Role of CEBP transcription factors in cell growth and tumorigenesis

CEBP转录因子在细胞生长和肿瘤发生中的作用

• 批准号: 10262069
• 负责人: peter f johnson
• 金额: $ 150.04万
• 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/10262069
• 关键词: 3' Untranslated Regions; Adaptor Signaling Protein; Antineoplastic Agents; Apoptotic; Automobile Driving; BRAF gene; Binding; Binding Proteins; Biology; Bypass; CCAAT-Enhancer-Binding Protein-beta; Cancer Etiology; Cell Cycle Arrest; Cell Proliferation; Cells; Cellular biology; Complex; Cytoplasm; DNA Binding; Data; Dependence; Development; Elements; Embryo; Endoplasmic Reticulum; Endosomes; Fibroblasts; Genes; Genetic Transcription; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Human; Inflammatory; Interleukin-1; Interleukin-6; KRAS2 gene; KSR gene; Kinetics; Knockout Mice; Knowledge; Laboratories; Link; Lung Neoplasms; MAP Kinase Gene; MAPK3 gene; MEKs; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Messenger RNA; Modeling; Molecular; Mus; Mutate; Mutation; Neoplastic Cell Transformation; Nonsense Codon; Normal Cell; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phase; Phenotype; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Proteins; Proteomics; Proto-Oncogenes; RAS genes; RNA Helicase; RNA-Binding Proteins; Ras/Raf; Regulation; Repression; Research; Resistance; Role; Serum; Signal Pathway; Signal Transduction; Stress; System; TP53 gene; Therapeutic; Transcript; Translational Activation; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; Untranslated Regions; cancer cell; cancer diagnosis; cancer therapy; cell growth; chemokine; cytokine; improved; insight; kinase inhibitor; mRNA Decay; mRNA Transcript Degradation; mutant; neoplastic cell; novel; novel anticancer drug; pancreatic neoplasm; phosphoproteomics; prevent; protein activation; ras Oncogene; response; scaffold; senescence; small molecule; trafficking; transcription factor; treatment strategy; tumor; tumorigenesis; tumorigenic

Cancer occurs when normal checks on cellular proliferation and survival are disrupted by activation of oncogenes and inactivation of tumor suppressors. RAS proto-oncogenes are often mutated in cancers, causing constitutive activation of the protein and dys-regulation of downstream signaling. Loss of tumor suppressor pathways, primarily p53 or RB, renders cells susceptible to transformation by RAS and other oncogenes. Mutations in tumor suppressor genes disrupt apoptotic or senescence responses to oncogenic stress. Acquiring detailed knowledge of these oncogenic and anti-oncogenic pathways is essential to understand how cancers develop and to identify molecular vulnerabilities that can be targeted by novel anti-cancer agents. Our research focuses on the transcription factor C/EBPbeta as a model for elucidating mechanisms by which RAS signaling activates downstream targets. Analysis of Cebpb null mice and mammalian tumor cells revealed that C/EBPb has oncogenic functions and is essential for the development of many cancers. However, in primary cells such as mouse embryo fibroblasts (MEFs), C/EBPb participates in oncogene-induced senescence (OIS). OIS is activated by oncogenic stresses and serves as an intrinsic barricade to tumor development. We are studying how the activity of the C/EBPb protein is controlled by oncogenic RAS signaling and the molecular basis for its dual pro- and anti-tumorigenic functions. C/EBPb is an auto-inhibited protein whose activity can be stimulated by oncogenic RAS signaling through the MEK-ERK cascade. C/EBPb de-repression involves multiple post-translational modifications (PTMs), including phosphorylation by the RAS effector kinases ERK1/2 and CK2. Activated C/EBPb is essential for cell cycle arrest in senescent cells. It also activates transcription of senescence-associated secretory phenotype (SASP) genes, which include pro-inflammatory cytokines/chemokines. An important finding from our lab was the discovery that the 3' untranslated region (3'UTR) of the Cebpb mRNA inhibits C/EBPb activity in tumor cells. This novel mechanism, termed 3'UTR regulation of protein activity (UPA), suppresses the DNA-binding, transcriptional and pro-senescence activities of C/EBPb. UPA requires a 3'UTR sequence containing several G/U rich elements (GREs) and the ARE/GRE-binding protein, HuR. Mechanistically, this system acts by restricting Cebpb mRNA transcripts to the peripheral cytoplasm, excluding them from a perinuclear region where the ERK1/2 and CK2 kinases reside. C/EBPb synthesized in the cell periphery is uncoupled from RAS signaling because it is inaccessible to its activating kinases. By contrast, 3'UTR inhibition and Cebpb mRNA compartmentalization are not observed in senescent primary mouse and human cells. Consequently, in these cells C/EBPb becomes activated by RAS signaling and OIS ensues to suppress tumorigenesis. We have used a proteomics approach to identify additional proteins that bind to the GRE and thus may be involved in Cebpb mRNA trafficking and UPA. One candidate, Upf1, is an RNA helicase involved in nonsense-mediated mRNA decay (NMD). NMD eliminates faulty transcripts that contain premature stop codons. However, Upf1 is also known to regulate degradation of normal mRNAs. We found that in tumor cells, Upf1 localizes to the perinuclear cytoplasm which is devoid of Cebpb transcripts. Depletion of Upf1 in human lung cancer cells increased Cebpb mRNA levels in the perinuclear cytoplasm and stimulated C/EBPb DNA binding. The cells also became senescent and expressed SASP genes such as IL-6 and IL-1 in a C/EBPb-dependent manner. Another GRE interactor, the RNA-binding protein staufen, has also been linked to mRNA localization and decay and acts together with Upf1 to degrade target mRNAs. These and other data indicate that perinuclear Upf1 and staufen, along with cytoplasmic HuR, bind to the Cebpb 3'UTR and promote perinuclear mRNA degradation, thus preventing C/EBPb activation. This C/EBPb-inhibiting system allows senescence bypass in tumor cells. We are currently investigating whether other pro- and anti-oncogenic transcription factors, including p53, are regulated by similar 3'UTR mechanisms. From a therapeutic standpoint, it may be possible to identify small molecules that disrupt the UPA system to activate latent senescence responses in tumor cells. A key aspect of the UPA mechanism involves subcellular compartmentalization of kinases. The RAS effector kinases p-ERK and CK2 become localized to "perinuclear signaling centers" (PSCs) in cells expressing oncogenic RAS or BRAF. We observed PSCs in all human tumor cell lines examined, irrespective of the driving oncogene, and in KRAS-induced mouse lung and pancreatic tumor tissues. These findings suggest that PSCs are a ubiquitous feature of cancer cells. ERK and CK2 PSCs are associated with perinuclear endosomes and require the MAPK scaffold KSR1 (kinase suppressor of Ras 1) for their formation. Accordingly, KSR1 also undergoes RAS-induced perinuclear compartmentalization, where it colocalizes with p-ERK and CK2. Thus, KSR1 plays a key role in the subcellular localization of RAS effector kinases. PSCs are also transiently induced by serum growth factors in normal cells with delayed kinetics (4-6 hr after GF stimulation). Mutant RAS and other oncogenes appear to persistently activate this late phase of GF signaling, localizing effector kinases such as ERK and CK2 to the perinuclear compartment in tumor cells. We propose that kinases tethered to perinuclear endosomes can access key substrates whose phosphorylation promotes neoplastic transformation and tumorigenesis. To understand how kinase localization drives tumorigenesis, we are using phosphoproteomics to identify substrates of PSC kinases. We are also elucidating the molecular basis for RAS-induced PSC formation, including investigating the role of signaling adaptor proteins known to regulate endosomal association with the perinuclear endoplasmic reticulum. Our preliminary data show that one such adapter, Tollip, is essential for CK2 PSC formation and tethers signaling complexes to endosomes via KSR1. Notably, Tollip is required for transformation caused by oncogenic KRAS but not HRAS, even though HRAS also induces PSCs. Thus, HRAS may use a different endosomal adapter protein to anchor downstream kinases to this critical signaling compartment. Normal cells are relatively unaffected by the absence of Tollip. The selective Tollip dependency of KRAS mutant tumors - a particularly lethal class of cancers - underscores the potential utility of developing anti-cancer drugs that inhibit Tollip and other PSC components. Such drugs may be better tolerated and have a wider therapeutic window than has been observed for RAS pathway kinase inhibitors.

当对细胞增殖的正常检查和生存受到激活和肿瘤抑制失活而破坏生存期时，就会发生癌症。 RAS原始基因通常在癌症中突变，从而导致蛋白质的本构激活和下游信号的DYS调节。肿瘤抑制途径（主要是p53或RB）的丧失使细胞容易受到RAS和其他癌基因的转化。肿瘤抑制基因的突变破坏凋亡或衰老对致癌应激的反应。获取有关这些致癌和抗结构途径的详细知识对于了解癌症如何发展和鉴定新型抗癌药物可以瞄准的分子脆弱性至关重要。我们的研究重点是转录因子C/EBPBETA，作为阐明RAS信号传导下游目标的机制的模型。 CEBPB无效小鼠和哺乳动物肿瘤细胞的分析表明，C/EBPB具有致癌功能，对于许多癌症的发展至关重要。但是，在小鼠胚胎成纤维细胞（MEF）等原代细胞中，C/EBPB参与了癌基因诱导的衰老（OIS）。 OI被致癌应力激活，并作为肿瘤发育的内在障碍。我们正在研究C/EBPB蛋白的活性如何受到致癌性RAS信号传导的控制以及其双重促肌和抗氧化函数的分子基础。 C/EBPB是一种自动抑制的蛋白质，可以通过MEK-ERK级联反应通过致癌性RAS信号来刺激其活性。 C/EBPB去抑制涉及多次翻译后修饰（PTM），包括RAS效应子激酶ERK1/2和CK2的磷酸化。激活的C/EBPB对于衰老细胞中的细胞周期停滞至关重要。它还激活与衰老相关的分泌表型（SASP）基因的转录，其中包括促炎性细胞因子/趋化因子。我们实验室的一个重要发现是，发现CEBPB mRNA的3'未翻译区（3'UTR）抑制肿瘤细胞中的C/EBPB活性。这种新型机制称为3'UTR调节蛋白活性（UPA），抑制了C/EBPB的DNA结合，转录和促染色活性。 UPA需要一个3'UTR序列，其中包含几个g/u丰富元素（GRE），并且是/gre-lagning蛋白，hur。从机械上讲，该系统通过将CEBPB mRNA转录物限制在周围细胞质中，将它们排除在ERK1/2和CK2激酶驻留的核周区域中。在细胞外围合成的C/EBPB与RAS信号传导未耦合，因为它无法与其激活激酶相关。相比之下，在衰老的原代小鼠和人类细胞中未观察到3'UTR抑制和CEBPB mRNA分室化。因此，在这些细胞中，C/EBPB被RAS信号传导激活，因此OIS随之而来抑制肿瘤发生。我们已经使用了蛋白质组学方法来鉴定与GRE结合的其他蛋白质，因此可能与CEBPB mRNA运输和UPA有关。一个候选者UPF1是参与胡说八道介导的mRNA衰变（NMD）的RNA解旋酶。 NMD消除了包含过早停止密码子的错误成绩单。但是，已知UPF1调节正常mRNA的降解。我们发现在肿瘤细胞中，UPF1定位于缺乏CEBPB转录本的核周细胞质。在人肺癌细胞中UPF1的耗竭增加了核周细胞质中的CEBPB mRNA水平和刺激的C/EBPB DNA结合。这些细胞还以C/EBPB依赖性方式变成了衰老，并表达了SASP基因，例如IL-6和IL-1。另一个GRE相互作用器，即RNA结合蛋白Staufen，也与mRNA定位和衰减有关，并与UPF1一起起作用，以降解靶标mRNA。这些和其他数据表明，核周UPF1和Staufen以及细胞质HUR结合到CEBPB 3'UTR并促进核周核mRNA降解，从而阻止C/EBPB激活。该C/EBPB抑制系统允许肿瘤细胞衰老旁路。我们目前正在研究其他促和抗疾病转录因子（包括p53）是否受相似的3'UTR机制调节。从治疗的角度来看，可以鉴定出破坏UPA系统以激活肿瘤细胞中潜在衰老反应的小分子。 UPA机制的一个关键方面涉及激酶的亚细胞隔室化。 Ras效应激酶P-ERK和CK2在表达致癌性RAS或BRAF的细胞中定位于“核周信号中心”（PSC）。我们在检查的所有人类肿瘤细胞系中观察到PSC，无论驱动癌基因以及KRAS诱导的小鼠肺和胰腺肿瘤组织。这些发现表明，PSC是癌细胞的普遍特征。 ERK和CK2 PSC与核周内体有关，需要MAPK支架KSR1（RAS 1的激酶抑制剂）以形成。因此，KSR1还经历了RAS诱导的核周分室化，并与P-ERK和CK2共定位。因此，KSR1在RAS效应激酶的亚细胞定位中起关键作用。 PSC还通过延迟动力学的正常细胞（GF刺激后4-6小时）的血清生长因子瞬时诱导。突变的RAS和其他癌基因似乎持续激活GF信号的后期，将效应子激酶（例如ERK和CK2）定位到肿瘤细胞中的核周室中。我们建议，激酶束缚在核周内体上可以访问磷酸化促进肿瘤转化和肿瘤发生的关键底物。为了了解激酶的定位如何驱动肿瘤发生，我们正在使用磷蛋白组学来鉴定PSC激酶的底物。我们还阐明了RAS诱导的PSC形成的分子基础，包括研究已知可调节内体与核周内核网状网状结合的信号转移蛋白的作用。我们的初步数据表明，这样的适配器Tollip对于CK2 PSC形成和Tethers信号复合物通过KSR1至关重要。值得注意的是，即使HRA还诱导了PSC，也需要Tollip进行致癌Kras而不是HRA引起的转化。因此，HRA可以使用不同的内体衔接蛋白将下游激酶锚定在此关键信号室中。正常细胞相对不受托利普的影响。 KRAS突变肿瘤的选择性Tollip依赖性（一种特别致命的癌症）强调了开发抑制Tollip和其他PSC成分的抗癌药物的潜在效用。与RAS途径激酶抑制剂相比，此类药物的耐受性可能更好，并且具有更宽的治疗窗口。