Characterizing plasma membrane-associated prohibitins in the regulation of RAS activation and tumourigenesis

抑制蛋白是进化上保守的蛋白质，其主要已知在嵴形态发生和线粒体功能的调节中的作用。它们是SPFH蛋白家族的旗舰成员，其特征在于存在保守的PHB结构域。最近的研究表明，在信号转导，病原体进入和免疫细胞功能的质膜相关的馏分的显着作用。我们发现，PHB 1在质膜上是必需的丝氨酸-苏氨酸激酶CRAF的激活。CRAF是RAS-RAF-MAPK级联的核心成员，其控制基本细胞过程。RAS蛋白是人类主要的癌基因之一，近30%的人类癌症携带RAS突变。由于RAS被认为几乎是“不可用药的”，因此迄今为止开发的抑制剂靶向下游激酶，并取得了一些临床成功。由于PHB 1是激活CRAF所必需的，因此破坏PHB 1-CRAF相互作用的小分子抑制剂具有重要意义。最近的研究表明，罗格列酰胺靶向这种相互作用，并防止CRAF-MAPK在一组细胞系中的激活。我们发现PHB 1在NSCLC中高度表达，并且用配体如罗格列胺和氟唑啉靶向PM相关的PHB 1可以防止RAS突变细胞系中的MEK 1活化。通过采用包括人NSCLC异种移植物和同种异体移植物的动物模型，我们还证明了罗格列胺治疗可预防KRAS介导的NSCLC肿瘤生长。有趣的是，我们发现用罗格列胺治疗可以阻止EGF刺激的细胞中的RAS-GTP加载，尽管最初的实验表明这并不是以直接的方式发生的。总之，我们的数据表明，罗格列胺可能作为RAS抑制剂，我们提出，靶向与化学配体的PHB导致抑制KRAS介导的肿瘤发生。有几个迫切的问题，我们想解决通过采用高度跨学科的方法，包括化学生物学方法：1）PHB 1如何有助于稳定的KRAS-GTP在细胞中？2)这些作用是否仅限于罗格列胺类药物，或者其他黄曲霉素类药物也有类似的作用？3)罗格列胺治疗是否会破坏RAS二聚化和RAS纳米簇的形成？4)罗格列酰胺是否抑制其他RAS亚型？5)PHB 1/2复合物如何靶向不同的亚细胞区室？在过去的30年里，靶向RAS一直是癌症研究的圣杯。我们确定了天然抗肿瘤药物rocaglamide，其在纳摩尔浓度下起作用，作为靶向细胞中RAS的潜在工具。因此，除了提供对PHB和RAS生物学的重要见解外，拟议的工作将为治疗RAS驱动的人类癌症开辟新的治疗途径。

Prohibitins are evolutionarily conserved proteins that are predominantly known for their role in the regulation of cristae morphogenesis and mitochondrial function. They are the flagship member of the SPFH family of proteins and characterized by the presence of a conserved PHB domain. Recent studies revealed a prominent role for the plasma membrane-associated fraction of prohibitins in signal transduction, pathogen entry and immune cell function. We discovered that PHB1 at the plasma membrane is required for the activation of the serine-threonine kinase CRAF. CRAF is the central member of the RAS-RAF-MAPK cascade that controls fundamental cellular processes. RAS proteins are among the major human oncogenes and nearly 30% of human carcinomas carry mutations in RAS. As RAS is considered almost "undruggable" the inhibitors that have been so far developed target the downstream kinases with some clinical success. As PHB1 is required for the activation of CRAF, small molecule inhibitors that disrupt PHB1-CRAF interaction are of significant interest. Recent work revealed that rocaglamides target this interaction and prevent CRAF-MAPK activation in a panel of cell lines. We discovered that PHB1 is highly expressed in NSCLCs and the targeting of PM–associated PHB1 with ligands like rocaglamide and fluorizoline prevented MEK1 activation in RAS-mutated cell lines. By employing animal models including human NSCLC xenografts and allografts, we also demonstrated that rocaglamide treatment prevented KRAS-mediated growth of NSCLC tumours. Interestingly, we discovered that treatment with rocaglamide prevented RAS-GTP loading in cells stimulated with EGF, though initial experiments suggest that this does not occur in a direct way. Altogether, our data suggest that rocaglamide could possibly function as a RAS inhibitor and we propose that targeting PHB with chemical ligands leads to inhibition of KRAS-mediated tumourigenesis. There are several pressing questions that we want to address by employing a highly interdisciplinary approach including chemical biology approaches:1) How does PHB1 contribute to the stability of KRAS-GTP in cells? 2) Are these effects confined to rocaglamides or do other flavaglines show similar effects? 3) Does rocaglamide treatment disrupt RAS dimerization and formation of RAS nanoclusters? 4) Do rocaglamides inhibit other RAS isoforms? 5) How is the PHB1/2 complex targeted to various subcellular compartments? Targeting RAS has been the holy grail of cancer research in the past 30 years. We identified the natural anti-tumour drug rocaglamide, which works in nanomolar concentrations, as a potential tool to target RAS in cells. Thus, apart from providing important insights into PHB and RAS biology the proposed work will open novel therapeutic avenues for treating RAS-driven human cancers.