相对恒定的器官大小是多细胞生物最显著的特征之一，其调控是发育生物学研究的重要内容。Hippo信号通路调控细胞增殖、凋亡及干细胞自我更新并因此高度保守地调控多细胞生物器官大小。申请人从2004年起致力于Hippo信号通路的研究，发表了20余篇论文，他引超过1500次，相继发现了Hippo通路抑制下游转录辅激活因子的分子机制、该通路调控基因表达及器官大小的关键下游转录因子、细胞粘附和细胞骨架对Hippo通路的调控、以及Hippo通路在细胞接触抑制、失巢凋亡、癌症、血管发育等生理病理过程中的作用。这些成果促进了Hippo通路及器官大小发育的研究。Hippo通路的上游信号及其作用的分子机制是器官大小调控研究的重要方向。本课题从Lats1/2新的上游激酶、磷酸酶及Hippo通路结合蛋白入手，阐明它们在Hippo通路中的作用和机制及其上游生理信号，并进一步研究这些新机制在体内调控器官大小的作用。

One of the most remarkable features of multicellular organisms is relatively constant organ size. The recently established Hippo signaling pathway plays an evolutionarily conserved role in organ size control by regulating cell proliferation, apoptosis, differentiation, and stem cell self-renewal. Research of the applicant has been focused on functions and mechanisms of the Hippo pathway ever since 2004, which has led to discoveries including: the mechanisms by which the Hippo pathway inhibits YAP/TAZ transcription co-activators; TEAD family as important transcription factor targets of the Hippo pathway; cell adhesion and cytoskeleton as robust upstream regulators of the Hippo pathway; and important functions of the Hippo pathway in cell contact inhibition, anoikis, cancer, and angiogenesis. These works promoted the progress of research on the Hippo pathway and organ size control. Despite rapid progress in the field, upstream signals of the Hippo pathway and the mechanisms by which they regulate the Hippo pathway kinases are largely unknown. This proposed study is based on potential Lats1/2 upstream kinases and phosphatases from screening of human kinase and phosphatase libraries, and also novel Hippo pathway interacting proteins from tandem affinity purifications. The functions and mechanisms of these molecules in the Hippo pathway as well as their roles in mediating mechanical stress or GPCR signaling will be examined. More importantly, we may discover novel physiological signals upstream of these molecules that regulate the Hippo pathway. Furthermore, we will examine the role of these Hippo pathway new components, mechanisms, and upstream signals in regulation of organ size in vivo. The study will provide molecular insights into the mystery of organ size control.

相对恒定的器官大小是多细胞生物最显著的特征之一，其调控是发育生物学研究的重要内容。Hippo信号通路调控细胞增殖、凋亡及干细胞自我更新进而高度保守地调控多细胞生物器官大小。更重要的是Hippo通路的失调导致多种人类疾病，特别是与癌症的发生发展具有密切的关系。本课题按计划从Lats1/2新的上游激酶、磷酸酶及Hippo通路结合蛋白入手，阐明它们在Hippo通路中的作用和机制及其上游生理信号，并进一步研究这些新机制在体内调控器官大小与癌症发生的作用。在本项目的支持下通过四年的潜心研究我们在多个方向上实现了突破，发现了Hippo通路的重要新机制及其在器官大小调控及癌症中的功能。首先我们通过蛋白激酶文库筛选发现SRC酪氨酸激酶作为一个原癌基因可以直接磷酸化LATS1并抑制其活性，从而激活YAP。并且在人乳腺癌样品中SRC的上调与YAP的激活呈正相关。其次通过串联亲和纯化我们发现了LATS2与去泛素化酶USP9X直接结合，并且USP9X通过直接去泛素化LATS2促进LATS2的蛋白水平，抑制YAP。在胰腺癌中USP9X通过LATS2发挥抑癌作用。通过对YAP激活的下游效应分子的研究我们发现YAP激活microRNA miR-130的表达，并且进一步通过抑制VGLL4的翻译调控YAP活性，形成YAP的正反馈回路。有意思的的是我们还发现YAP的激活通过直接促进LATS2的表达形成负反馈机制。通过以小鼠肝脏为模型的体内研究我们阐明了这些正负反馈机制在器官大小调控和肿瘤发生过程中发挥的重要作用。在阐明YAP激活导致癌症发生的机制方面，我们发现在肝细胞中激活YAP能够通过促进趋化因子CCL2和CSF1的表达募集巨噬细胞到肿瘤起始细胞周围。敲减CCL2和CSF1不但阻止了巨噬细胞的招募还阻止了YAP诱导的肝肿瘤形成。进一步研究表明招募的巨噬细胞为2型巨噬细胞，他们通过对T细胞的抑制使得肿瘤起始细胞能够逃逸免疫监视，避免被清除。这一研究首次通过对肝脏肿瘤起始细胞的遗传操作证明了巨噬细胞的特异性招募在单个肿瘤起始细胞阶段对肝脏肿瘤发生的关键作用，并证明了Hippo通路是这一功能的关键分子机制，为预防和治疗肝癌提供了新的思路。