Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expressionof genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

The technical objectives of this project were to: (1) transfect mutant P53s commonly mutated in human breast cancer into normal human mammary epithelial cells obtained from different donors and isolate clones; (2) characterize the clones for extension of lifespan and immortalization; (3) determine if expression of any of the mutant p53s provide a growth advantage to breast epithelial cells prior to immortalization; (4) determine in breast epithelial cells immortalized and expressing mutant p53 if expression of the mutant p53 is necessary for the maintenance of growth; and (5) determine downstream genomic targets of p53 that may be important in the development and progression of breast cancer. The technical objectives accomplished include the determination of the effectiveness of different p53 mutants in extension of breast epithelial lifespan and immortalization; the spontaneous immortalization of Li-Fraumeni breast epithelial cells; the determination of the effectiveness of different promoters for achieving extension of lifespan and immortalization; and the determination of the role of telomerase in the development of human breast cancer.

This book provides the readers with an overview of research on p53, which has been shown to play a role in numerous crucial biological pathways in normal and cancer cells. Leading scientist in the field, who have all made direct contributions to the understanding of the molecular events underpinning p53 function, have been invited to contribute the various chapters, which discuss the current knowledge of the signaling cascades that are activated by mutations in p53 and overexpression of MDM2, frequently found in human cancer and are major causes of oncogenesis. This book features chapters on the molecular basis of oncogenesis induced by gain of function mutation of p53, signaling pathways induced by MDM2 overexpression, control of mutant or wild-type p53 function by MDM2 and MDMX, p53 mutation in hereditary cancer and structural aspects that activate mutant p53 which can be targeted by drug therapy. This book should be useful for scientists at all levels.

The current year (2004) marks the Silver Anniversary of the discovery of the p53 tumor suppressor. The emerging ?eld ?rst considered p53 as a viral antigen and then as an oncogene that cooperates with activated ras in transforming primary cells in culture. Fueling the concept of p53 acting as a transforming factor, p53 expression was markedly elevated in various transformed and tumorigenic cell lines when compared to normal cells. In a simple twist of fate, most of the studies conducted in those early years inadvertently relied on a point mutant of p53 that had been cloned from a normal mouse genomic library. A bona ?de wild-type p53 cDNA was subsequently isolated, ironically, from a mouse teratocarcinoma cell line. A decade after its discovery, p53 was shown to be a tumor suppressor that protects against cancer. It is now recognized that approximately half of all human tumors arise due to mutations within the p53 gene. As remarkable as this number may seem, it signi?cantly underrepresents how often the p53 pathway is targeted during tumorigenesis. It is my personal view, as well as many in the p53 ?eld, that the p53-signaling pathway is corrupted in nearly 100% of tumors. If you are interested in understanding cancer and how it develops, you must begin by studying p53 and its pathway. After demonstrating that p53 functions as a tumor suppressor the ?eld exploded and p53 became a major focus of scientists around the world.

Approximately 50% of human cancers have accumulated missense mutations in the gatekeeping tumor suppressor protein p53, usually resulting in genomic instability and a very poor prognosis. The wild-type p53 protein is required for assessing DNA damage in cells and making the decision to either induce cell cycle arrest to facilitate DNA repair, or to induce a suicide response in those cells with irreparable damage. In human tumors, many hot-spot mutations are found within the DNA-binding domain of p53, rendering it incapable of sequence-specific transactivation of target genes such as p2l, bax, and mdm2. Some of these mutants, in addition to having dominant-negative functions, also gain novel functions by interacting with proteins differently from the wild-type p53 protein. One such gain-of-function p53 mutant possesses an Arg to His substitution at codon l75 (l72 in mice) and has been shown to be involved in the dysregulation of centrosome duplication leading to abnormal mitoses and subsequent aneuploidy. Because centrosome abnormalities and aneuploidy are often seen in high-grade breast tumors, unraveling the mechanism behind the involvement of p53l72 R-H in centrosome dysregulation will help us to understand the progression of mammary carcinogenesis. In order to identify potential indirect target genes regulated by this mutant, we employed a suppressive subtractive hybridization technique to generate a cDNA library specific to p53 null mammary epithelial cells (MECs) expressing the l72 R-H mutant. cDNA made from p53 null mammary epithelial cells transiently transfected with wild-type p53 was subtracted from cDNA made from mutant p53 transfected cells. The subtraction procedure generated a pool of cDNAs differentially expressed in the presence of the mutant protein; many interesting genes were revealed to be candidates for regulation by mutant p53. Some of these include developmental, metabolic, transcriptional,

Mutation of the p53 gene is very frequent in breast cancer. In normal cells induction of wild-type p53 function leads to either cell cycle arrest or cell death. Loss of this function can contribute to oncogenic cell transformation. Additionally the presence of mutant forms of p53 in breast tumor cells may actually facilitate the process of tumorigenesis. The properties of mutant p53 proteins in vitro and in breast tumor cell lines will be studied, experiments will focus on analysis of the structure and modification of mutant p53 proteins as well as the effect of cellular signalling on p53 function. Additionally it is planned to establish breast cell lines expressing inducible mutant p53 to determine the effect of such mutants on parameters of cell cycle, growth and death. We have discovered that all mutant forms of pS3 tested are capable of binding specifically to p53 response elements present in p53 target genes at lower but not at physiological temperatures. Furthermore, we have identified a means by which such binding is stabilized at the higher temperature. This will allow us to explore means to develop molecules that might have the outcome of converting pS3 in breast tumor cells from mutant to wild-type in function. One approach will be to develop a yeast- based screen for mutant p53 modifying genes. Such reagents would have clear therapeutic advantages.