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: 9556264
• 负责人: peter f johnson
• 金额: $ 138.66万
• 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/9556264
• 关键词: 3' Untranslated Regions; Adaptor Signaling Protein; Antineoplastic Agents; Apoptotic; BRAF gene; Binding Proteins; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Cancer Etiology; Cell Death; Cell Proliferation; Cells; Cellular biology; Complex; Cytoplasm; Cytostatics; DNA Binding; Development; Disabled Persons; Elements; Endosomes; Fibroblasts; Future; Genetic Transcription; Goals; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Homodimerization; Human; Hydrophobicity; KRAS2 gene; KSR gene; Kinetics; Knockout Mice; Knowledge; Laboratories; Laboratory Study; Leucine Zippers; Location; Lung Neoplasms; MAP Kinase Gene; MAPK3 gene; MEKs; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Molecular; Molecular Biology; Monitor; Mus; Mutate; Mutation; N-terminal; Normal Cell; Nuclear; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Peripheral; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Premalignant Cell; Protein Family; Proteins; Proto-Oncogenes; Ras/Raf; Regulation; Research; Resistance; Rodent; Role; Serum; Signal Pathway; Signal Transduction; Site; Stress; Structure; TP53 gene; Tissue Sample; Transactivation; Transcript; Translating; Translational Activation; Translations; Tumor Suppression; Tumor Suppressor Proteins; Tumorigenicity; cancer cell; cancer diagnosis; cancer therapy; cell growth; cell transformation; improved; insight; mutant; neoplastic cell; novel; novel marker; protein activation; ras Oncogene; response; ribosomal protein S6 kinase kinase; scaffold; senescence; targeted cancer therapy; trafficking; transcription factor; tumor; tumorigenesis; tumorigenic

Cancer involves aberrant control of cellular proliferation due to activation of oncogenes and inactivation of tumor suppressors. Tumor suppressors 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, since such mutations disrupt apoptotic or senescence responses to oncogenic stress. 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 targeted by novel anti-cancer 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. Analysis of Cebpb null mice and 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 mouse fibroblasts (MEFs), C/EBPb is also required for oncogene-induced senescence (OIS). In senescing cells, C/EBPb arrests 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 are studying the mechanisms by which C/EBPb expression and activity are controlled by oncogenic RAS signaling and the molecular basis for its dual role in suppressing and promoting cancer. 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 cascade. C/EBPb 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 the DNA-binding and transactivation functions of C/EBPb. C/EBPb becomes activated by RAS signaling via several inducible post-translational modifications (PTMs). C/EBPb was previously shown to be phosphorylated by ERK kinase. We identified a RSK kinase site in the leucine zipper that regulates C/EBPb DNA binding and homodimerization, and recently mapped a CK2 phosphorylation site 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/EBPb protein, thereby suppressing its pro-senescence and cytostatic activities in tumor cells. The 3'UTR blocks the DNA-binding and transcriptional activities of C/EBPb, which are otherwise induced by oncogenic RAS. The 3'UTR inhibitory effect was mapped to a region bearing G/U rich elements (GREs), and required the ARE/GRE-binding protein, HuR. These components act by directing Cebpb transcripts to the peripheral cytoplasm, excluding them from a perinuclear region where the C/EBPb kinases p-ERK1/2 and CK2 reside in RAS-transformed cells. In this location, newly-translated C/EBPb is uncoupled from RAS signaling and fails to undergo phosphorylation and activation by ERK and CK2. Thus, the intracellular site of C/EBPb translation is critical for RAS-induced activation of the protein via effector kinases such as p-ERK. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization are not observed in primary mouse and human fibroblasts. Consequently, RAS-induced activation of C/EBPb is permitted 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 whether the activities of other pro-oncogenic and anti-oncogenic transcription factors are controlled by 3'UTR sequences. In addition, we are identifying proteins (in addition to HuR) that regulate Cebpb mRNA trafficking and are required for UPA regulation. In cells expressing oncogenic RAS or BRAF, p-ERK and CK2 become re-localized to nuclear-proximal structures we call "perinuclear signaling complexes" or PSCs. PSCs are associated with endosomes and require the MAPK scaffold KSR1 (kinase suppressor of Ras 1) for their formation. KSR1 also undergoes RAS-induced targeting to PSCs and colocalizes with p-ERK and CK2. Thus, besides its known ability to facilitate RAF-MEK-ERK signaling, KSR1 plays a key role in regulating subcellular localization of RAS effector kinases. We found that PSCs are also induced by serum growth factors in normal cells with delayed kinetics (4-6 hr after GF stimulation). We propose that mutant RAS constitutively activates this late phase of GF signaling, where effector kinases become localized to a perinuclear compartment and access key substrates that drive cell proliferation. We have detected PSCS in several kinds of human tumor cells and in KRAS-induced mouse lung tumors, suggesting that this localized complexes are a ubiquitous feature of the cancer signaling landscape. Our current studies are aimed at elucidating the molecular basis for RAS-induced formation of PSCs, focusing on the role of signaling adaptor proteins associated with specific classes of perinuclear endosomes. In the future, PSC components may prove to be effective targets for cancer therapy, as well as providing novel biomarkers to identify tumor cells in tissue samples. In addition, PSCs could be used to monitor tumor responses to anti-cancer drugs that inhibit the RAS-ERK pathway.

癌症涉及因癌基因激活和肿瘤抑制失活而导致细胞增殖的异常控制。肿瘤抑制剂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）提供了固有的障碍。 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易受RAS和其他肿瘤基因转化的转化，因为这种突变破坏了对肿瘤应激的凋亡或衰老反应。获取有关各种致癌和抗结构途径的详细知识对于理解癌症如何发展和鉴定肿瘤细胞的独特脆弱性至关重要，而肿瘤细胞的独特脆弱性可以由新颖的反癌策略瞄准。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及其作为RAS信号的下游目标的作用。对CEBPB无效小鼠以及人类和啮齿动物肿瘤细胞的分析表明，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，在原代小鼠成纤维细胞（MEF）中，癌基因诱导的衰老（OIS）也需要C/EBPB。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。由于它在对RA的细胞反应中起重要作用，因此我们正在研究C/EBPB表达和活性受致癌性RAS信号传导控制的机制，以及其在抑制和促进癌症中双重作用的分子基础。 C/EBPB是一种本质上抑制（自动抑制）蛋白的蛋白质，可以通过RAF-Mek-ERK级联反应通过致癌Ras或生长因子信号传导刺激其活性。 C/EBPB受到蛋白质N末端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C/EBPB的DNA结合和反式激活功能。 C/EBPB通过几种诱导后翻译后修饰（PTM）激活RAS信号。 C/EBPB先前被证明被ERK激酶磷酸化。我们在亮氨酸拉链中鉴定了一个RSK激酶位点，该位点调节C/EBPB DNA结合和同构化，并且最近绘制了RAS诱导的DNA结合所需的CK2磷酸化位点。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/EBPB蛋白的RAS诱导的翻译后激活，从而抑制了其在肿瘤细胞中的启发性和细胞稳定活性。 3'UTR阻止了C/EBPB的DNA结合和转录活性，否则它们是由致癌Ras诱导的。将3'UTR抑制作用映射到带有g/u富元素（GRES）的区域，并需要res/gre-lage蛋白，hur。这些组件通过将CEBPB转录物引导到周围细胞质，将它们排除在核周区域，其中C/EBPB激酶P-ERK1/2和CK2位于RAS转化的细胞中。在此位置，新翻译的C/EBPB与RAS信号脱在一起，无法通过ERK和CK2进行磷酸化和激活。因此，C/EBPB翻译的细胞内部位对于通过效应激酶（例如P-ERK）诱导RAS诱导的蛋白质激活至关重要。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分室化。因此，允许RAS诱导的C/EBPB激活，并可以实施OIS来抑制肿瘤发生。我们预计UPA样机制可能会调节许多蛋白质以协调对RAS信号的细胞反应。我们目前正在研究其他亲核和抗结构转录因子的活性是否由3'UTR序列控制。此外，我们正在确定调节CEBPB mRNA运输的蛋白质（除HUR），并且是UPA调节所必需的。在表达致癌性RAS或BRAF的细胞中，P-ERK和CK2被重新定位到我们称为“核核信号传导复合物”或PSC的核能结构。 PSC与内体相关，需要MAPK支架KSR1（RAS 1的激酶抑制器）进行形成。 KSR1还经历了RAS诱导的靶向PSC，并与P-ERK和CK2共定位。因此，除了其已知的促进Raf-Mek-ERK信号传导的能力外，KSR1在调节RAS效应激酶的亚细胞定位方面起着关键作用。我们发现，PSC也由延迟动力学的正常细胞中的血清生长因子诱导（GF刺激后4-6小时）。我们提出，突变的RAS组成型激活GF信号的后期，其中效应激酶始于核周室，并访问驱动细胞增殖的关键基板。我们已经在几种人肿瘤细胞和KRAS诱导的小鼠肺肿瘤中检测到PSC，这表明这种局部复合物是癌症信号景观的无处不在的特征。我们目前的研究旨在阐明RAS诱导的PSC形成的分子基础，重点是与特定类别的核周内体相关的信号转移蛋白的作用。将来，PSC成分可能被证明是癌症治疗的有效靶标，并且提供了新型的生物标志物来鉴定组织样品中的肿瘤细胞。此外，PSC可用于监测抑制RAS-ERK途径的抗癌药物的肿瘤反应。