Abstract 777: ELTD1/ADGRL4, a novel adhesion GPCR regulator of tumour angiogenesis, suppresses lipid metabolism in endothelial cells, and is upregulated in breast cancer endothelium and epithelium
Authors: David M. Favara, Madhulika Nambiar, Helen Sheldon, Massimo Masiero, Demin Li, Ali Jazayeri, Alison H. Banham, Adrian L. Harris
Journal: Cancer Research (2017)

Abstract Background: We identified ELTD1/ADGRL4, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells &amp; vascular smooth muscle cells. Expression in the tumour vasculature is significantly increased. Our aims were to analyse ELTD1’s function in endothelial cells &amp; its role in breast cancer. Method: After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) &amp; validated with qPCR &amp; confocal microscopy. We investigated ELTD1 signalling by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis’. For this, truncated forms of ELTD1 &amp; peptides analogous to the proposed tethered agonist region were designed. FRET-based 2nd messenger (Cisbio IP-1; cAMP) &amp; luciferase-reporter assays (NFAT; NFKB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were qPCR screened. Furthermore, primary human breast cancers (n=245) &amp; matched primary &amp; nodal secondary breast cancers (n=79) were stained for ELTD1 expression. Staining intensity was then scored &amp; compared with survival. Results: HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of SLC24A1, which transports citrate from the mitochondria to the cytoplasm &amp; ACLY, which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. We validated this at RNA &amp; protein expression level &amp; showed that ELTD1 inhibited lipid droplet formation. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi; Gαs; Gαq; Gα12/13). In breast cancer, we found that no representative cell line screened expressed ELTD1. Breast cancer immunohistochemistry revealed higher intensity vascular ELTD1 staining within the tumour stroma contrasted to normal stroma &amp; expression within tumour epithelial cells (15%). Higher ELTD1 expression in both the tumour stroma vasculature (n=241; HR=0.68; p=0.04) &amp; within the subset of tumour positive cases (n=24; HR=0.3; p=0.02) correlated with improved relapse free survival (RFS). Conclusion: ELTD1’s regulation of lipid synthesis through suppression of ACLY &amp; SLC25A1 highlights ELTD1’s role as a novel regulator of endothelial metabolism. Unlike other aGPCRs, ELTD1 does not signal through canonical G protein pathways. The good prognosis of ELTD1 expression may be related to inducing a quiescent endothelial population. It will be of interest to relate this to activity of anti-angiogenic therapy. Nevertheless, a relevant fraction of patients expressing ELTD1 still relapsed &amp; this may be a suitable target for this population. Citation Format: David M. Favara, Madhulika Nambiar, Helen Sheldon, Massimo Masiero, Demin Li, Ali Jazayeri, Alison H. Banham, Adrian L. Harris. ELTD1/ADGRL4, a novel adhesion GPCR regulator of tumour angiogenesis, suppresses lipid metabolism in endothelial cells, and is upregulated in breast cancer endothelium and epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2017-777