A neuploidy (aberrant chromosome number) is a hallmark feature of human malignancies (1, 2) and has also been proposed as a necessary event for tumorigenesis (2). Although there have been many proposed hypotheses, there is no general agreement as to why aneuploidy is so highly prevalent in cancer cells, and how it contributes to tumor progression (3, 4). Importantly, if aneuploidy forms an underlying cause of human cancer, it has not been fully substantiated. The mechanisms of aneuploidy also remain a fundamental unresolved problem in cancer biology.To understand how aneuploidy might originate in mammalian tissues, we have focused on the elements that regulate chromosomal segregation, particularly those involved in sister chromatid cohesion and separation, because chromosome missegregation, for example during mitosis, can lead to aneuploidy. A key gene in our analysis is ESPL1, which encodes an endopeptidase called Separase that separates sister chromatids by cleaving cohesin Rad21/Mcd1/Scc1 during the metaphase to anaphase transition. The hypothesis we tested is that hormonal stimulation of the p53-null mouse mammary gland results in misexpression of the ESPL1 gene, thus promoting aneuploidy and breast cancer formation. Dysregulation of the mitotic machinery that helps maintain chromosomal stability in mammary cells can result in aneuploidy and subsequently, cancer formation. We focused on Separase for the following reasons that have important implications for breast cancer: (i) Separase plays a central role in promoting faithful chromosome segregation; (ii) our previous studies strongly indicated that hormonal stimulation of p53-null mice mammary gland results in overexpression of the ESPL1 and Separase protein, which may be a direct cause of aneuploidy (5); and (iii) siRNA-mediated knockdown of Separase and Separase deficient mouse embryonic fibroblasts results in genomic instability (6-8).An evolutionarily conserved protein complex called cohesin and an endopeptidase named Separase play pivotal roles in the accurate segregation of sister chromatids into two daughter cells. Cohesion along the length of the sister chromatids is formed during DNA replication in S phase. Cohesion along the chromosomal arms is removed during prophase and from centromeric regions at the metaphase-to-anaphase transition when Separase is activated after its inhibitory chaperone securin is degraded (9, 10).To understand how aberration in sister chromatid separation may contribute to chromosomal missegregation, we investigated the role of Separase overexpression in mouse mammary cells by using a mammary epithelial transplant model (11) as well as various biochemical and functional assays. Our results indicate that conditional overexpression of Separase alone in mammary epithelial cells with a p53 mutant background is sufficient to induce aneuploidy and tumorigenesis in vitro and in vivo. Results Conditional Expression of Mouse Separase (mSeparase) Results inAneuploidy in Mouse Mammary Epithelial Cells. To examine the direct effect of ...
Background: Aneuploidy is a common feature of human tumors, often correlating with poor prognosis. Several proteins involved in the mitotic spindle checkpoint are thought to play a major role in aneuploidy suppression. Specifically, the cohesin proteins that regulate sister chromatid separation and Separase, the endopeptidase that cleaves Rad21 to remove cohesion and allow timely cell cycle progression are thought to play key roles in maintaining normal cellular ploidy and preventing accumulation of abnormal cellular karyotypes. Studies using a transplant model showed that over expression of Separase in diploid mouse mammary epithelial cells resulted in the rapid accumulation of aneuploidy, and when transplanted in mice, there was rapid development of mammary tumors. It was also shown before that a sub set of human breast cancers show significant over expression of Separase, suggesting a possible role of Separase in the development and/or progression of breast cancer. Material and Methods: Using genetically engineered mouse models we studied the direct effect of Separase over expression in the mouse mammary epithelium. Two separate mammary gland specific promoters, WAP and MMTV, were used to drive Separase expression in the mouse mammary gland. The role of loss of p53 combined with over expression of Separase was studied by crossing the Tg-MMTV and WAP-Separase mice with p53 knockout mice in the same genetic background. Results: We found that over expression of Separase in the mouse mammary epithelium resulted in aberrant proliferation as early as day one lactation. The increase in proliferation was more acute in a p53 heterozygous background. Also, the Separase over expressing mice showed a significant delay in involution compared to wild type mammary epithelium and an accumulation of DNA damage. In a p53 heterozygous background, some of these mice developed spontaneous mammary tumors over a period of 7-12 months. Ongoing studies are evaluating the histopathological nature of these mammary tumors. Discussion: Previous studies suggested that Separase is over expressed in a sub set of human breast cancers. An understanding of the specific biological pathways involved in the initiation and progression of cancer in the mammary epithelium from aberrant expression of Separase can establish Separase as a novel diagnostic marker for breast cancer screening. Our studies suggest that Separase over expression in the mammary epithelium results in accumulation of aberrant proliferation and delayed involution post lactation, as well as results in the accumulation of DNA damage. These factors combined with loss of a key tumor suppressor like p53 can lead to genetic instability and breast cancer development. Further classification of the sub type of breast cancer in this animal model using array analysis can help establish Separase as a new prognostic marker in breast cancer research. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-05.
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