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

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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.