Role of Cdc25A in breast cancer

描述（由申请人提供）：Cdc25A是一种双特异性蛋白磷酸酶，在乳腺癌中经常过表达，并与低生存率相关。该项目的长期目标是了解Cdc25A在乳腺癌中的确切作用，并为针对该蛋白的抗癌治疗建立科学基础。Cdc25A通过在G1到S期间激活细胞周期蛋白E (A)/Cdk2，以及在G2/M边界激活细胞周期蛋白B(A)/Cdk1来促进细胞周期进程。Cdc25A的异位表达缩短了通过G1的通道，并与H-ras合作转化小鼠胚胎成纤维细胞，而Cdc25A的沉默导致在G1和G2时阻滞。我们已经证明，在乳腺形态发生和肿瘤抑制中起重要作用的转化生长因子β (tgfβ)通过转录和翻译后方式下调Cdc25A。这些观察结果表明，Cdc25A可能是乳腺癌的一个关键致癌基因，尽管它尚未在动物模型中得到体内证实。这一建议是基于我们的以下初步数据：(i)我们产生的cdc25a杂合敲除小鼠对MMTV-ras诱导的乳腺肿瘤发生表现出明显的抗性；（ii） MMTV-Cdc25A基因与MMTV-ras基因在小鼠肿瘤发生中的协同作用；（iv） tgf - β以smad3依赖的方式促进Cdc25A蛋白降解，该途径在MCF7和其他人类乳腺癌细胞系中可能存在缺陷；（iii） Cdc25A过表达抑制凋亡信号调节激酶-1 (ASK1)，降低细胞对氧化应激诱导的凋亡的敏感性。本提案评估的中心假设是Cdc25A蛋白降解缺陷限制了乳腺癌的发生和/或进展，导致细胞周期进展和凋亡失控。具体目的是：(1)确定tgf - β /Smad信号失调是否参与人乳腺癌细胞系中Cdc25A的稳定；(2)利用Cdc25A siRNA和四环素诱导的Cdc25A突变体表达的MCF-10a细胞，确定Cdc25A表达减少或Cdc25A稳定性改变如何影响细胞周期进程、细胞对氧化应激的敏感性以及ras和new的恶性转化；(3)利用我们的新型Cdc25A敲除和MMTV-Cdc25A转基因小鼠系，确定Cdc25A表达改变如何影响MMTV-neu、MMTV-myc或MMTV-Wnt-1转基因小鼠的乳腺肿瘤发生。我们期望这些研究将为我们提供Cdc25A稳定在乳腺癌发展中的作用的重要见解，并为开发针对癌基因的有效治疗提供分子处理。

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.