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

描述(由申请人提供)：BRAF突变发生在相当大比例的人类肿瘤中，代表了MAPK通路的一种结构性激活机制。我们已经证明，BRAF的激活突变增加了对该途径的小分子抑制剂的敏感性。相反，我们发现HER2过表达的乳腺癌对MEK抑制具有抵抗力，尽管药物有效地抑制了MAPK的活性。我们的初步数据表明，HER激酶激活的肿瘤(和WT BRAF)和致癌BRAF的肿瘤具有相似的磷酸化ERK水平；然而，BRAF突变的肿瘤具有更高水平的磷酸化MEK和特定的MEK-ERK依赖的转录本。此外，MEK抑制剂诱导的反馈上调通路仅见于受体激活的肿瘤，而不是在具有激活BRAF突变的肿瘤中。我们假设，与HER激酶激活的肿瘤相比，B-RAF突变肿瘤中MAPK通路的输出增加是由于RAF上游和/或在RAF水平上该通路的反馈抑制的损害。我们假设，在BRAF突变的肿瘤中，上游反馈的禁用会导致途径吞吐量的增加，导致ERK效应物和负责途径反馈的靶点(DUSP、SPRY蛋白)的表达增加。DUSP(MAP激酶磷酸酶)的这种增加可能是ERK下调到生理耐受水平的关键。反馈蛋白和效应蛋白的增加可能共同导致了转化表型的某些方面。在这项提案中，我们描述了支持这些断言的更多初步数据，并描述了一项研究计划，以确定MEK抑制的反馈反应机制。我们将确定磷酸化的ERK是否代表了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干扰来确定关键蛋白质在反馈程序中的作用。最后，我们将建立由致癌的BRAF和激活的受体酪氨酸激酶转化的等基因系统，以确定是否可以产生特定的反馈和效应蛋白表达模式。这些研究的翻译目标是确定调节对RAF和MEK抑制剂的反应的反馈途径，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。这项研究的目标是了解调节这一通路激活的反馈机制。了解这种调控机制将有助于研究人员使用针对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(11/07版)页面续格式页面

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