Role of Cdc25A in breast cancer

描述(申请人提供)：CDC25A，一种双特异性蛋白磷酸酶，在乳腺癌中经常过度表达，并与不良生存有关。该计划的长期目标是了解CDC25A在乳腺癌中的确切作用，并建立针对该蛋白的抗癌治疗的科学基础。CDC25A通过S激活细胞周期蛋白E(A)/CDK2和G2/M交界处的细胞周期蛋白B(A)/CDK1，从而促进细胞周期进程。在转化小鼠胚胎成纤维细胞的过程中，异位表达的CDc25A缩短了通过G1的通道，并与H-ras协同作用，而沉默CDC25A则导致G1和G2的停滞。我们已经证明，转化生长因子-β(TGFbeta)对于乳腺的形态发生和肿瘤抑制非常重要，它通过转录和翻译后方式下调CDc25A。这些观察表明，CDC25A可能是乳腺癌中的一个关键癌基因，尽管它还没有在动物模型中得到活体证明。这一建议基于我们的初步数据：(I)我们构建的CDc25A杂合基因敲除小鼠在MMTV-ras诱导的乳腺肿瘤发生中显示出明显的耐药性；(Ii)MMTV-CDc25A转基因与MMTV-ras转基因在小鼠乳腺肿瘤发生中具有协同作用；(Iv)转化生长因子-β以SMAD3依赖的方式促进CDc25A蛋白的降解，而这一途径在MCF7和其他人乳腺癌细胞系中可能存在缺陷；(Iii)CDc25A过表达抑制细胞凋亡信号调节激酶-1(ASK1)，降低细胞对氧化应激诱导的凋亡的敏感性。在这项建议中评估的中心假设是，CDC25A蛋白的缺陷降解对乳腺癌的开始和/或进展是限速的，导致细胞周期进展和细胞凋亡的非调控控制。这些研究的具体目的是：(1)确定非调控的转化生长因子-β/Smad信号转导通路是否参与了人乳腺癌细胞系中CDc25A的稳定表达；(2)利用携带CDc25A siRNA的MCF-10a细胞和四环素诱导的CDc25A突变体表达，确定CDc25A表达降低或稳定性改变如何影响细胞周期进展、细胞对氧化应激的敏感性以及ras和neu诱导的恶性转化；(3)利用我们新型的CDc25A-基因敲除和MMTV-CDc25A转基因小鼠株系，确定CDc25A表达变化对乳腺肿瘤发生的影响。我们希望这些研究将为我们提供对CDC25A稳定在乳腺癌发展中的作用的重要洞察，并为开发针对癌基因的有效治疗方法提供一个分子处理。

DESCRIPTION (provided by applicant): Cdc25A, a dual-specific protein phosphatase, is frequently overexpressed in breast cancer, and associated with poor survival. The long-term goal of this program is to understand the exact role of Cdc25A in breast cancer and establish a scientific basis for anti-cancer therapies targeted on this protein. Cdc25A promotes cell cycle progression by activating cyclin E (A)/Cdk2 during G1 through S, and also cyclin B(A)/Cdk1 at the G2/M boundary. Ectopic expression of Cdc25A shortens the passage through G1 and cooperates with H-ras in transforming mouse embryonic fibroblasts, while silencing of Cdc25A results in arrest at both G1 and G2. We have shown that transforming growth factor-beta (TGFbeta), which is important for morphogenesis and tumor suppression of the mammary gland, downregulates Cdc25A in both transcriptional and posttranslational manners. These observations suggest that Cdc25A could be a critical oncogene in breast cancer, although it has not been demonstrated in vivo using animal models. This proposal is based on our following preliminary data: (i) Cdc25A-heterozygous knockout mice we generated show marked resistance to MMTV-ras induced mammary tumorigenesis; (ii) An MMTV-Cdc25A transgene synergistically cooperates with the MMTV-ras transgene in murine tumorigenesis; (iv) TGF-beta promotes Cdc25A protein degradation in a Smad3-dependent manner and this pathway could be defective in MCF7 and other human breast cancer cell lines; (iii) Cdc25A overexpression inhibits apoptosis signal-regulating kinase-1 (ASK1) and diminishes cellular sensitivity to oxidative stress-induced apoptosis. The central hypothesis evaluated in this proposal is that defective degradation of Cdc25A protein is rate-limiting for initiation and/or progression of breast cancer, causing deregulated control of cell cycle progression and apoptosis. The specific aims are: (1) Determine whether deregulated TGF-beta/Smad signaling is involved in Cdc25A stabilization in human breast cancer cell lines; (2) Determine how reduced Cdc25A expression or altered stability ofCdc25A affects cell cycle progression, cellular sensitivity to oxidative stress, and malignant transformation in response to ras and neu, using MCF-10a cells with Cdc25A siRNA and tetracycline-inducible expression ofCdc25A mutants; (3) Determine how altered Cdc25A expression affects breast tumorigenesis in mice with MMTV-neu, MMTV-myc or MMTV-Wnt-1 transgene, using our novel Cdc25A-knockout and MMTV-Cdc25A transgenic mouse lines. We expect that these studies will provide us with significant insight into the role of Cdc25A stabilization in breast cancer development and a molecular handle to develop effective therapies to target the oncogene.