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: 8552666
• 负责人: peter f johnson
• 金额: $ 132.71万
• 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/8552666
• 关键词: 3' Untranslated Regions; Address; Affect; Animals; Antineoplastic Agents; Apoptosis; Binding Proteins; Biological Process; Bypass; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; Carcinogens; Cell Aging; Cell Cycle Arrest; Cell Proliferation; Cell Survival; Cells; Clinical; Colon Carcinoma; Cytostatics; DNA Binding; Databases; Defect; Development; Disabled Persons; Down-Regulation; EP300 gene; Elements; Ethylnitrosourea; Exhibits; Family; Family member; Fibroblasts; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Growth; Growth Factor; Heterodimerization; Homodimerization; Human; In Vitro; Incidence; Inflammatory; 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; Mediating; Messenger RNA; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; N-terminal; Neoplastic Cell Transformation; Normal Cell; Oncogene Activation; Oncogenes; Oncogenic; Outcome; Pathway interactions; Phenotype; Phosphotransferases; Play; Post-Translational Protein Processing; Post-Translational Regulation; Premalignant; Protein Family; Proteins; Proto-Oncogenes; Protocols documentation; Regulation; Regulator Genes; Regulatory Pathway; Reporting; Repression; Research; Resistance; Rodent; Role; Signal Transduction; Signal Transduction Pathway; Site; Skin Neoplasms; Structure of thyroid parafollicular cell; System; Transactivation; Transcription Coactivator; Transforming Growth Factor beta; Translational Activation; Translations; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Proteins; Work; base; cancer cell; cancer therapy; carcinogenesis; cell growth; cell transformation; cellular targeting; chemokine; cytokine; dimer; immortalized cell; in vivo; interest; member; neoplastic cell; new therapeutic target; novel; p19ARF; prevent; programs; rapid growth; response; senescence; transcription factor; tumor; tumorigenesis; tumorigenic

The development of cancer involves aberrant control of cellular proliferation, owing mainly to the activation of oncogenes and inactivation of tumor suppressors. The latter proteins provide an intrinsic barrier to de-regulated growth and cancer by inducing apoptosis or permanent growth arrest (senescence) in 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 used to develop novel anti-cancer agents and strategies.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 the role of C/EBPbeta as a downstream effector of Ras signaling. Studies using Cebpb null mice as well as analysis of human and rodent tumor cells have shown that C/EBPb has pro-oncogenic functions and is essential for the development of many cancers. However, in primary fibroblasts (MEFs) C/EBPb is also required for oncogene-induced senescence (OIS), an intrinsic tumor suppression mechanism that prevents neoplastic transformation in vitro and in vivo. In senescing cells, C/EBPb acts to arrest cellular proliferation through 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 have undertaken studies to elucidate the mechanisms by which C/EBPb expression and its activity are controlled by oncogenic Ras signaling and to understand the molecular basis for its dual role in both suppressing and promoting cancer.Post-translational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed (auto-inhibited) protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through the Raf-MEK-ERK signal transduction pathway. 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 folded core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb becomes activated by Ras signaling through several inducible post-translational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper that serves 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, probably due to decreased circulating levels of the growth factor IGF-1 in 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 cell cycle arrest and senescence 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. Our studies indicate that members of the Egr family of transcription factors are critical regulators of CEBPb gene transcription and their levels decrease upon expression of RasV12 in transformed fibroblasts. Regulation of C/EBPb activity by heterodimerization with C/EBPgamma: C/EBPg is a ubiquitously-expressed C/EBP protein that preferentially heterodimerizes with C/EBPb and other family members. However, its biological functions are not well understood. We discovered that C/EBPg-deficient MEFs have reduced proliferative potential and display increased replicative senescence in vitro. This defect is in part due to the ability of C/EBPg to form heterodimers with C/EBPb and suppress its cytostatic activity. C/EBPg knockout cells also display elevated levels of senescence-associated secretory phenotype (SASP) genes, which encode pro-inflammatory cytokines (e.g., IL-6, CXCL chemokines, etc.) that are involved in senescence induction. Furthermore, by analyzing gene expression databases we found that increased CEBPG levels are linked to poorer clinical outcomes in several human cancers, including lung tumors. Accordingly, depletion of CEBPG in a human lung tumor cell line caused impaired cell growth and increased senescence. Thus, C/EBPg is a key regulator of cell proliferation and survival and may have a critical role in promoting the rapid growth of many tumor cells.A novel function for the Cebpb 3'UTR in regulating C/EBPb protein activity: We recently made the unexpected discovery that the Cebpb 3' untranslated region (3'UTR) inhibits Ras-induced post-translational activation of C/EBPb, thereby suppressing its pro-senescence and cytostatic activities specifically in tumor cells. The 3'UTR blocks activation of C/EBPb DNA-binding and transcriptional activities that are otherwise induced by oncogenic Ras. The 3'UTR also prevented the C/EBPb-mediated induction of SASP genes, while promoting expression of genes linked to cancers and TGFbeta signaling. The 3'UTR inhibitory effect was mapped to an A/U rich element (ARE)-containing region and required the ARE-binding protein, HuR. Notably, these components excluded Cebpb mRNA from a perinuclear cytoplasmic region where the C/EBPb kinases p-ERK1/2 and CK2 reside in Ras-transformed cells. These findings indicate that the intracellular site of C/EBPb translation controls de-repression by Ras signaling via effector kinases such as ERK. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were not observed in primary mouse and human fibroblasts. Consequently, in these cells Ras-induced activation of C/EBPb proceeds and OIS is implemented to suppress tumorigenesis. IWe are currently addressing whether other anti-oncogenic and oncogenic proteins are regulated by UPA-like mechanisms acting through 3'UTR sequences. We are also attempting to identify additional components of the 3'UTR regulatory pathway, as we anticipate that the UPA system will provide an attractive target for new therapeutic strategies aimed at reactivating intrinsic tumor suppression programs in cancer cells.

癌症的发展涉及对细胞增殖的异常控制，这主要是由于致癌基因的激活和抑制肿瘤抑制剂的失活。 后一种蛋白质通过诱导凋亡或永久性生长停滞（衰老）在恶性细胞中诱导了下调生长和癌症的内在障碍。 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。 获取各种致癌和抗结合途径的详细知识对于理解癌症如何发展和确定可用于开发新型抗癌剂和策略的肿瘤细胞的独特脆弱性至关重要。 我们的研究主要关注C/EBPBETA作为RAS信号的下游效应子的作用。 使用CEBPB无效小鼠以及对人和啮齿动物肿瘤细胞的分析的研究表明，C/EBPB具有促疾病的功能，对于许多癌症的发展至关重要。 但是，在原代成纤维细胞（MEFS）中，癌基因诱导的衰老（OIS）也需要C/EBPB，这是一种固有的肿瘤抑制机制，可防止体外和体内肿瘤转化。 在渗透细胞中，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。 因为它在细胞对RAS的反应中起着重要作用，所以我们进行了研究，以阐明C/EBPB表达及其活性受致癌性RAS信号控制的控制，并了解其在抑制和促进癌症的c/eBPB活性的c/eBPB的c/ebpb ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins in Is ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins ins：c/ebpb。通过RAF-MEK-ERK信号转导途径刺激致癌RAS或生长因子信号传导。 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。 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通过几种诱导后翻译后修饰（PTM）通过RAS信号传导激活。 我们以前鉴定出亮氨酸拉链中的RSK激酶位点，该位点是C/EBPB DNA结合和均匀化的重要调节剂。 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的疾病作用：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展具有抵抗力。 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生长因子IGF-1的循环水平降低所致。 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 cell cycle arrest and senescence by a mechanism requiring 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。 我们的研究表明，转录因子EGR家族的成员是CEBPB基因转录的关键调节剂，其水平在转化的成纤维细胞中的RASV12表达后降低。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C/EBP蛋白，优先与C/EBPB和其他家庭成员进行异质化。 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，并且在体外表现出了增加的复制性衰老。 该缺陷部分是由于C/EBPG与C/EBPB形成异二聚体并抑制其细胞抑制活性的能力。 C/EBPG基因敲除细胞还显示出与衰老诱导有关的促炎性细胞因子（例如IL-6，CXCL趋化因子等）的衰老相关分泌表型（SASP）基因的升高水平。 此外，通过分析基因表达数据库，我们发现CEBPG水平升高与包括肺部肿瘤在内的几种人类癌症的临床结局较差有关。因此，在人肺肿瘤细胞系中CEBPG的耗竭导致细胞生长受损并增加衰老。 Thus, C/EBPg is a key regulator of cell proliferation and survival and may have a critical role in promoting the rapid growth of many tumor cells.A novel function for the Cebpb 3'UTR in regulating C/EBPb protein activity: We recently made the unexpected discovery that the Cebpb 3' untranslated region (3'UTR) inhibits Ras-induced post-translational activation of C/EBPb, thereby抑制其在肿瘤细胞中专门抑制其促染色性和细胞抑制活性。 3'UTR阻止了C/EBPB DNA结合和转录活性的激活，这些活性是由致癌Ras引起的。 3'UTR还防止了C/EBPB介导的SASP基因的诱导，同时促进了与癌症和TGFBETA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，并需要结合蛋白，HUR。 值得注意的是，这些成分将CEBPB mRNA排除在核周细胞质区域，其中C/EBPB激酶P-ERK1/2和CK2位于Ras转换的细胞中。 这些发现表明，C/EBPB翻译的细胞内部位通过效应子激酶（例如ERK）来控制RAS信号传导的抑制。 值得注意的是，在原代小鼠和人成纤维细胞中未观察到3'UTR抑制和CEBPB mRNA隔室化。 因此，在这些细胞中，RAS诱导的C/EBPB流量的激活和OIS被实施以抑制肿瘤发生。 IWE目​​前正在解决其他抗疾病和致癌蛋白是否受3'UTR序列作用的UPA样机制调节。 我们还试图确定3'UTR调节途径的其他组成部分，因为我们预计UPA系统将为旨在重新激活癌细胞内固有肿瘤抑制程序的新治疗策略提供有吸引力的目标。