Molecular Basis of Aneuploidy

描述（由申请人提供）：本提案关注一个重要但尚未探索的问题-类固醇激素是如何与p53突变相互作用产生非整倍体和恶性肿瘤的，以及染色体分离蛋白分离酶是如何参与的？我们的性类固醇依赖性p53小鼠肿瘤前乳腺癌模型允许一个独特的方法来解决这个问题。在该模型中，类固醇诱导p53突变乳腺导致染色体不稳定、非整倍体和肿瘤形成，类似于大多数人类乳腺癌。我们提出了一种范式，即存在一组蛋白质，其解除管制可促进非整倍体（称为PRAN；非整倍体的启动子），包括导致整个或部分染色体丢失或获得的染色体不稳定性，并且PRAN蛋白受类固醇激素和肿瘤抑制因子p53的相互调节。我们发表的和新的初步数据提供了第一个证据，证明类固醇激素在有丝分裂蛋白参与姐妹染色单体内聚和分离的调节中起作用。我们提出肿瘤抑制因子p53突变和类固醇激素信号的联合作用通过影响参与染色体分离的关键蛋白的表达而在乳腺癌中产生非整倍体。我们关注的是调节染色体分离的元件，特别是姐妹染色单体内聚/分离蛋白，作为候选的PRAN蛋白，因为在有丝分裂过程中染色体错分离会导致非整倍体。该分析中的一个关键基因是ESPL1，该基因编码一种称为分离酶的内肽酶，该酶通过在中期到后期转变期间切割内聚蛋白Rad21/SCC1/MCD1来分离连接的姐妹染色单体。假设是激素刺激p53小鼠乳腺导致ESPL1基因错误表达，从而促进非整倍体和乳腺癌的形成。本研究将稳定转染ESPL1的p53突变型和野生型（WT）乳腺细胞进行体内移植，并建立ESPL1转基因小鼠模型，以检验激素治疗后的PRAN模式。通过表征ESPL1启动子区域，研究了ESPL1在转录水平上的类固醇和p53调控。这些目标将通过追求两个特定目标来实现：1)分离酶过表达在非整倍体中的功能作用，以及2)ESPL1基因表达的转录调控。这项研究不仅阐明了激素诱导的非整倍体的潜在机制，这是癌症生物学中一个尚未解决的基本问题，而且还可能发现一类新的蛋白质，这些蛋白质负责染色体不稳定性和乳腺癌的进展。

DESCRIPTION (provided by applicant): 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.