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: 7733000
• 负责人: PETER JOHNSON
• 金额: $ 136.65万
• 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/7733000
• 关键词: Acetylation; Adopted; Affect; Animals; Apoptosis; Binding; Biological; Bone Density; Bypass; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-gamma; CDKN2A gene; Carcinogens; Cell Aging; Cell Cycle Arrest; Cell Proliferation; Cells; Cellular Morphology; Class; Colon Carcinoma; Complex; DNA Binding; Defect; Development; Dimerization; Diploidy; Dominant-Negative Mutation; Down-Regulation; E2F1 gene; Environment; Event; Family member; Fatty acid glycerol esters; Fibroblasts; Frequencies; Genes; Goals; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Heterodimerization; Hormones; Human; Incidence; Insulin; Insulin-Like Growth Factor I; Knockout Mice; Laboratories; Leptin; Leucine Zippers; Lung Neoplasms; Lymphoma; Lysine; Malignant Neoplasms; Mediating; Medical Surveillance; Methylation; Mitotic; Modification; Mus; Mutagenesis; Mutation; N-terminal; NIH 3T3 Cells; Oncogene Activation; Oncogenes; Oncogenic; Pathway interactions; Phenotype; Phosphorylation Site; Phosphotransferases; Physiological; Post-Translational Protein Processing; Post-Translational Regulation; Premalignant; Proliferating; Property; Protein Family; Proteins; Proto-Oncogenes; RBL2 gene; Regulation; Repression; Research; Resistance; Role; Signal Transduction; Site; Skin Neoplasms; Specificity; Structure; TP53 gene; Transactivation; Transplantation; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Proteins; Tumorigenicity; United States National Institutes of Health; Work; arginyllysine; autocrine; c-myc Genes; cancer cell; cell growth; cell growth regulation; cyclin A2; dimer; enhancer binding protein; human RBL2 protein; immortalized cell; in vivo; interest; leukemia; macrophage; monocyte; mutant; neoplastic cell; p19ARF; response; senescence; transcription factor; tumor; tumor progression; tumorigenesis; tumorigenic

Cancers arise when cells escape intrinsic controls on proliferation due to activation of oncogenes, together with loss/inactivation of tumor suppressors that normally promote apoptosis or senescence of pre-malignant cells. Proto-oncogenes encoding Ras GTPases are frequently activated in cancer cells and the p53 and RB tumor suppressor pathways are disrupted in nearly all tumors. Elucidating the components of these oncogenic and anti-oncogenic pathways is essential for understanding how cancers develop and thus to identify unique vulnerabilities of tumor cells that can be exploited therapeutically. Our laboratory is interested in the C/EBP (CCAAT/enhancer binding protein) family of bZIP transcription factors, especially their roles in regulating cell proliferation and tumorigenesis. Our research focuses on the function and regulation of C/EBPbeta as a downstream effector of Ras. Our studies have also implicated C/EBPb in oncogene-induced senescence (OIS) in primary mouse fibroblasts, where it acts together with RB:E2F complexes to inhibit proliferation of cells expressing oncogenic Ras. C/EBPb may therefore possess both pro- and anti-tumorigenic properties. We seek to understand these opposing functions in more mechanistic detail and to illuminate their contributions to cancer development or tumor suppression in vivo. Role of C/EBPb in tumorigenesis: Work from our laboratory and others revealed a critical role for C/EBPb in the development of certain cancers. For example, C/EBPb-deficient mice are completely resistant to skin tumors induced by carcinogens that cause Ras mutations. C/EBPb is also essential for Myc/Raf-induced transformation of monocyte/macrophages and is required for growth factor-independent suppression of apoptosis (self-sufficiency), a hallmark of leukemias and other tumor cells. We identified the IGF-1 gene as a transcriptional target of C/EBPb in transformed macrophages and demonstrated that the survival defect of C/EBPb-deficient cells is caused by impaired autocrine IGF-1 signaling. To determine whether C/EBPb affects other kinds of cancers we are investigating the consequences of C/EBPb deficiency on ENU-induced tumorigenesis in mice. Thus far the results indicate that C/EBPb KO animals develop fewer lymphomas than WT mice and show reduced incidence/malignancy of several other cancers. To extend these findings we are crossing the C/EBPb KO mice to a latent activatable K-ras strain that develops lung tumors at high frequency. Preliminary results show that animals lacking C/EBPb develop fewer and less malignant tumors. Finally, we are examining the expression and function of C/EBPb in human tumor cell lines to extend our observations to human cancers. C/EBPb regulates systemic insulin/IGF-1 and the tumor environment: To further investigate regulation of IGF-1 by C/EBPb, we examined IGF-1 levels and related physiological phenotypes of C/EBPb KO mice. The mutant animals display decreased fat content and bone density and contain reduced circulating levels of IGF-1, insulin and leptin. Transplanted colon cancer cells showed diminished tumorigenicity in C/EBPb null mice compared to WT animals, indicating that C/EBPb contributes to a more favorable tumor environment. This effect may be due to the lower levels of tumor-promoting growth factors and hormones in C/EBPb-deficient mice. Our findings indicate that C/EBPb promotes tumor development through both cell-autonomous and non-cell-autonomous mechanisms. Role of C/EBPb in cellular senescence: In many primary (non-immortalized) cells, elevated levels of activated Ras or other oncogenes induce senescence, a stable form of cell cycle arrest that requires induction of the ARF-p53 and p16Ink4a-RB tumor suppressor pathways. Recent work indicates that oncogene-induced senescence (OIS) serves as a bonafide tumor surveillance mechanism in vivo. We found that C/EBPb null MEFs fail to undergo Ras-mediated cell cycle arrest, demonstrating a requirement for C/EBPb in RasV12-induced cellular senescence. In addition, C/EBPb over-expression in normal MEFs or human diploid fibroblasts induces cell cycle arrest and a senescent cellular morphology. In contrast, expression of C/EBPb in cells lacking all three RB family members (pRb, p107 and p130) or expressing dominant negative E2F1 stimulates proliferation. These findings indicate that RB:E2F complexes act in concert with C/EBPb to regulate growth of fibroblasts and possibly other cells. Consistent with this idea, C/EBPb binds to and represses E2F-regulated genes such as c-Myc and cyclin A2. Whereas oncogenic Ras provokes premature senescence in primary fibroblasts, it transforms immortalized cells such as NIH 3T3 fibroblasts, which carry a deletion that removes the p16, p15 and p19Arf tumor suppressors. We found that Ras increases C/EBPb levels in MEFs but silences C/EBPb expression in NIH 3T3 cells. This difference was traced to p19Arf, since re-expression of Arf but not p16 or p53 restored C/EBPb levels in Ras-expressing NIH 3T3 cells. Ras also decreased C/EBPb expression in p16/p19Arf null MEFs. Forced expression of C/EBPb blocked Ras-induced transformation and proliferation, showing that C/EBPb is capable of inducing senescence in NIH 3T3 cells. These observations indicate that C/EBPb is a p53-independent target of the Arf tumor suppressor pathway in fibroblasts, and Ras-induced down-regulation of C/EBPb may be an important event in bypassing OIS and allowing tumor progression in some cell contexts. Post-translational regulation of C/EBPb activity: Because C/EBPb mediates cellular responses to oncogenic Ras, we are investigating post-translational regulation of C/EBPb activity by Ras signaling. We have found that the DNA-binding activity of C/EBPb is auto-inhibited and becomes activated in cells expressing oncogenic Ras or stimulated with growth factors. C/EBPb auto-inhibition requires three regions in the N-terminal half of the protein, including the transactivation domain, which are predicted to adopt secondary structure. Mutagenesis studies support the idea that these auto-inhibitory sequences participate in folding interactions that reciprocally inhibit C/EBPb DNA-binding and transactivation functions. A parallel objective is to characterize Ras-induced post-translational modifications (PTMs) on C/EBPb that mediate signal-dependent de-repression. We identified a RSK kinase site in the leucine zipper that serves as an important regulator of C/EBPb DNA-binding activity and also modulates dimerization specificity. We are presently investigating how other phosphorylation sites and different classes of modifications such as lysine acetylation and Lys/Arg methylation control C/EBPb DNA-binding, dimerization, and transactivation function. Ultimately we seek to determine how specific PTMs affect the biological and oncogenic properties of C/EBPb. Involvement of C/EBPgamma in cell growth regulation through heterodimerization with C/EBPb: C/EBPg is a ubiquitously-expressed protein that forms heterodimers with other C/EBP family members, including C/EBPb. We are investigating its role in cell growth regulation by analyzing C/EBPg-deficient mice and cells derived from these animals. C/EBPg-deficient MEFs proliferate very poorly in culture and express reduced levels of pro-mitotic r [summary truncated at 7800 characters]

当细胞因癌基因的激活而逃脱固有的对照时，癌症就会出现，以及通常促进肿瘤抑制子的丧失/灭活，通常会促进细胞凋亡或衰老前的细胞。编码RAS GTPase的原始基因在癌细胞中经常激活，并且在几乎所有肿瘤中，p53和RB肿瘤抑制途径都被破坏。阐明这些致癌性和抗疾病途径的成分对于理解癌症如何发展，从而确定可以通过治疗方法剥削的肿瘤细胞的独特脆弱性至关重要。我们的实验室对BZIP转录因子的C/EBP（CCAAT/增强子结合蛋白）家族感兴趣，尤其是它们在调节细胞增殖和肿瘤发生中的作用。我们的研究重点是C/EBPBETA作为RAS下游效应子的功能和调节。我们的研究还暗示了c/eBPB在原代小鼠成纤维细胞中致癌基因诱导的衰老（OIS）中，其中它与RB：E2F复合物一起作用以抑制表达致癌性Ras的细胞的增殖。因此，C/EBPB可能具有促肿瘤和抗肿瘤特性。我们试图以更机械的细节来理解这些相反的功能，并阐明它们对体内癌症发展或抑制肿瘤的贡献。 C/EBPB在肿瘤发生中的作用：我们实验室和其他人的作品揭示了C/EBPB在某些癌症发展中的关键作用。例如，C/EBPB缺乏的小鼠完全抵抗引起RAS突变的致癌物诱导的皮肤肿瘤。 C/EBPB对于MYC/RAF诱导的单核细胞/巨噬细胞的转化也是必不可少的，对于非生长因子非依赖性抑制细胞凋亡（自给自足），白血病和其他肿瘤细胞的标志所必需。我们将IGF-1基因确定为转化巨噬细胞中C/EBPB的转录靶标，并证明C/EBPB缺陷型细胞的存活缺陷是由自分泌IGF-1信号传导受损引起的。 为了确定C/EBPB是否影响其他类型的癌症，我们正在研究C/EBPB缺乏对小鼠ENU诱导的肿瘤发生的后果。到目前为止，结果表明C/EBPB KO动物的淋巴瘤比WT小鼠少，并且显示出其他几种癌症的发病率/恶性肿瘤。为了扩展这些发现，我们将C/EBPB KO小鼠越过了潜在的可激活K-RAS菌株，该菌株以高频发展肺部肿瘤。初步结果表明，缺乏C/EBPB的动物出现较少，恶性肿瘤。最后，我们正在研究人类肿瘤细胞系中C/EBPB的表达和功能，以将我们的观察结果扩展到人类癌症。 C/EBPB调节系统性胰岛素/IGF-1和肿瘤环境：为了进一步研究C/EBPB对IGF-1的调节，我们检查了C/EBPB KO小鼠的IGF-1水平以及相关的生理表型。突变动物显示脂肪含量和骨密度降低，并含有循环水平降低的IGF-1，胰岛素和瘦素。与WT动物相比，移植的结肠癌细胞在C/EBPB无效小鼠中显示出肿瘤性降低，表明C/EBPB有助于更有利的肿瘤环境。这种影响可能是由于C/EBPB缺陷型小鼠的肿瘤生长因子和激素的水平较低。我们的发现表明，C/EBPB通过细胞自主和非细胞自治机制促进肿瘤的发育。 C/EBPB在细胞衰老中的作用：在许多原发性（非弱化）细胞中，活化的Ras或其他癌基因的水平升高会诱导衰老，这是一种稳定的细胞周期停滞形式，需要诱导ARF-P53和P16INK4A-RB TUMOR抑制途径。 最近的工作表明，癌基因诱导的衰老（OIS）在体内充当真正的肿瘤监测机制。我们发现C/EBPB无效MEF无法进行RAS介导的细胞周期停滞，这表明RASV12诱导的细胞衰老的C/EBPB需要。此外，正常MEF或人二倍体成纤维细胞中的C/EBPB过表达会诱导细胞周期停滞和衰老的细胞形态。相反，在缺乏所有三个RB家族成员（PRB，P107和P130）或表达显性负E2F1的细胞中C/EBPB的表达刺激了增殖。这些发现表明，RB：E2F复合物与C/EBPB一致作用，以调节成纤维细胞和其他细胞的生长。 与这个想法一致，C/EBPB与E2F调节的基因（例如C-Myc和Cyclin A2）结合并抑制。尽管致癌性RA会激发原代成纤维细胞中的过早衰老，但它会改变永生的细胞，例如NIH 3T3成纤维细胞，这些细胞带有缺失，可去除p16，p15和p19arf肿瘤抑制子。我们发现RAS增加了MEF中的C/EBPB水平，但是NIH 3T3细胞中的C/EBPB表达沉默。这种差异可追溯到P19ARF，因为在表达RAS的NIH 3T3细胞中，ARF重新表达了ARF，而不是P16或P53恢复了C/EBPB水平。 RAS还降低了p16/p19arf null MEF中的C/EBPB表达。 C/EBPB的强迫表达阻断了RAS诱导的转化和增殖，表明C/EBPB能够在NIH 3T3细胞中诱导衰老。这些观察结果表明，C/EBPB是成纤维细胞中ARF肿瘤抑制途径的p53独立于靶标，而RAS诱导的C/EBPB的下调可能是绕过OIS和允许在某些细胞环境中允许肿瘤进展的重要事件。 C/EBPB活性的翻译后调节：由于C/EBPB介导细胞对致癌性RA的反应，因此我们正在研究通过RAS信号传导C/EBPB活性的翻译后调节。我们发现，C/EBPB的DNA结合活性自动抑制，并在表达致癌性RA的细胞中被激活或被生长因子刺激。 C/EBPB自动抑制需要在蛋白质的N末端一半中的三个区域，包括反式激活结构域，预计将采用二级结构。诱变研究支持了这些自身抑制序列参与相互作用的折叠相互作用的观念，这些相互作用会相互抑制C/EBPB DNA结合和反式激活功能。一个平行的目标是表征RAS诱导的C/EBPB上介导信号依赖性抑制的C/EBPB上的翻译后修饰（PTM）。 我们确定了亮氨酸拉链中的RSK激酶位点，它是C/EBPB DNA结合活性的重要调节剂，还调节了二聚化特异性。我们目前正在研究其他磷酸化位点和不同类别的修饰类别，例如赖氨酸乙酰化和Lys/arg甲基化控制C/EBPB DNA结合，二聚化和反式激活功能。最终，我们试图确定特定PTM如何影响C/EBPB的生物学和致癌特性。 C/EBPGAMMA通过与C/EBPB的异二聚化参与细胞生长调节：C/EBPG是一种普遍表达的蛋白质，与其他C/EBP家族成员（包括C/EBPB）形成异二聚体。我们正在通过分析C/EBPG缺陷型小鼠和来自这些动物的细胞来研究其在细胞生长调节中的作用。 C/EBPG缺陷的MEF在培养中非常差，并表达降低的促麦片R水平[汇总以7800个字符]

项目成果

Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription.

• DOI: 10.1016/j.molimm.2006.05.004
• 发表时间: 2007-02
• 期刊: Molecular immunology
• 影响因子: 3.6
• 作者: C. Spooner;Xiangrong Guo;P. Johnson;R. C. Schwartz

C/EBPbeta serine 64, a phosphoacceptor site, has a critical role in LPS-induced IL-6 and MCP-1 transcription.

C/EBPbeta 丝氨酸 64 是一个磷酸受体位点，在 LPS 诱导的 IL-6 和 MCP-1 转录中具有关键作用。

• DOI: 10.1016/j.cyto.2007.03.001
• 发表时间: 2007
• 期刊: Cytokine
• 影响因子: 3.8
• 作者: Spooner,ChaunceyJ;Sebastian,Thomas;Shuman,JonD;Durairaj,Srinivasan;Guo,Xiangrong;Johnson,PeterF;Schwartz,RichardC
• 通讯作者: Schwartz,RichardC

Critical prosurvival roles for C/EBP beta and insulin-like growth factor I in macrophage tumor cells.

C/EBP β 和胰岛素样生长因子 I 在巨噬细胞肿瘤细胞中发挥重要的促生存作用。

• DOI: 10.1128/mcb.24.8.3238-3250.2004
• 发表时间: 2004
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Wessells,Jennifer;Yakar,Shoshana;Johnson,PeterF
• 通讯作者: Johnson,PeterF