potentially fatal disease.

DESCRIPTION: (adapted from the investigator's abstract) Germline alterations in BRCA1, a tumor suppressor gene of unknown function, are associated with an 85-90% lifetime risk of developing female breast cancer. Data from our laboratory and others suggest that BRCA1 is a nuclear phosphoprotein that produces growth inhibition when overexpressed in cultured cell lines. There are additional data that suggest that BRCA1 expression peaks at the G1/S boundary and is barely detectable at other phases of the cell cycle, and that BRCA1 G1/S expression is induced by mitogen exposure. Recent data for our group suggests that BRCA1 activates transcription of the cell growth inhibitor p21, and that p21 is required for the growth inhibitory effects of BRCA1 in a cell culture model. Additionally, they have preliminary evidence that BRCA1 binds to p53, a tumor suppressor known to function as a checkpoint at G1/S. Based on these data, we hypothesize that BRCA1 is a G1/S checkpoint molecule, and at least some of its effects are mediated by p21. The work outlined in this proposal will test this hypothesis by addressing the following specific aims: 1) to define the role of BRCA1 in p21 expression and function in cell cycle regulation, 2) to demonstrate interaction between BRCA1 and p53 and to determine the role of this interaction in p21-dependent and -independent BRCA1 function, and 3) to use the association between BRCA1, p21, and p53 to define the response to DNA damage and regulation of apoptosis. These experiments will define the endpoints of tumor "preventor" gene that helps maintain genome stability through a p21-dependent pathway. They will extend the observations made in cell culture to a series of 150 archival breast cancers we have collected from BRCA1 mutation carriers to determine the potential clinical relevance of the findings. As an exponentially increasing body of literature suggests that alterations in cell cycle regulation may be a unifying feature of malignant transformation, the studies we propose should provide insight into the role played by BRCA1 in tumor suppression. Identification of the specific lesions in growth regulatory pathways that are altered in BRCA1-associated breast cancer also will define potential preventative and therapeutic targets.

DESCRIPTION: (Applicant's Description) Hereditary cancer predisposition has been denoted by the National Cancer Institute as a high priority area of investigation, and the development of a Cancer Genetics Network has been proposed as a critical component of this effort. When established, the Cancer Genetics Network will provide a resource for the study of patients with a family history of cancer, some of which will have documented mutations in the known cancer susceptibility genes. The University of Pennsylvania Cancer Center is extremely well-suited to serve as a contributing member of the Cancer Genetics Network. Barbara Weber, MD, an internationally recognized expert in cancer genetics will serve as the PI. Timothy Rebbeck, PhD, a Co-Investigator, is one of a handful of molecular epidemiologists with expertise in cancer. The University of Pennsylvania meets all basic Network criteria with an established breast cancer family database of more than 1000 families with extensive family, demographic and exposure history, a registry of more than 200 known BRCA1 and BRCA2 mutation carriers, and a registry of more than 3000 patients from the Pigmented Lesion Clinic. Established clinical cancer risk evaluation programs provide ongoing accrual of patients with a family history of breast, ovarian and colon cancer as well as melanoma. Protocols for pre and post testing counseling for individuals who opt for genetic susceptibility testing are in place and have served as models for many developing programs in the US. Mechanisms for the tracking and follow-up of patients are well-established, biological specimens are in hand for many of the individuals in the registries, specimen collection is ongoing, with newly-entered patients, and an informatics system that will interface easily with other Network sites is in place. Unique capabilities at The University of Pennsylvania include a molecular diagnostic laboratory that provides CLIA-approved analysis of BRCA1 and BRCA1 and is establishing protocols for HNPCC testing, a Cancer Center network of community hospitals that refer all patients for consideration of genetic testing, the Center for Bioethics that has been in the forefront of research in genetic testing for cancer susceptibility, and state-of the art communication capabilities with management of both OncoLink (an online resource for all cancer patients that has been nationally recognized for excellence) and the BIC website (a repository for all known BRCA1 and BRCA2 alterations). In addition, the investigators who will direct Network activities at the University of Pennsylvania have an extensive track record of participation in large collaborative studies requiring sharing of specimens and data.

The overall goal of this Program Project is to increase our understanding of the contribution of estrogen to the development of breast cancer. We will address the use of breast cancer risk determinants in both African American and Caucasian women using a genetic, as well as a biochemical approach. We will address the issue of breast cancer risk determinants in both African American and Caucasian women using a genetic, as well as a biochemical approach. We also will evaluate the clinical utility of modifying breast epithelial exposure to estrogen by assessing the response of women at increased risk to the Selective Estrogen Receptor Modifier (SERM) Tamoxifen in two inter-related clinical trials. Thus the overall goals of the project are: 1) To develop a genetic model of breast cancer risk by analyzing a panel of proposed breast cancer susceptibility alleles related to hormone metabolism and response to DNA damage in African American and Caucasian women with breast cancer and a matched set of controls; 2) To develop a biochemical model of breast cancer risk by analyzing interindividual variability in estrogen metabolism in African American and Caucasian women with breast cancer and a matched set of controls, all of whom have been genotyped for the susceptibility alleles related to hormone metabolism; 3) To develop a pharmacogenetic model of breast cancer risk by combining genotypic data on the proposed breast cancer susceptibility alleles with the biochemical risk profile developed by studying estrogen metabolism in cases vs. controls; 4) To evaluate the response of MRI-defined alteration in breast volume as endpoints; 5) To identify biologic markers of response to Tamoxifen using markers of oxidative damage in peripheral blood and immunohistochemical evaluation of regions of increased density seen with MRI. At the conclusion of this study, we will have developed a comprehensive model for breast cancer risk based on a range of measures of estrogen effect that is applicable to both African Americans and Caucasians and we will have tested the ability of Tamoxifen to alter surrogate measures of risk. In addition, we will have evaluated the ability of Tamoxifen to alter surrogate measures of risk. In addition, we will have evaluated the potential of MRI-detected breast changes as surrogate endpoints and accumulated data on a range of histopathologic lesions that may be used as surrogate endpoints as well.

DESCRIPTION (provided by applicant): BRCA1 is for the cellular to DNA and for reco`mbination, although required response damage homologous it is not yet clear exactly what role it plays in these critical processes. BRCA1 also acts as a co-activator of p53-responsive promoter elements, suggesting that modulating expression of genes that mediate apoptosis and cell cycling is part of this process. Yet, given the universal nature of these cellular processes, it is difficult to explain the striking differences in cancer risk between breast and other tissues in women with BRCA1 germline mutations. The answer will lie in a meticulous dissection of events that initiate and drive BRCAl-associated tumorigenesis. One important clue is that of hormone responsiveness in the tissues at highest risk. During the last funding period, we developed a murine model that recapitulates a known disease-associated human germline BRCA1 mutation with a conditional deletion of the C-terminus of Brca1. We will use that model to ask: What genetic events are necessary for BRCA1-related tumors to develop? The specific aims of this proposal are: Aim 1: To generate murine mammary hyperplasia and carcinomas in mice with a homozygous deletion of the Brcal BRCT domain. Aim 2: To define the early genetic changes in BRCA1 related tumorigenesis using genomic analyses of murine and human tissue Aim3: To define the role of estrogen in the initiation and progression of Brcal-associated mammary tumors. Aim 4: To evaluate the role of haploinsufficiency in BRCA1 mut/wt cells. The completion of this work will define pathways that are altered in BRCAl-associated cancers and will determine whether ER-negative breast cancers predominate in women with BRCA1 mutations because they arise from cells that are intrinsically ER negative or because other proliferative advantages replace ER signaling. We will determine whether heterozygous BRCA1 wt/mut cells have higher mutation rates than BRCA1 wt/wt cells, and whether this effect, if present, is enhanced by estrogen. A complete understanding of these events will lead to testable strategies for prevention, early diagnosis and treatment for women with BRCA1 mutations as well as an enhanced understanding of the molecular events that drive cancer development in general.