hRPF1在AKT激活中的作用

It has been reported that E3 ubiquitination ligase hRPF1 is frequently over-expressed in human cancers. The molecular mechanism underlying hRPF1 dependent tumorigenesis, however, is controversial and far from clear. AKT activity is both positively and negatively regulated. For instance, Insulin-like Growth Factor-1 (IGF-1) binding to the IGF-1 receptor (IGF-1R) activates the PI3K-AKT pathway via phosphorylation of the intermediate adaptor protein IRS1. hRPF1 is reported to increase PI3K-AKT activity during embryonic development via maintenance of cell surface IGF-1R protein levels although the molecular mechanism underling this observation is not clear. In contrast to IGF-1R, PTEN inhibits the PI3K-AKT pathway. Although hRPF1 can target PTEN for ubiquitination dependent degradation no obvious effect of hRPF1 on PTEN stability was observed in several mouse hRPF1 knockout models. Our preliminary studies support these results since hRPF1 dependent AKT-P increases were independent of PTEN but dependent on IGF 1R. Moreover, we have made the novel discovery that hRPF1 directly binds to replication protein A, a single strand DNA (ssDNA) binding protein which is generally believed to play a critical role in DNA metabolism. The role of hRPF1 on AKT activation is inhibited by replication protein A. Thus, the hypothesis to be tested is that increased hRPF1 expression activates AKT via facilitation of IGF-1R signaling in human cancer cells. IGF-1R inhibition suppresses cancer cell growth, particularly in cancer cells with hRPF1 over-expression. Furthermore, hRPF1 function is suppressed via a direct interaction with replication protein A. The importance of our proposed work is that it would not only improve our understanding of the molecular mechanisms by which hRPF1 promotes tumor development but also provide guidance for future clinical trials. Furthermore, the expected results from these studies will also facilitate the identification and development of biomarkers and drug targets in human cancer. The innovation of the proposed study is (1) linking hRPF1 oncogenic activity to IGF-1R signaling, (2) revealing a novel function of replication protein A in signal transduction with potential applicability for new chemotherapeutic drug development. (3) providing improved guidance for cancer treatment planning based on better predictions of tumor response to IGR-1R inhibition by identification of cancers patients with hRPF1 over-expression.

hRPF1是一种常在人类癌症中过表达的E3泛素化连接酶。尽管已报导hRPF1具有促进Akt活化的致癌基因功能，但是有关hRPF1活化Akt的分子机制尚不明确。我们检验的主要假说是：hRPF1通过活化IGF-1R信号而激活Akt，从而在细胞增殖中起重要作用。并且一种通常只在DNA代谢中起作用的单链DNA结合蛋白-复制蛋白A（RPA），却抑制IGF-1R-Akt信号通路。因此，通过IGF-1R抗体或RPA抑制IGF-1R信号通路可以抑制过表达hRPF1癌细胞的生长。对这一假说的检验将加深对潜在hRPF1相关癌症发生发展分子机制的理解。再者，RPA抑制IGF-1R-Akt信号的新功能发现将有利于研发靶向hRPF1或IGF-1R的新药物。此外，通过发现过表达hRPF1癌细胞对IGF-1R-Akt信号抑制的高敏感反应性，选择合理靶向IGF-1R基因治疗的敏感人群，为临床癌症治疗提供科学依据。

我们实验证实了本项目的主要假说，即hRPF1通过活化IGF-1R信号而激活Akt。首先，hRPF1维持肿瘤细胞IGF-1R蛋白的相对稳定性以促进IGF-1R信号网络，其中Grb10具有重要作用；而hRPF1通过调控蛋白酪氨酸磷酸酶(PTP)促进IGF-1R的下游关键因子IRS1的活化过程。其次，RPA阻断AKT活化过程并不依赖于IGF-1R，这与我们原设想不同，提示 RPA通过其它途径阻断AKT的激活；进一步数据支持该结果，即与hRPF1作用结合的RPA1位点对IGF-1R-P及AKT-P具有不同的调控功能。再者，我们发现另一种E3泛素连接酶RNF126亦与hRPF1相互作用结合，在细胞DNA的同源重组HR修复中具有重要作用；并且RNF126和hRPF1二者均促进细胞周期检测点激酶CHK1的表达；遂我们从DNA代谢、损伤及修复方面探讨hRPF1如何激活AKT，以及RPA如何抑制与hRPF1相关的AKT活化过程。之后我们惊喜发现RNF126在mRNA转录水平调控BRCA1的蛋白表达以促进HR修复；而BRCA1本身就是调控AKT活性的重要蛋白，因此hRPF1与RNF126相互作用的内在联系，为深入研究hRPF1激活AKT的机制提供了新方向。本项目首次提出并以证实了E3泛素连接酶hRPF1可激活IGF-IR-AKT信号通路的观点，并对该激活机制展开了深入探讨（部分数据已发表于2015年《Oncogene》杂志），该课题揭示了hRPF1、RNF126与CHK1间的相互关系，为hRPF1/RNF126相关肿瘤的研究提供科学依据；提出了CHK1抑制剂可作为过表达hRPF1/RNF126相关肿瘤的一种潜在临床治疗方法。