Androgen Receptor at the Intersection of Steroid, Thyroid and Lipid Signaling pathways: Structure /Function Studies

PHD项目总结：细胞使用一系列相互连接的信号转导途径，对代谢物、激素和脂质介导的细胞内和细胞外信息做出反应。这些途径依赖于蛋白质效应器(受体、酶、转录因子)来传递信号信息，并改变细胞的功能和反应。解开这些分子机制对于了解正常和疾病细胞中的生物途径非常重要，而CRISPR Cas9基因组编辑有助于精确分析这些过程中的关键基因。本项目将专注于雄激素受体与脂蛋白激酶PIP5K1A新的相互作用的分子基础。人PIP5K1A蛋白是一种脂酶，能产生信号分子磷脂酰肌醇4，5-二磷酸(PIP2)的细胞池。这种膜相关的脂质及其衍生物在内吞作用、细胞-细胞相互作用和受体介导的信号转导中具有不同的作用，此外，还通过调节染色质调节在细胞核中发挥拟议的功能。PIP2是PIP3和其他信号分子的前体，驱动癌细胞中AKT的磷酸化。与瑞典的研究小组合作，我们已经证明PIP5K1A调节雄激素受体蛋白(Sarwar等人，2016年OncoTarget)，并且对于癌细胞和巨噬细胞之间的细胞材料交换是必不可少的，这有助于建立骨和其他组织中的转移(Miftakhova等人，2016年癌症研究；Karlsson等人，提交癌症研究)。此外，我们的合作者(Semenas等人，PNAS 2014)发现的一种小分子抑制剂会导致AR和其他类固醇受体的破坏，尽管其分子基础尚不清楚。Heery实验室在核激素受体的结构和功能以及它们在与辅助激活蛋白相互作用方面的作用方面拥有长期的专业知识(例如Heery等人，1997；2001；Chan等人，2014；Fulton等人2017年)以及它在不同细胞类型中的作用(Bevan等人，1997；Nilsson等人，2015 OncoTarget；Karlsson等人，提交；Aksoy等人，正在准备中；Ngee等人正在准备中)。我们最近使用CRISPR Cas9基因组编辑来产生PIP5K1A的单等位基因和双等位基因敲除，以及PIP5K1A的N末端的缺失，移除了一个潜在的膜靶向结构域。初步结果显示，在细胞系中禁用PIP5K1A功能会导致大量增殖障碍、细胞形态改变，并且在动物实验中，对于LNCaP C4-2细胞，肿瘤起始能力降低，这与使用PIP5K1A激酶抑制剂的研究一致。因此，该项目将研究AR和PIP5K1A的物理相互作用，并对PIP5K1A消融引起的转录组变化进行RNA序列分析。为了评估AR与PIP5K1A的潜在相互作用，我们将利用酵母双杂交和其他蛋白质-蛋白质相互作用技术，评估PIP5K1A中的LxxLL基序在这些复合体中的作用。将探索有针对性的CRISPR方法，使用碱基编辑在不同的细胞系中产生激酶Dead PIP5K1A，从而在禁用功能的同时保存蛋白质相互作用。最后，该项目将与我们小组与维也纳医科大学合作研究AR与甲状腺受体的功能串扰的最新研究相吻合，这在该领域是一个全新的观察结果。

Summary of the PhD project:Cells use a host of interconnected signal transduction pathways to respond to intracellular and extracellular information mediated by metabolites, hormones and lipids. These pathways rely on protein effectors (receptors, enzymes, transcription factors) to relay the signalling information and modify cell function and response. Unravelling these molecular mechanisms is important to understand biological pathways in both normal and diseased cells, and CRISPR CAS9 genome editing facilitates precise analysis of key genes in these processes.This project will focus on the molecular basis of novel interactions of androgen receptors with the lipid kinase PIP5K1A. Human PIP5K1A protein is a lipid kinase that generates the cellular pool of the signalling molecule phosphatidylinositol 4,5-bisphosphate (PIP2). This membrane associated lipid and its derivatives have diverse roles in endocytosis, cell-cell interactions and receptor-mediated signalling, in addition to proposed functions in the nucleus through modulating chromatin regulators. PIP2 is a precursor of PIP3 and other signalling molecules and drives phosphorylation of AKT in cancer cells. In collaboration with groups in Sweden, we have shown that PIP5K1A regulates androgen receptor protein (Sarwar et al, 2016 Oncotarget) and is essential for exchange of cellular materials between cancer cells and macrophages, which helps establish metastases in bone and other tissues (Miftakhova et al., 2016 Cancer Research; Karlsson et al., submitted Cancer Research). Moreover, a small molecule inhibitor discovered by our collaborators (Semenas et al., PNAS 2014) leads to destruction of AR and other steroid receptors, although the molecular basis of this is unknown.The Heery lab has longstanding expertise in the structure and function of Nuclear Hormone receptors and their actions in terms of interaction with coactivators proteins (e.g. Heery et al., 1997; 2001; Chan et al., 2014; Fulton et al 2017 plus ~40 other publications) including the AR and its action in different cell types (Bevan et al, 1997; Nilsson et al., 2015 Oncotarget; Karlsson et al., submitted; Aksoy et al, in preparation; Ngee et al in preparation). We have recently used CRISPR CAS9 genome editing to generate single and double allele knockouts of PIP5K1A, as well as a deletion of the N-terminus of PIP5K1A removing a potential membrane-targeting domain. Preliminary results show that disabling PIP5K1A function in cell lines leads to substantial impediment of proliferation, changes in cell morphology and in the case of LnCaP C4-2 cells, reduced tumour initiating capacity in animal studies, consistent with studies using the PIP5K1A kinase inhibitor. Thus, the project will investigate the physical interactions of AR and PIP5K1A, as well as perform RNA seq analysis of transcriptome alterations due to ablation of PIP5K1A. To evaluate potential interactions of AR with PIP5K1A we will utilise yeast two hybrid and other protein-protein interaction techniques, assessing the role of LXXLL motifs in PIP5K1A in these complexes. Targeted CRISPR approaches using base editing to generate kinase dead PIP5K1A in different cell lines will be explored, thus conserving protein interactions while disabling function. Finally, the project will dovetail with recent studies from our group in collaboration with the Medical University of Vienna to study functional crosstalk of AR with thyroid receptors, a completely novel observation in this field.