1658 Biochemical Society Transactions (2014) Volume 42, part 6

A review of ELTD1, a pro-angiogenic adhesionGPCRDavid M. Favara*†1, Alison H. Banham‡ and Adrian L. Harris**Department of Oncology, University of Oxford, Oxford, U.K.

†Balliol College, University of Oxford, Oxford, U.K.

‡NDCLS-Radcliffe Department of Medicine, University of Oxford, Oxford, U.K.

AbstractEpidermal growth factor, latrophilin and seven-transmembrane domain-containing 1 (ELTD1), an orphanG-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has recently been identifiedas a potential cancer biomarker and a novel regulator of angiogenesis. In this mini-review, we presentan overview of the current literature on ELTD1 and present bioinformatics data showing ELTD1’s sequenceconservation, its expression in cancer cell lines and its mutational frequency in human cancers. Additionally,we present sequence homology alignment results confirming ELTD1 to be a hybrid comprising motifs sharedwith individual members in both adhesion GPCR subfamilies 1 and 2. Finally, we discuss why tumourendothelial ELTD1 expression may confer a good prognosis yet still represent a therapeutic target.

IntroductionAngiogenesis is the process by which new blood vesselsform from pre-existing vessels. This tightly regulatedprocess occurs physiologically throughout life during periodsof controlled tissue growth, tissue repair and woundhealing, as well as during the menstrual cycle, duringplacental implantation in the uterus and in embryogenesis[1]. Pathological angiogenesis refers to inappropriate andexcessive angiogenesis and is associated with a spectrumof diseases, most notably cancer [2]. Cancer-associatedangiogenesis provides the tumour with oxygen and essentialnutrients needed for promoting tumour growth, expansionand metastasis and is thus an attractive therapeutic target [2].

Since the first association between angiogenesis andtumour growth over 40 years ago [3], much work hasbeen invested into the search and development of anti-angiogenic therapies. The majority of anti-angiogenicapproaches developed to date target components of eitherthe vascular endothelial growth factor (VEGF) pathwayor the NOTCH intercellular signalling pathway, whichtogether make up the two most studied angiogenic pathways[4]. Translation of these therapies into clinical practicehas, however, proved difficult. For example, despite manypositive results achieved in preclinical models with VEGFinhibition, its translation into clinical practice has yielded

Key words: adhesion G-protein-coupled receptor (adhesion GPCR), angiogenesis, biomarker,

cancer, epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1

(ELTD1).

Abbreviations: DLL4, Delta-like ligand 4; ECD, extracellular domain; EGF, epidermal growth

factor; ELTD1, epidermal growth factor, latrophilin and seven-transmembrane domain-containing

1; GAIN, GPCR autoproteolysis inducing domain; GPCR, G-protein-coupled receptor; GPS, GPCR

proteolytic site; ICD, intracellular tail; 7TM, seven-transmembrane domain; VEGF, vascular

endothelial growth factor.1To whom correspondence should be addressed (email david.favara@balliol.ox.ac.uk).

poor overall survival regardless of its broad-spectrumanti-tumour activity. This may be because of tumourresistance, either preceding therapy or developing followingprolonged therapy and leading to the restoration of tumourgrowth [5]. It can thus be hypothesized that tumoursundergoing selective anti-angiogenic pressure may over timebe able to recruit alternative undiscovered parallel angiogenicpathways unaffected by the given therapy. The search forsuch novel parallel pathways is an active area of currentresearch.

Recently, epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1 (ELTD1) was reportedas a novel and previously largely uncharacterized regulator oftumour angiogenesis. Cross-talk with VEGF and NOTCHpathways was identified via either stimulation (VEGF) [6,7]or repression [Delta-like ligand 4 (DLL4)] [6] of ELTD1expression. The present mini-review is a summary of currentknowledge regarding ELTD1.

ELTD1 and the adhesion GPCR familyDiscovered in 2001, ELTD1 is an orphan member of theG-protein-coupled receptor (GPCR) superfamily [8]. Thissuperfamily comprises the largest receptor family in thehuman proteome with over 900 members and is dividedinto five groups according to the sequence homology of adomain present in all members, the ‘seven-transmembranedomain’ (7TM) [9] (Figure 1). Within the GPCR superfamily,ELTD1 belongs to the 33 member ‘adhesion family’, so namedfor their large extracellular domains containing adhesionmotifs, a feature not found in other GPCR families [10].In general, the ‘adhesion family’ remains the most poorlystudied and understood of the five GPCR families [11].Within the ‘adhesion family’, ELTD1 is grouped into the‘family 1/latrophilin-like’ subfamily. This grouping is based

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Figure 1 An overview of the GPCR superfamily focusing on the adhesion family

GPCRs can be classified into five groups according to ‘seven-transmembrane domain’ sequence homology. The adhesion

family forms the second largest group and is the most poorly understood of the GPCRs. Members of each adhesion subfamily

are listed. Dotted lines surround members with a known ligand. ELTD1 is circled in black. Adhesion motifs expressed by

members from each subfamily are listed. Note that not all motifs listed for each subfamily are expressed by every member

of that group. CAD, cadherin motif; CALX-β, calnexin-β motif; CUB, Cs1 and Csr/Uegf/BMP1 motif; IG, immunoglobulin

motif; LAM, laminin motif; LRR, leucin-rich repeat; OLF, olfactomedin motif; EGF, epidermal growth factor motif; EEAR,

epitempin/epilepsy-associated repeat; HRM, hormone receptor motif; PTX, pentraxin motif; RBL, rhamnose-binding lectin

motif; SEA, sperm protein, enterokinase, agrin module; TSP, thrombospondin motif.

solely on 7TM sequence homology [10]. Latrophilin 1–3 formthe other members of this group and are so named becauseof their ability to bind α-latrotoxin, a toxin produced by theblack widow spider (Latrodectus mactans) [12].

Located on chromosome 1p31.1, ELTD1 encodes a3527 nucleotide transcript (AT content 64 %) translatedas a 690 amino acid protein [13]. ELTD1 contains 15exons, expressed as two splice variants: one full-lengthand the other a 149 amino acid truncated C-terminalsegment of the transmembrane domain. Like all adhesionGPCRs, ELTD1 can be topographically divided into threecomponents (Figure 2A): (i) an extracellular domain (ECD)containing receptor-specific ecto-domains as well as the‘GPCR autoproteolysis inducing domain’ (GAIN), (ii) a7TM and (iii) an intracellular tail (ICD).

In humans, bioinformatic analysis reveals that the 430-amino-acid ELTD1 ECD encodes an epidermal growth factor(EGF) repeat, an EGF–Ca2 + binding repeat and a GAINdomain (Figure 2A). ELTD1’s EGF adhesion motifs are not

found in other ‘family 1/latrophilin-like’ subfamily members.Ligands to ELTD1’s extracellular domains have yet to beidentified. Adhesion GPCRs are the only GPCRs to containGAIN domains. These conserved domains are importantin adhesion GPCR assembly and are found in all but oneadhesion GPCR [14]. Within ELTD1’s GAIN domain, amotif termed the ‘GPCR proteolytic site’ (GPS) undergoesautoproteolytic cleavage during protein assembly in theendoplasmic reticulum before rejoining non-covalently (atthe cleavage site) and being exported to the Golgi complexand then to the cell membrane [15] (Figure 2B). Althoughthe GPS motif makes up a small area of the larger GAINdomain (53 out of 263 amino acids respectively in ELTD1),the entire GAIN domain itself is required for autoproteolysisto occur [11]. In some adhesion GPCRs, it is hypothesizedthat autoproteolytic cleavage may allow for swapping ofextracellular domain fragments between receptors once atthe cell surface [16]. Although this has been noted withlatrophilin 1, EMR2 and GPR56, [17–19] it remains unproven

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1660 Biochemical Society Transactions (2014) Volume 42, part 6

Figure 2 ELTD1’s putative structure and conservation

(A) ELTD1’s bioinformatically derived putative structure. (B) Close-up of ELTD1’s GPS motif within ELTD1’s extracellular GAIN

domain. Autoproteolytic cleavage point indicated by dotted red line. (C) ELTD1’s conservation: ELTD1 exhibits high-level

conservation across vertebrates (black bars) and moderate conservation in C. elegans (white bar), signifying evolutionary

importance. Sequence data obtained from the Ensembl genome browser [13]. His, histidine; Leu, leucine; Thr, threonine;

AA, amino acid(s).

for the majority of adhesion GPCRs, including ELTD1.Other postulated roles for cleavage include involvement inadhesion GPCR ligand–receptor interactions, signalling andprotection of receptor/membrane integrity during periodsof mechanical stress [16]. The amino acids involved inthis autoproteolytic cleavage have been identified [16]in all cleaved GPCRs with ELTD1’s being histidine (position405), leucine (position 406) and threonine (position 407)(Figure 2B). ELTD1’s cleavage occurs between the leucineand threonine residues. Although a mass spectrometry basedstudy of the human plasma N-glycoproteome has identifieda circulating N-terminal ELTD1 peptide [20], it remainsunknown whether ELTD1 commonly disassociates at itsproteolytic cleavage site once it is expressed on the cellsurface.

Canonical GPCR signalling involves ligands binding toextracellular regions of the 7TM which initiate G-proteinsignalling. Whether this also occurs in members of the‘adhesion family’ remains unclear [16]. ELTD1’s signallingability has yet to be established. ELTD1’s 7TM (Figure 2A)

comprises 237 amino acids and its ICD comprises 22 aminoacids, the shortest ICD among members of the ‘family1/latrophilin-like’ subfamily. Bioinformatic analysis revealsonly a single predicted phosphorylation site on ELTD1’sICD. ELTD1’s crystal structure remains unsolved.

When comparing homology with human ELTD1 acrossspecies, we found that Eltd1 is highly conserved acrossvertebrates (50–99 %) with moderate conservation in non-vertebrates such as Caenorhabditis elegans (16 %) (Figure 3).This conservation signifies an evolutionarily important rolefor ELTD1.

When compared with other ‘adhesion family’ GPCRs,ELTD1 appears to be a hybrid comprising motifs fromadhesion subfamilies 1 and 2 and lacking the adhesionmotifs found in other subfamily 1 members. Interestingly,ELTD1 expression does not follow the tissue distribution ofother subfamily 1 members {predominantly expressed in thecentral nervous system (CNS) [21]} or subfamily 2 members(predominantly expressed by cells of the immune system[22]). We performed sequence homology comparisons which

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Angiogenesis and Vascular Remodelling: New Perspectives 1661

Figure 3 ELTD1 in cancer

(A) ELTD1 expression levels in 1036 cancer cell lines: ELTD1 is poorly expressed in the majority of cancer cell lines. Data

obtained from the Cancer Cell Line Encyclopedia [39]. (B) ELTD1’s alteration frequency in 48 curated cancer-genome datasets

obtained from cBioPortal for Cancer Genomics [41]. The number of altered cases and total cases per dataset is listed above

each column. ELTD1 is mutated in �10 % of melanoma and lung squamous cell carcinoma cases. Amplification without

other ELTD1 alterations occurs in bladder carcinoma, sarcoma and uterine carcinosarcoma datasets. ACyC, adenoid cystic

carcinoma; adeno, adenocarcinoma; AML, acute myeloid leukaemia; ccRCC, kidney renal clear cell carcinoma; CCLE, Cancer

Cell Line Encyclopedia; CML, chronic myeloid leukaemia; DLBC lymphoma, diffuse large B-cell lymphoma; GBM, glioblastoma

multiforme; MM, multiple myeloma; NCI60, National Cancer Institute 60 human tumour cell line screen; pRCC, renal papillary

cell carcinoma; SC, squamous carcinoma.

reveal that ELTD1’s EGF repeats share closest homologywith EMR3 (subfamily 2) and that ELTD1’s GAIN, 7TMand ICD are closest to LAT3 (subfamily 1). Although ligandsto EMR3’s EGF motifs remain unknown, the EGF motif

ligands of other subfamily 2 members are known, namely:dermatan sulfate [23] for both CD97 and EMR2; and CD55[24], CD90 [25] and integrins α5β1, avβ3 [26] for CD 97.These are not known to associate with ELTD1.

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1662 Biochemical Society Transactions (2014) Volume 42, part 6

ELTD1 as an angiogenic regulator

When first discovered, ELTD1 expression was noted only inrat bronchiolar and vascular smooth muscle cells and in bothrat and human cardiomyocytes [8]. In 2008, two separatestudies found ELTD1 to be part of the normal endothelialtranscriptome in humans [27] and mice [28] respectively. In2012, a study aiming to characterize the transcriptome ofblood vessels associated with grade IV glioblastoma foundELTD1 to be one of 95 genes up-regulated in these vessels[7]. The same study found that VEGF-A and transforminggrowth factor β2 (TGF-β2) up-regulated ELTD1 expressionin these vessels but did not specifically associate ELTD1as a regulator of angiogenesis [7]. In 2013, ELTD1 wascharacterized as a novel regulator of both physiological andtumour angiogenesis [6].

ELTD1’s association as a regulator of tumour angiogenesiswas identified using a bioinformatic screen for previouslyuncharacterized genes associated with tumour angiogenesis[6]. This was performed by analysing the expression profileof approximately 1000 primary human tumour samples fromthree different tumour types and comparing expression with acore signature of well-recognized angiogenic and endothelialcell associated ‘seed’ genes. ELTD1 was found to be thehighest ranked non-seed gene which was not an establishedregulator of angiogenesis. This role was then functionallyvalidated both in vitro and in vivo [6].

ELTD1 was found to be highly expressed in vascularendothelial cells and vascular smooth muscle cells, withhigher endothelial expression being observed in peri-tumoralvessels (across a range of tumours) than in vessels frommatched normal tissues. ELTD1 was also found to beregulated by two key angiogenic ligands, namely VEGF,up-regulating ELTD1, and DLL4, down-regulating ELTD1.VEGF’s effect on ELTD1 validated previous findings inglioblastoma-associated vessels [7]. ELTD1 and DLL4 werealso found to antagonize each other. A separate study focusingon the breast cancer perivascular niche further validatedthis antagonism by showing that NOTCH1 silencing ofneovascular sprouts increased extracellular ELTD1 proteinenrichment [29].

ELTD1 silencing in human umbilical vein endothelialcells demonstrated its important role in tip cell functionduring sprouting angiogenesis. Additionally, through useof the zebrafish (Danio rerio) embryo, Eltd1 was shownto be important in vasculogenesis. In this model, Eltd1silencing using morpholinos caused severe vascular defectsand prevented effective intersegmental vessel formation fromthe dorsal aorta.

When human ovarian and colorectal tumour xenograftswere implanted in mice, systemic anti-mouse Eltd1 silencingin the murine stroma was found to substantially inhibittumour growth. In addition to having smaller and fewertumours, Eltd1 silenced mice survived longer and had farfewer sites of metastasis when compared with controls. Nosignificant toxicity was observed as body weight and heart tobody weight ratios were unaffected.

Finally, high endothelial cell ELTD1 expression was foundto correlate with improved overall survival in a number ofcancer types, namely renal cancer, colorectal cancer, headand neck squamous cell carcinoma and ovarian cancer. Thesefindings implicate ELTD1 as a putative prognostic markerof favourable outcome for these cancers when treated withchemotherapy.

The ELTD1 ‘vessel maturity’ hypothesisTumour-associated vessels are functionally and structurallyabnormal, being immature, tortuous and very leaky [30].These factors all result in poor drug delivery to the tumoursite. In contrast, mature vessels are less leaky and tortuous,which facilitates better drug delivery to the tumour site. Thisis the principle behind vascular maturation/normalization asa strategy for the treatment of cancer [31].

In the light of the findings that higher vascularELTD1 expression correlates with improved survival innumerous cancer types, we hypothesize that ELTD1 maybe important in regulating tumour vessel maturation, withhigher expression promoting higher microvessel density,more mature and differentiated and/or less leaky vessels.This is analogous to vascular normalization after anti-VEGFtherapy [32]. The consequence of this is improved perfusion,thus aiding drug delivery to the tumour, as well as producinga less aggressive tumour (through a reduction in tumourhypoxia which acts as a driver of metastasis).

Previously, propanolol (a non-specific β-adrenergicreceptor inhibitor) was shown to be selectively toxicto malignant vascular endothelial tumour cells versusnormal endothelial cells when administered to the SVRangiosarcoma-forming cell line in mice. In this context,transcriptional profiling revealed that propanolol similarlyincreased endothelial expression of both Eltd1 and Tek, a geneencoding the Tie2 angiogenic receptor which promotes vesselquiescence, stability and maturation [33,34]. This associationadds further weight to ELTD1’s vessel maturity hypothesis.Should our hypothesis be proven correct, therapies whichincrease endothelial ELTD1 expression (like propanolol) maybecome important in the management of certain cancers.

In summary, ELTD1’s roles in sprouting angiogenesis andits putative association with vessel maturity may explainwhy higher endothelial ELTD1 expression correlates withimproved survival in numerous tumour types and yet alsorepresents a therapeutic target.

Other roles for ELTD1Other groups have shown ELTD1 expression to be importantin rodent cardiomyocyte development [8], and in protectingagainst pathological hypertrophy in response to increasedmajor vessel pressure loading in mice [35]. This study alsofound that failed hypertrophied human hearts in patientsawaiting cardiac transplantation had significantly lowercardiac ELTD1 expression than healthy controls [35]. Inthis context, ELTD1 polymorphisms have been found to be

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positively selected in a cohort of high-altitude living Andeanpeople, possibly as a cardioprotective measure against lifelonglow oxygen induced increased cardiac effort [36]. This cohortwas also found to have an additional muscle layer in theirpulmonary arteries [36]. Thus, in some contexts, there maybe benefits derived from increasing ELTD1 expression.

In addition to its angiogenic role in cancer, ELTD1 has alsobeen found up-regulated in the tumour vasculature of highgrade gliomas [37] and has been found to be part of a panelof nine genes up-regulated in patients with ulcerative colitisharbouring an occult colorectal cancer [38].

Using publically available bioinformatic data, we note thatthe majority of cancer cell lines do not highly express ELTD1[39] (Figure 3A). We found that ELTD1 mutations occur in awide range of human cancers. These mutations do not clusterin any specific region but rather occur randomly across all ofELTD1’s domains [40]. The frequency of ELTD1 mutations,however, is low, with only melanoma and lung squamouscarcinoma having a mutational frequency of 10 % or morein 48 curated cancer genome datasets [41] (Figure 3B). Thesemutations are thus unlikely to be relevant functionally.

We reviewed genes in the vicinity of ELTD1 for thepresence of known cancer driver genes and found thatthe known cancer gene FUBP1 (a causative gene for botholigodendrogliomas and oligoastrocytomas [42]) is presentin the same band as ELTD1 (1p31.1). Looking within 10gb ofeither side of ELTD1, we found an additional cancer driver,BCL10 (a causative gene in mucosa-associated lymphoidtissue lymphoma [43]). Analysis of ELTD1, FUBP1 andBCL10 in a range of cancer genome datasets reveals thatmutations in these three genes occur mostly in ELTD1 andthat amplifications or deletions usually affect all three geneswhen present in a patient. This suggests that ELTD1 mayfunction as a passenger gene in these circumstances or be anovel cancer driver.

Other associations include ELTD1 polymorphisms con-ferring vulnerability to cannabis dependence [44] andincreased event-free longevity [from a meta-analysis of ninegenome-wide association studies (GWAS) on aging] [45].ELTD1 has also been described as one of eight genesresponsible for subcutaneous fat thickness in humans and pigs[46]. Finally, ELTD1 polymorphisms have been identifiedin patients at risk of developing graft-versus-host diseasefollowing haemopoietic stem cell transplants [47], and incattle who are resistant to therapy against tick parasites[48]. In all these cases, however, the mechanisms underlyingELTD1’s association remain to be identified.

ConclusionELTD1 is an adhesion GPCR with roles which includethe regulation of physiological and tumour angiogenesis,involvement in cardiac development and cardioprotection,and regulation of expression in certain cancers. In all thesevaried roles, ELTD1 warrants further investigation as both abiomarker and therapeutic target.

Funding

D.M.F. is funded by the Rhodes Scholarship. A.H.B. and A.L.H. are

funded by Cancer Research UK.

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Received 6 August 2014doi:10.1042/BST20140216

C©The Authors Journal compilation C©2014 Biochemical Society