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: 8763075
• 负责人: peter f johnson
• 金额: $ 120.7万
• 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/8763075
• 关键词: 3' Untranslated Regions; Address; Affect; Amino Acids; Anabolism; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Binding Proteins; Binding Sites; Biological Process; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; Carcinogens; Cell Aging; Cell Death; Cell Proliferation; Cells; Cellular Stress; Clinical; Colon Carcinoma; Cytoplasm; Cytostatics; DNA Binding; Databases; Defect; Development; Disabled Persons; EP300 gene; Elements; Ethylnitrosourea; Exhibits; Family member; Fibroblasts; Gene Expression; Genes; Glutathione; 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; Lead; 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; Neoplastic Cell Transformation; Normal Cell; Oncogene Activation; Oncogene Proteins; Oncogenes; Oncogenic; Outcome; Oxidative Stress; Pathway interactions; Phenotype; Phosphotransferases; Play; Post-Translational Protein Processing; Post-Translational Regulation; Premalignant; Protein Family; Proteins; Proto-Oncogenes; Protocols documentation; Reactive Oxygen Species; Regulation; Regulatory Pathway; Research; Resistance; Rodent; Role; Signal Transduction; Site; Skin Neoplasms; Stress; System; Transactivation; Transcript; Transcription Coactivator; Transforming Growth Factor beta; Translational Activation; Translations; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Proteins; Work; activating transcription factor; bZIP Protein; base; biological adaptation to stress; cancer cell; carcinogenesis; cell growth; cell transformation; cellular targeting; chemokine; cytokine; deprivation; dimer; immortalized cell; in vivo; interest; neoplastic cell; new therapeutic target; novel; outcome forecast; prevent; programs; rapid growth; response; senescence; transcription factor; tumor; tumorigenesis; tumorigenic

Cancer development involves aberrant control of cellular proliferation, resulting from activation of oncogenes and inactivation of tumor suppressors. The latter provide an intrinsic barrier to de-regulated growth and cancer by promoting cell death or inducing 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 nearly universally disabled. Loss of tumor suppressor pathways renders cells susceptible to transformation by Ras and other oncogenes by disrupting cell death or senescence responses. 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 roles in cell proliferation and tumorigenesis. Our research focuses primarily on C/EBPbeta as a downstream target of Ras signaling. Studies using Cebpbeta 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/EBPbeta 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/EBPbeta acts to arrest cellular proliferation through a pathway requiring RB:E2F. Thus, C/EBPbeta 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/EBPbeta 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/EBPbeta activity: C/EBPbeta is an intrinsically repressed (auto-inhibited) protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through the Raf-MEK-ERK cascade. C/EBPbeta is auto-inhibited by 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/EBPbeta 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/EBPbeta DNA-binding and homodimerization. We also showed that the activated, homodimeric form of C/EBPbeta is 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. Pro-oncogenic role of C/EBPbeta: Previous studies showed that C/EBPbeta-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/EBPbeta 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/EBPbeta KO mice than in WT animals, probably due to decreased circulating levels of the growth factor IGF-1 in mutant mice. Thus, C/EBPbeta exerts both cell-autonomous and non-cell-autonomous effects on tumorigenesis. Regulation of C/EBPbeta activity by heterodimerization with C/EBPgamma: C/EBPgamma is a ubiquitously-expressed C/EBP protein that heterodimerizes with C/EBPbeta and other C/EBP family members. However, its biological functions are not well understood. We found that C/EBPgamma-deficient MEFs have reduced proliferative potential and display increased replicative senescence in vitro. This defect is partly due to the ability of C/EBPgamma to form heterodimers with C/EBPbeta and suppress its cytostatic activity. C/EBPgamma 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 and are activated by C/EBPbeta homodimers. Furthermore, by analyzing gene expression databases for several human cancers we found that increased CEBPG levels are frequently linked to poorer clinical outcomes, including lung tumors. Depletion of CEBPG in a human lung tumor cell line caused impaired cell growth and increased senescence. Thus, C/EBPgamma positively regulates cell proliferation/survival and may have a critical role in promoting the rapid growth of many tumor cells. We also found that Cebpg-/- MEFs exhibit elevated levels of reactive oxygen species (ROS) and increased apoptosis. This phenotype is due to reduced levels of the cellular antioxidant, glutathione, due to defective expression of genes regulating glutathione biosynthesis. Notably, we observed that C/EBPgamma forms heterodimers with the stress-induced bZIP protein, ATF4. ATF4 is a central regulator of the integrated stress response, which regulates cellular adaptation to several types of stress including oxidative stress, amino acid deprivation, and ER stress. Many stress-induced genes contain binding sites for C/EBP:ATF heterodimers (CARE elements), and it has been widely believed that CARE sites bind C/EBPbeta:ATF4 heterodimers. Our work demonstrates that C/EBPgamma is in fact the critical partner of ATF4 in stressed cells. This regulatory role of C/EBPgamma may also be important for its pro-oncogenic activity. A novel function for the Cebpb 3'UTR in regulating C/EBPbeta protein activity: We recently discovered that the Cebpb 3' untranslated region (3'UTR) inhibits Ras-induced post-translational activation of the C/EBPbeta protein, thereby suppressing its pro-senescence and cytostatic activities in tumor cells. The 3'UTR blocks activation of C/EBPbeta DNA-binding and transcriptional activities that are otherwise induced by oncogenic Ras. The 3'UTR also prevented C/EBPbeta-driven expression of SASP genes, while promoting expression of genes linked to cancers and TGFbeta signaling. The 3'UTR inhibitory effect was mapped to a region bearing A/U rich elements (AREs) and also required the ARE-binding protein, HuR. Notably, these components excluded Cebpb transcripts from a perinuclear region of the cytoplasm where the C/EBPbeta kinases p-ERK1/2 and CK2 reside in Ras-transformed cells. These findings indicate that the intracellular site of C/EBPbeta translation is critical for Ras-induced activation via effector kinases such as p-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/EBPbeta proceeds and OIS can be implemented to suppress tumorigenesis. We 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. 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，这是由于癌基因的激活和抑制肿瘤抑制剂的失活而产生的。后者通过促进细胞死亡或诱导恶性细胞中的永久生长停滞（衰老），为下调生长和癌症提供了内在障碍。 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。肿瘤抑制途径的丧失使细胞通过破坏细胞死亡或衰老反应而易受RAS和其他癌基因转化的细胞。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。我们的实验室研究转录因子的C/EBP（CCAAT/增强子结合蛋白）及其在细胞增殖和肿瘤发生中的作用。我们的研究主要关注C/EBPBETA作为RAS信号的下游目标。使用CEBPBETA NULL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具有亲核功能，对于许多癌症的发展至关重要。但是，在原发性成纤维细胞（MEFS）中，癌基因诱导的衰老（OIS）也需要C/EBPBETA，这是一种内在的肿瘤抑制机制，可防止体外和体内肿瘤转化。在渗透细胞中，C/EBPBETA通过需要RB的途径来阻止细胞增殖：E2F。因此，C/EBPBETA具有促肿瘤活性和抗肿瘤活性。由于它在对RA的细胞反应中起重要作用，因此我们进行了研究，以阐明C/EBPBETA表达及其活性受致癌性RAS信号传导控制的机制，并了解其在抑制癌症和抑制癌症中的双重作用的分子基础。 C/EBPBETA活性的翻译后调节：C/EBPBETA是一种本质上抑制（自动抑制）蛋白的蛋白质，可以通过RAF-MEK-MEK-ERK CASCADE通过致癌Ras或生长因子信号传导刺激其活性。 C/EBPBETA在蛋白质的N末端一半中的三个短区域自动抑制，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/EBPBETA通过几种诱导后翻译后修饰（PTM）通过RAS信号传导激活。我们先前鉴定出亮氨酸拉链中的RSK激酶位点，该位点是C/EBPBETA DNA结合和同构化的重要调节剂。我们还表明，C/EBPBETA激活的同型二聚体形式能够与转录共激活因子P300/CBP关联，并且这种相互作用需要三个N末端自动抑制元件。因此，这些调节序列具有双功能角色。 C/EBPBETA的亲核作用：先前的研究表明，C/EBPBETA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/EBPBETA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。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/EBPBETA 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。因此，C/EBPBETA对肿瘤发生既对肿瘤发生的细胞自主和非细胞自治作用均发挥作用。通过用C/EBPGAMMA异二聚化来调节C/EBPBETA活性：C/EBPGAMMA是一种普遍表达的C/EBP蛋白，可与C/EBPBETA和其他C/EBP家族成员异二聚体。但是，它的生物学功能尚不清楚。我们发现缺乏C/EBPGAMMA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/EBPGAMMA与C/EBPBETA形成异二聚体并抑制其细胞抑制活性的能力。 C/EBPGAMMA基因敲除细胞还显示出与衰老相关的分泌表型（SASP）基因的水平升高，这些表型（SASP）基因编码促炎性细胞因子（例如IL-6，CXCL趋化因子等），这些因子涉及衰老诱导并被C/EBPBETA同型胶合体激活。此外，通过分析几种人类癌症的基因表达数据库，我们发现CEBPG水平升高通常与包括肺部肿瘤在内的较差的临床结局有关。 CEBPG在人肺肿瘤细胞系中的耗竭导致细胞生长受损并增加衰老。因此，C/EBPGAMMA积极调节细胞增殖/存活，并且可能在促进许多肿瘤细胞的快速生长中起关键作用。我们还发现，CEBPG - / - MEF表现出升高的活性氧（ROS）和凋亡增加。该表型是由于细胞抗氧化剂谷胱甘肽的水平降低，这是由于调节谷胱甘肽生物合成的基因表达缺陷所致。值得注意的是，我们观察到C/EBPGAMMA与应力诱导的BZIP蛋白ATF4形成异二聚体。 ATF4是综合应力反应的中心调节剂，它调节细胞适应几种类型的应激，包括氧化应激，氨基酸剥夺和ER应激。许多应激诱导的基因包含C/EBP的结合位点：ATF异二聚体（护理元件），并且人们普遍认为护理位点结合C/EBPBETA：ATF4异二聚体。我们的工作表明C/EBPGAMMA实际上是ATF4在压力细胞中的关键合作伙伴。 C/EBPGAMMA的这种调节作用对于其促疾病的活性也可能很重要。 CEBPB 3'UTR在调节C/EBPBETA蛋白活性中的一个新功能：我们最近发现，CEBPB 3'未翻译区域（3'UTR）抑制RAS诱导的C/EBPBETA蛋白的翻译后激活，从而抑制了其在thumor ciltercentic ciltentic stostictic cilterctic cilterctic cill tamor cy tumor cyor tumor tamor tamor tamor tamor的抑制。 3'UTR阻止了C/EBPBETA 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SASP基因的C/EBPBETA驱动的表达，同时促进了与癌症和TGFBETA信号相关的基因的表达。将3'UTR抑制作用映射到带有A/U富元素（ARES）的区域，并且还需要结合蛋白，HUR。值得注意的是，这些成分排除了CEBPB转录本，来自细胞质的核周区域，其中C/EBPBETA激酶P-ERK1/2和CK2位于Ras转化的细胞中。这些发现表明，C/EBPBETA翻译的细胞内部位对于通过效应激酶（如P-ERK）诱导RAS诱导的激活至关重要。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/eBPBETA的激活可以实施，以抑制肿瘤发生。我们目前正在解决其他抗结构性和致癌蛋白是否受到通过3'UTR序列作用的UPA样机制调节。我们还试图确定3'UTR调节途径的其他组件。我们预计，UPA系统将为旨在重新激活癌细胞内在肿瘤抑制程序的新治疗策略提供一个有吸引力的目标。