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: 9153546
• 负责人: peter f johnson
• 金额: $ 132.19万
• 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/9153546
• 关键词: 3' Untranslated Regions; Aging; Antineoplastic Agents; Antioxidants; BRAF gene; Binding; Binding Proteins; Biochemistry; Biological Markers; Biological Process; Biology; Birth; Breast Cancer cell line; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; Cancer Cell Growth; Cancer Etiology; Cell Aging; Cell Death; Cell Proliferation; Cells; Cellular Stress; Cellular Stress Response; Cellular biology; Clinical; Complex; Cysteine; Cytoplasm; Cytostatics; DNA Binding; Data; Defect; Development; Disabled Persons; Drug Targeting; Economic Inflation; Elements; Endosomes; Exhibits; Family member; Fibroblasts; Future; Gene Expression; Gene Expression Profiling; Genes; Genomics; Glutathione; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Heterodimerization; Homodimerization; Human; Hypoxia; In Vitro; Kinetics; Knock-out; Knockout Mice; Knowledge; Laboratories; Laboratory Study; Leucine Zippers; Link; Lung; Lung Neoplasms; MAP Kinase Gene; MAPK3 gene; MEKs; Malignant - descriptor; Malignant Neoplasms; Maps; Mediating; Melanoma Cell; Messenger RNA; Molecular; Monitor; Mus; Mutate; N-terminal; Neoplastic Cell Transformation; Nutrient; Oncogene Activation; Oncogenes; Oncogenic; Outcome; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Phase; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Premalignant; Protein Family; Proteins; Proto-Oncogenes; Ras/Raf; Regulation; Research; Resistance; Respiratory Failure; Response Elements; Rodent; Role; Signal Pathway; Signal Transduction; Site; Solid Neoplasm; Stress; Structure; Transactivation; Transcript; Transforming Growth Factor beta; Translational Activation; Translations; Tumor Suppression; Tumor Suppressor Proteins; Work; activating transcription factor; base; biological adaptation to stress; cancer cell; cancer gene expression; cell growth; cell transformation; deprivation; factor C; human KSR protein; in utero; in vivo; insight; neoplastic cell; novel; prevent; ras Oncogene; response; scaffold; senescence; targeted cancer therapy; transcription factor; transmission process; tumor; tumorigenesis; tumorigenic

Cancer involves aberrant control of cellular proliferation, resulting from activation of oncogenes and inactivation of tumor suppressors. The latter provide an intrinsic barrier to cell 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 and its role 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. 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 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). C/EBPbeta was previously shown to be phosphorylated by activated ERK kinase, and we identified a RSK kinase site in the leucine zipper that serves as an important regulator of C/EBPbeta DNA-binding and homodimerization. We have also mapped a CK2 phosphorylation site that is required for Ras-induced DNA binding. An important finding from our lab was the discovery 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. Hence, C/EBPbeta is uncoupled from Ras signaling and fails to undergo phosphorylation and activation by ERK and CK2. 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 anticipate that UPA-like mechanisms may regulate many proteins to coordinate cellular responses to Ras signaling. We are currently investigating this possibility by studying the regulation of other pro-oncogenic and anti-oncogenic transcription factors by their 3'UTR sequences. In cells expressing oncogenic Ras, p-ERK and CK2 are present in structures that we call "perinuclear signaling complexes" or PSCs. PSCs are associated with endosomes and require the MAPK scaffold, KSR1 (kinase suppressor of Ras 1). Our research shows that in addition to its known ability to facilitate Raf-MEK-ERK signal transmission, KSR1 plays a key role in directing perinuclear localization of Ras effector kinases. We found that PSCs also form in response to growth factor signals, but with delayed kinetics (4-6 hr after GF stimulation). We propose that oncogenic Ras signaling mirrors this late phase of GF signaling in which effector kinases become localized to a perinuclear compartment, where they access key substrates such as C/EBPbeta in a UPA-regulated manner. We have observed similar localized signaling complexes in several kinds of human tumor cells and in KRas-induced mouse lung tumors, suggesting that PSCs are a ubiquitous feature of the signaling landscape in cancer cells. In the future, PSC components may prove to be effective targets for cancer therapies. Our preliminary data from BRAF-driven human melanoma cells indicates the potential utility of PSCs as a biomarker to monitor the efficacy of anti-cancer drugs targeting the Ras-Raf pathway. In a separate project we are investigating the regulatory and biological functions of the small C/EBP family member, C/EBPgamma. C/EBPgamma is a dimeric partner of C/EBPbeta, and one of its roles is to modulate the activity of C/EBPbeta through heterodimerization. Cebpg knockout MEFs display severe proliferative defects, increased senescence, and elevated expression of senescence-associated secretory phenotype (SASP) genes, effects that are at least partly due to increased levels of C/EBPbeta homodimers. Cebpg KO cells also exhibit oxidative stress that was linked to defective synthesis of the cellular anti-oxidant, glutathione. The growth defects in these cells were reversed by addition of the anti-oxidant, N-acetyl cysteine (NAC). Many adverse conditions, including redox imbalances and ER stress, induce the bZIP transcription factor ATF4, which serves as a "master" regulator of many cellular stress responses. We found that ATF4:C/EBPgamma heterodimeric complexes are induced in stressed cells and bind to genomic C/EBP:ATF response elements (CAREs), which regulate numerous stress response genes. Our studies have identified C/EBPgamma as a novel and essential C/EBP partner of ATF4. Cebpg knockout mice die shortly after birth due to defective lung inflation and respiratory failure. These defects could be substantially reversed by in utero administration of NAC to alleviate oxidative stress. C/EBPgamma also has an important role in cancer, and gene expression analysis suggests correlations between elevated CEBPG mRNA levels and increased malignancy in several human cancers. Furthermore, depletion of C/EBPgamma led to senescence and oxidative stress in human lung and breast tumor cell lines. Cebpg-/- mice also develop significantly fewer malignant solid tumors than WT mice upon aging, consistent with the anti-oxidant functions of C/EBPgamma promoting cancer. In summary, our data indicate that cancer cells rely on C/EBPgamma:ATF heterodimers to regulate genes involved in mitigating stresses arising from increased ROS, hypoxia, and nutrient deprivation.

Cancer involves aberrant control of cellular proliferation, resulting from activation of oncogenes and inactivation of tumor suppressors.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），为细胞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。 Our research focuses primarily on C/EBPbeta and its role as a downstream target of Ras signaling.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是一种本质上抑制（自动抑制）蛋白，其活性可以通过RAF-MEK-MEK-ERK CASCADE通过致癌性RA或生长因子信号刺激。 C/EBPBETA受到蛋白质N末端一半的三个短区域的抑制，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。 C/EBPBETA先前被活化的ERK激酶磷酸化，并且我们在Leucine拉链中鉴定了一个RSK激酶位点，该位点是C/EBPBETA DNA结合和同型二聚化的重要调节剂。 We have also mapped a CK2 phosphorylation site that is required for Ras-induced DNA binding.我们实验室的一个重要发现是，发现CEBPB 3'未翻译区域（3'UTR）抑制了C/EBPBETA蛋白的RAS诱导的翻译后激活，从而抑制了其促肿瘤细胞中的促染色和细胞抑制活性。 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。 Hence, C/EBPbeta is uncoupled from Ras signaling and fails to undergo phosphorylation and activation by ERK and CK2.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。 We anticipate that UPA-like mechanisms may regulate many proteins to coordinate cellular responses to Ras signaling.我们目前正在通过研究其他3'UTR序列来调节其他促疾病的转录因子，从而研究这种可能性。 In cells expressing oncogenic Ras, p-ERK and CK2 are present in structures that we call "perinuclear signaling complexes" or PSCs. PSCs are associated with endosomes and require the MAPK scaffold, KSR1 (kinase suppressor of Ras 1).我们的研究表明，除了其已知促进RAF-Mek-ERK信号传播的能力外，KSR1还在指导RAS效应激酶的核周定位方面起着关键作用。 We found that PSCs also form in response to growth factor signals, but with delayed kinetics (4-6 hr after GF stimulation).我们提出，致癌性RAS信号反映了GF信号的后期，在该阶段中，效应子激酶位于核周室中，它们以UPA调节的方式访问诸如C/EBPBETA之类的关键基板。我们已经观察到在几种人类肿瘤细胞和KRAS诱导的小鼠肺肿瘤中相似的局部信号传导复合物，这表明PSC是癌细胞信号景观的无处不在特征。 In the future, PSC components may prove to be effective targets for cancer therapies.我们来自BRAF驱动的人类黑色素瘤细胞的初步数据表明，PSC作为生物标志物的潜在效用，以监测针对RAS-RAF途径的抗癌药物的功效。 In a separate project we are investigating the regulatory and biological functions of the small C/EBP family member, C/EBPgamma. C/EBPgamma is a dimeric partner of C/EBPbeta, and one of its roles is to modulate the activity of C/EBPbeta through heterodimerization. CEBPG基因敲除MEF显示出严重的增殖缺陷，衰老增加以及衰老相关分泌表型（SASP）基因的表达升高，这些作用至少部分是由于C/EBPBETA同型二聚体水平升高所致。 Cebpg KO cells also exhibit oxidative stress that was linked to defective synthesis of the cellular anti-oxidant, glutathione. The growth defects in these cells were reversed by addition of the anti-oxidant, N-acetyl cysteine (NAC).许多不利条件，包括氧化还原失衡和ER应力，都会引起BZIP转录因子ATF4，它是许多细胞应力反应的“主”调节剂。我们发现ATF4：C/EBPGAMMA异二聚体复合物在应力细胞中诱导并与基因组C/EBP结合：ATF响应元件（CARES），该元件（CARES）调节了许多应力响应基因。 Our studies have identified C/EBPgamma as a novel and essential C/EBP partner of ATF4. Cebpg knockout mice die shortly after birth due to defective lung inflation and respiratory failure. These defects could be substantially reversed by in utero administration of NAC to alleviate oxidative stress. C/EBPGAMMA在癌症中也具有重要作用，基因表达分析表明CEBPG mRNA水平升高与几种人类癌症的恶性肿瘤之间的相关性。 Furthermore, depletion of C/EBPgamma led to senescence and oxidative stress in human lung and breast tumor cell lines. CEBPG - / - 小鼠在衰老后的恶性实体瘤的发生率也明显少于WT小鼠，这与促进癌症的C/EBPGAMMA的抗氧化功能一致。总而言之，我们的数据表明，癌细胞依赖于C/EBPGAMMA：ATF异二聚体来调节与ROS，缺氧和营养剥夺增加有关的减轻应激所涉及的基因。