Feedback regulation and functional output of the RAS/RAF/MEK/MAPK pathway in huma

人RAS/RAF/MEK/MAPK通路的反馈调节和功能输出

• 批准号: 8534035
• 负责人: Christine Anne Pratilas
• 金额: $ 11.84万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534035
• 关键词: Accounting; BRAF gene; Binding; Biological Process; Breast Carcinoma; Cell Line; Cell Nucleus; Cell Proliferation; Cells; ChIP-on-chip; Clinical Trials; Data; Disabled Persons; Down-Regulation; Drug usage; ERBB2 gene; ETV1 gene; ETV4 gene; Effectiveness; Epidermal Growth Factor Receptor; Event; Exposure to; Family; Family member; Feedback; Fractionation; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Growth; Human; Immunoblotting; Impairment; JUN gene; MAP Kinase Gene; MAP Kinase Kinase Kinase 1; MAPK1 gene; MEK inhibition; MEKs; Measurement; Measures; Mediator of activation protein; Mitogen-Activated Protein Kinases; Mus; Mutation; Nuclear; Oncogenic; Output; Pathway interactions; Patients; Pattern; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Principal Investigator; Protein Kinase Inhibitors; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Refractory; Regulation; Research; Research Personnel; Research Proposals; Resistance; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; System; Testing; Tetracyclines; Transcript; Transfection; Up-Regulation; base; cell growth; cell transformation; gain of function; human MAP3K1 protein; immunocytochemistry; in vivo; in vivo Model; inhibitor/antagonist; melanoma; mutant; neoplastic cell; overexpression; programs; promoter; protein expression; protein kinase inhibitor; public health relevance; receptor; response; selective expression; small hairpin RNA; small molecule; tissue culture; transcription factor; tumor; tumor growth; tumor xenograft; vector control

DESCRIPTION (provided by applicant): BRAF mutations occur in a significant proportion of human tumors, and represent a mechanism of constitutive activation of the MAPK pathway. We have demonstrated that activating mutations of BRAF confer sensitivity to small molecule inhibitors of the pathway. In contrast, we showed that HER2-overexpressing breast carcinomas were resistant to MEK inhibition, despite effective pharmacologic inhibition of MAPK activity. Our preliminary data suggest that tumors with HER kinase activation (and WT BRAF) and those with oncogenic BRAF have similar levels of phosphorylated ERK; however, BRAF mutant tumors have higher levels of phosphorylated MEK and selected MEK-ERK dependent transcripts. Further, MEK inhibitor-induced feedback upregulation of the pathway is seen only in the receptor-activated tumors, but not in tumors with activating BRAF mutation. We hypothesize that increased output of the MAPK pathway in B- RAF mutant tumors compared to HER kinase-activated tumors is due to the impairment of feedback inhibition of the pathway, upstream of, and/or at the level of, RAF. We hypothesize that disabling of upstream feedback in BRAF mutant tumors causes an increase in pathway throughput, resulting in increased expression of ERK effectors, and targets responsible for pathway feedback (DUSP, SPRY proteins). This increase in DUSPs (MAP kinase phosphatases) may be critical for the downregulation of ERK to physiologically tolerated levels. The increase in both feedback and effector proteins may together be responsible for aspects of the transformed phenotype. In this proposal, we describe further preliminary data which support these assertions, and describe a research plan to determine the mechanism of feedback response to MEK inhibition. We will determine whether phosphorylated ERK represents an accurate reflection of pathway activation. We will use small molecule inhibitors of the pathway, as well as RNA interference to determine the role of critical proteins in the feedback program. Finally, we will develop isogenic systems transformed by oncogenic BRAF and activated receptor tyrosine kinases to determine if specific feedback and effector protein expression patterns can be generated. The translational goals of these studies are to identify feedback pathways modulating the response to RAF and MEK inhibitors which will impact the effectiveness of these compounds in clinical trials. PUBLIC HEALTH RELEVANCE: The RAS-RAF-MEK-ERK signaling pathway is an important regulator of tumor cell growth and proliferation. The goals of this research proposal are to understand feedback mechanisms which regulate the activation of this pathway. Understanding this regulatory mechanism will help investigators use drugs which target this pathway to select patients whose tumors are most likely to respond to them. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

描述（由申请人提供）：BRAF突变在相当大比例的人类肿瘤中发生，代表MAPK通路的组成性激活机制。我们已经证明，激活突变的BRAF赋予敏感性的途径的小分子抑制剂。相反，我们发现HER 2过表达的乳腺癌对MEK抑制有抵抗力，尽管MAPK活性的有效药理学抑制。我们的初步数据表明，HER激酶激活（和WT BRAF）的肿瘤和致癌BRAF的肿瘤具有相似的磷酸化ERK水平;然而，BRAF突变肿瘤具有更高水平的磷酸化MEK和选定的MEK-ERK依赖性转录物。此外，仅在受体激活的肿瘤中观察到MEK受体诱导的通路反馈上调，但在具有激活BRAF突变的肿瘤中未观察到。我们推测，与HER激酶激活的肿瘤相比，B- RAF突变型肿瘤中MAPK通路的输出增加是由于RAF上游和/或RAF水平上的通路反馈抑制受损。我们假设BRAF突变肿瘤中上游反馈的失能导致通路通量的增加，导致ERK效应子和负责通路反馈的靶点（DUSP，SPRY蛋白）的表达增加。这种DUSPs（MAP激酶磷酸酶）的增加可能对ERK下调至生理耐受水平至关重要。反馈蛋白和效应蛋白的增加可能共同负责转化表型的各个方面。在这个建议中，我们描述了进一步的初步数据，支持这些断言，并描述了一个研究计划，以确定反馈响应MEK抑制的机制。我们将确定磷酸化的ERK是否代表通路激活的准确反映。我们将使用该途径的小分子抑制剂以及RNA干扰来确定反馈程序中关键蛋白质的作用。最后，我们将开发由致癌BRAF和激活的受体酪氨酸激酶转化的同基因系统，以确定是否可以产生特定的反馈和效应蛋白表达模式。这些研究的转化目标是确定调节RAF和MEK抑制剂反应的反馈途径，这将影响这些化合物在临床试验中的有效性。 公共卫生相关性：RAS-RAF-MEK-ERK信号通路是肿瘤细胞生长和增殖的重要调节因子。本研究的目的是了解调节该途径激活的反馈机制。了解这种调节机制将有助于研究人员使用靶向这一途径的药物来选择肿瘤最有可能对其产生反应的患者。PHS 398/2590（Rev. 11/07）