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，癌症就会发生。由RasGTPase调控的信号通路就是其中之一，它的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/苏氨酸激酶家族成员是Ras通路的关键中间体，直接与活化的Ras结合，并作为ERK级联的启动激酶，由Raf、MEK和ERK蛋白激酶组成。哺乳动物有三种Raf蛋白，A-Raf， B-Raf和C-Raf（也称为Raf-1）。正如我们所期望的那样，对于参与细胞信号传导的蛋白质，Raf激酶可以直接促进致癌转化和其他人类疾病状态。例如，Raf上游调控因子（如受体酪氨酸激酶和Ras）的突变或扩增，通常会在含有这些等位基因的肿瘤中通过Raf/MEK/ERK级联导致组成性信号传导。此外，Raf蛋白本身的突变可以作为疾病驱动因素发挥作用。C-Raf种系突变可导致Noonan和LEOPARD RASopathy综合征，而B-Raf突变见于Noonan、LEOPARD和CFC RASopathy综合征，其中75%的CFC患者发生B-Raf突变。此外，在70%的恶性黑色素瘤以及许多结直肠癌、卵巢癌、肺癌和甲状腺乳头状癌中都观察到B-Raf的体细胞突变。多年来，我们研究团队的主要目标是阐明调节Raf催化活性的机制。这些研究已经确定了导致Raf激活/失活周期的关键磷酸化事件和蛋白质相互作用。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化调控回路，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，可以被癌症治疗药物如rigosertib、紫杉醇和vincristine参与，通过氧化和有丝分裂应激激活JNK/MAPK级联。在本综述期间，我们开发了生物发光共振能量转移（BRET）技术来研究活细胞条件下Raf的调节相互作用。利用该系统，我们评估了KIT受体酪氨酸激酶与B-Raf V600E之间的信号串扰，发现KIT信号激活WT-B-Raf可以干扰B-Raf V600E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技术进行高通量筛选，以鉴定可以破坏或阻止Raf二聚化或Ras/Raf在活细胞中相互作用的天然产物化合物。在此基础上，研究人员从菲律宾巨水藻中分离出了新的巨philontype pyrololoiminoquines，并发现了两个新鉴定的化合物可以破坏Raf二聚化和ERK级联信号。这些发现表明巨噬细胞的化学支架可以为人类疾病状态下靶向ERK级联提供小分子治疗先导。

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.