hGPR87 contributes to viability of human tumor cells

Sebastian Glatt1, Daniel Halbauer

1, Stefan Heindl

1, Andreas Wernitznig

1, Daniela Kozina

1, Kuan-Chung Su

1,

Christina Puri2, Pilar Garin-Chesa1,2 and Wolfgang Sommergruber1*

1Boehringer-Ingelheim Austria GmbH, Dr. Boehringer-Gassse 5-11, Vienna, Austria2Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria

Emerging in vitro and in vivo data underline the crucial role of G-protein-coupled receptors (GPCRs) in tumorigenesis. Here, wereport the contribution of hGPR87, a predicted member of theP2Y subfamily of GPCRs, to proliferation and survival of humantumor cell lines. hGPR87 mRNA transcript was found to be pref-erentially overexpressed in squamous cell carcinomas (SCCs) ofdifferent locations and in their lymph node metastases. Up-regula-tion of both, transcript and protein, was detected in samples ofSCC of the lung, cervix, skin and head and neck (pharynx, larynxand epiglottis). In addition to the expression of hGPR87 in tumorswhich originate from stratified epithelia, we identified otherhGPR87-positive tumor types including subsets of large cell andadenocarcinomas of the lung and transitional cell carcinomas ofthe urinary bladder. Loss of function studies using siRNA inhuman cancer cell lines lead to antiproliferative effects and induc-tion of apoptosis. Like other known P2Y receptors, hGPR87 wasfound to be mainly located on the cell surface. The overexpressionof hGPR87 preferentially in SCCs together with our functionaldata suggests a common molecular mechanism for SCC tumori-genesis and may provide a novel intervention site for mechanism-based antitumor therapies.' 2008 Wiley-Liss, Inc.

Key words: squamous cell carcinoma; GPR87; immunohisto-chemistry; survival; apoptosis; protein localization

G-protein-coupled receptors (GPCRs) account for more than2% of all genes encoded by the human genome and are thereforeone of the largest gene families known within the human genome.1

The characteristic seven-transmembrane-spanning (7TM) a-heli-ces of GPCRs harboring extracellular N- and intracellular C-ter-mini in addition to their structurally highly complex ligand bind-ing sites fulfill the criteria of specificity and ‘‘drugability.’’2 Themolecular characterization of putative GPCRs3 and the identifica-tion of their natural occurring ligands (‘‘de-orphanization’’) willallow the elucidation of the molecular mechanisms of GPCR-driven diseases and the development of novel target-mediatedtherapies.4

Aberrant overexpression of GPCRs can induce a transformationprocess leading to a neoplastic cell5 and thereby the function ofthe endogenous GPCR is incorporated into an essential part of tu-mor progression and survival program.1 The incorporation ofGPCR functionality by overexpression is also exploited by severaltumor-associated DNA viruses.6 GPCR-mediated signaling cansupport tumorigenesis by promoting tumor growth, angiogenesis,survival, migration, metastasis and drug resistance.7–12

The orphan hGPR87 was first described in an ‘‘EST Data Min-ing Strategy’’ for novel GPCR encoding genes, and has beenpreliminarily assigned to the P2Y receptor family by wholesequence homology13 and the presence of conserved amino acids,shown to be important for ligand binding and specificity in relatedfamily members.13–15 By utilizing a ligand docking softwareUDP-Glucose and cysteinyl-leukotrienes (CysLTs) have been pro-posed as potential ligand candidates for hGPR87, supporting theprediction based on conserved amino acid residue homology.14,16

However, despite the intense work in different laboratories,de-orphanization of hGPR87 still remains unresolved.

In this study, we investigated the tissue distribution of hGPR87utilizing different mRNA expression databases including laser-capture-micro-dissected (LCM) tissue samples and immunohisto-chemistry in a panel of human tumors. Our analysis showed that

normal tissues generally lack RNA signals, with the exception ofthe head and neck region. Analysis of tumor samples revealedprominent hGPR87 RNA expression in SCCs. This expressionwas further confirmed by immunohistochemistry in head andneck, cervix, skin and lung carcinoma samples. Additionally, weused several human tumor cell lines to study the effect of hGPR87on proliferation and survival in vitro. We depleted hGPR87 inhuman tumor cells using siRNA, which resulted in the specificdecrease of proliferation rates accompanied by the induction ofapoptosis. Analysis of the cellular localization of hGPR87 usingGFP/RFP fusion proteins and fluorescence-activated cell sorting(FACS) revealed a predominant cell surface localization ofhGPR87. Altogether, overexpression of hGPR87 and its functionmay represent an essential step for the development and mainte-nance of tumors, particularly of SCCs.

Material and methods

Cell lines and cell culture

Human HN5 head-and-neck squamous cell carcinoma cellswere grown in DMEM supplemented with 10% fetal bovine se-rum, L-Glutamine, nonessential amino acids and 1mmol/l sodiumpyruvate (all from Invitrogen, Carlsbad, CA). All other cell lineswere obtained from ATCC and were propagated according to pro-vider’s instructions. Cell numbers and viability were analyzed acc-ording to manufacturer’s instructions using a Vi-CellXR device(Beckman Coulter, Fullerton, CA).

Tissue samples

Human tissues were collected at the Institute of Pathology,Medical University of Vienna, Austria, following institutionalguidelines. In addition, 10 cases of cervical SCCs were obtainedfrom Oridis Biomed (Graz, Austria).

Transcription profiling

For expression analysis, box-and-whisker plots were generatedas described.17,18 The respective hybridizations were performed onthe Affymetrix HG-U133A/B oligonucleotide chip set (Affyme-trix, Santa Clara, CA). These chips are based on 25-mer oligonu-cleotides and allow the detection of more than 39,000 human tran-scripts, with probe sets of 11 oligonucleotides used per transcript.

Chip data were normalized with the statistical algorithm imple-mented in the Microarray Suite version 5.0 (mas5; Affymetrix).Briefly, the raw expression intensity for a given chip experiment ismultiplied by a global scaling factor to allow comparisonsbetween chips. This factor is calculated by removing the highest2% and the lowest 2% of the values of the non-normalized expres-

During submission of this manuscript, hGPR87 was reported to be acti-vated by lysophophatidic acid (Tabata et al., Biochem Biophys Res Com-mun, epub ahead of print).*Correspondence to: Oncology Research, Boehringer Ingelheim Aus-

tria GmbH, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria. Fax:143-1-80105-2782.E-mail: wolfgang.sommergruber@vie.boehringer-ingelheim.comReceived 31 August 2007; Accepted after revision 8 November 2007DOI 10.1002/ijc.23349Published online 8 January 2008 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 122, 2008–2016 (2008)' 2008 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

sion values and calculating the mean for the remaining values, astrimmed mean. One hundred divided by the trimmed mean givesthe scaling factor, where 100 is the standard value used by Gene-Logic. For oligonucleotide-specific background normalizationindividual absent- and present-calls are calculated by comparingthe absolute signal intensities for corresponding perfect-match andmismatch oligonucleotides.

siRNA analyses

A SMARTpool-plus set of 4 individual siRNAs targeting thehGPR87 mRNA sequence was obtained from Dharmacon (Lafay-ette, CO). The pool and the individual siRNAs were tested sepa-rately for efficient down-regulation of hGPR87 mRNA and protein.The siRNA duplex targeting the following sequence 50-GATCG-GAAGTTCGCATATATT-30 showed the most efficient silencingeffect. siCONTROL Non-Targeting siRNA #1 was also purchasedfrom Dharmacon. For each siRNA transfection, �2 3 106 cellswere plated in a 15 cm tissue culture dish (GreinerBioOne, Frick-enhausen, Germany) and cultured for 24 hr to yield 40–50% con-fluence. siRNA duplexes were transfected with DharmaFect (#4)reagent at indicated concentrations. The cells were analyzed 24–72 hr after siRNA transfection. Cellular proliferation rates weremeasured 72 hr after transfection according to manufacturer’sinstructions using AlamarBlue reagent (AbD Serotec, Oxford, UK).

PCR

Total RNA was isolated using Trizol (Invitrogen, Carlsbad,CA) reagent, following manufacturer’s protocol including DNA-seI digest of genomic DNA. One microgram of the total RNAextraction was immediately reverse transcribed into ssDNA usingthe PowerScript PrePrimed kit (Clontech, Mountain View, CA).After ssDNA synthesis the nucleotide concentration was measuredand equal amounts of ssDNA were used for subsequent PCR steps.For quantitative RT-PCR we used commercially available gene-specific TaqMan expression assays (Applied Biosystems, FosterCity, CA) for hGPR87 and beta-actin. Quantification and analyseswere performed using the Mx4000 device from Stratagene (LaJolla, CA) using the provided software package. Verification ofthe expressed fusion construct was carried out using conventionalPCR with rTaq polymerase and specific primers for hGPR87 (50-GGCCAAGAGAGTCACAATT CAGG-30), GFP (50-CGGCGGCGGTCACGAAGCCG-30; 50-GGTGAGCAAGGGCGCCGAGC-30) and beta-actin (50-AGG CCGGCTTCGCGGGCGAC-30; 50-CTCGGGAGCCACACGCAG CTC-30) at 63�C annealing tem-perature and 35 cycles.

Apoptosis assay

siRNA treated cells for ApoDirect (BD Pharmingen, FranklinLakes, NJ) analysis were rinsed with cold phosphate-buffered sa-line (PBS; 17 mmol/l KH2PO4, 50 mmol/l Na2HPO4 and 150mmol/l NaCl) and scraped off using a rubber policeman. Nonad-herent cells were collected by centrifugation and combined withthe adherent cell fraction. After centrifugation the cells werewashed twice in PBS, resuspended in 1% paraformaldehyde andincubated on ice for 15 min. After fixation cells were washedtwice in PBS and then resuspended in ice-cold 70% ethanol andincubated on ice for 30min. The cells were stored at 220�C o/nand stained the following day. For the detection of cells containingDNA double-strand breaks the ApoDirect kit was performed fol-lowing manufactorer’s instructions. The stained samples were sub-sequentially analyzed with the FACSCalibur device and the Cell-Quest software package (BD Pharmingen, Franklin Lakes, NJ).

FACS analysis

Briefly, the cells were washed with PBS and harvested by theaddition of versene at 37�C. Subsequently, the cells were washedwith staining buffer (PBS, 0.5% BSA) and incubated with the pri-mary antibody (10 lg/ml) for 45 min at 4�C. The staining wascontinued by repeated washing and incubation with the secondary

antibody (2 lg/ml) for 45 min at 4�C. Finally, the cells werewashed again 3 times and resuspended in staining buffer. Themeasurement was performed on the BD FACSCanto device anddata were analyzed using the WinMDI software.

Generation of fusion proteins

The ORFs of hGPR87-GFP and/or -RFP fusions were clonedinto the pcDNA5/FRT vector, carrying a Flp Recombination Tar-get (FRT) site and a hygromycin resistance gene. The Flp-In-293cell line, which is derived from HEK 293 cells carrying also anintegrated FRT site, was obtained from Invitrogen (Carlsbad, CA).The Flp-In-293 cells were cotransfected with pOG44, which enco-des the Flp recombinase, and pcDNA5/FRT hGPR87-GFP or -RFP. Only cells transfected with both vectors could accomplish theintegration of hGPR87-GFP or -RFP into the genomic FRT site,which disturbs the zeocin-resistance ORF. Therefore, we selectedfor stably transfected cell clones with hygromycin (200 lg/ml) andconfirmed site specific recombination by zeozin sensitivity.Approximately 80 cell clones were analyzed for doxycycline-de-pendent expression of the fusion constructs and sequence identityof the expressed construct. Those clones with the best signal tonoise ratio (uninduced/induced) were further used for analyses.Expression was induced by the addition of 100 ng/ml doxycycline.

Antibody generation

The abGPR87N antibody was generated by immunizing rabbitswith a peptide corresponding to the N-terminus of hGPR87(MGFNLTLAKLPNNELHGQESHNSGNRSDGPGKNTTLHNEFDTIVLP). abGPR87 was affinity purified using Sulfo-Link-Gelcoupled corresponding peptide. The antiserum was eluted with0.2 mol/l Glycine/HCl, pH 2.7, and 150 mmol/l NaCl.

Immunoblotting

Cells were rinsed in ice-cold PBS and harvested. After centrifu-gation the cell pellet was resuspended in lysis buffer containing20 mmol/l Tris (pH 7.5), 5 mmol/l EDTA, 2 mmol/l EGTA, 0,5%Triton X-100, 30 mmol/l sodium fluoride, 20 mmol/l sodium py-rophosphate, 40 mmol/l b-glycerophosphate, 1 mmol/l sodiumorthovanadate, 10 lmol/l leupeptin, 5 lmol/l pepstatin A, 3 mmol/l benzamidine and supplemented with 1 mmol/l phenylmethylsul-fonyl fluoride. Precasted 4–12% gradient Tris-glycine gels (BIO-RAD, Hercules, CA) were used for electrophoresis. After transferto Hybond nitrocellulose membranes (Amersham, Buckingham-shire, UK), the blots were blocked in 5% dried milk in TBS-T(Tris-buffered-saline containing Tween) for 1 hr at RT and incu-bated at 4�C o/n with the primary antibodies. For peptide competi-tion the antibodies were preincubated in 5% milk in TBS-T withthe indicated amounts of peptide for 4 hr at RT. abGPR87C wasfrom Acris antibodies (Herford, Germany), antibody for GAPDHfrom Abcam (Cambridge, UK), anti-PARP from Cell SignalingTechnology (Boston, MA). Detection of the bound antibody wasperformed using ECL reagent (GE Healthcare, Bucks, UK).

Immunohistochemistry

An indirect immunoperoxidase assay was used as follows.Briefly, 5-lm thick paraffin sections were cut and mounted onpoly-(L-lysine)-coated slides. After deparaffinization and epitoperetrieval with Proteinase K in TrisEDTA buffer, pH 8.0, for15 min at 37�C, the sections were blocked with 10% normal goatserum. Subsequently, the primary antibody (abGPR87C) wasapplied for 1 hr at RT followed by incubation with goat anti-rabbithorseradish peroxidase labeled polymer, Dako Envision System(Dako, Carpinteria, CA) for 30 min at RT. The final reaction prod-uct was visualized with DAB (0.06% 3,30-diaminobenzidine inPBS, 0.003% H2O2) for 2–5 min. The slides were then dehydratedin alcohol, and counterstained with hematoxylin. The specificityof the staining was confirmed by preincubating the primary Abwith a synthetic peptide (PSL GmbH, Heidelberg, Germany) for

2009hGPR87 CONTRIBUTES TO VIABILITY OF TUMOR CELLS

2 hr at RT at the final concentration of 25 lg/ml of Ab, whichresulted in no staining of the tumor cells.

Results

Expression profiling reveals preferential overexpression ofhGPR87 in SCC

We recently identified a number of genes which were overex-pressed in a tumor-specific manner by combining cDNA subtrac-tion and cDNA microarray analyses of different tumor types.19 Asa part of this study, we observed that hGPR87 was overexpressedin SCCs of the lung in comparison to adenocarcinomas and to thecorresponding normal lung tissue.

On the basis of these findings in lung SCCs and tumor-specificgenomic amplification patterns,20–26 we analyzed the expressionof hGPR87 in more detail utilizing transcription profiling data-bases,17 which are based on the Affimetrix GeneChip technology.In agreement with the genomic amplification of 3q24, we foundthe hGPR87 transcript significantly up-regulated in squamous cellcarcinomas of different locations, as well as in their lymph node(LN) metastasis and in transitional cell carcinomas (TCC) of theurinary bladder. In addition to SCC-specific expression, we foundthe hGPR87 transcript up-regulated in skin samples adjacent toprimary tumors and in psoriasis sections in comparison to samplestaken from healthy individuals (Fig. 1a). The presence of non-transformed tumor stroma consisting of activated fibroblasts,blood vessels and inflammatory cells in whole tissue preparations

very often leads to misinterpretation of ex vivo tissue expressiondata.27 To exclude a putative relevant contribution of GPCRexpression from the tumor stroma, we analyzed the expressionpattern of hGPR87 in several microdissected (LCM) tissue sam-ples of lung carcinomas and compared it with intact tumor sam-ples and with nonneoplastic lung tissue samples. For a better com-parability total RNA from all cell types and LCM-dissected epi-thelial tumor cells was analyzed from different sections of theidentical frozen tissue samples. Using this approach, we were ableto confirm the tumor-specific expression of hGPR87 (Fig. 1b).Additional support for the tumor-specific expression of hGPR87comes from the transcription profiling analyses of human carci-noma cell lines. With the exception of a gastric cancer cell line(NCI-N87), no expression of hGPR87 was detected in nonsqua-mous carcinoma cell lines whereas profound expression wasdetected in SCC-derived cell lines (Fig. 1c). All the squamous cellcarcinoma cell lines with an increased expression level ofhGPR87 also carried genomic amplifications on 3q24, therebyreflecting the genomic and the transcriptional status describedin vivo (data not shown).

Cellular localization of hGPR87

To study the cellular localization of hGPR87, we generated apolyclonal rabbit antiserum (abGPR87N) directed against a pep-tide corresponding to the extracellular N-terminus of hGPR87. Acommercially available polyclonal antibody to the N-terminus(GPR87N, LifeSpan Biosciences, WA), was tested in parallel.

FIGURE 1 – Analyses of hGPR87 mRNA expression in human normal tissues, neoplastic diseases and human tumor cell lines. hGPR87mRNA expression was analyzed using Affimetrix Chip technology. The expression levels of hGPR87 mRNA are shown in box-plots (a). Thevertical center line in the box indicates the median; the vertical dotted line refers to the mean value. The box itself represents the interquartilerange (IQR) between the first and third quartiles. Whiskers extend to 1.5 times the IQR; the position of extreme values, if above the upperwhisker limits, are marked by open circles. The human sample collection has been described by the originator of the BioExpress database.17

Numbers in brackets indicate the sample numbers. The Affymetrix identification code for hGPR87 is 219936_s_at. For a set of lung cancer sam-ples both LCM-purified epithelial cells (LCM1) and non-LCM tissue sections (-LCM) were available and subjected to transcription profilinglikewise (b). LCM was carried out essentially as previously described.27 hGPR87 mRNA expression levels in human tumor cell lines are shownas bar blots of their relative expression values (c). Individual absent (A) and present (P) calls are indicated (for details see Material and meth-ods). TCC, transitional cell carcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung cancer; Adenoca, adenocarcinoma; AdSq Ca, ad-enosquamous carcinoma; LCC, large cell carcinoma; Normal TA, normal tumor-associated; Mel (P), melanoma primary; BCC, basal cell carci-noma; Mel, melanoma; normal (green), cancerous (red), metastatic (blue), tumor-associated normal (yellow), psoriasis (grey).

2010 GLATT ET AL.

Both antisera were used to analyze the presence of endogenoushGPR87 on the cell surface of HN5 cells by FACS analysis usinga nonmembrane-permeabilizing protocol (Fig. 2a). To monitor thedynamics and intracellular trafficking of hGPR87, a doxycycline-inducible expression system in HEK-293 cells with hGPR87-GFP/RFP fusion proteins was generated and stable cell clones wereestablished. GFP expression as a nonfusion protein served as con-trol. In the HEK-293 cells the localization of transiently trans-fected GFP was strikingly different (cytoplasm) in comparison tohGPR87 fusion constructs (cell surface) (Fig. 2c). The identity ofthe doxycycline-induced fusion transcripts was confirmed bysemiquantitative RT-PCR (Fig. 2b) and subsequent sequencing ofthe RT-PCR products. It was shown that hGPR87-GFP wasmainly localized on the cell surface of different cell lines (e.g.,HEK293, HeLa), which seems to reflect the in vivo localization intumor cells (Figs. 3b and 4). Interestingly, we observed hGPR87internalization (redistribution into vesicle structures) under certaincell culture conditions (data not shown) in our cell line models.Whether this redistribution of hGPR87 into vesicles is due to acti-vation by agonists, possible present under those cell culture condi-tions, remains to be determined as the natural ligand of hGPR87 isstill unknown.

Immunohistochemistry revealed strong expression of hGPR87 in ahigh proportion of different SCCs and lung adenocarcinomas

As the biological function of hGPR87 seems to be essential forsurvival of tumor cell lines, we next addressed the question ofwhether a correlation exists between the up-regulation of RNAtranscripts (Figs. 1a and 1b) and the protein expression in the cor-responding tumor tissue samples by immunohistochemistry.

Fifty-eight paraffin-embedded tumor samples were analyzedusing a rabbit polyclonal-antiserum directed to the C-terminal in-tracellular domain of hGPR87 (abGPR87C). The specificity of theantiserum was demonstrated on Western blots (Fig. 3a) and on tis-sue samples (Fig. 3b) by absorption experiments with the immu-nizing peptide. For all tumor tissue samples, the correspondingnonneoplastic tissues were used as staining controls.

For several lung cancer samples, paraffin-embedded and fresh-frozen samples were available, thus, allowing the correlationbetween mRNA expression levels by qPCR and immunohisto-chemistry (IHC) staining from adjacent tissues. In the majority ofcases, a good correlation was observed, in general the mRNA lev-els were lower in adenocarcinomas than in SCCs, whereas byIHC, the hGPR87 expression was similar in both tumor types,regarding the intensity of the immunostaining and the percentageof positive cells in the cases analyzed (Fig. 3b). Similarly, SCCsfrom other locations (cervix, larynx, pharynx and skin) showedstrong and homogeneous expression in the tumor cells (Fig. 4).The essential contribution of hGPR87 to tumorigenesis is sup-ported by the fact that a high proportion of analyzed samples arehGPR87-positive in IHC such as 10/17 lung SCCs and 9/10 cervixSCCs to name but a few. The results of the IHC analyses are sum-marized in Table I.

hGPR87 protein is essential for proliferation and viability ofhuman tumor cell lines

Interestingly, we found elevated hGPR87 mRNA transcript lev-els not only in primary SCCs, but also in their lymph node metas-tases, suggesting that hGPR87 may transduce signals that promotetumor cell proliferation and survival. To test this hypothesis we

FIGURE 2 – The cellular localization of hGPR87. Presence of hGPR87 at the cell surface is shown by FACS analysis using GPR87N (GPR87N,blue), anti-E. coli antibody (E. coli, green) and secondary antibody alone (2nd Ab, black) (a). PCR analysis of stable cell clones of HEK-293 cellsexpressing C-terminally GFP-tagged fusion proteins of hGPR87 under the regulation of a doxycycline-dependent promoter. Doxycycline-depend-ent induction of expression was confirmed by conventional PCR (b). In contrast to GFP alone (GFP) which is localized in the cytoplasm, theinduced fusion protein hGPR87-GFP (1doxy) shows a predominant localization on the cell surface (c). Original magnification:363.

2011hGPR87 CONTRIBUTES TO VIABILITY OF TUMOR CELLS

used an siRNA approach in a hGPR87-positive cell line (HN5;Fig. 1c) and in two hGPR87-negative cell lines (MDA-MB453and T47D; Fig. 1C) for the detection of possible off-target effectsof the siRNA duplex. Out of a predesigned siRNA pool (see Mate-rials and methods), we then identified a single siRNA duplex,which reduced hGPR87 mRNA to below 10% in HN5 cells overthe entire observation period of 3 days (Fig. 5a). Reduced levelsof hGPR87 protein remained detectable after 24 hr, but werenearly undetectable at 48 and 72 hr (Fig. 5b). In addition, weincluded control siRNA duplexes and mock controls to excludeany off-target effects of the siRNA treatment on the cells. Usingincreasing amounts of siRNA, a dose-dependent decrease in pro-liferation rates in HN5 cell lines was detected, whereas prolifera-tion rates remained unchanged in the hGPR87-negative MDA-MB-453 cell line (Figs. 5c and 5d). Additionally, no changes inproliferation rates were detected in mock-transfected controls(data not shown). The decrease in proliferation rates coincideswith the induction of apoptosis, which was shown by caspase-de-pendent PARP cleavage (Figs. 5c and 6b) and the occurrence ofdouble-strand breaks (Fig. 5d). The decrease in proliferation rateand the induction of apoptosis was accompanied by the reductionof overall viability of HN5 cells (Fig. 6c). In contrast, hGPR87-

negative T47D cells showed no changes in the growth curve, upontreatment with either GPR87-1 or siCTRL siRNA even 72 hr aftertransfection (Fig. 6c). In addition, reduction of HN5 cellular via-bility starting at 48 hr after siRNA transfection coincides with sig-nificant reduction of hGPR87 protein levels at that time point(Figs. 6c and 5b). Accordingly, the reduction of overall viabilitycoincides with the induction of apoptosis in HN5 cells, but not inhGPR87-negative T47D cells, as shown by caspase-dependentPARP cleavage (Fig. 6d).

Discussion

There is now a large body of evidence that GPCRs indeed playa crucial role in tumorigenesis covering all important steps of can-cer progression from transformation,28,29 growth and survival30,31

to metastasis.32,33 An additional important contribution of GPCRsto tumorigenesis comes from the intensive crosstalk with canoni-cal pathways. For instance, it has been shown that in colon cancerthe EP2 receptor can promote the transactivation of EGFRexpressed on colon cancer cells through Src,34 GPCR ligands likeprostaglandin E2 (PEG2) and bradykinin (BK) activate EGFR sig-

FIGURE 3 – hGPR87 expression in lung cancer by immunohistochemistry and Western blot analysis. The specificity of the antibody was deter-mined by preabsorption of the antibody with the immunizing peptide and shown on Western blots (a) and on tissue sections (b) of the corre-sponding tumors. The peptide concentrations used to optimize the blocking experiments are indicated. Strong and homogeneous staining of thetumor cells is shown in the adenocarcinoma and in the squamous cell carcinoma samples, with a membrane/cytoplasmic pattern. Notice that theimmunostaining signal is completely abolished after incubation with the blocking peptide. Original magnification: 320.

2012 GLATT ET AL.

naling30 or peptide growth factor (PGF) receptor tyrosine kinase-RAS-RAF-MEK/PI3K-AKT signaling stimulates the HH/SMOpathway via activation of GLI in melanoma.35

Here, we identified hGPR87 as a GPCR, which is essential forthe viability of several human squamous cell carcinoma cell linesand which is predominantly overexpressed in SCCs of differentlocations and in their lymph node metastases. Expression ofhGPR87 is highly restricted in differentiated normal tissues (Fig.1a), suggesting that the transcription of hGPR87 is under strictcontrol. In tumors, however, induction and/or overexpression hasbeen observed. Consistently, the genomic region containing thehgpr87 gene on human chromosome 3q24,13 is found to be ampli-fied in neoplastic cells of different locations, including lung carci-nomas (squamous and adenoca),36–38 cervical squamous carcino-mas,20–23 head and neck squamous cell carcinomas (i.e., larynx,pharynx, tonsil, tongue)24,25 and urinary bladder carcinomas.26

Possibly, these genomic amplifications of 3q24 represent a majorroute for hGPR87 deregulation. However, other yet unknown clo-nal effects within transformed cells, which might select forhGPR87 overexpression, cannot be excluded. As the expressionlevels of hGPR87 mRNA and hGPR87 protein correlate in ourRT-PCR/IHC analyses, we assume that hGPR87 protein expres-sion levels are not posttranscriptionally up-regulated. The utiliza-tion of transcription profiles derived from LCM-prepared humantumor tissue samples and IHC analyses clearly show that hGPR87is specifically expressed in tumor cells and not in cells of the tu-mor stroma. The overall incidence of hGPR87 (over)expressionamong all IHC-analyzed tumor types is very high (Table I), how-ever, the absence of hGPR87 protein in 40% of lung SCCs indi-cates a different molecular mechanism for squamous cell carcino-genesis in those tumors. Although the absolute hGPR87 expres-sion levels are varying (e.g., in SCCs of lung and cervix), thefactor of induction (based on mean expression values) betweennormal and cancerous tissue is very similar within different tumorspecies (Fig. 1a).

The whole genomic organization of the chromosomal locus ofhGPR87 on human chromosome 3q24 is highly conserved in man,

rat and mouse. The amino acid sequence of GPR87 is nearly iden-tical in chimpanzee (100%) and rhesus monkey (99%), whereasthe orthologs of mouse, rat and dog exhibit strong amino-acidsequence variation in the N-terminal extracellular domain, butbeing nearly identical in the remaining sequence. However, themurine expression pattern of Gpr87 in normal tissues resemblesthat in humans (data not shown) pointing towards a conserved bio-logical function.

Several P2 receptors have been shown to be expressed in tumortissues39 and to harbor transforming activity.40 On the basis of ouranalyses, none of the previously described receptors has such aprofound tumor-specific expression pattern like hGPR87. In addi-tion, we identified upregulation of hGPR87 transcript at inflamma-tion sites such as in psoriasis (Fig. 1a). Interestingly, it was shownthat arachidonic acid metabolism-related genes are deregulated inesophageal SCCs41 and that inhibiting leukotriene synthesis inSCCs cell lines leads to decrease in proliferation rates.42 As thehGPR87 protein sequence shows high similarities to a P2Y-sub-family (P2Y12, 13, 14) and also high similarity to CysLTR1 andCysLTR2, hGPR87 may add a yet unidentified link between

FIGURE 4 – IHC analysis of hGPR87 expression in human SCC samples. Examples of hGPR87 expression in SCCs of the lung, cervix, larynxand pharynx are shown. Original magnification: 320.

TABLE I – SUMMARY OF IHC-ANALYZED MALIGNANT TUMORS SAMPLES

Tumor type nhGPR87 expression

Positive Negative

Lung cancer total 31 21 10Lung SCC* 17 10 7Lung Adenoca* 14 11 3Head and neck cancer total 12 9 3Larynx SCC 7 7 0Pharynx SCC 5 2 3Skin SCC 5 5 0Cervix SCC 10 9 1

*qPCR was performed from all samples where corresponding cryo-samples were available. In those samples a high correlation of mRNAexpression and intensity of IHC staining was observed.

2013hGPR87 CONTRIBUTES TO VIABILITY OF TUMOR CELLS

FIGURE 5 – siRNA-mediated knock down of hGPR87 in human head and neck SCC tumor cell line HN5. The levels of hGPR87 mRNA arestrongly reduced in siRNA-treated cells (full box) in comparison to cells treated with siCTRL (open box) during a 3-day period (a). Proteinexpression levels of hGPR87 were analyzed on Western blots utilizing GAPDH as loading control. siCONTROL#1 (Dharmacon, CO) was usedas control (b). Induction of apoptosis was most prominent at 48 hr posttransfection and was monitored by a caspase-induced PARP-cleavage(PARP-cleavage product is marked with an asterisk). As controls a commercially available control siRNA (siCONTROL#1; Dharmacon), amock transfection and untreated HN5 cells were used (c). FACS analysis showing the percentage of cells with DNA double strand breaks (fullbox) or without (open box) in relation to total cell numbers (d).

FIGURE 6 – Effects of hGPR87 siRNA-mediated knock down on proliferation and overall viability of human tumor cells. The effects of vary-ing GPR87-1 siRNA concentrations were measured by AlamarBlue staining in a hGPR87-positive (HN5) and a HGPR87-negative (MDA-MB-453) cell line (a); in parallel, caspase-dependent PARP cleavage product (asterisk) was analyzed on Western blots (b). Overall cellular viabilitywas measured by counting viable cells (Trypan blue staining) at the indicated time points after transfection in a hGPR87-positive (HN5) andhGPR87-negative (T47D) cell line (c); in parallel, caspase-dependent PARP cleavage product (asterisk) was analyzed on Western blots at 48and 72 hr after transfection (d).

2014 GLATT ET AL.

CysLT-mediated chronic inflammation response and the develop-ment of SCCs.

On the basis of knock down experiments using siRNA, hGPR87facilitates proliferation and is responsible for survival of humantumor cell lines. The cellular localization of hGPR87 seems to besimilar to other P2Y family members, as P2Y1, P2Y2, P2Y11 andP2Y12 were described to localize to the cell surface under normalcell culture conditions and internalize after ligand stimulation.43–46

As the inducible promoter of our hGPR87-GFP and/or -RFPfusion-proteins allows a dose-dependent expression of differentlevels of hGPR87, we can exclude that the localization is renderedby the artificial overexpression; the localization is not dependenton the expression level in HEK293 cells. The internalization ofhGPR87-GFP/RFP could be observed under specific cell cultureconditions (data not shown). Therefore, it might be possible thatligand-stimulation of hGPR87, as known from other GPCRs,results in intracellular localization. Perhaps the presence or ab-

sence of ligand was responsible for the differences in our IHCstainings, ranging from a clear cell surface to a diffuse cytoplas-matic localization of hGPR87. However, a more detailed under-standing of the molecular signaling mechanism will be availablewhen hGPR87 becomes de-orphanized.

In summary, our results indicate that hGPR87 plays an essentialrole for proliferation and survival of tumor cells. As overexpres-sion is predominately found in tumor cells and tumor samples ofsquamous cell origin, hGPR87 might be specifically involved insquamous cell carcinogenesis.

Acknowledgements

We are grateful to Dr. Michael Seewald and Dr. ChristianHaslinger for their assistance with bioinformatics. We also thankMag. Christian Rupp and Dr. Helmut Dolznig for their criticalreading of the manuscript.

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