Role of CEBP transcription factors in regulating cell growth and tumorigenesis

CEBP转录因子在调节细胞生长和肿瘤发生中的作用

• 批准号: 7965252
• 负责人: peter f johnson
• 金额: $ 121.29万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965252
• 关键词: Accounting; Address; Adult; Affinity; Animals; Apoptosis; Arginine; Biological; Biological Process; Bypass; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; CDK6-associated protein p18; Carcinogens; Cell Aging; Cell Proliferation; Cells; Colon Carcinoma; Cyclin-Dependent Kinase Inhibitor; Cytostatics; DNA Binding; DNA-Binding Proteins; Development; Disabled Persons; Down-Regulation; Enzymes; Ethylnitrosourea; Exhibits; Family member; Fibroblasts; Future; Gene Expression Profiling; Gene Silencing; Gene Targeting; Genomics; Goals; Growth; Growth Factor; Heterodimerization; Homodimerization; IL8 gene; Incidence; Insulin-Like Growth Factor I; Interleukin-6; Knock-out; Knockout Mice; Knowledge; Laboratory Study; Leucine Zippers; Lymphomagenesis; Malignant Epithelial Cell; Malignant Neoplasms; Mediating; Methods; Modeling; Modification; Mus; Mutant Strains Mice; N-terminal; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Phenotype; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Post-Translational Regulation; Premalignant; Protein C; Protein Family; Proteins; Proto-Oncogenes; Protocols documentation; RNA Interference; Regulation; Reporting; Research; Resistance; Role; Signal Transduction; Site; Skin Neoplasms; TP53 gene; Therapeutic Intervention; Transactivation; Tumor Suppressor Proteins; Work; cancer cell; carcinogenesis; cell growth; cell transformation; chemokine; cytokine; dimer; enhancer binding protein; in vivo; interest; lung carcinogenesis; metaplastic cell transformation; neoplastic cell; novel; p19ARF; receptor; response; senescence; transcription factor; tumor; tumorigenesis; tumorigenic

Development of cancer involves aberrant control of cellular proliferation due to activation of oncogenes as well as inactivation of tumor suppressors, which protect against unregulated growth by inducing apoptosis or growth arrest (senescence) of pre-malignant cells. Ras proto-oncogenes are often mutationally activated in cancer cells, while the p53 or RB tumor suppressor pathways are almost universally disabled. Detailed knowledge of oncogenic and anti-oncogenic pathways is essential for understanding how cancers develop and to identify unique vulnerabilities of tumor cells that can be exploited for therapeutic intervention. Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) transcription factors, especially their involvement in regulating cell proliferation and tumorigenesis. Our research focuses primarily on C/EBPbeta and its role as a downstream effector of oncogenic Ras signaling. Tumor studies using Cebpb null mice as well as analysis of C/EBPb-deficient cells revealed that C/EBPb has pro-oncogenic functions. However, in RasV12-expressing fibroblasts (MEFs) C/EBPb is also required for oncogene-induced senescence (OIS), an intrinsic tumor suppressive mechanism. In the latter context C/EBPb acts to arrest cellular proliferation by a pathway requiring RB:E2F. Thus, C/EBPb possesses both pro- and anti-tumorigenic activities. Because it plays an important role in cellular responses to Ras, we wish to elucidate the mechanisms by which C/EBPb expression and activity are controlled by oncogenic and physiological Ras signaling and thus to better understand the dual activities of C/EBPb in suppressing or promoting cancer in vivo. Posttranslational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed protein whose activity can be stimulated by oncogenic Ras or growth factor signals. C/EBPb auto-inhibition involves three regions in the N-terminal half of the protein that, together with sequences at the C terminus, are predicted to form a hydrophobic core. The folded core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb activation by Ras signaling involves several inducible posttranslational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper as an important regulator of C/EBPb DNA-binding and homodimerization. Recently, we identified two Ras-induced phosphorylation sites and a methylated arginine within an auto-inhibitory domain that are required for functional of C/EBPb activation by Ras. Ongoing work includes identifying the effector pathways and modifying enzymes downstream of Ras that regulate these PTMs and understanding their roles in controlling the biological and oncogenic functions of C/EBPb. Pro-oncogenic role of C/EBPb: Previous studies showed that C/EBPb-deficient mice are resistant to development of carcinogen-induced skin tumors. We have extended these findings by using other carcinogenesis protocols and mouse tumor models. C/EBPb knockout mice exposed to ENU exhibit impaired lymphomagenesis and reduced incidence/malignancy of a broad spectrum of other cancers compared to WT animals. Also, growth of mouse colon carcinoma cells is less efficient in C/EBPb KO mice than in WT animals, possibly due to decreased circulating levels of IGF-1 in the mutant mice. C/EBPb null mice also develop fewer tumors than WT mice in a K-ras lung carcinogenesis model. Future studies will address the possible involvement of proinflammatory cytokines (IL-6), chemokines (IL-8), and their receptors as oncogenic targets of C/EBPb in Ras-induced and other cancers. Tumor suppressor functions of C/EBPb: We previously found that C/EBPb is required for RasV12-induced cellular senescence in MEFs and showed that C/EBPb over-expression alone induces senescent-like growth arrest by a mechanism requiring RB:E2F. Cells lacking the tumor suppressor p19ARF bypass OIS and are transformed by Ras. We found that ARF, but not p53, is required to maintain C/EBPb levels in Ras-expressing fibroblasts, indicating that C/EBPb is part of an ARF-dependent tumor suppressor network. C/EBPb expression requires the Egr-1 and 2 transcription factors and activity of GSK3 kinases. Oncogenic Ras signaling inhibits GSK3 and decreases Egr levels, which at least partly explains down-regulation of C/EBPb in Ras-transformed cells. We are continuing to investigate how loss of ARF alters cellular responses to Ras and regulates Cebpb gene silencing. We are also using gene expression profiling and functional RNAi screens to identify target genes that mediate the cytostatic effects of C/EBPb. Regulation of C/EBPb activity by heterodimerization with C/EBPgamma: C/EBPg is a ubiquitously expressed protein that preferentially heterodimerizes with C/EBPb and other C/EBP family members. However, its biological functions are not well understood. We found that C/EBPg-deficient MEFs have reduced proliferative potential; this is in part due to the ability of C/EBPg to suppress the growth-inhibitory effects of C/EBPb by forming heterodimers. C/EBPg knockout cells display elevated levels of cyclin-dependent kinase (Cdk) inhibitors such as p15, p18, and p27, which may account for the decreased proliferative potential. C/EBPg null mice were originally reported to be nonviable; however, we found that the mutant mice survive when crossed onto a mixed strain background. Therefore, current work focuses on characterizing the phenotypes of adult C/EBPg deficient mice and investigating the role of C/EBPg in cell proliferation and tumorigenesis in vivo. Since C/EBPg alters the biological activity of C/EBPb, we are interested in identifying genomic targets of C/EBPb homo- and heterodimers. To that end we are developing a novel protein complementation/affinity tag (PCAT) method to enrich for chromosomal targets of various C/EBPb dimers. In principle, this method can be used to determine the interactome for any dimeric DNA-binding protein.

癌症的发展涉及对癌基因激活以及抑制肿瘤抑制因素的异常控制，这些控制因诱导肿瘤前细胞的细胞凋亡或生长停滞（衰老）而防止不受管制的生长。 RAS原始基因通常在癌细胞中被突变激活，而p53或RB肿瘤抑制途径几乎是普遍残疾的。详细了解致癌和抗结构途径对于理解癌症如何发展和鉴定可用于治疗干预的肿瘤细胞的独特脆弱性至关重要。我们的实验室研究C/EBP（CCAAT/增强子结合蛋白）转录因子，尤其是它们参与调节细胞增殖和肿瘤发生。我们的研究主要关注C/EBPBETA及其作为致癌RAS信号的下游效应因子的作用。使用CEBPB无效小鼠的肿瘤研究以及对C/EBPB缺陷细胞的分析表明，C/EBPB具有促性功能。但是，在表达RASV12的成纤维细胞（MEFS）中，C/EBPB也需要致癌基因诱导的衰老（OIS），这是一种固有的肿瘤抑制机制。在后一种情况下，C/EBPB通过需要RB的途径来阻止细胞增殖：E2F。因此，C/EBPB具有促肿瘤和抗肿瘤活性。 由于它在对RA的细胞反应中起着重要作用，因此我们希望阐明C/EBPB表达和活性通过致癌和生理RAS信号控制的机制，从而更好地了解C/EBPB在抑制VIVO抑制或促进癌症中C/EBPB的双重活性。 C/EBPB活性的翻译后调节：C/EBPB是一种本质上抑制的蛋白质，可以通过致癌性RA或生长因子信号刺激其活性。 C/EBPB自动抑制作用涉及蛋白质N末端的三个区域，这些区域与C末端的序列一起被预测形成疏水性核心。折叠的核心隔离基本区域和反式激活结构域，抑制了DNA结合和反式激活。 C/EBPB通过RAS信号传导激活涉及几种诱导后翻译后修饰（PTMS）。我们先前鉴定出亮氨酸拉链中的RSK激酶位点是C/EBPB DNA结合和同构化的重要调节剂。最近，我们确定了两个RAS诱导的磷酸化位点和一个自身抑制结构域内的甲基化精氨酸，这是RAS功能c/eBPB激活所必需的。正在进行的工作包括识别效应子途径并修改RAS下游的酶，该酶调节这些PTMS并了解它们在控制C/EBPB的生物学和致癌功能中的作用。 C/EBPB的亲构作用：先前的研究表明，C/EBPB缺陷小鼠对致癌物诱导的皮肤肿瘤的发育有抵抗力。我们通过使用其他致癌方案和小鼠肿瘤模型扩展了这些发现。与WT动物相比，暴露于ENU的C/EBPB敲除小鼠表现出受损的淋巴作用和降低其他癌症的发病率/恶性肿瘤。同样，小鼠结肠癌细胞的生长在C/EBPB KO小鼠中的效率低于WT动物，这可能是由于突变小鼠中IGF-1水平降低所致。 在K-Ras肺癌发生模型中，C/EBPB无效小鼠的肿瘤也比WT小鼠少。 未来的研究将解决促炎细胞因子（IL-6），趋化因子（IL-8）及其受体作为C/EBPB的致癌靶标的C/EBPB在RAS诱导的和其他癌症中的参与。 C/EBPB的肿瘤抑制功能：我们先前发现C/EBPB是MEFS中RASV12诱导的细胞衰老所必需的，并表明仅C/EBPB过表达就可以通过需要RB：E2F的机制诱导衰老样生长。缺乏肿瘤抑制剂p19arf旁路OI的细胞并被RAS转化。我们发现，在表达RAS的成纤维细胞中维持C/EBPB水平需要ARF而不是P53，这表明C/EBPB是ARF依赖性肿瘤抑制器网络的一部分。 C/EBPB表达需要EGR-1和2个转录因子以及GSK3激酶的活性。致癌性RAS信号传导抑制GSK3并降低EGR水平，这至少部分解释了RAS转换细胞中C/EBPB的下调。我们正在继续研究ARF的损失如何改变对RA的细胞反应并调节CEBPB基因沉默。我们还使用基因表达分析和功能性RNAi筛选来识别介导C/EBPB胞质效应的靶基因。通过用C/EBPGAMMA的异二聚化来调节C/EBPB活性：C/EBPG是一种普遍表达的蛋白质，优先与C/EBPB和其他C/EBP家族成员优先进行异二聚体。但是，它的生物学功能尚不清楚。我们发现C/EBPG缺陷的MEF降低了增殖潜力。这部分是由于C/EBPG通过形成异二聚体来抑制C/EBPB的生长抑制作用的能力。 C/EBPG基因敲除细胞显示出较高的细胞周期蛋白依赖性激酶（CDK）抑制剂，例如p15，p18和p27，这可能是增殖潜力降低的。据报道，C/EBPG无效小鼠是不可行的。但是，我们发现，当交叉在混合应变背景上时，突变小鼠生存。因此，当前的工作着重于表征成年C/EBPG缺乏小鼠的表型，并研究C/EBPG在体内细胞增殖和肿瘤发生中的作用。由于C/EBPG改变了C/EBPB的生物学活性，因此我们有兴趣识别C/EBPB均和异二聚体的基因组靶标。为此，我们正在开发一种新型的蛋白质互补/亲和力标签（PCAT）方法，以丰富各种C/EBPB二聚体的染色体靶标。原则上，该方法可用于确定任何二聚体DNA结合蛋白的相互作用组。