Biological role of modifications to C/EBPbeta-1 and -2

C/EBPbeta-1 和 -2 修饰的生物学作用

• 批准号: 6704051
• 负责人: LINDA SEALY
• 金额: $ 30.96万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6704051
• 关键词: biological signal transduction; breast neoplasms; cell growth regulation; cell line; chemical conjugate; enhancer binding protein; genetically modified animals; guanine nucleotide binding protein; laboratory mouse; mammary epithelium; phenotype; phosphorylation; posttranslational modifications; transcription factor

DESCRIPTION (provided by applicant): The basic leucine zipper transcription factor, C/EBPbeta, is critical for growth and differentiation of the mammary gland. Epithelial cell proliferation in early pregnancy and differentiation at late pregnancy are severely impaired in C/EBPbeta null mice, which fail to lactate. Alternative translation of the intronless C/EBPbeta gene produces three different protein isoforms. C/EBPbeta-1 and -2 are transactivators, whereas C/EBPbeta-3 lacks a transactivation domain and inhibits transcription. Although differences between C/EBPbeta-1 and -2 have received little attention, our studies show that they are functionally distinct. Ectopic C/EBPbeta-2 expression transforms mammary epithelial cells (MECs) in vitro. The cells become anchorage independent, undergo an epithelial to mesenchymal transition (EMT), and gain invasive growth characteristics. In contrast, C/EBPbeta-1 expression blocks the invasive growth of metastatic breast cancer cell lines in culture. In this application we propose to examine the mechanistic basis underlying the functional dichotomy in C/EBPbeta-1 vs. -2 expression. These two transactivators differ by only 21 N-terminal amino acids present in C/EBPbeta-1, but absent from C/EBPbeta-2. We hypothesize that C/EBPbeta-1 and -2 undergo isoform-specific posttranslational modifications essential for their different functions. In support of this hypothesis, we have recently shown that C/EBPbeta-1, but not C/EBPbeta-2, is conjugated to the small ubiquitin-like modifier proteins, SUMO2 and SUMO3. We will determine whether SUMO-conjugation is necessary for the inhibition of invasive growth by C/EBPbeta-1. We will also investigate whether SUMO2,3 modification is required for protein-protein interactions with the Swi/Snf chromatin remodeling complex and/or affects the localization of C/EBPbeta-1 is subnuclear speckles. In contrast, C/EBPbeta-2 is targeted by multiple protein kinases, some of which are downstream of Ras activation. Indeed, C/EBPbeta has been shown to be an essential target of oncogenic Ras signaling in skin tumorigenesis and NIH 3T3 transformation. We will examine the requirement for C/EBPbeta-2 phosphorylation by ERKI/2 and p90Rsk-2 in mammary epithelial cell transformation. In human breast cancer, Ras-dependent signaling pathways are often activated by alterations in receptor tyrosine kinases. Therefore, we plan to determine whether receptor tyrosine kinase activation will synergize with C/EBPbeta-2 in transforming mammary epithelial cells. Because C/EBPbeta-2 promotes, whereas C/EBPbeta-1 inhibits, the invasive growth of MECs, misregulated expression of these two transactivator isoforms could contribute to growth and metastasis in breast cancer. Interestingly, C/EBPbeta-2 is undetectable in normal human breast tissue (obtained from reduction mammoplasty) where only C/EBPbeta-1 is expressed. However, 60% of primary breast tumors examined showed a high level of C/EBPbeta-2 expression, and moderate expression was detected in another 30% of the samples. Moreover, all breast cancer cell lines in culture express C/EBPbeta-2, but none express C/EBPbeta-1. We have recently generated mice carrying an MMTV-driven C/EBPbeta-2 transgene, and significantly, virgin females exhibit precocious, hyperplastic mammary gland development. We will continue to study these animals to determine if females with hyperplasia go on to develop neoplasia and/or have accelerated development of metastatic carcinoma when crossed with other mouse models of breast cancer. Once cancer cells have metastasized, breast cancers are largely incurable even with state-of-the-art approaches. These studies will provide important insights into how misregulated C/EBPbeta isoform expression may contribute to the development of metastatic mammary carcinoma.

描述（由申请人提供）：基本的亮氨酸拉链转录因子C/EBPBETA对于乳腺的生长和分化至关重要。 在C/EBPBETA NULL小鼠中，妊娠早期妊娠早期的上皮细胞增殖受到严重损害，这些小鼠无法乳酸。内在C/EBPBETA基因的替代翻译产生三种不同的蛋白质同工型。 C/EBPBETA-1和-2是反式激活剂，而C/EBPBETA-3缺少反式激活域并抑制转录。尽管C/EBPBETA -1和-2之间的差异很少受到关注，但我们的研究表明它们在功能上是不同的。异位C/EBPBETA-2表达在体外转化乳腺上皮细胞（MEC）。这些细胞成为锚定的独立性，经历了间充质转变（EMT）的上皮，并获得侵入性生长特征。相反，C/EBPBETA-1表达阻碍了培养中转移性乳腺癌细胞系的侵入性生长。在此应用中，我们建议检查C/EBPBETA -1与-2表达中功能性二分法的机械基础。这两个反式激活剂仅在C/EBPBETA-1中存在21个N末端氨基酸，但C/EBPBETA-2不存在。我们假设C/EBPBETA-1和-2经历了同种异体特异性的翻译后修饰，对它们的不同功能必不可少。为了支持这一假设，我们最近表明，C/EBPBETA-1（而不是C/EBPBETA-2）与小型泛素样修饰蛋白SUMO2和SUMO3偶联。我们将确定Sumo结合是否对于C/EBPBETA-1抑制侵入性生长是必需的。我们还将研究SUMO2,3是否需要与SWI/SNF染色质重塑络合物相互作用和/或影响C/EBPBETA-1的定位是亚核斑点。相反，C/EBPBETA-2由多种蛋白激酶靶向，其中一些激酶是RAS激活的下游。实际上，C/EBPBETA已被证明是皮肤肿瘤发生和NIH 3T3转化中致癌性RAS信号传导的基本目标。我们将研究ERKI/2和P90RSK-2在乳腺上皮细胞转化中对C/EBPBETA-2磷酸化的需求。在人类乳腺癌中，RAS依赖性信号通路通常会因受体酪氨酸激酶的改变而激活。因此，我们计划确定受体酪氨酸激酶激活是否会在转化乳腺上皮细胞中与C/EBPBETA-2协同作用。由于C/EBPBETA-2会促进C/EBPBETA-1抑制，而MEC的侵入性生长，这两种反式激活剂同工型的表达不正常可能有助于乳腺癌的生长和转移。有趣的是，在正常的人类乳房组织（从还原乳腺成形术中获得）C/EBPBETA-2在仅表达C/EBPBETA-1的正常乳房组织中是无法检测的。然而，检查的原发性乳腺肿瘤中有60％表现出高水平的C/EBPBETA-2表达，在另外30％的样品中检测到中度表达。此外，培养物中的所有乳腺癌细胞系都表达C/ebpbeta-2，但没有表达C/ebpbeta-1。我们最近产生了携带MMTV驱动的C/EBPBETA-2转基因的小鼠，并且显着的，处女雌性表现出早熟，增生的乳腺发育。我们将继续研究这些动物，以确定患有增生的女性是否继续发展肿瘤和/或与其他乳腺癌小鼠模型交叉时，转移性癌的发展加速。一旦癌细胞转移，即使采用最先进的方法，乳腺癌也会在很大程度上无法治愈。这些研究将提供重要的见解，即C/EBPBETA同工型表达如何有助于转移性乳腺癌的发展。