This proposal focuses on an important but unexplored problem - how steroid hormones, which are well known risk factors, interact with p53 mutations to produce aneuploidy and malignancy, and how the chromosomal segregation protein Separase is involved? Our sex-steroid dependent p53-mice preneoplastic breast cancer model allows a unique approach to this problem. In this model, steroidal induction in p53 mutant mammary glands results in chromosomal instability, aneuploidy and tumor formation analogous to that seen in majority of human breast cancers. We propose a paradigm that there is a set of proteins whose deregulation promotes aneuploidy (termed PR AN; Promoter of Aneuploidy) including chromosomal instability which results in loss or gain of whole or parts of chromosomes, and that PRAN proteins are interactively regulated by steroid hormones and the tumor suppressor p53. Our published and new preliminary data provide the first evidence that steroid hormones play a role in the regulation of mitotic proteins involved in sister chromatid cohesion and separation. We propose that the combined effect of mutation of the tumor suppressor p53 and signaling by steroid hormones produces aneuploidy in breast cancer by affecting expression of key proteins involved in chromosomal separation. We focuses on the elements that regulate chromosomal segregation, particularly sister chromatid cohesion/separation proteins, as candidate PRAN proteins, since chromosome missegregation during mitosis can lead to aneuploidy. A key gene in this analysis is ESPL1, which encodes an endopeptidase called Separase that separates joined sister chromatids by cleaving cohesin Rad21/SCC1/MCD1 during the metaphase to anaphase transition. The hypothesis is that hormonal stimulation of p53 null mouse mammary glands results in misexpression of the ESPL1 gene, thus promoting aneuploidy and breast cancer formation. This proposal applies in vivo transplantation of p53 mutant and wild type (WT) mammary cells that are stably transfected with ESPL1, and an ESPL1 transgenic mice model to test the PRAN paradigm following hormone treatment. Steroid and p53 regulation of ESPL1 at the transcriptional level is studied by characterizing the ESPL1 promoter region. These objectives will be accomplished by pursuing two specific aims: 1) Functional role of Separase overexpression in aneuploidy, and 2) Transcriptional regulation of ESPL1 gene expression. The proposed study not only elucidate underlying mechanisms of hormone-induced aneuploidy, a fundamental unresolved question in cancer biology, but also likely to identify a new class of proteins that are responsible for chromosomal instability and breast cancer progression. ? ?
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