Regulation of Ras-Dependent Signal Transduction Pathways

当正常细胞功能的控制由于关键信号转导通路的缺陷而出错时，癌症通常会出现。由RasGT 3调节的信号传导途径是这样的途径之一，并且其功能是调节重要的细胞过程，包括增殖、分化、存活和衰老。Raf丝氨酸/苏氨酸激酶家族的成员是Ras途径中的关键中间体，直接结合活化的Ras并充当ERK级联中的起始激酶，所述ERK级联由Raf、MEK和ERK蛋白激酶组成。存在三种哺乳动物Raf蛋白，A-Raf、B-Raf和C-Raf（也称为Raf-1）。正如可以预期的那样，Raf激酶可以直接导致致癌转化和其他人类疾病状态。例如，Raf的上游调节因子（如受体酪氨酸激酶和Ras）的突变或扩增经常导致通过携带这些等位基因的肿瘤中的Raf/MEK/ERK级联的组成型信号传导。此外，Raf蛋白本身的突变可以作为疾病驱动因素。C-Raf的种系突变可能是努南和LEOPARD RAS病综合征的病因，而B-Raf突变见于努南、LEOPARD和心面皮肤（CFC）RAS病综合征，其中B-Raf突变发生在75%的CFC患者中。此外，在70%的恶性黑色素瘤以及许多结直肠癌、卵巢癌、肺癌和乳头状甲状腺癌中观察到B-Raf的体细胞突变。多年来，我们研究团队的一个主要目标是阐明调节Raf催化活性的机制。这些研究导致了关键的磷酸化事件和蛋白质相互作用，有助于Raf激活/失活循环的识别。此外，我们的工作已经证明了Raf二聚体在正常和疾病相关信号传导中的重要性，并确定了Raf二聚体界面作为治疗靶点。最近进行的研究发现了一种新的磷酸调节回路，可以在细胞应激条件下抑制Raf激活和Ras信号传导。该回路作为应激激活的信号检查点发挥作用，并且可以被癌症治疗剂如rigosertib、紫杉醇和长春新碱参与，其通过氧化和有丝分裂应激激活JNK/MAPK级联。在此审查期间，我们开发了生物发光共振能量转移（BRET）技术，研究Raf在活细胞条件下的调节相互作用。使用该系统，评估了KIT受体酪氨酸激酶和B-Raf V600 E之间的信号传导串扰，发现KIT信号传导对WT-B-Raf的激活可干扰B-Raf V600 E驱动的黑色素瘤形成。在与分子靶标实验室的合作中，我们还使用BRET技术进行了高通量筛选，以鉴定可以破坏或阻止Raf二聚化或活细胞中Ras/Raf相互作用的天然产物化合物。作为这项努力的结果，新的macrophilone型吡咯亚氨基喹从海洋水螅Macrorhynchia philippina中分离出来，发现两种新鉴定的化合物破坏Raf二聚化和ERK级联信号。这些发现表明，macrophilones的化学支架可以为靶向人类疾病状态中的ERK级联提供小分子治疗线索。

Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway, and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, binding directly to activated Ras and serving as the initiating kinases in the ERK cascade, comprised of the Raf, MEK and ERK protein kinases. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf can be are causative for Noonan and LEOPARD RASopathy syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) RASopathy syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. Over the years, a major goal of our research team has been to elucidate the mechanisms that regulate Raf catalytic activity. These studies have led to the identification of critical phosphorylation events and protein interactions that contribute to the Raf activation/inactivation cycle. In addition, our work has demonstrated the importance of Raf dimerization in normal and disease-associated signaling and has identified the Raf dimer interface as a therapeutic target. Studies conducted more recently have led to the discovery of a new phospho-regulatory circuit that can suppress Raf activation and Ras signaling under conditions of cellular stress. This circuit functions as a stress-activated signaling checkpoint and can be engaged by cancer therapeutics such as rigosertib, taxol, and vincristine, that activate the JNK/MAPK cascade through oxidative and mitotic stress. During this review period, we developed bioluminescence resonance energy transfer (BRET) technologies to study Raf regulatory interactions under live cell conditions. Using this system, signaling cross-talk between the KIT receptor tyrosine kinase and B-Raf V600E was evaluated, resulting in the discovery that activation of WT-B-Raf by KIT signaling could interfere with melanoma formation driven by B-Raf V600E. In collaboration with the Molecular Targets Laboratory, we have also used the BRET technology to conduct a high-throughput screen to identify natural product compounds that can disrupt or prevent Raf dimerization or the Ras/Raf interaction in live cells. As a result of this effort, new macrophilone-type pyrroloiminoquines were isolated from the marine hydroid Macrorhynchia philippina, and two of the newly identified compounds were found to disrupt Raf dimerization and ERK cascade signaling. These findings indicate that the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads for targeting the ERK cascade in human disease states.