Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry Into S-Phase and Supports

Published OnlineFirst August 21, 2012; DOI: 10.1158/1541-7786.MCR-12-0139

Molecular Cancer Cell Cycle, Cell Death, and Senescence Research

Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry into S-Phase and Supports

G2–M Cell-Cycle Progression

Stefan Kastner1, Tilman Voss1, Simon Keuerleber2, Christina Glockel€ 2, Michael Freissmuth2, and Wolfgang Sommergruber1

Abstract It has long been known that G protein-coupled receptors (GPCR) are subject to illegitimate expression in tumor cells. Presumably, hijacking the normal physiologic functions of GPCRs contributes to all biologic capabilities acquired during tumorigenesis. Here, we searched for GPCRs that were expressed in lung cancer: the mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found frequently overexpressed in tissue samples obtained from patients with small cell lung cancer. Several observations indicate that overexpression of Gpr19 confers a specific advantage to lung cancer cells by accelerating transition through the cell- cycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell – lines. (ii) Cell-cycle progression through G2 M-phase was impaired in cells transfected with siRNAs directed against Gpr19 and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. (iii) The expression levels of Gpr19 mRNA varied along the cell-cycle with a peak observed in S-phase. (iv)TheputativecontrolofGpr19 expression by E2F transcription factors was verified by chromatin immunoprecipitation: antibodies directed against E2F-1 to -4 allowed for the recovery of the Gpr19 promoter. (v) Removal of E2F binding sites in the Gpr19 promoter diminished the expression of a luciferase reporter. (vi) E2f and Gpr19 expression correlated in lung cancer patient samples. To the best of knowledge, this is the first example of a GPCR showing cell-cycle-specific mRNA expression. Our data also validate GPR19 as a candidate target when overexpressed in lung cancer. Mol Cancer Res; 10(10); 1343–58. 2012 AACR.

Introduction with SCLC exhibiting the most aggressive and metastatic phenotype (2). Lung cancer is among the leading causes of cancer- fi related deaths worldwide and is strongly associated with The de ning features of the family of G protein-coupled smoking. On the basis of pathologic features lung cancers receptors (GPCR) are the hydrophobic core of 7 transmem- are classified into 2 major groups. Small cell lung cancer brane-spanning a-helices and their eponymous action, i.e., (SCLC) accounts for 20% of all cases and displays char- the activation of their cognate heterotrimeric G protein(s). acteristics of neuroendocrine tumors (1). Non-small cell The human complement of GPCRs consists of more than lung cancer (NSCLC) is further subdivided into squamous 800 members (3). GPCRs are intimately involved in the cell carcinoma (30%–40%), adenocarcinoma (30%– control of virtually all cell types, their structure allows for 40%), and large cell carcinoma (<10%). Both types of binding of highly diverse ligands, thus they are considered lung cancer not only differ in their cellular morphology the most druggable family of proteins (4). However, only but also in the available treatment and their prognosis— about 60 GPCRs are currently targeted by drugs approved for clinical use (5). Several arguments support the assumption that GPCRs Authors' Affiliations: 1Boehringer Ingelheim RCV GmbH & Co KG, may also be specifically targeted to limit the growth of cancer Department of Lead Discovery; and 2Institute of Pharmacology, Center cells: signaling pathways controlled by GPCRs promote of Physiology and Pharmacology, Medical University of Vienna, Vienna, fl Austria proliferation, survival, cell migration, angiogenesis, in am- mation, and subversion of the immune system (4, 6). Their Note: Supplementary data for this article are available at Molecular Cancer — Research Online (http://mcr.aacrjournals.org/). uncontrolled exploitation mostly seen by overexpression of GPCRs—could in turn contribute to the malignant Corresponding Author: Wolfgang Sommergruber, Boehringer Ingelheim RCV GmbH & Co KG, Department of Lead Discovery, Dr. Boehringer-Gasse transformation of cells (6, 7). It has long been known that 5-11, 1121 Vienna, Austria. Phone: 0043-180105-2399; Fax: 0043-180105- illegitimate expression of GPCRs occurs in tumor cells (8) 32399; E-mail: [email protected] and an involvement in cell-cycle control has been shown for doi: 10.1158/1541-7786.MCR-12-0139 several GPCRs and GPCR-associated proteins. For instance, 2012 American Association for Cancer Research. H4 histamine receptors mediate a reversible cell-cycle arrest

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in growth factor-induced hematopoietic progenitor cells (9) Cell proliferation and GPCR kinase 5 was assigned an essential role in the Theeffectofdoxorubicin (1mmol/L)orsiRNA(20nmol/L) regulation of prostate tumor growth by affecting cell-cycle on cell proliferation was determined by monitoring the control (10). number of viable cells using the alamarBlue detection kit (Life In the present work, we searched for GPCRs that are Technologies). Experiments were done in 96-well culture aberrantly expressed in lung cancer with a particular focus on plates. Fluorescence was recorded using a 2030 Multilabel SCLC. The underlying rationale was the observation that ReaderVICTORX5(PerkinElmer).Inaddition,proliferation GPR87 was found to be overexpressed in lung squamous cell of cells was assessed by determining cellular confluence using carcinoma (11) and contributed to the viability of tumor a CloneSelect Imager (Molecular Devices). The surface cov- cells (12). Similarly, the chemokine receptor CXCR4 was ered by cells (96-well plate) was recorded on regular time proposed to contribute to lung cancer metastasis (13). In points. Besides, phase contrast microscopy pictures of cells SCLC cells, it was suggested that an autocrine loop sustained were recorded (20 magnification). RhoA activation via activation of a GPCR (14). Our search identified G protein-coupled receptor 19 Flow cytometry and cell-cycle analysis (GPR19), which was overexpressed predominantly in sam- Cells were fixed with 70% ethanol (–20C), washed with ples from patients with SCLC. Currently very little is known PBS and subject to RNase A treatment (1 mg/mL; Life about the biologic functions of GPR19 in normal and Technologies) for 5 minutes followed by propidium iodide diseased tissues (15). RNA interference established a link DNA staining (50 mg/mL; Sigma Aldrich). Cellular fluo- to cell proliferation and transition through the cell-cycle rescence was quantified using a FACSCalibur (Becton – fl as progression through the G2 M-phase was impaired in Dickinson) ow cytometer. Fluorescence histograms were cells transfected with Gpr19 siRNAs. In fact, we observed analyzed with FlowJo software 7.5 (Tree Star). The Watson that the levels of Gpr19 mRNA varied along the cell-cycle curve-fitting model was chosen for cell-cycle analysis. with a peak in S-phase. This might be accounted for by the binding of E2F transcription factors to the promoter region RNA isolation and cDNA synthesis of Gpr19. The ABI Prism 6100 Nucleic Acid PrepStation (Life Technologies) was used to isolate RNA from cell lysates following the manufacturer's instructions. Contaminating Materials and Methods DNA was removed by DNase treatment. cDNA was Cell culture and cell-cycle arrest synthesized in a GeneAmp PCR System 9700 (Life Tech- Human lung-derived cell lines were cultured in normal nologies) using the iScript cDNA Synthesis Kit (Bio-Rad) growth medium as suggested by the provider (Supple- following the manufacturer's protocol and diluted with mentary Table S1) and maintained in logarithmic growth nuclease-free water (1:5–1:8) for direct use in quantitative phase. They were authenticated using short tandem repeat PCR applications. analysis and tested for mycoplasma contamination on a regular basis. Cells were arrested at different stages of the Quantification of gene expression by real-time PCR cell-cycle as follows: 100,000 cells per well were seeded in Reverse transcription quantitative real-time PCR (RT- 6-well culture plates, allowed to adhere for 16 hours in the qPCR) was done on an Mx2005P qPCR machine (Strata- respective growth medium and subsequently incubated in gene) using the TaqMan technology (Life Technologies). A the presence of hydroxyurea (Sigma Aldrich; 1 mmol/L) or total of 10 mL of TaqMan Gene Expression Master Mix (Life aphidicolin (Sigma Aldrich; 3 mmol/L). After 24 hours, Technologies) were mixed with 1 mL of both FAM- (gene of the medium was exchanged for normal growth medium to interest) and VIC-labeled (reference gene) TaqMan assays remove the compounds and to thereby release the cells (Life Technologies; Supplementary Table S2) and 8 mLof from the arrest. diluted cDNA. PCR conditions were 2 minutes at 50C and 10 minutes at 95C followed by 40 cycles of 15 seconds at RNA interference 95C and 1 minute at 60C (data collection). siRNA oligonucleotides were purchased from Sigma The expression of genes of interest was normalized (geo- Aldrich (GPR19 #1 (50-CUGACCUUCUCAUCAGCG- metric averaging) against multiple most stably expressed U-30), GPR19 #2 (50-CUCAGUCUACCACCAACUA- reference genes. Reference genes B2m, Actb, Cypa, Gapdh, 30), CTL #2: (50-CAGCAUCUUC UUUGGGAUU-30), Hprt1, Rplp0, Rnase P, and Tfr were tested for stable polo-like kinase 1 [PLK1; 50-GUCUCAAGGCCUCCU- expression as described (16). The final selection of reference AAUA-30]), and Thermo Fisher Scientific (CTL #1, pool genes was based on this stable expression, a low difference in of nontargeting siRNAs). An additional control siRNA quantification cycles and a PCR efficiency comparable to the (CTL #2) was used which had been designed against Gpr19 ones for the genes of interest. Pairwise duplexing was done butdidnotcauseGpr19 mRNA downregulation. The for all genes of interest against all selected reference genes. final siRNA concentration was 20 nmol/L and transfec- Relative expression ratios were normalized against a control tionsweredoneusingLipofectamineRNAiMAX(Life sample (17). Assay controls (either devoid of template or Technologies) following the manufacturer's instructions lacking reverse transcriptase) were included in all experi- (reverse transfection). ments and gave negative results.

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Immunofluorescence labeling described (23). Rabbit polyclonal immunoglobulin G (IgG) Cells seeded in black poly-D-lysine 96-well culture plates antibodies against E2F family members—E2F-1 (sc-193X), were fixed with formaldehyde and blocked with 1% goat E2F-2 (sc-633X), E2F-3 (sc-878X; detects both iso- serum (Dako) and 2% bovine serum albumin in PBS. They forms), E2F-4 (sc-866X), E2F-5 (sc-999X), E2F-6 (sc- were immunostained for cyclin B1 (BD Biosciences) and 22823X), E2F-7 (sc-66870X)—were from Santa Cruz Bio- phosphorylated histone H3 (Ser10; Upstate) proteins. DNA technology (24–26) and the isotype control was from Cell was stained using Hoechst 33342 (Life Technologies). Signaling. DNA from the precipitate and the sheared chromatin input (1/100th the material used for ChIP) was High-content screening recovered by phenol/chloroform/isoamyl alcohol extraction, Fluorescence-based cell detection was done with an precipitated using isopropanol/sodium acetate and resus- ArrayScan VTI HCS reader (Thermo Fisher Scientific). Six pended in 100 mL of 10 mmol/L Tris. wells per reaction condition and 36 fields (maximum) per well ChIP PCR was conducted in a GeneAmp PCR System were recorded at 20 magnification. The total number of 9700 (Life Technologies) using 2 mL of DNA solution cells monitored per well was in the range of 300 to 1,000 and 200 nmol/L of forward and reverse primer, respectively, depending on siRNA treatment and time post-siRNA trans- in a JumpStart REDTaq ReadyMix Reaction Mix (Sigma fection. For Plk1 siRNA-transfected samples this was only Aldrich). PCR conditions were 45 seconds at 95C followed valid on the second day after transfection due to the strong by 32 cycles of 15 seconds at 95C, 15 seconds at 59,40 antimitotic effect of the knockdown. The data set was seconds at 72C, and completed by 3 minutes at 72C. processed and visualized by Spotfire DecisionSite 9.1.2 Nuclease-free water instead of DNA was used as nontemplate (TIBCO) and iView 1.0.182.1 (Thermo Fisher Scientific). control. PCR products were separated by electrophoresis on Cells were detected on the basis of Hoechst 33342 DNA 2% agarose gels and visualized by ethidium bromide staining. staining. Intensities of immunostaining for both, phosphorylated histone H3 and cyclin B1 were examined by a Kol- Luciferase reporter assay mogorov–Smirnov goodness-of-fit analysis as described (18). DMS 53, HEK-293, and NCI-H1703 cells were transfected by electroporation using the Amaxa Cell Line Nucleo- Transcription profiling and hierarchical clustering fector Kit V (Lonza; 3 mg of plasmid for DMS 53 and NCI- RNA from human NSCLC, SCLC, and normal lung H1703, 5 mg of plasmid for HEK-293) and 10,000 cells samples was purchased from Origene Technologies. were seeded in 96-well culture plates. At 14 hours (DMS 53), Detailed information on patient samples is listed in Sup- 13 hours (HEK-293), or 26 hours (NCI-H1703) after plementary Table S3. Affymetrix microarray analysis was transfection, cells were washed with PBS, lysed using 100 mL conducted on the Human Exon 1.0 ST Array platform. of Beetle-Lysis-Juice (shaking at 500 rpm for 5 minutes; Samples were processed following the Affymetrix GeneChip PJK) and the light signal was measured in an EnVision 2101 Whole Transcript Sense Target Labeling Assay manual. Raw Multilabel Reader (2 seconds; PerkinElmer) after 5 minutes. (single intensity value per probe on a microarray) files were Robust Multichip Average-normalized using the affy pack- Results age in R/Bioconductor (19). Seven hundred seventy-three Gpr19 mRNA expression is high in SCLC patient genes encoding GPCRs were selected on the basis of Gene samples and cell lines Ontology number 0004930. Their expression values in Many GPCRs control signaling pathways that regulate NSCLC, SCLC, and normal lung samples were extracted cell growth, proliferation, and differentiation. It has long from the microarray data. Geometric mean values across all been known that tumor cells may express GPCRs in an normal lung samples were used as reference for the values aberrantfashion(8).Inanunbiasedapproachweinves- from NSCLC and SCLC patient samples. Hierarchical tigated gene expression levels of all known GPCRs in a clustering (average linkage, Euclidean distance) of the log series of human SCLC, NSCLC, and normal lung RNA 2-transformed normalized expression data were conducted samples. Our survey also included odorant receptors, as described (20). Gene expression levels of Gpr19 (probe set because these may be expressed in tissues other than the 207183_at) in various human normal and cancerous tissues olfactory epithelium; in fact, they may occur in neoplasms were investigated by using the BioExpress database (Gene such as prostate cancers (27). Hierarchical clustering Logic; 12). identified Gpr19 among the GPCRs that showed a mes- Gene expression data of human lung-derived samples sage overexpression mostly in SCLC but not in NSCLC from Hou and colleagues (21) and Rohrbeck and colleagues samples relative to normal controls (Fig. 1A). High Gpr19 (22) were extracted from the Oncomine data platform mRNA levels in SCLC samples were verified by a direct (Compendia Bioscience) and analyzed using Spotfire Deci- comparison with those seen in samples from NSCLC and sionSite 9.1.2 (TIBCO). normal lung (Fig. 1B). This was confirmed by RT-qPCR analysis (Fig. 1C). Chromatin immunoprecipitation and chromatin We further quantified Gpr19 expression levels by RT- immunoprecipitation PCR qPCR in a set of NSCLC, SCLC, and normal lung-derived Chromatin immunoprecipitation (ChIP) analyses were cell lines. The expression pattern of Gpr19 was recapitu- carried out with 5 mg of respective E2F antibodies as lated, i.e., cell lines derived from SCLC had higher Gpr19

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1.0 B Microarray CDRT-qPCR 0.8 40 *** ** ** *** 0.6 1.0 30 0.4

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Figure 1. High Gpr19 expression in SCLC patient samples and cell lines. A, heat map for differential expression of genes that encode for proteins with GPCR activity (Gene Ontology number 0004930) in human NSCLC and SCLC patient samples (Affymetrix Human Exon 1.0 ST Array). Cases are represented in columns and single genes in rows. Intensity values (pseudo color scale) are expression values normalized against normal lung samples and log 2-transformed. Enlargement shows the Gpr19-containing cluster. B and C, results for Gpr19 gene expression from microarray (probe set NM_006143_at) and RT-qPCR analysis of human NSCLC, SCLC, and normal lung samples combined per histologic group [box (interquartile range) and whiskers (min-max) plot with median (central bar)]. For each sample RT-qPCR was conducted for Gpr19 normalized against reference genes Actb, Cypa, Hprt1,andRplp0 and geometric mean values were calculated. Statistically signiﬁcant differences were determined by a Kruskal–Wallis test followed by Dunn's multiple comparison (, P < 0.01; , P < 0.001). D, RT-qPCR results for Gpr19 gene expression (normalized against reference genes Actb, Cypa, Hprt1,andRplp0; geometric mean) in various human NSCLC, SCLC, and normal lung cell lines. E, Gpr19 gene expression determined by Affymetrix GeneChip analysis (chip sets HG-U133 A and HG-U133 B, probe set 207183_at) in human tissues (green, normal; red, cancer; yellow, metastasis), box (interquartile range), and whiskers (extend to 1.5 times the interquartile range) plot with median (central bar) and outliers (open circles). Sample numbers are indicated in brackets. Data were extracted from the BioExpress database (Gene Logic). expression than those derived from NSCLC and from normal ﬁbrosis patient (IB3-1). This may be related to the fact that the lung (Fig. 1D). However, high levels of Gpr19 mRNA were IB3-1 cell line was immortalized by the large T antigen (28). also seen in a NSCLC cell line (NCI-H1703) and an We used the BioExpress database for examination of immortalized lung epithelial cell line derived from a cystic Gpr19 expression in various human normal and cancerous

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tissues. This search revealed high Gpr19 mRNA expression days 3 and 6 after treatment using phase contrast microscopy predominantly in the central nervous system (cerebral cor- (Supplementary Fig. S1B). NCI-H1703 showed a similar tex, cerebellum, spinal cord; Fig. 1E) as described (29). In amount of dead cells when cells were Gpr19 siRNA-trans- addition, Gpr19 mRNA levels were prominent in samples fected compared with Plk1 knockdown and doxorubicin from SCLC and pancreas islet cell carcinoma. treatment. This was already the case on day 3 after treatment and became also apparent for DMS 53 cells on day 6. The RNA interference-mediated knockdown of Gpr19 effective knockdown of Gpr19 mRNA by Gpr19-targeting reduces cell proliferation siRNAs was verified using RT-qPCR (Fig. 2C). We The prominent expression of Gpr19 preferentially in also attempted to confirm these experiments by using anti- SCLC and in several lung cancer-derived cell lines sug- bodies against GPR19. However, neither commercially avail- gested that this receptor may play a crucial role in cell able antibodies from different providers nor in-house gener- proliferation and/or survival. We investigated the effect of ated antisera toward 9 different GPR19 peptides allowed Gpr19 knockdown on proliferation in lung cancer cell specific detection on Western blots (data not shown, 31). lines NCI-H1703 (NSCLC) and DMS 53 (SCLC). These Gpr19 Gpr19 – cell lines were chosen based on high mRNA expres- Knockdown of induces G2 M arrest sion and because they grow as monolayers. Most SCLC cell Knockdown of Gpr19 reduced cell proliferation in NCI- lines including COR-L88, NCI-H209, NCI-H345, NCI- H1703 and DMS 53 cells. Hence, we examined which phase H446, and SHP-77 failed to grow as monolayers but of the cell-cycle was affected by the loss of Gpr19 mRNA. formed (floating) clusters, which rendered them less ame- NCI-H1703 cells were either left untreated or transfected with nable to experimental manipulations. GPR19 is an orphan control (CTL #1, CTL #2), Gpr19 (#1, #2), or Plk1 siRNAs. receptor, its physiologic ligand is not known and there is no On days 2, 3, and 4 after transfection flow cytometry was antagonist. Accordingly, we used RNA interference to done to analyze the distribution of cells in different stages of examine the role of this receptor in supporting cell growth. the cell-cycle (Fig. 3A and B). Effective knockdown of Gpr19 Cells were either treated with siRNAs targeting Gpr19 mRNA was confirmed by RT-qPCR (Fig. 3C). The number – mRNA for destruction (GPR19 #1, GPR19 #2), control of cells in G2 M-phase increased over time when cells had siRNA (CTL #1, CTL #2), or transfection reagent only been treated with Gpr19 siRNAs in comparison to untreated – (mock) or left untreated. Additional positive controls or control siRNA-treated cells. This increase in the G2 M included the cytotoxic anthracycline doxorubicin and an population occurred at the expense of cells that were in S-phase Plk1 siRNA directed against (30). We monitored the whereas the proportion of cells in G1 was less affected. As Plk1 – extent of well surface covered by adherently growing anticipated, knockdown of caused a pronounced G2 M cells (confluence) to quantify cell proliferation. Images of arrest (30). This experiment was also conducted in DMS 53 wells covered by NCI-H1703 and DMS 53 cells were cells using untreated cells or cells transfected with control captured and analyzed at regular intervals after treatment siRNA CTL #1, Gpr19 siRNA GPR19 #2 (most effective using the CloneSelect Imager (Fig. 2A). Well thumbnails depletion of Gpr19 mRNA), or Plk1 siRNA which yielded (pictures of the well bottom and cellular detection by the similar results (Supplementary Fig. S2A and S2B; knockdown selected detection algorithm) were the basis for confluence of Gpr19 mRNA documented in Supplementary Fig. S2C). calculation (Supplementary Fig. S1A). Measurements at We verified the observations obtained from flow cytome- regular intervals started 10 hours after treatment and lasted try by an independent approach that relied on high-content fi – until day 6 (see gure insets for data points from 3 experi- screening of cells stained for the G2 M-phase marker cyclin ments; Fig. 2A). At this time point, untreated, mock and B1 and the mitosis marker phosphorylated histone H3. This control siRNA-treated cells had already reached a high experiment was only conducted with NCI-H1703 cells extent of confluence. Plk1 siRNA-transfected and doxo- because DMS 53 cells were not suitable for imaging with rubicin-treated cells did not grow (absent gain in conflu- the ArrayScan VTI HCS reader. NCI-H1703 cells were ence over time). For cells transfected with either Gpr19- transfected with control (CTL #1, CTL #2), Gpr19 (#1, #2), targeting siRNA #1 or #2, the cell growth was inhibited as or Plk1 siRNAs or left untreated and analyzed on days 2, 3, gain in well confluence was modest. and 4 after transfection. Knockdown of Gpr19 mRNA by We confirmed the growth inhibition by siRNAs directed Gpr19-targeting siRNAs was effective as monitored by RT- against Gpr19 in an additional proliferation assay (alamar- qPCR (not shown). We used a Kolmogorov–Smirnov test to Blue), which recorded the number of viable, i.e., metabol- evaluate the distribution of cells that stained positive for ically active cells. Cells were transfected with siRNAs or phosphorylated histone H3 and cyclin B1 for each time treated with doxorubicin and the metabolic activity of cells point after siRNA transfection. This evaluation confirmed – was determined 3 and 6 days later (Fig. 2B). There was a the increase in G2 M-arrested cells (Fig. 4A and B). Figure clear-cut reduction in metabolically active cells transfected 4C and D show representative pictures from the high- with siRNAs targeting Gpr19 compared with untreated, content screen based on Hoechst 33342 DNA staining and mock and control siRNA-treated samples. In both cell lines cells stained positive for phosphorylated histone H3 and the effect was more pronounced on day 6 than on day 3. cyclin B1, respectively, on day 3 after transfection. The level To directly visualize the effect on proliferation, we recorded of both, immunoreactivity for phosphorylated histone H3 pictures of cells from all different experimental conditions on and cyclin B1 increased in cells transfected with the Gpr19

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A 100 100 NCI-H1703 100 100 DMS 53 80 80 Untreated 60 60 ** ** 80 ** *** 80 40 40 Mock

20 20 Confluence [%] day 6 [%] day Confluence Confluence [%] day 6 [%] day Confluence CTL #1 0 0 60 60 Dox Dox Mock PLK1 Mock PLK1 CTL #2 CTL #1 CTL #2 CTL #1 CTL #2 Untreated GPR19 #1 GPR19 #2 Untreated GPR19 #1 GPR19 #2 GPR19 #1 40 40 GPR19 #2 Confluence [%] Confluence Confluence [%] Confluence

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0 0 level mRNA GPR19 Relative 0.0 Dox Dox Mock PLK1 Mock PLK1 Mock CTL #2 CTL #2 CTL #1 CTL #1 CTL #1 CTL #2 Untreated Untreated GPR19 #1 GPR19 #2 GPR19 #1 GPR19 #2 Untreated GPR19 #1 GPR19 #2

Figure 2. Inhibition of cell proliferation by siRNAs directed against Gpr19. A, cell proliferation was determined by measuring confluence, i.e., percentage of total well area covered by cells: shown is the time course after siRNA transfection (20 nmol/L) or addition of doxorubicin (1 mmol/L); data are means SD of 3 wells per condition (96-well plate) from a representative experiment for untreated, mock (transfection reagent only)-, siRNA (CTL #1 and #2, Gpr19-targeting #1 and #2, Plk1-targeting)-, and doxorubicin (Dox)-treated NSCLC cell line NCI-H1703 and SCLC cell line DMS 53. Two additional experiments gave similar results. The figure insets illustrate the results from the last confluence measurement on day 6 posttransfection from the 3 individual experiments (arithmetic means are indicated by the lines). B, cell proliferation was assessed by measuring cell viability with alamarBlue on day 3 and 6 after siRNA transfection and doxorubicin application for NCI-H1703 and DMS 53 cells in 3 independent experiments (done in triplicates); arithmetic means are indicated by the lines. Statistically significant differences across all experimental groups were determined by one-way ANOVA followed by Tukey test. GPR19 #1 and GPR19 #2 P values refer to the least significant one from the comparison with CTL #1 and CTL #2 (, P < 0.05; , P < 0.01; , P < 0.001). C, relative Gpr19 mRNA levels on day 3 after siRNA transfection (normalized against reference genes Cypa and Hprt1, geometric mean of triplicates per condition; data are means þ SD of 3 experiments).

siRNA. This effect was most prominent on days 3 and 4 after case. High-content screening was indicative of abnormal – Plk1 transfection. The G2 M block induced by the siRNA DNA content in cells that had been treated with siRNAs also resulted in increased levels of phosphorylated histone directed against Gpr19. Visual inspection revealed an excess of H3 and cyclin B1. In addition, high-content screening polylobed and binucleated cells (Supplementary Fig. S3). ﬁ – con rmed the G2 M arrest and the reduced proliferative capacity of cells treated with Gpr19 siRNA on the basis of Gpr19 mRNA expression is cell-cycle dependent and Hoechst 33342 DNA staining (data not shown). peaks during S-phase Manipulations that interfere with transition of cells Observations from Gpr19 mRNA knockdown experi- – through G2 M are predicted to result in aberrant cell division. ments are consistent with the hypothesis that this GPCR Gpr19 – Accordingly, we examined whether transfection with plays a role during the G2 M-phase of the cell-cycle. We siRNAs elicited morphologic changes that were consistent therefore investigated whether Gpr19 mRNA was differen- with impaired chromosomal segregation. This was indeed the tially expressed during the course of the cell-cycle.

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A Untreated CTL #1 CTL #2 GPR19 #1 GPR19 #2 PLK1 100 100 100 100 100 100

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0 level mRNA Relative GPR19 0.0 CTL #1 CTL #2 Untreated GPR19 #1 GPR19 #2 PLK1 day 2 PLK1 day 3 PLK1 day 4 CTL #2 day 3 CTL #2 day 4 CTL #1 day 2 CTL #2 day 2 CTL #1 day 3 CTL #1 day 4 Untreated day 2 Untreated day 3 Untreated day 4 GPR19 #1 day 2 GPR19 #1 day 3 GPR19 #2 day 3 GPR19 #2 day 4 GPR19 #2 day 2 GPR19 #1 day 4

Figure 3. Gpr19 siRNA-induced increase in relative number of cells with G2–M-phase DNA content. A and B, the cell-cycle distribution (G1-, S-, G2–M-phase) of NCI-H1703 cells (means þ SD of 3 experiments) was determined staining the cellular DNA with propidium iodide and quantifying the distribution by ﬂow cytometry on days 2, 3, and 4 after siRNA transfection (untreated cells; CTL #1 and #2, GPR19 #1 and #2, PLK1; 20 nmol/L). Cellular debris was excluded in a forward versus side scatter dot plot. The proﬁles of DNA content were analyzed using the cell-cycle tool (Watson model) of

FlowJo 7.5 software (A); this model approximates G1-(green) and G2–M-(blue)-phase populations with Gaussian curves and calculates the S-phase population (yellow) exactly based on the DNA content histogram [FL2-Area (FL2-A); purple line represents calculated sum of G1,S,G2–Mcells based on the Watson model]. For each day and cell-cycle phase, differences across all experimental groups were tested for signiﬁcance using one-way ANOVA followed by Tukey test (B). GPR19 #1 and GPR19 #2 P values refer to the least signiﬁcant one from the comparison with CTL #1 and CTL #2 (, P < 0.05; , P < 0.01). C, relative Gpr19 mRNA levels on day 2 after transfection (normalized against reference genes Cypa and Hprt1, geometric mean of triplicates percondition;dataaremeansþ SD of 3 experiments).

– NCI-H1703 and DMS 53 cells were treated with at the G1 S boundary and subsequently declined to reach a hydroxyurea for 24 hours to synchronize them at the nadir after 12 hours in both, DMS 53 and NCI-H1703 cells. – cyclin B1 G1 S transition. Samples were taken at various time points In contrast, a peak in mRNA expression was seen post release from cell-cycle arrest for DNA content analysis after 12 hours, i.e., coincident with the accumulation of cells ﬂ – by ow cytometry. Upon removal of hydroxyurea, DMS 53 in the G2 M-phase of the cell-cycle. These results were (Fig. 5A, left panel) and NCI-H1703 cells (Fig. 5B, left anticipated as cyclin B1 and cyclin E1 show a known cyclic panel) moved subsequently through the cell-cycle in an pattern of mRNA expression over the course of the cell-cycle. essentially synchronous fashion: the number of cells in S- Expression levels of cyclin E1 are highest before S-phase – phase rapidly accumulated and the DNA content shifted entry, fall during S-phase, and remain low during G2 M- cyclin B1 from 2n to 4n within 8 hours. After 12 hours, the vast phase whereas message is low in G1-phase, increases – – majority of cells had reached the G2 M-phase of the cell- during S-phase and peaks at G2 M-phase (32). Both pat- cycle. At later time points (24, 36, and 48 hours) the effect of terns were recapitulated in our experiments. The mRNA of hydroxyurea was lost and the cell-cycle distribution in the Rpl32 encodes a ribosomal protein of the 60S large ribosomal population approached that seen in an asynchronously subunit and does not vary through the cell-cycle (32). growing culture. RNA was also extracted at these time points Accordingly, we used Rpl32 as an internal control. In and the message levels of several genes were determined by addition, we examined the cell-cycle-dependent changes in RT-qPCR (Fig. 5A and B, right-hand panels). The levels of mRNAs encoding 2 different GPCRs, namely Lpar1 for mRNA encoding cyclin E1 were elevated although cells were NCI-H1703 cells and Chrm3 for DMS 53 cells. These

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A 0.8 B 0.8 Day 2 posttransfection pH3 Day 2 posttransfection cyclinCCNB1 B1 Day 3 posttransfection Day 3 posttransfection Figure 4. Gpr19 siRNA-induced Day 4 posttransfection 0.6 Day 4 posttransfection 0.6 increase in immunoreactivity for phosphorylated histone H3 (pH3) 0.4 0.4 ** and cyclin B1. The levels of cyclin *** B1 (marker for G2–M) and of KS statistic KS statistic KS ** ** 0.2 * 0.2 * phosphorylated histone H3 (marker for mitosis) were 0.0 0.0 determined by high-content screening after transfection with PLK1 PLK1 siRNA (untreated cells; CTL #1 and CTL #2 CTL #1 CTL #1 CTL #2 #2, GPR19 #1 and #2, PLK1; Untreated GPR19 #1 GPR19 #2 Untreated GPR19 #1 GPR19 #2 20 nmol/L). A and B, Kolmogorov– C Day 3 posttransfection (pH3) D Day 3 posttransfection (cyclin B1) Smirnov (KS) statistic for pH3- (A) and cyclin B1-positive (B) cellular staining on days 2, 3, and 4 after GPR19 GPR19 siRNA transfection (means þ SD of 3 experiments). For each day,

#1 #1 differences across experimental Untreated Untreated groups—except for PLK1 (strong antimitotic effect of the mRNA knockdown)—were tested for signiﬁcance using one-way ANOVA followed by Tukey test. GPR19 GPR19 GPR19 #1 and GPR19 #2 P values refer to the least signiﬁcant one

#2 #2 CTL #1

CTL #1 from the comparison with CTL #1 and CTL #2 (, P < 0.05; , P < 0.01; , P < 0.001). C and D, images of immunostaining for pH3 (C, yellow) and cyclin B1 (D, red) in one representative ﬁeld of 36 (maximal) ﬁelds per well (96-well culture PLK1

PLK1 plate). Objects encircled in green represent cells detected based on

CTL #2 CTL #2 Hoechst 33342 staining.

mRNAs remained essentially constant at the time points in Gpr19 mRNA levels recapitulated those seen in hydroxy- examined. The mRNA encoding Gpr19, however, was urea-treated cells. subject to cell-cycle-dependent regulation: Gpr19 mRNA We explored the correlation between Gpr19 expression – levels rose when the synchronized cells moved through S- levels and the relative number of cells in G1-, S-, and G2 M- phase, peaked after 6 to 8 hours, and started to decline as cells phase for both hydroxyurea-synchronized DMS 53 and – reached G2 M-phase, i.e., after 10 hours (Fig. 5A and B, NCI-H1703 cells (Fig. 6). The proportion of cells at right panels). At later time points (24, 36, and 48 hours) different stages of the cell-cycle was determined using the mRNA levels did not differ appreciably from those seen in Watson curve-fitting model from FlowJo. As hydroxyurea – untreated control cells (open bar in the right-hand panel arrested cells at the G1 S transition, it was impossible to of Fig. 5A and B), regardless of which mRNA was examined. precisely discriminate between G1- and S-phase populations This is consistent with the fact that the cell population grew at 0 and 2 hours after release of the hydroxyurea-induced again asynchronously at this stage. block. These data points were therefore omitted. In both cell We addressed the possibility that differential Gpr19 lines Gpr19 message levels correlated with the relative mRNA expression was because of an off-target effect of number of cells in S-phase. Conversely, Gpr19 mRNA levels hydroxyurea, i.e., unrelated to its effect on the cell-cycle. For were inversely related to the fraction of G1 cells. In contrast, this reason we repeated the synchronization study with DMS we did not observe any correlation between the amount of – Gpr19 53 cells using the DNA replication inhibitor aphidicolin. cells in G2 M-phase and mRNA expression. Cell-cycle profiles and the degree of synchronization were comparable to those of hydroxyurea-treated cells (Fig. 5C, Transcription factors E2F-1, E2F-2, E2F-3, and E2F-4 left panel). Similarly, mRNA profiles were also comparable are recruited to the Gpr19 promoter. (Fig. 5C, right panel). Most importantly, following aphidi- The data in Figs. 5 and 6 suggested that accumulation of colin-induced synchronization, the time-dependent changes the mRNA encoding Gpr19 was initiated as soon as cells

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A DMS 53 hydroxyurea arrest 5 Untr 10 h 0 h 12 h Untreated 0 h 2 h 4 h 6 h 8 h 2 h 24 h 4 ** 4 h 36 h * * ** 6 h 48 h 3 8 h ** ** * ** 10 h 12 h 24 h 36 h 48 h 2

Relative mRNA level 1

0 RPL32 Cyclin E1 Cyclin B1 GPR19 CHRM3 B NCI-H1703 hydroxyurea arrest 6

Untreated 0 h 2 h 4 h 6 h 8 h *

* ** * 4

** ** *

10 h 12 h 24 h 36 h 48 h 2 Relative mRNA level

0 RPL32 Cyclin E1 Cyclin B1 GPR19 LPAR1 C DMS 53 aphidicolin arrest 8 ** ** Untreated 0 h 2 h 4 h 6 h 8 h ** 6

**** ** 4 ** 10 h 12 h 24 h 36 h 48 h **

Relative mRNA level mRNA Relative 2

0 RPL32 Cyclin E1 Cyclin B1 GPR19 CHRM3

Figure 5. Differential expression of Gpr19 mRNA over the course of the cell-cycle. DMS 53 cells (A and C) and NCI-H1703 (B) were treated with hydroxyurea (1 mmol/L; A and B) or aphidicolin (3 mmol/L; C) for 24 hours. Following release of the block, DNA content was determined at regular time points by propidium iodide staining and flow cytometry (left-hand panels) and the corresponding expression profiles of the indicated genes (encoding cyclin B1 and cyclin E1, the ribosomal protein RPL32, and the GPCRs GPR19, LPAR1, or CHRM3) by RT-qPCR (right-hand panels). The gene of interest mRNA expression was normalized against 4 reference genes (Actb, Cypa, Hprt1, Rplp0). Gene expression plots show mRNA levels (geometric mean) relative to control samples [untreated (untr) cells] with error bars indicating 95% confidence intervals. Gpr19 mRNA expression at each time point post release was compared with the mRNA expression of nondifferentially expressed Rpl32 using a t test with Welch correction (, P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001). Experiments were repeated yielding concurrent results.

Cdc6 albumin progressed from G1- to S-phase. Members of the E2F family DMS 53 cells (Fig. 7). The promoters of and of transcription factors are known to boost transcription served as positive and negative controls (34, 35), respectively. from E2F-targeted promoter sites during late G1- or early S- Chromatin fragmentation was achieved by sonication and phase (33), e.g., the component of the prereplicative com- shearing resulted in DNA fragments of 300 to 600 base pairs. plex cell division cycle 6 (Cdc6; 34). It was therefore impossible to discriminate between recruit- The Gpr19 promoter region (5 kb of the 50 upstream ment of E2F-1, E2F-2, E2F-3, and E2F-4 to either binding sequence ahead of the open reading frame) was examined for sites 15 or 185 and/or to both of them. Primer pairs for prospective E2F binding sites using ConSite and the Uni- PCR amplification of all predicted E2F binding sites were versity of California, Santa Cruz (UCSC) genome browser. chosen in a way that the PCR product did not contain any Four potential E2F binding sites (15, 185, 3184, and other site—except for primer pair GPR19 (15; 185). 3769 bp upstream of the Gpr19 open reading frame) were With this primer pair the resulting PCR product covered both retrieved from the ConSite search. In contrast, only the one at these predicted E2F binding sites of the Gpr19 promoter. 15 was identified by the UCSC genome browser search. We Several facts suggest that the binding of E2F-1, confirmed the presence of E2F binding sites in the Gpr19 E2F-2, E2F-3, and E2F-4 to the promoter sites at posi- promoter by ChIP. ChIP assays identified the presence of tion 15 and/or 185 of the Gpr19 gene were specific: E2F-1, E2F-2, E2F-3, and E2F-4 in both NCI-H1703 and (i) immunoprecipitation of these E2F-family members

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3 3 3

2 2 2 DMS 53

1 1 1

R 2 : 0.8598 R 2 : 0.6124 R 2 : 0.0065 0 0 0 0 20 40 60 0 20 40 60 80 100 0 20 40 60 80 4 4 4

3 3 3 NCI-H1703

Relative GPR19 mRNA level 2 2 2

1 1 1

R 2 : 0.8728 R 2 : 0.6209 R 2 : 0.0102 0 0 0 0 10 20 30 40 50 0 20 40 60 80 0 10 20 30 40 50

Cells in G1-phase [%] Cells in S-phase [%] Cells in G2-M-phase [%]

Figure 6. Correlation of Gpr19 mRNA levels with the amount of cells in S-phase. Gpr19 expression levels were determined over the course of the cell- cycle from hydroxyurea-arrested DMS 53 and NCI-H1703 cells (see Fig. 5) and plotted as a function of the relative number of cells in G1-, S-, and G2–M- phase. Shown is the result of linear regressions; squared correlation coefﬁcient values (R2) are given for each plot.

did neither pull down the coding sequence of Gpr19 Both E2F binding sites at positions 15 and 185 of the nor (ii) the promoter region of albumin. (iii) Several Gpr19 promoter are crucial for luciferase reporter gene E2F-family members with repressor activity (E2F-5, expression. E2F-6, E2F-7; 36) did not pull down the Gpr19 promot- A fragment of the Gpr19 promoter (300 bp upstream er, either. of the Gpr19 open reading frame) was introduced upstream

DMS 53 NCI-H1703 No AB IgG E2F-1 E2F-2 E2F-3 E2F-4 Input 1:25 Input 1:125 Input Water E2F-5 E2F-6 E2F-7 1:5 Input No AB IgG E2F-1 E2F-2 1:125 Input E2F-4 E2F-5 1:25 Input Water E2F-3 Input E2F-6 E2F-7 1:5 Input

GPR19 (-15)

GPR19 (-185)

GPR19 (-15; -185)

GPR19 (-3184)

GPR19 (-3769)

GPR19 (CDS)

Albumin

CDC6

Figure 7. Binding of E2F-1 to -4 to the promoter of Gpr19. Chromatin immunoprecipitation was conducted with antibodies against E2F family members (E2F-1, 2, 3, 4, 5, 6, 7) and IgG isotype control. As an additional negative control the immunoprecipitation was done in the absence of any antibody (no AB). DNA from the sheared chromatin input was serially diluted with water (undiluted, 1:5, 1:25, 1:125). Internal controls also included ampliﬁcation of the promoter of a known E2F target gene (Cdc6) and of an E2F-independent gene (albumin) and to a region lying in the coding sequence (CDS) of Gpr19. Primer pair GPR19 (15; 185) covered both predicted E2F binding sites of the Gpr19 promoter in the PCR product. Shown are the amplicons from a representative experiment, which was repeated twice with similar results. Full-length agarose gels for NCI-H1703 and PCR primer sequences are presented in Supplementary Fig. S4.

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HEK-293 NCI-H1703 DMS 53

2,500 20,000 800

2,000 * 15,000 600 ** 1,500 * 10,000 ** 400 *** 1,000 *** *** 5,000 200 500 Relative light units light Relative units light Relative units light Relative

0 0 0 300GPR19 300GPR19 300GPR19 pGL3-Basic pGL3-Basic pGL3-Basic 300 (mut 15) 300 (mut 15) 300 (mut 15) 300 (mut 185) 300 (mut 185) 300 (mut 185) 300 (mut 15+185) 300 (mut 15+185) 300 (mut 15+185)

Figure 8. The importance of both E2F binding sites in the Gpr19 promoter at positions 15 and 185 upstream of the Gpr19 open reading frame for luciferase reporter gene expression. HEK-293, NCI-H1703, and DMS 53 cells were transiently transfected with a control (pGL3-Basic) or a Gpr19 promoter fragment-containing luciferase reporter construct (pGL3-Basic-300GPR19; 300 bp of the Gpr19 promoter upstream of the Gpr19 open reading frame). Besides, cells were transfected with constructs in which the putative E2F transcription factor binding sites at position 15 and/or 185 upstream of the Gpr19 open reading frame had been eliminated [pGL3-Basic-300GPR19 (mut 15), pGL3-Basic-300GPR19 (mut 185), pGL3-Basic-300GPR19 (mut 15þ185)]. Data are means þ SD of 6 replicates per condition (96-well plate) from a representative experiment. Differences across all experimental groups were determined by one-way ANOVA followed by Tukey test. P values of GPR19-mutated constructs 15 and/or 185 refer to the comparison with the nonmutant promoter construct pGL3-Basic-300GPR19 (, P < 0.05; , P < 0.01; , P < 0.001). The experiments were repeated yielding concurrent results. of a firefly luciferase reporter gene to evaluate its influence on of Gpr19 and E2F transcription factors E2f-1 to E2f-3 could gene expression (Fig. 8). In addition, the putative E2F be detected (Fig. 9). The in-house study revealed a corre- transcription factor-binding sites at positions 15 and/or lation coefficient of 0.863 for E2f-1, 0.682 for E2f-2, and 185 had been selectively removed. The removal of the 0.554 for E2f-3 whereas the correlation coefficient for E2f-4 putative E2F binding site at position 15 alone or in with Gpr19 was 0.350. Similar correlations were seen in the combination with the one at position 185 negatively other 2 studies. affected reporter gene expression in HEK-293 and NCI- The study by Rohrbeck and colleagues and our in-house H1703 cells (Fig. 8, left and centerfold graphs). In contrast, study identified high Gpr19 expression predominantly in the knockout of the putative E2F binding site at position SCLC patient samples (Fig. 9B and C). SCLC samples were 185 alone did not diminish luciferase expression. The not included in the study described by Hou and colleagues opposite was true for DMS 53 cells: Removal of the E2F but high Gpr19 expression was observed in a subset of consensus site at position 185 from the Gpr19 promoter NSCLC samples here, namely large cell lung carcinoma sequence diminished luciferase expression here (Fig. 8, right (Fig. 9A). This was also true for a few lung cancer samples graph). This signal reduction was further augmented when classified as adenocarcinoma or squamous cell carcinoma. the E2F consensus site at position 15 had also been erased. Hence, the consensus motif for the recruitment of E2F Discussion transcription factors to the promoter of Gpr19 at both positions 15 and 185 upstream of the Gpr19 open The orphan receptor GPR19 was first identified in the reading frame seemed to be important for luciferase reporter central nervous system (29, 37); it is also found in ovary and gene expression. The preferred usage of each position might testis (37) and abundantly expressed in human embryonic be cell line dependent. stem cells (38). The gene encoding human Gpr19 resides on the short arm of chromosome 12 in a region that is frequently E2F transcription factor gene expression correlates with rearranged in childhood leukemia and to a lesser extent in Gpr19 expression in human lung cancer samples. other neoplasms; accordingly, GPR19 has been postulated The recovery of the Gpr19 promoter in E2F ChIP assays to play a role in cancer development (39). In fact, high levels and the results from Gpr19 promoter fragment-controlled of mRNA coding for GPR19 were found in metastatic luciferase reporter expression indicated a cause-and-effect melanoma (7, 40). Here we showed that GPR19 is associated relation between the action of E2F transcription factors and with lung cancer. This conclusion is based on (i) an unbiased the expression of Gpr19. We addressed the question whether approach that surveyed GPCRs specifically for their over- coexpression of E2F transcription factors and Gpr19 could expression in SCLC, (ii) database mining, and (iii) the be observed in patient samples as well. Indeed, on the basis of presence of high Gpr19 mRNA levels in several human lung 2 published studies (21, 22) and our in-house study cancer-derived cell lines. High levels of Gpr19 mRNA were described in Fig. 1A–C, a good correlation of the expression not only detected in patient samples from SCLC but also

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AB C Lung adenocarcinoma Large cell lung carcinoma Lung Squamous cell lung carcinoma Lung Lung adenocarcinoma Small cell lung carcinoma Squamous cell lung carcinoma Large cell lung carcinoma Lung Lung adenocarcinoma Small cell lung carcinoma Squamous cell lung carcinoma

Figure 9. Coexpression of transcription factors E2f-1 to -4 and Gpr19 in 3 independent lung cancer patient sample sets. A, study by Hou and colleagues (21): 19 large cell lung carcinoma, 65 normal lung (Lung), 45 lung adenocarcinoma, and 27 squamous cell carcinoma samples were compared. The study was conducted on the U133 Plus 2 Array. B, study by Rohrbeck and colleagues (22): 5 normal lung (Lung), 16 lung adenocarcinoma, 9 small cell lung carcinoma, and 15 squamous cell lung carcinoma samples were compared. The study was conducted on the Human Genome Focus Array. C, in-house study (all patient samples had been ordered from Origene Technologies, details are described in Materials and Methods and in Supplementary Table S3): 3 large cell lung carcinoma, 14 normal lung (Lung), 8 lung adenocarcinoma, 8 small cell lung carcinoma, and 5 squamous cell lung carcinoma samples were analyzed. The study was conducted on the Human Exon 1.0 ST Array. For the generation of the row dendrogram, the following settings were used: clustering method, unweighted pair group method with arithmetic mean (UPGMA); distance measure, Euclidean; ordering weight, average value; normalization, none; and empty value replacement, constant value: 0. For the generation of the column dendrogram, the same settings were applied except for that correlation was used as the distance measure. Max, Average, and Min refer to the expression level of the respective gene. The probe set for each gene used is indicatedon the X-axis, the various tumor types are indicated by a color code in the column tissue type.

from pancreas islet cell cancer. Both these carcinoma types crine large cell lung carcinoma. The NSCLC cell line NCI- are often characterized by the presence of neuroendocrine H1703 also showed high levels of Gpr19 mRNA expression. markers (41, 42). This is also true for a subset of NSCLC It is not clear whether this reflects a neuroendocrine phe- called large cell neuroendocrine carcinoma (2) and approx- notype or de novo illegitimate expression. Thus, the discrim- imately 30% of SCLC is associated with NSCLC elements ination between Gpr19 high and Gpr19 low expression lung (43). In fact, an increase in Gpr19 message was observed in cancers might not strictly follow histologic boundaries. In large cell lung carcinoma relative to normal lung samples addition, we confirmed the presence of high Gpr19 mRNA when using the BioExpress database (Fig. 1E). In addition, expression in melanoma (Fig. 1E). high levels of Gpr19 expression could also be attributed to We examined if expression of Gpr19 conferred a growth some NSCLC patient samples (mainly large cell lung car- advantage in lung cancer cells. The results were unequivocal: cinoma) in the lung cancer gene expression study described regardless of the histologic classification of the cell line by Hou and colleagues (21; Fig. 9A). These data sets did not (SCLC or NSCLC), knockdown of Gpr19 mRNA depressed discriminate between neuroendocrine and nonneuroendo- cell proliferation. This finding is consistent with earlier

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observations that indicate a role of GPR19 during embryo- stimulated NCI-H1703 cells that highly express Gpr19 genesis, in particular in the developing brain: high levels of mRNA (data not shown). Because pertussis toxin-catalyzed Gpr19 mRNA are initially observed in germ cell layers of the ADP-ribosylation of Gai/Gao blocks access of receptors to embryo. Subsequently Gpr19 mRNA accumulates in the their C-terminus, this manipulation ought to have neural plate, the subventricular zone and other sites, from unmasked constitutive inhibition of cAMP accumulation. which neuronal cells emerge in the differentiating central Similarly, we failed to find any effect of conditioned medium nervous system (15). (supernatants from cell lines NCI-H1703 and NCI-H345 GPCRs control signaling pathways that are typically which highly express Gpr19 mRNA) on cAMP accumula- associated with recruitment of quiescent cells into the tion in HEK-293 cells that had been transfected with Gpr19 – cell-cycle (G0 G1 transition) or with accelerated progression expression plasmids (data not shown). Thus, it seems Gpr19 – through G1 (4, 6). Surprisingly, knockdown of unlikely that the action of GPR19 in the G2 M-phase of affected progression through the cell-cycle at a later stage, the cell-cycle arises from coupling to Gi/Go. Further progress – fi namely in G2 M. This conclusion is based on 2 independent in this area is contingent on the identi cation of the cognate lines of evidence, namely the determination of DNA content agonist(s) of GPR19. Finally, a function other than direct fl fi – by ow cytometry and the quanti cation of the G2 M signaling via G proteins might also be conceivable for marker cyclin B1 and the mitosis marker phosphorylated GPR19, e.g., recruiting G protein-independent pathways histone H3 by immunocytochemistry. Cyclin B1 exerts its via b-arrestins (50), via direct binding of tyrosine kinases function in conjunction with cyclin-dependent kinase 1 (51), or by acting as a scaffolding protein (52). Gpr19 during late G2-phase and early mitosis (44). Histone H3 Our experiments showed that the expression of was phosphorylation at Ser10 correlates with chromosome con- regulated during the cell-cycle. This finding provides addi- densation during mitosis with phosphorylation levels start- tional—albeit circumstantial—evidence for a role of GPR19 ing to increase in late G2-phase (45). Thus, our observations in the regulation of the cell-cycle. The levels of mRNA are consistent with a model where GPR19 impinges on encoding GPR19 peaked when most cells were in S-phase. checkpoint controls that allow for transition through G2 and For soluble proteins, this typically implies that the proteins entry into mitosis or for initiation of the separation of are required for S-phase transition. GPR19, however, is a daughter cells. Earlier findings also suggest that GPR19 may membrane protein, which must mature through the secre- play a role in cell division: Gpr19 gene expression levels are tory pathway to reach its presumed site of action, the cell high in murine spermatocytes undergoing meiotic cell divi- surface. Although transcription and translation are rapid (less sion (46). In this context it is of interest that NCI-H1703 than 1 minute for a protein of 400 amino acids), folding in cells treated with siRNAs directed against Gpr19 exhibited a the endoplasmic reticulum imposes a time lag of up to several binuclear or polylobed nuclear phenotype (Supplementary hours (53). Besides, subsequent trafficking through and Fig. S3). maturation steps in the secretory pathway result in an The mechanistic link between a GPCR and cell division additional delay before insertion of the protein into the cell – during G2 M is not intuitively evident. It is, however, worth membrane. Pharmacochaperone-triggered folding of the pointing out that heterotrimeric G proteins of the Gai/Gao- A1-adenosine receptor suggests that more than 5 hours are subfamily are involved in the control of the mitotic spindle; required for the receptor to reach the cell surface (54). Thus, they are thought to regulate the mitotic force generator and a delay of several hours is to be anticipated between induc- thus to promote chromosomal segregation (47). The expres- tion of mRNA expression and accumulation of GPR19 at sion of a GPCR does not per se render a cell susceptible to the cell surface. Given this inherent delay, expression regulation by the receptor—the ligand must also be present. of Gpr19 is timely for a membrane protein required for the – Alternatively, the receptor has a high level of basal activity G2 M-phase. and engages its cognate G protein(s) in the absence of an GPR19 is not the only GPCR whose expression is cell- agonist (48). The growth inhibitory action of Gpr19-direct- cycle-dependent. In primary cultures of human microvas- ed siRNAs could also be accounted for by these 2 scenarios cular endothelial cells, the protein levels of the chemokine that are not necessarily mutually exclusive: (i) when over- receptor CXCR3 were found to be highest when cells exited – expressed, GPR19 displays strong constitutive activity. (ii) S-phase and entered G2 M (55). We stress that our data refer GPR19-expressing cells also synthesize and release the ago- to mRNA levels of Gpr19 rather than accumulation of the nist; knockdown of the mRNA encoding the receptor protein at the cell surface. CXCR3 was also shown to be disrupts the resulting autocrine loop. GPR19 was previously directly involved in the proliferation of endothelial cells: its proposed to couple to Gi because it engaged a fusion protein ligands interferon-g-inducible protein of 10 kDa (IP-10) composed of Gaq and the last 5 amino acids of Gai (49). On and monokine induced by interferon-g (Mig) stalled cell the basis of sequence alignments of the transmembrane core, proliferation. This effect could be reversed by a CXCR3- GPR19 was classified as a class A (rhodopsin-like) receptor in specific antibody. spite of the absence of an NPxxY-motif in transmembrane The promoter region of the gene encoding GPR19 con- helix 7; the cognate ligand was proposed to be a peptide (37). tains several candidate-binding sites for E2F transcription We attempted to verify Gi-dependent inhibition of cAMP factors. We provide conclusive evidence that at least one of formation via GPR19. However, we failed to detect an effect these was in fact occupied by E2F family members E2F-1, of pertussis toxin on cAMP accumulation in forskolin- E2F-2, E2F-3, and E2F-4 in both DMS 53 and

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NCI-H1703 cells. E2F transcription factors are known to proliferation. Transiently transfected GPR19 expression did – activate the expression of genes at the G1 S transition not result in gain of proliferation in neither HEK-293 nor necessary for cell-cycle progression (33, 35) including, NCI-H1703 cells (data not shown). However, these studies e.g., Cdc6, Cdc25 (34), thymidine kinase,ordihydrofolate have not been conducted in nonmalignant cells. Whether reductase (35). The family of E2F transcription factors has this indicates that GPR19 might lack the ability to transform been subdivided into activators (E2F-1, E2F-2, E2F-3) and cells into a malignant phenotype needs further investigation. repressors (E2F-4, E2F-5, E2F-6, E2F-7, E2F-8) of gene Hence, the present data do not allow the categorization of transcription (33, 36). Transactivation and repression result Gpr19 as an oncogene (56). Furthermore, its overexpression from interactions with different cofactors. E2F-4 and E2F-5 particularly seen in lung cancer-derived cell lines and SCLC both prevent quiescent cells from entering G1-phase. Upon patient samples might be accounted for by the deregulated mitogenic stimulation, E2F-1, E2F-2, and E2F-3 accumu- action of members from the E2F family of transcription late in late G1-phase and cause a pattern of gene expression factors, which is very often observed in lung cancer (57, 58). that drives cells into S-phase. During late S-phase this signal is Taken together, our observations highlight GPR19 as a attenuated by E2F-6, E2F-7, and E2F-8 (36). Classification candidate drug target for the treatment of a subset of lung of E2Fs into activators and repressors of gene transcription is cancers. To the best of our knowledge, GPR19 is the first an oversimplification, because some isoforms can mediate GPCR whose mRNA has been shown to be expressed in a – both transactivation and repression. However, we documen- cell-cycle-dependent manner and to impinge on G2 M ted binding of 3 activating E2F isoforms to the Gpr19 transition. Retinoblastoma- and E2F-dependent transcrip- promoter. Furthermore, the cloning of Gpr19 promoter tion may not only regulate S-phase-specific genes but also fragments of different length with and without E2F binding control the expression of genes that are required at later – site integrity at positions 15 and/or 185 upstream of a stages, e.g., in G2 M, particularly if these are membrane luciferase reporter identified the importance of both sites for proteins subject to intracellular trafficking. Finally, we gene expression. It might be possible that both E2F binding believe that the insights from our current work should also sites are in use for the activation of Gpr19 gene expression in a be of relevance to understand the physiological role of cell-specific manner. In all 3 cell lines tested, the combined GPR19 during embryogenesis and brain development. abolition (positions 15 and 185) led to the most pronounced reduction of luciferase reporter expression. Disclosure of Potential Conflicts of Interest Besides, the expression of activator E2F transcription No potential Conflicts of interest were disclosed. factors E2f-1, E2f-2, and E2f-3 could be correlated with the expression of Gpr19 in human lung cancer samples from Authors' Contributions Conception and design: S. Kastner, M. Freissmuth, W. Sommergruber independent lung cancer gene expression studies. Only a Development of methodology: S. Kastner, T. Voss weak correlation was observed between Gpr19 and E2f-4. Acquisition of data (provided animals, acquired and managed patients, provided Gpr19 facilities, etc.): S. Kastner, T. Voss, M. Freissmuth The observed promoter binding of the repressor E2F- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- 4 might reflect its recruitment to the promoter during early tational analysis): S. Kastner, T. Voss, M. Freissmuth, W. Sommergruber Writing, review, and/or revision of the manuscript: S. Kastner, T. Voss, M. G1-phase and subsequent displacement with activator E2Fs Gpr19 Freissmuth, W. Sommergruber in late G1- and early S-phase leading to transcription. Administrative, technical, or material support (i.e., reporting or organizing data, Thus, it seems justified to conclude that there might be a constructing databases): S. Kastner, T. Voss, S. Keuerleber, C. Gl€ockel cause-and-effect relation between E2F binding to the Gpr19 Study supervision: M. Freissmuth, W. Sommergruber Assisted with experiments: S. Keuerleber, C. Gl€ockel promoter and the observed accumulation of the mRNA coding for GPR19 during S-phase. Our conclusion is further Acknowledgments supported by the observation that Gpr19 mRNA was present The authors thank Andreas Bernthaler and Andreas Wernitznig for excellent help at high levels in the immortalized cell line IB3-1. This cell with microarray gene expression analysis and Anja Ebert for kindly providing the ChIP assay protocol. line was derived from the lung epithelium of a patient fi suffering from cystic brosis with the help of a hybrid virus. Grant Support Accordingly, IB3-1 cells contain high levels of SV40 large T This work was supported by Boehringer Ingelheim, the FWF-funded doctoral antigen (28), which inactivates the retinoblastoma protein program CCHD (Cell Communication in Health and Disease) and PLACEBO (Platform Austria for Chemical Biology) funded by the Austrian Gen-Au (Genome and hence derepresses E2F-dependent transcription. Research in Austria) program. When the Gpr19 message in Gpr19-expressing lung The costs of publication of this article were defrayed in part by the payment of page cancer-derived cell lines was attenuated by the use of RNA charges. This article must therefore be hereby marked advertisement in accordance with interference, proliferation of these cells was diminished (Fig. 18 U.S.C. Section 1734 solely to indicate this fact. — 2). Thus, GPR19 might be a vulnerable target, which Received March 8, 2012; revised July 19, 2012; accepted August 9, 2012; when overexpressed in lung cancer cells—is involved in cell published OnlineFirst August 21, 2012.

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1358 Mol Cancer Res; 10(10) October 2012 Molecular Cancer Research

Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry into S-Phase and Supports G 2− M Cell-Cycle Progression

Stefan Kastner, Tilman Voss, Simon Keuerleber, et al.

Mol Cancer Res 2012;10:1343-1358. Published OnlineFirst August 21, 2012.

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