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: 8348976
• 负责人: peter f johnson
• 金额: $ 108.98万
• 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/8348976
• 关键词: 3' Untranslated Regions; Accounting; Affect; Animals; Apoptosis; Binding Proteins; Biological Process; Bypass; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; Cancerous; Carcinogens; Cell Aging; Cell Proliferation; Cells; Colon Carcinoma; Cytostatics; DNA Binding; Defect; Development; Disabled Persons; Down-Regulation; EP300 gene; Elements; Ethylnitrosourea; Exhibits; Family; Family member; Fibroblasts; Functional RNA; Gene Expression; Gene Silencing; Gene Targeting; Growth; Growth Factor; Heterodimerization; Homodimerization; Human; In Vitro; Incidence; Insulin-Like Growth Factor I; Interleukin-6; Knock-out; Knockout Mice; Knowledge; Laboratory Study; Leucine Zippers; Link; Lung Neoplasms; Lymphomagenesis; MAPK3 gene; MEKs; Malignant Epithelial Cell; Malignant Neoplasms; Maps; Messenger RNA; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; N-terminal; Normal Cell; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Phosphotransferases; Play; Post-Translational Protein Processing; Post-Translational Regulation; Premalignant; Protein Family; Proteins; Proto-Oncogenes; Protocols documentation; RNA Interference; Regulation; Reporting; Repression; Research; Resistance; Rodent; Role; Signal Transduction; Site; Skin Neoplasms; Stress; System; Therapeutic; Transactivation; Transcription Coactivator; Translations; Tumor Suppression; Tumor Suppressor Proteins; Work; base; cancer cell; carcinogenesis; cell growth; cytokine; dimer; in vivo; neoplastic cell; novel; novel therapeutic intervention; p19ARF; programs; response; senescence; therapy design; 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. Acquiring detailed knowledge of the various 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 purposes. Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) family of transcription factors and 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 human and rodent tumor cells has 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 suppression mechanism in vitro and in vivo. In the context of OIS C/EBPb acts to arrest cellular proliferation by a pathway requiring RB:E2F. C/EBPb thus 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 Ras signaling and understand the basis for C/EBPbs dual activities in suppressing or promoting cancer. Post-translational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through a pathway critically dependent on MEK-ERK signaling. C/EBPb auto-inhibition involves three short regions in the N-terminal half of the protein that, together with sequences at the C terminus, are predicted to fold into a hydrophobic core. The core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb activation by Ras signaling involves several inducible post-translational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper as an important regulator of C/EBPb DNA-binding and homodimerization. We also showed that the activated, homodimeric form of C/EBPb is specifically capable of associating with the transcriptional coactivator p300/CBP, and this interaction requires the three N-terminal auto-inhibitory elements. Thus, these regulatory sequences have bifunctional roles in C/EBPb regulation. 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. For example, C/EBPb knockout mice treated with the carcinogen ENU exhibit impaired lymphomagenesis and reduced incidence/malignancy of a broad spectrum of other cancers compared to WT animals. C/EBPb null mice also develop fewer lung tumors than WT mice in a K-ras-induced carcinogenesis model. Furthermore, mouse colon carcinoma cells grow less efficiently in C/EBPb KO mice than in WT animals, possibly due to decreased circulating levels of IGF-1 in the mutant mice. Thus, C/EBPb exerts both cell-autonomous and non-cell-autonomous effects on tumorigenesis. Tumor suppressor functions of C/EBPb: We have reported 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 Egr family transcription factors and oncogenic Ras signaling decreases Egr levels in transformed fibroblasts, which at least partly explains down-regulation of C/EBPb in these cells. We are continuing to investigate how loss of ARF alters cellular responses to Ras and regulates Cebpb gene silencing. 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 display reduced proliferative potential in vitro. This defect 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 also display elevated levels of proinflammatory cytokines such as IL-6, which are involved in senescence induction and may account for the decreased proliferative potential of C/EBPg-deficient cells. Thus, C/EBPg is a key modulator of C/EBPb cytostatic activity. Role of the Cebpb 3'UTR in regulating C/EBPb protein activity: We recently made the unexpected discovery that the Cebpb 3'UTR inhibits the Ras-induced post-translational of C/EBPb, thus quelling its pro-senescence and cytostatic activities specifically in tumor cells. The 3'UTR suppresses the activation of C/EBPb DNA-binding and transcriptional potential in response to oncogenic Ras signaling. The 3UTR blocked the induction of senescence-associated C/EBPb target genes, while promoting expression of genes linked to cancers and TGFb signaling. The 3'UTR inhibitory effect was mapped to an A/U rich element (ARE)-containing region and requires the ARE-binding protein, HuR. These components also excluded Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPb translation controls de-repression by Ras signaling via effector kinases such as ERK. Notably, 3UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPb activation and OIS to proceed. In summary, our findings reveal a novel mechanism, termed 3'UTR regulation of protein activity or UPA, whereby non-coding mRNA sequences selectively regulate C/EBPb activity and suppress its anti-oncogenic functions. The UPA system may represent an attractive target for novel therapeutic interventions designed to reactivate tumor suppression programs in cancer cells.

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。获取有关各种致癌和抗疾病途径的详细知识对于理解癌症如何发展和鉴定可用于治疗目的的肿瘤细胞的独特脆弱性至关重要。我们的实验室研究转录因子的C/EBP（CCAAT/增强子结合蛋白）家族及其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人和啮齿动物肿瘤细胞的分析表明，C/EBPB具有亲核功能。但是，在表达RASV12的成纤维细胞（MEFS）中，癌基因诱导的衰老（OIS）也需要C/EBPB，这是一种内在的体外和体内固有肿瘤抑制机制。在OIS C/EBPB的背景下，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性RAS信号传导控制的机制，并了解C/EBPBS双重活动在抑制或促进癌症方面的基础。 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RAS或生长因子信号通过途径刺激其活性，这可以通过严格依赖MEK-ERK信号传导来刺激。 C/EBPB自动抑制作用涉及蛋白质N端一半中的三个短区域，这些区域与C末端的序列一起被预测折叠成疏水性核心。核心隔离基本区域和反式激活结构域，抑制DNA结合和反式激活。 RAS信号传导的C/EBPB激活涉及几种诱导后翻译后修饰（PTM）。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。我们还表明，C/EBPB激活的同二聚体形式特别能够与转录共激活因子p300/cbp相关，并且这种相互作用需要三个N末端自动抑制元件。因此，这些调节序列在C/EBPB调节中具有双功能作用。 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。例如，与WT动物相比，用致癌物治疗的C/EBPB敲除小鼠表现出受损的淋巴作用和其他癌症的发病率/恶性。在K-RAS诱导的癌变模型中，C/EBPB无效小鼠的肺部肿瘤也比WT小鼠少。此外，小鼠结肠癌细胞在C/EBPB KO小鼠中的生长效率低于WT动物，这可能是由于突变小鼠中IGF-1的循环水平降低所致。因此，C/EBPB对肿瘤发生既对细胞自主和非细胞自主影响。 C/EBPB的肿瘤抑制功能：我们报告说，RASV12诱导的MEF中的细胞衰老需要C/EBPB，并表明仅C/EBPB过表达就可以通过需要RB的机制诱导衰老样生长，需要RB：E2F。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家族转录因子和致癌性RAS信号传导降低了转化的成纤维细胞中的EGR水平，这至少部分解释了这些细胞中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。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。我们发现缺乏C/EBPG的MEF在体外表现出降低的增殖潜力。该缺陷部分是由于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基因敲除细胞还显示出参与衰老诱导的促炎细胞因子（例如IL-6）的水平升高，可能造成C/EBPG缺陷细胞的增殖潜力降低。因此，C/EBPG是C/EBPB细胞抑制活性的关键调节剂。 CEBPB 3'UTR在调节C/EBPB蛋白活性中的作用：我们最近提出了一个意外的发现，CEBPB 3'UTR抑制了RAS诱导的C/EBPB的翻译后，因此在肿瘤细胞中特别是在肿瘤细胞中抑制其促肾上腺和细胞抑制作用。 3'UTR抑制了C/EBPB DNA结合和转录电位的激活，以响应致癌性RAS信号传导。 3UTR阻止了与衰老相关的C/EBPB靶基因的诱导，同时促进了与癌症和TGFB信号相关的基因的表达。将3'UTR抑制作用映射到含有A/U的元素（IS）区域，需要结合蛋白质，HUR。这些成分还从包含活化的ERK1/2的核周细胞质区域排除了CEBPB mRNA，表明C/EBPB翻译的位点通过ras信号通过效应激酶（例如ERK）来控制RAS信号的抑制。值得注意的是，在原发性成纤维细胞中不存在3UTR抑制和CEBPB mRNA隔室化，从而可以进行RAS诱导的C/EBPB激活和OIS。总而言之，我们的发现揭示了一种新的机制，称为蛋白质活性或UPA的3'UTR调节，在该调节中，非编码mRNA序列选择性地调节C/EBPB活性并抑制其抗恢复功能。 UPA系统可能是旨在重新激活癌细胞抑制肿瘤抑制程序的新型治疗干预措施的有吸引力的目标。