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: Favara David M. , Nambiar Madhulika , Sheldon Helen , Masiero Massimo , Li Demin , Jazayeri Ali , Banham Alison H. , Harris Adrian L. 
Published: July 2017

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 & vascular smooth muscle cells. Expression in the tumour vasculature is significantly increased. Our aims were to analyse ELTD1’s function in endothelial cells & its role in breast cancer.

Method: After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) & validated with qPCR & confocal microscopy. We investigated ELTD1 signalling by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis’. For this, truncated forms of ELTD1 & peptides analogous to the proposed tethered agonist region were designed. FRET-based 2nd messenger (Cisbio IP-1; cAMP) & 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) & matched primary & nodal secondary breast cancers (n=79) were stained for ELTD1 expression. Staining intensity was then scored & compared with survival.

Results: HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of SLC24A1, which transports citrate from the mitochondria to the cytoplasm & ACLY, which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. We validated this at RNA & protein expression level & 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 & expression within tumour epithelial cells (15%). Higher ELTD1 expression in both the tumour stroma vasculature (n=241; HR=0.68; p=0.04) & 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 & 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 & 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