RGS12 Is a Novel Tumor-Suppressor Gene in African American Prostate

Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0669 Cancer Molecular and Cellular Pathobiology Research

RGS12 Is a Novel Tumor-Suppressor Gene in African American Prostate Cancer That Represses AKT and MNX1 Expression Yongquan Wang1,2, Jianghua Wang2, Li Zhang2,3, Omer Faruk Karatas2, Longjiang Shao2, Yiqun Zhang4, Patricia Castro2, Chad J. Creighton4,5,and Michael Ittmann2

Abstract

African American (AA) men exhibit a relatively high incidence epithelial cells. Notably, RGS12 exhibited potent tumor-suppres- and mortality due to prostate cancer even after adjustment for sor activity in prostate cancer and prostate epithelial cell lines in socioeconomic factors, but the biological basis for this disparity is vitro and in vivo. We found that RGS12 expression correlated unclear. Here, we identify a novel region on chromosome 4p16.3 negatively with the oncogene MNX1 and regulated its expression that is lost selectively in AA prostate cancer. The negative regulator in vitro and in vivo. Further, MNX1 was regulated by AKT activity, of G-protein signaling RGS12 was deﬁned as the target of 4p16.3 and RGS12 expression decreased total and activated AKT levels. deletions, although it has not been implicated previously as a Our ﬁndings identify RGS12 as a candidate tumor-suppressor tumor-suppressor gene. RGS12 transcript levels were relatively gene in AA prostate cancer, which acts by decreasing expression of reduced in AA prostate cancer, and prostate cancer cell lines AKT and MNX1, establishing a novel oncogenic axis in this showed decreased RGS12 expression relative to benign prostate disparate disease setting. Cancer Res; 77(16); 1–11. 2017 AACR.

Introduction expression in AA and EA prostate cancer using large-scale expression microarrays (2–5) including a study from our group (6). A African American (AA) men have a significantly higher inci- number of studies have focused on a smaller set of preselected dence of prostate cancer compared with European American (EA) genes (7–9). All of these studies indicate that there is differential men (1) and are twice as likely to die from prostate cancer gene expression between AA and EA prostate cancers. The compared with EA men. The biological basis for this difference TMPRSS2/ERG fusion gene is much less frequent in AA prostate in prostate cancer mortality is unclear. Because AA men account cancer based on studies of DNA, RNA, and protein (8–16). for a significant fraction of all prostate cancer–related deaths in the Elevated SPINK1 expression appears to be more common in AA United States, it is important to understand the basis for this prostate cancer (8, 9, 17–19). Among other genes upregulated in higher mortality in order to optimize prevention and treatment AA prostate cancer, inflammatory genes are prominent (2, 4, 7). strategies for this higher risk group of men. We have recently identified MNX1 as an oncogene that is There have been a number of studies comparing prostate cancer expressed at significantly higher levels in AA prostate cancer tissues from AA and EA men. Several studies have compared gene compared with EA prostate cancer (6). We further demonstrated that MNX1 is regulated by AKT and androgen receptor activity and 1Department of Urology, Southwest Hospital, Third Military Medical University, upregulates lipid synthesis, which has been linked to aggressive Chongqing, China. 2Department of Pathology and Immunology, Baylor College disease (20, 21), and thus, MNX1 may contribute to disease of Medicine and Michael E. DeBakey Department of Veterans Affairs Medical aggressiveness in AA prostate cancer. Center, Houston, Texas. 3Department of Biochemistry and Molecular Biology, We have published (22) a study of allelic loss and gain in 20 College of Basic Medical Sciences, Third Military Medical University, Chongqing, fi 4 AA prostate cancers using Affymetrix 500k SNP arrays to de ne China. Dan L. Duncan Cancer Comprehensive Cancer Center Division of regions of recurrent copy-number gain and loss in localized Biostatistics, Baylor College of Medicine, Houston, Texas. 5Department of Medicine, Baylor College of Medicine, Houston, Texas. prostate cancer and compared the pattern of copy-number alterations (CNA) with that of a similar cohort of EA men Note: Supplementary data for this article are available at Cancer Research (23). We found multiple cytobands with a statistically higher Online (http://cancerres.aacrjournals.org/). frequency of CNAs in our AA cohort over the EA cohort. The fi Y. Wang and J. Wang contributed equally to this article as co rst authors. only unique CNA identified in this initial analysis that had not Current address for O.F. Karatas: Department of Molecular Biology and Genetics, been previously linked to prostate cancer was loss of chromo- Erzurum Technical University, Erzurum, Turkey. some 4p16.3. Corresponding Author: Michael Ittmann, Baylor College of Medicine and We have now extended our original CNA studies to a new set Michael E. DeBakey Veterans Association Medical Center, One Baylor Plaza, of 40 highly tumor-enriched primary prostate cancers and Mailstop: BCM315, Houston, TX 77030. Phone: 713-798-6196; Fax: 713-798-5838; matched benign prostate tissues from AA men using high- E-mail: [email protected] resolution Affymetrix 6.0 SNP arrays and expression array doi: 10.1158/0008-5472.CAN-17-0669 analysis using RNAs from the same tissues. We have confirmed 2017 American Association for Cancer Research. the specific loss of 4p16.3 described previously (22) and

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identified a novel tumor-suppressor gene, RGS12, at this locus used are listed in Supplementary Table S1. Differences in mRNA that shows significantly decreased expression in AA prostate levels were analyzed using the DDCt method normalized to cancer but not EA prostate cancer. Both in vitro and in vivo b-actin expression. Each measurement point was repeated at least data show that RGS12 is a tumor-suppressor gene, as would be in triplicate. predicted from its known ability to negatively regulate pro- oncogenic signal transduction. Furthermore, we have found Cell culture that loss of RGS12 increases expression of MNX1 at least in part Human immortalized normal prostate epithelial cell line by regulating AKT protein levels. Our findings establish a novel PNT1A and prostate cancer cell lines LNCaP, DU145, and oncogenic axis in AA prostate cancer. PC3 were all maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS (Invitrogen). LAPC4 cells were Materials and Methods cultured in RPMI-1640 medium with 10% FBS supplemented with 10 nmol/L R1881 (Sigma). VCaP and 293T cells were Prostate and prostate cancer tissue maintained in DMEM (Invitrogen) with 10% FBS. All cell culture Tissue samples were obtained from the Human Tissue Acqui- medium contained 1x Antibiotic-Antimycotic (Gibco). PNT1A sition and Pathology Core of the Dan L. Duncan Cancer Center cells were obtained from the European Type Culture Collection. and were collected from fresh radical prostatectomy specimens PNT1A with myristoylated AKT and controls have been described after obtaining informed consent under a Baylor College of previously (24). All other cell lines were obtained from the – Medicine Institutional Review Board approved protocol and as American Type Culture Collection. Cell were obtained between such followed the principals of the Declaration of Helsinki and 2001 and 2012, expanded, frozen, and stored as stocks in liquid the Belmont Report. Cancer tissues include at least 70% tumor nitrogen. All cell lines are authenticated by STR analysis at MD tissue, and benign tissues were free of cancer on pathologic Anderson Cancer Center Characterized Cell Line Core Facility. examination. DNAs and RNAs were extracted using a Qiagen Cells are tested monthly for mycoplasma contamination. DNA/RNA mini kit following the manufacturer's protocol. Stable knockdown of RGS12 Affymetrix 6.0 SNP and Agilent 60K expression arrays LNCaP cells with stable knockdown of RGS12 were produced DNAs from AA prostate cancer tissues and matched benign by utilizing RGS12-shRNAs in lentiviral vector pGFP-C-shLenti tissue were analyzed using Affymetrix 6.0 SNP arrays by the Albert (Origene). Four unique human shRNAs (A–D) for RGS12 con- Einstein College of Medicine Genomics Core. SNP array data were structs in lentiviral GFP vector were purchased from Origene (Cat processed using the crlmm package in Bioconductor, with the # TL302015). Another 3 unique shRNA-RGS12 constructs preprocessing steps for copy-number estimation as follows: (1) (V3LHS_310594; 310595; and 310599) were purchased from quantile normalization of the raw intensities (quantile normal- the Baylor College of Medicine C-Bass core. Lentiviruses carrying izing the SNPs and nonpolymorphic markers separately), (2) these stable shRNAs were produced in 293T cells using a Lenti- genotyping, and (3) for total copy number, translating the nor- vpak Packaging kit (Origene) following the manufacturer's malized intensities to an estimate of raw copy number by adding instruction. LNCaP and PNT1A cells were infected by these viruses the allele-specific summaries. For each of the 1M SNP probes, each and were selected with 0.5 mg/mL puromycin (Sigma). tumor profile was centered on the paired normal, in order to generate tumor:normal ratios. Tumor:normal logged values were Plasmid construction and transfection averaged by gene, and each profile was centered on the median of Primers used to amplify three human RGS12 isoforms (Gen- log ratios across all genes. For heat map presentation, gene-level Bank accession NM_198229, NM_002926, and NM_198227) are tumor:normal values were further collapsed into cytobands. listed in Supplementary Table 2. Three RGS12 isoforms were When combining datasets from multiple studies, values for each cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen) con- dataset were binned as gain or loss or no change, using a similar taining CMV promoter. Constructs were sequenced and con- approach to that of our previous study (22). For the Lapointe firmed their accuracies before transfection into cells. The trans- dataset, the SD of the tumor profile with the smallest SD across fection was performed using Fugene 6 reagent (Promega), and cytobands was used as the reference for defining gain or loss events transfected cells were selected and maintained in media contain- within each cytoband; cytobands with average values greater than ing 200 mg/mL G418. þ3SD were called as gain, and cytoband values less than 3SD were called as loss. Gene-level copy alterations for the Taylor Cell proliferation assay dataset were previously binned in that study, with average cyto- Cells (5 103) were plated in each well of 96-well plates. > band log2 ratio 0.6 or 0.6 being called here as gain or loss, Proliferation was determined using the Cell Counting Kit-8 Cell respectively. For our own SNP array datasets [present study and Proliferation Assay Kit (Dojindo Molecular Technologies) as Castro and colleagues (22)], a log2 of 0.2 was used as the cutoff described by the manufacturer. The absorbance was read at [similar to that of Castro and colleagues (22)]. Expression array 450 nm with VERSAmax Tunable microplate reader. analysis has been described previously (6). GEO accession number is pending. Matrigel invasion assays Cells (2.5 104) were plated in the top chamber of Matrigel- Quantitative real-time PCR coated membrane (24-well insert; pore size, 8 mm; BD Bio- Gene expression levels were tested using quantitative real-time sciences). The cells on the apical side of each insert were then PCR (qRT-PCR) on an Applied Biosystem (StepOne, Lifetechnol- scraped off after 24 hours. The wells were washed with PBS, fixed ogies). Total RNAs were extracted using the RNasy Kit (Qiagen). with 100% methanol, and stained with DAPI. After staining, cDNAs were synthesized as described previously. TagMan probes membranes were removed from the insert and mounted on slides,

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and the invading cells were counted under the Nikon Eclipse and samples without data on race. A total of 32 cytobands showed TE2000-U microscope. Matrigel assays were performed in significantly higher loss or gain (P < 0.01, one-sided Fisher exact triplicate. test) in AA prostate cancer when compared with EA prostate cancer (Fig. 1B and Supplementary Table S3). Of note, we con- Soft-agar growth assay firmed the specific loss of 4p16.3 described previously (P < 0.001). Six-well plates with 0.5% base agar layer mixed with 1X It is well known that hereditary cancer loci often show somatic culture media plus 10% FBS were prepared before the seeding alterations as well, so it is noteworthy that 6 of the 32 cytobands of cells. PNT1A cells (5 104) with stable knockdown of we identified have been implicated in hereditary AA prostate RGS12 or vector controls were plated in 0.35% top agar layer cancer by linkage analysis, including 8q24 (26–28),11q13 each agar dishes. Cell colonies were counted after incubation at (28), 12q24 (29), 14q32 (30), 17p11 (31), and 17q21 (32). We 37C in an incubator for 3 weeks and staining with 1 mg/mL of then carried out a cluster analysis of the cytobands that were idonitrotetrazolium chloride for 8 hours. This experiment was significantly different between AA and EA prostate cancer. Most of repeated twice. the AA prostate cancers cluster into four groups in this analysis as indicated in Fig. 1B. Group A has no losses in these cytobands. Western blot Group B shows multiple gains and some losses, whereas Group D Total cellular protein lysate was prepared as described previ- shows more focal gains. Group C shows loss in multiple cyto- ously. Anti–b-actin was obtained from Sigma-Aldrich (dilution bands preferentially lost in AA prostate cancer. Most of the AA 1:5,000). Anti-MNX1 was purchased from Origene (Cat# prostate cancer cases with loss of 4p16.3 cluster in this group. TA337035) and used at a dilution of 1:3,000. Anti-RGS12 was Of note, Group C clusters adjacent to the metastatic prostate purchased from Santa Cruz (sc-514173). Antibodies to total AKT cancers, which are predominantly derived from EA men, and the and phospho-AKT-T308 and S473 were from Cell Signaling. metastatic cases also show loss at 4p16.3, indicating that loss of Western blotting procedures were described previously (6). 4p16.3 is likely to be associated with aggressive disease in primary prostate cancer. Mouse xenograft studies All procedures were approved by the Baylor College of Med- Identification of a novel tumor suppressor in AA prostate cancer icine Institutional Animal Use and Care Committee. Experiments We have also carried out expression array analysis using RNAs were carried out on 8- to 10-week-old male SCID mice. Tumor from the same cancers used for CNA analysis (6). We identified a xenografts were established by subcutaneous injection over each total of 4,341 probes altered in prostate cancer versus benign (P < flank in 50 mL volume mixed with 50 mL Matrigel (BD Bioscience). 0.01) in AA prostate cancer, and the overall quality of the data has Tumors were harvested 8 weeks after inoculation, and the tumor been confirmed as described previously (6). To identify the weights were recorded. Tumor tissues were snap frozen for further potential tumor suppressor on 4p16.3 that was preferentially lost mRNA and protein expression studies. in AA prostate cancer, we systematically examined expression of genes on 4p16.3 in AA prostate cancer in the expression array data. Statistical analysis We found two genes that are adjacent on 4p16.3 (HTT and Numerical values from two groups were compared by the RGS12), which both show downregulation of mRNA in AA Mann–Whitney test, with P < 0.05 considered significant. For prostate cancers. Detailed examination of deletions showed that more than two groups, ANOVA was used followed by pairwise losses are more concentrated in RGS12, and CNAs correlate with r ¼ P ¼ comparison to controls, which were considered significant if expression levels for RGS12 ( 0.59; 0.016) but not HTT P < 0.05. (Supplementary Table S4). RGS12 has three alternatively spliced protein coding isoforms as shown in Supplementary Fig. S2. The proteins Results encoded by isoforms 1 and 2 are almost identical and encode CNAs in AA prostate cancer full-length proteins. Isoform 3 lacks the amino terminal PTB We have extended our original CNA studies of AA prostate and PDZ domains. Because of the extensive overlap of the three cancer (22) to a new set of 40 highly tumor-enriched primary isoforms, we were not able to measure the mRNA levels of all prostate cancers and matched benign prostate tissues from AA threeisoformsindividually.Wewereabletoanalyzetotal men using high-resolution Affymetrix 6.0 SNP arrays (906K RGS12 using a probe from the common region of all three SNPs). We combined these new data, our published data [Castro isoforms, total isoforms 1þ2, total isoforms 1þ3, and isoform and colleagues (22)] and the predominantly EA data sets of 3 alone. qRT-PCR analysis using a TaqMan probe that detects Lapointe and colleagues (23) and Taylor and colleagues (total all three RGS12 isoforms showed significantly decreased RGS12 AA: n ¼ 89; EA: n ¼ 169; ref. 25) for analysis. Data organized by expression in AA prostate cancer (Fig. 2A; P < 0.001, Mann– race are shown in Supplementary Fig. S1. Cluster analysis of these Whitney). Analysis using primers detecting both isoforms 1 þ 2 data revealed that the vast majority of AA prostate cancers clus- or isoform 3 only also showed significantly decreased expres- tered in two major groups (Fig. 1A, right), indicating that AA and sion levels in AA prostate cancers compared with benign EA prostate cancers have different patterns of CNAs than EA prostate tissues (Fig. 2B and C; both P < 0.001; Mann–Whit- prostate cancer. The AA cluster to the far right shows a distinct ney). However, no loss was seen in EA prostate cancer using pattern of CNAs. A smaller cluster of AA prostate cancers clusters primers detecting all three isoforms (Fig. 2D). Scatter plots of adjacent to the metastatic samples and has significant similarities this data are shown in Supplementary Fig. S3. Overall, the CNA to CNAs in these samples. and expression analysis data show that there is loss of RGS12 We then compared frequencies of CNAs at all cytobands alleles and/or gene expression in AA prostate cancer that is not between AA and EA patients after excluding metastatic samples seen in EA prostate cancer.

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Figure 1. CNA in AA prostate cancer. A, Cluster analysis of CNAs in AA and EA prostate cancer. Yellow is gain and blue is loss for each cytoband on chromosomes as shown on the left. Cases are in columns and cases from the current study are gray on upper bar, whereas cases from Lapointe are red, Taylor are blue, and Castro are black. Race is shown with AA in black, EA in gray, and white not available. Metastatic lesions are also identified (black). The majority of AA cases cluster in two groups to the right. B, Cluster analysis of cytobands with loss and gain in AA prostate cancer identified using SNP arrays of DNAs from 40 highly tumor-enriched tissues and matched benign tissues, our prior analysis of 20 AA prostate cancers (Castro) and two primarily EA datasets (Taylor and Lapointe). Cytobands that were more commonly altered in AA prostate cancer (P < 0.01, one-sided Fisher exact test) were identified. Yellow, gain; blue, loss. Most of the AA prostate cancers cluster into four groups, as indicated by horizontal red lines. Group A has no losses in these cytobands; Group B shows multiple gains and some losses; Group C shows loss in multiple cytobands, whereas Group D shows more focal gains. Note that both groups B and C cluster adjacent to blocks of metastatic cancers. Losses at 4p16.3 were concentrated in Group C.

We then compared expression of RGS12 in PNT1A cells, an Biological effects of RGS12 knockdown in vitro immortalized normal prostate epithelial cell line to LNCaP, VCaP, In order to study RGS12's potential tumor-suppressor function, LAPC4, DU145, and PC3 prostate cancer cell lines. As shown we knocked down RGS12 expression in PNT1A cells with three in Fig. 2E, all five prostate cancer cell lines showed decreased different RGS12-shRNAs using a lentiviral vector (pGFP-C- RGS12 relative to PNT1A. The differences were highly statistically shLenti). After stable selection, knockdown of RGS12 in each significant (P < 0.001; ANOVA) in all prostate cancer cell line group was confirmed using a TaqMan probe that detects all three except DU145 where the decrease was relatively small (P < 0.05, isoforms (Fig. 3A). Knockdown of RGS12 significantly increased ANOVA). Analysis of isoform expression in the same cell lines cell proliferation (P < 0.001 at 4 days, ANOVA) in all three groups revealed that isoform 1þ2 and isoform 1þ3 expression was compared with scrambled control (Fig. 3B). We also tested colony decreased in all prostate cancer cell lines except DU145 (Fig. formation in soft agar, a hallmark of transformation. PNT1A cells 2F). Isoform 3 had relatively low expression in PNT1A compared are not fully transformed and form only rare small colonies in this with isoforms 1 and 2, and although there was a trend for lower assay. As shown in Fig. 3C, knockdown of RGS12 markedly isoform 3 expression in the prostate cancer cell lines, this was not increased colony formation in soft agar (P < 0.001, ANOVA). statistically significant. The overall colony sizes in RGS12 knockdown lines were also

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Figure 2. RGS12 expression in prostate cancer. A–C, Relative RGS12 mRNA expression by qRT-PCR in benign prostate and prostate cancer from AA men. A, All isoforms. B, Isoforms 1 and 2. C, Isoform 3. Mean SD is shown. , P < 0.001, Mann–Whitney. D, Relative RGS12 mRNA expression by qRT-PCR in benign prostate and prostate cancer from EA men, all isoforms. E, Relative RGS12 expression (all isoforms) in prostate cancer cell lines and the benign prostate epithelial cell line PNT1A. Mean SD; , P < 0.05; , P < 0.001; ANOVA pairwise comparisons versus PNT1A. F, RGS12 expression using probes recognizing specific isoforms 1 and 2, 1 and 3, or 3 only is shown. Mean SD; , P < 0.05; , P < 0.001; ANOVA pairwise comparisons versus same probe in PNT1A. much larger compared with controls (Fig. 3D). As a positive transfection of the three isoforms. Using an anti-RGS12 antibody, control, we used PNT1A cells expressing Huntington-interacting we were able to confirm the overexpression of different RGS12 protein-1, which formed colonies in soft agar (data not shown) as isoforms, although based on the relative band intensity in anti-V5 we have shown previously (33). We also knocked down RGS12 in and anti-RGS12 Westerns, the affinity of the anti-RGS12 antibody LNCaP using four different shRNAs (Fig. 3E). As in PNT1A, for isoform 3 was higher than for isoforms 1 and 2. After stable proliferation was significantly increased with all four shRNAs transfection of all three isoforms into LNCaP cells, we confirmed (Fig. 3F). significantly increased RGS12 expression at RNA level in LNCaP We then cloned all three major isoforms of RGS12 from LNCaP cells (Fig. 4B). Overexpression of each isoform significantly cells into pCDH-CMV-MCS-EF1-Neo lentiviral vector. As seen decreased cell proliferation (Fig. 4C). Isoform 3 had a lower in Fig. 4A, V5 antibody detected all three bands with correct effect on growth compared with the other two isoforms (P < estimated sizes using lysate from 293T cells following transient 0.05 vs. Iso1 or Iso2, ANOVA). Overexpression of each isoform

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Figure 3. Biological effects in vitro of RGS12 knockdown. A, RGS12 mRNA levels in PNT1A cells or PNT1A with scrambled shRNA or three different shRNAs targeting the RGS12 common region. B, Proliferation as determined by MTT assay in PNT1A cell lines or PNT1A with scrambled or RGS12 targeting shRNAs. C, Colony formation in PNT1A or PNT1A treated with scrambled or RGS12-targeting shRNAs. NS, not signiﬁcant; , P < 0.05; , P < 0.001; , P < 0.001; ANOVA pairwise versus scrambled. D, Typical colonies in PNT1A cell lines with or without RGS12 knockdown. Note that PNT1A without RGS12 knockdown form very small colonies. E, RGS12 mRNA levels in LNCaP cells with scrambled shRNA or four shRNAs targeting RGS12. F, Proliferation, as assessed by MTT assay in LNCaP with scrambled or RGS12-targeting shRNAs. Mean SD; , P < 0.001; ANOVA.

dramatically inhibited cell invasion (Fig. 4D), and again isoform growth was monitored twice weekly. At end of 5 weeks, mice 3 showed a weaker effect on invasion (P < 0.01 vs. Iso1 or Iso2, were euthanized and primary tumors were excised, weighed, and a ANOVA). portion of the tumor was frozen in liquid nitrogen for molecular analysis and another portion fixed and paraffin-embedded. The Biological effects of RGS12 expression in vivo difference of tumor weight between shD group and controls was To evaluate tumor-suppressive activities in vivo, we carried out statistically significant (Fig. 5A, P < 0.05, Mann–Whitney), with xenograft experiments in SCID mice. In the first experiment, two higher tumor weights in tumors with RGS12 knockdown. In the groups of mice (10 mice/group) were injected subcutaneously second experiment, we used LNCaP cells overexpressing RGS12 with LNCaP with vector or LNCaP-shD, respectively. Tumor isoform 2 or 3 or control cells with scrambled vector. Tumor

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Figure 4. Biological effects in vitro of RGS12 overexpression. A, Western bots of lysates from 293T cells transiently transfected with V5-tagged RGS12 isoforms 1–3. Antibodies targeting V5, RGS12, or actin (loading control) were used for Western blotting. RGS12 isoform 3 is smaller because it lacks the amino terminal domains. B, RGS12 mRNA levels in LNCaP cell and cell lines expressing RGS12 vector control or RGS12 isoforms 1–3. C, Proliferation as measured by MTT assay in LNCaP cell lines expressing RGS12 isoforms 1– 3 versus control LNCaP and vector controls. D, Invasion in LNCaP cell lines expressing RGS12 isoforms 1–3 versus control LNCaP and vector controls. Mean SD is shown. , P < 0.001; ANOVA pairwise versus vector control.

weights were significantly decreased in both isoform expressing datasetofGrassoandcolleagues(34).Thesedataarefroma groups compared with control cells (Fig. 5B, P < 0.001, Mann– predominantly EA cohort but have a mixture of localized (59 Whitney). Surprisingly, isoform 3 tumors were smaller than cases) and metastatic prostate cancer (35 cases), unlike our isoform 2–expressing tumors given that isoform 3 appeared to data, which are all from localized disease. These correlations are be less tumor suppressive than isoform 2 in vitro. Analysis of shown in Supplementary Fig. S4. We hypothesized that RGS12 RGS12 mRNA expression in the final tumors revealed that isoform may repress MNX1 expression. 3 tumors had approximately 4-fold higher levels of RGS12 com- We examined expression of MNX1 in the LNCaP cell lines with pared with isoform 2 tumors (data not shown). This implies there overexpression of RGS12 isoforms 1 to 3. As seen in Fig. 6A, MNX1 may have been preferential growth of tumor cells with lower protein expression was completely lost in isoform 1–expressing RGS12 knockdown during the in vivo growth, and this effect was cells and markedly diminished in isoform 2– and 3–expressing more profound in the isoform 2–expressing cells than the isoform cells. Conversely, knockdown of RGS12 markedly increased 3–expressing cells. Finally, because PNT1A cells with RGS12 MNX1 expression in LNCaP cells in vitro (Fig. 6B). Similar knockdown formed colonies in soft agar, we injected PNT1A-shD increased levels of MNX1 protein were seen in xenograft tumors or shE cells or control PNT1A cells into mice. After 8 weeks, there from mice with RGS12 knockdown (Fig. 6C). Examination of were no tumors found in the PNT1A control group, whereas MNX1 in LNCaP xenografts expressing RGS12 isoform 3 or vector obvious tumor masses were seen in both groups with RGS12 controls significant downregulation of MNX1 protein (Fig. 6D). knockdown. The average weight of tumors collected was 68 and Thus, RGS12 significantly decreases MNX1 protein expression. 84 mg in the shD and shE groups, respectively (Fig. 5C). To We have shown previously using inhibitors of the PI3K confirm that the tumors were from PNT1A cells, we used SV40 T- (LY294002) and AKT (AZD5363) that MNX1 is strongly regulated antigen immunohistochemistry, because PNT1A cells were orig- by AKT activity (6). We have now confirmed this observation inally immortalized with SV40 large T-antigen. As shown in Fig. using PNT1A cells expressing myristoylated AKT. As shown in Fig. 5D, tumor cells were positive for SV40 T-antigen. Overall, our data 7A, these cells show increased levels of total and phosphorylated show that RGS12 is tumor-suppressor gene in vivo for prostate AKT compared with vector controls and also show increased cancer and prostate epithelial cells. MNX1 protein levels. We then examined the impact of RGS12 expression on AKT activity. As shown in Fig. 7B, isoform 1 almost RGS12 represses expression of MNX1, an AKT-regulated completely abolishes AKT protein expression as well as expression oncogene of phosphorylated AKT. Expression of isoforms 2 and 3 resulted in We have recently shown that MNX1 is an oncogenic tran- lesser decreases in total and phosphorylated AKT. Knockdown of scription factor whose expression is preferentially increased in RGS12 in PNT1A cells showed increase in S473 phosphorylated AA prostate cancer. Examination of the gene expression data in AKT (Supplementary Fig. S5). Of note, PNT1A are PTEN wild type. our AA prostate cancers (6) revealed a significant negative Examination of AKT mRNA in cells overexpressing RGS12 correlation between MNX1 and RGS12 mRNA expression in showed increased mRNA that was statistically significant for iso- cancer tissues (0.278; P ¼ 0.028, Spearman). We saw a similar forms 1 and 2 (Fig. 7C). This strongly suggests that RGS12 negative correlation (0.230; P ¼ 0.01, Spearman) in the regulates AKT posttranscriptionally, with a feedback upregulation

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Figure 5. RGS12 is a tumor-suppressor gene in vivo. A, Final tumor weight of LNCaP vector controls and LNCaP with RGS12 knockdown. Mean SD. , P < 0.05, Mann–Whitney. B, Final tumor weights of LNCaP vector controls and LNCaP with expressing RGS12 isoform 2 or isoform 3. Mean SD. , P < 0.001; ANOVA pairwise versus vector control. C, Final tumor weight of PNT1A controls and PNT1A with RGS12 knockdown by two different shRNAs. Mean SD is shown. Controls had no tumors. D, Immunohistochemistry with antibody to SV40 T-antigen in PNT1A tumor with RGS12 knockdown to conﬁrm origin from PNT1A cells. Negative control (no primary antibody) is also shown.

of AKT mRNA. Consistent with this, we observed a positive with overall survival in lung cancer (35), but the mechanism correlation between RGS12 and AKT mRNA levels in our expres- for this association is unknown. RGS12 is a negative regulator of sion microarray data (0.338, P ¼ 0.009; Supplementary Fig. S6). G-protein signaling that acts via enhancing GTP hydrolysis (36). As such, it can inhibit signal transduction from G-protein–coupled receptors, a number of which have been implicated in the Discussion pathogenesis of prostate cancer (37–42), although the exact In this report, we have shown that RGS12 is preferentially targets of RGS12 are not clear. RGS12 has been shown to interact deleted in AA compared with EA prostate cancer, and there is with the IL8 receptor (43). It also contains a phospho-tyrosine significantly lower RGS12 mRNA expression in prostate cancer binding domain and has been shown to interact with MAPK/ERK compared with benign tissues in AA but not EA prostate cancer. and PI3K signaling in various contexts (44–46) and thus may act We observed a significant correlation of genomic deletion and as link between G-protein–coupled signaling and other signaling decreased expression (r ¼ 0.59; P ¼ 0.016) in AA prostate cancer, pathways (36). but at this level of correlation, it is likely that other factors may also We have shown previously that MNX1 is an oncogenic tran- affect RGS12 mRNA expression in AA prostate cancer in addition scription factor whose expression is markedly increased in AA to genomic deletion. Such factors will require further studies to prostate cancer and to a much lesser extent in EA prostate cancer elucidate. (6). Remarkably, examination of gene expression data in our AA Our in vitro and in vivo data show that RGS12 is a tumor- prostate cancers revealed a significant negative correlation suppressor gene. Of note, decreased RGS12 is by itself capable of between MNX1 and RGS12 mRNA expressions in AA prostate fully transforming immortalized normal prostate epithelial cells cancer tissues. Both knockdown and overexpression studies con- such that they form large colonies in soft agar and tumors in SCID firm that RGS12 can strongly inhibit expression of MNX1. AKT mice, indicating that it has a strong tumor-suppressive effect in activity strongly regulates MNX1 expression based on our pub- this context. It can also affect tumorigenesis and transformation- lished data (6), and analysis of PNT1A cells expressing myristoy- related cellular phenotypes in vitro and in vivo in fully transformed lated AKT confirms this observation. Our data indicate that RGS12 prostate cancer cells. significantly negatively regulates AKT protein levels and activity. It RGS12 has not been previously implicated as a tumor-suppres- should be noted that levels of phosphorylated AKT were roughly sor gene. A SNP in RGS12 has been shown to be associated proportional to AKT protein levels, indicating that activation of

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establishing a novel oncogenic axis in AA prostate cancer. MNX1 enhances lipid synthesis, which has been shown to be associated with disease aggressiveness (20, 21). The increased MNX1 result- ing from RGS12 loss is mediated at least in part by its effect on AKT protein levels, but other mechanisms may also be involved in RGS12 regulation of MNX1 expression. Of course, increased AKT protein will almost certainly affect other AKT targets as well. Whether decreased RGS12 also affects other cellular targets that can enhance transformed phenotypes is unclear. Further studies are needed to fully clarify the mechanism of action of RGS12 as a tumor-suppressor gene in prostate cancer. Our current and previous CNA studies in AA prostate cancer have shown that there are signiﬁcant quantitative and qualitative differences in CNAs between AA and EA prostate cancer. Of the 32 cytobands with CNAs identiﬁed as being more common in AA

Figure 6. RGS12 represses MNX1 expression. A, Western blot with anti-MNX1 antibody of lysates from LNCaP cells and cell lines expressing RGS12 isoforms 1–3or vector control. Actin was used as a loading control. B, Western blot with anti- MNX1 antibody of lysates from LNCaP cell lines with RGS12 knockdown versus control LNCaP and scrambled control. Actin was used as a loading control. C, Western blot of protein lysates of LNCaP tumors with RGS12 knockdown or control tumors with anti-MNX1 antibody. D, MNX1 protein in LNCaP xenografts expressing RGS12 isoform 3 and vector controls.

AKT was not inhibited, but with lower AKT protein, total phosphorylated AKT was also decreased. The exact mechanism by which RGS12 can regulate AKT protein levels is yet to be determined but appears to be posttranscriptional since AKT mRNA is actually increased by RGS12 overexpression. The regulation of AKT protein levels has not been as inten- sively studied as its activation by phosphorylation, although increased AKT protein may enhance AKT signaling, particularly in the context of dysregulated AKT activation. As reviewed by Liao and Hung (47), there are multiple posttranscriptional mechanisms potentially affecting AKT protein levels. AKT can be phosphorylated at threonine-450, and this phosphorylation affects protein stability. This site is phosphorylated during AKT translation and may be important in modulating interactions with Pin1, which can regulate AKT stability. It should be noted that Pin1 is increased in prostate cancer and is associated with aggressive disease (48). Interactions with heat shock proteins Figure 7. can also increase AKT stability. On the other hand, ubiquitin- RGS12 represses AKT protein expression. A, Western blots of cell lysates from mediated proteolysis or degradation by caspases of AKT has PNT1A cells expressing myristoylated AKT (m-AKT) or vector controls (Vec) for also been described (47). Additional studies are needed to MNX1, phosphorylated AKT (S473 and T308), or total AKT. Actin was used as a loading control. B, Western blots of lysates from LNCaP cell lines expressing understand the mechanism by which AKT protein levels are RGS12 isoforms 1–3, control LNCaP and vector controls for MNX1, total AKT, and controlled by RGS12. phosphorylated AKT (T308). Actin was used as a loading control. C, AKT mRNA Our data indicate that decreased RGS12 enhances transformed in LNCaP cells expressing RGS12 isoforms 1–3 and vector controls. Mean SD. phenotypes at least in part via increasing expression of MNX1, , P < 0.05, ANOVA.

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Wang et al.

prostate cancer, 6 coincide with a region linked to familial AA Analysis and interpretation of data (e.g., statistical analysis, biostatistics, prostate cancer. However, the majority of CNAs we have identified computational analysis): Y. Wang, J. Wang, L. Zhang, Y. Zhang, C.J. Creighton, have not been previously linked to AA prostate cancer. Although M. Ittmann Writing, review, and/or revision of the manuscript: Y. Wang, J. Wang, we have focused on 4p16.3 in these studies, other areas of CNAs C.J. Creighton, M. Ittmann that are more frequently present in AA prostate cancer identified in Administrative, technical, or material support (i.e., reporting or organizing our studies may also harbor novel tumor suppressors or onco- data, constructing databases): Y. Wang, L. Zhang, M. Ittmann genes relevant to AA prostate cancer. Future studies will hopefully Study supervision: J. Wang, C.J. Creighton, M. Ittmann identify additional genes that affect initiation and/or progression in AA prostate cancer and provide new insights into the optimal Grant Support strategies for prevention and treatment of prostate cancer in AA This work was supported by grants from the Department of Defense Prostate men. Cancer Research Program (W81XWH-12-1-0046 to M. Ittmann); the National Cancer Institute supporting the Dan L. Duncan Cancer Center (P30 CA125123) Disclosure of Potential Conflicts of Interest Human Tissue Acquisition and Pathology and Genomic and RNA Profiling Shared Resources; the Prostate Cancer Foundation (M. Ittmann), and by the use No potential conflicts of interest were disclosed. of the facilities of the Michael E. DeBakey VAMC. Authors' Contributions The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Conception and design: J. Wang, L. Zhang, M. Ittmann accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: J. Wang, L. Zhang Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Wang, L. Zhang, O.F. Karatas, L. Shao, P. Castro, Received March 5, 2017; revised May 16, 2017; accepted June 8, 2017; M. Ittmann published OnlineFirst June 13, 2017.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 14. Farrell J, Young D, Chen Y, Cullen J, Rosner IL, Kagan J, et al. Predominance 2015;65:5–29. of ERG-negative high-grade prostate cancers in African American men. Mol 2. Wallace TA, Prueitt RL, Yi M, Howe TM, Gillespie JW, Yfantis HG, et al. Clin Oncol 2014;2:982–86. Tumor immunobiological differences in prostate cancer between African- 15. Powell IJ, Dyson G, Chinni SR, Bollig-Fischer A. Considering race and the American and European-American men. Cancer Res 2008;68:927–36. potential for ERG expression as a biomarker for prostate cancer. Per Med 3. Reams RR, Agrawal D, Davis MB, Yoder S, Odedina FT, Kumar N, et al. 2014;11:409–12. Microarray comparison of prostate tumor gene expression in African- 16. Kelly GM, Kong YH, Dobi A, Srivastava S, Sesterhenn IA, Pathmanathan R, American and Caucasian American males: a pilot project study. Infect et al. ERG oncoprotein expression in prostate carcinoma patients of Agent Cancer 2009;4Suppl 1:S3. different ethnicities. Mol Clin Oncol 2015;3:23–30. 4. Kinseth MA, Jia Z, Rahmatpanah F, Sawyers A, Sutton M, Wang-Rodriguez J, 17. Johnson MH, Ross AE, Alshalalfa M, Erho N, Yousefi K, Glavaris S, et al. et al. Expression differences between African American and Caucasian SPINK1 defines a molecular subtype of prostate cancer in men with more prostate cancer tissue reveals that stroma is the site of aggressive changes. rapid progression in an at-risk, natural history radical prostatectomy Int J Cancer 2014;134:81–91. cohort. J Urol 2016;196:1436–44. 5. Wang BD, Ceniccola K, Yang Q, Andrawis R, Patel V, Ji Y, et al. Identification 18. Faisal FA, Sundi D, Tosoian JJ, Choeurng V, Alshalalfa M, Ross AE, et al. and functional validation of reciprocal microRNA-mRNA pairings in Racial variations in prostate cancer molecular subtypes and androgen African American Prostate Cancer Disparities. Clin Cancer Res 2015;21: receptor signaling reflect anatomic tumor location. Eur Urol 2016; 4970–84. 70:14–7. 6. Zhang L, Wang J, Wang Y, Zhang Y, Castro P, Shao L, et al. MNX1 is 19. Khani F, Mosquera JM, Park K, Blattner M, O'Reilly C, MacDonald TY, oncogenically upregulated in African-American Prostate Cancer. Cancer et al. Evidence for molecular differences in prostate cancer between Res 2016;76:6290–8. African American and Caucasian men. Clin Cancer Res 2014;20: 7. Powell IJ, Dyson G, Land S, Ruterbusch J, Bock CH, Lenk S, et al. Genes 4925–34. associated with prostate cancer are differentially expressed in African 20. Migita T, Ruiz S, Fornari A, Fiorentino M, Priolo C, Zadra G, et al. Fatty acid American and European American men. Cancer Epidemiol Biomarkers synthase: a metabolic enzyme and candidate oncogene in prostate cancer. Prev 2013;22:891–7. J Natl Cancer Inst 2009;101:519–32. 8. Tomlins SA, Alshalalfa M, Davicioni E, Erho N, Yousefi K, Zhao S, et al. 21. Huang WC, Li X, Liu J, Lin J, Chung LW. Activation of androgen receptor, Characterization of 1577 primary prostate cancers reveals novel biological lipogenesis, and oxidative stress converged by SREBP-1 is responsible for and clinicopathologic insights into molecular subtypes. Eur Urol 2015; regulating growth and progression of prostate cancer cells. Mol Cancer Res 68:555–67. 2012;10:133–42. 9. Yamoah K, Johnson MH, Choeurng V, Faisal FA, Yousefi K, Haddad Z, et al. 22. Castro P, Creighton CJ, Ozen M, Berel D, Mims MP, Ittmann M. Genomic Novel biomarker signature that may predict aggressive disease in African profiling of prostate cancers from African American Men. Neoplasia American Men with prostate cancer. J Clin Oncol 2015;33:2789–96. 2009;11:305–12. 10. Lindquist KJ, Paris PL, Hoffmann TJ, Cardin NJ, Kazma R, Mefford JA, et al. 23. Lapointe J, Li C, Giacomini CP, Salari K, Huang S, Wang P, et al. Genomic Mutational landscape of aggressive prostate tumors in African American profiling reveals alternative genetic pathways of prostate tumorigenesis. men. Cancer Res 2016;76:1860–8. Cancer Res 2007;67:8504–10. 11. Bianchi-Frias D, Basom R, Delrow JJ, Coleman IM, Dakhova O, Qu X, et al. 24. Zhang L, Shao L, Creighton CJ, Zhang Y, Xin L, Ittmann M, et al. Function of Cells comprising the prostate cancer microenvironment lack recurrent phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis. clonal somatic genomic aberrations. Mol Cancer Res 2016;14:374–84. Oncotarget 2015;6:6281–94. 12. Borromeo MD, Savage TK, Kollipara RK, He M, Augustyn A, Osborne JK, 25. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. et al. ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroen- Integrative genomic profiling of human prostate cancer. Cancer Cell docrine tumors and regulate distinct genetic programs. Cell Rep 2016; 2010;18:11–22. 16:1259–72. 26. Freedman ML, Haiman CA, Patterson N, McDonald GJ, Tandon A, 13. Feibus AH, Sartor O, Moparty K, Chagin K, Kattan MW, Ledet E, et al. Waliszewska A, et al. Admixture mapping identifies 8q24 as a prostate Clinical use of PCA3 and TMPRSS2:ERG urinary biomarkers in African- cancer risk locus in African-American men. Proc Natl Acad Sci U S A American men undergoing prostate biopsy. J Urol 2016;196:1053–60. 2006;103:14068–73.

OF10 Cancer Res; 77(16) August 15, 2017 Cancer Research

RGS12 in African American Prostate Cancer

27. Han Y, Rand KA, Hazelett DJ, Ingles SA, Kittles RA, Strom SS, et al. Prostate 38. Pi M, Quarles LD. Multiligand specificity and wide tissue expression of cancer susceptibility in men of African Ancestry at 8q24. J Natl Cancer Inst GPRC6A reveals new endocrine networks. Endocrinology 2012;153: 2016;108. 2062–9. 28. Hooker S, Hernandez W, Chen H, Robbins C, Torres JB, Ahaghotu C, et al. 39. Brennan SC, Thiem U, Roth S, Aggarwal A, Fetahu I, Tennakoon S, et al. Replication of prostate cancer risk loci on 8q24, 11q13, 17q12, 19q33, and Calcium sensing receptor signalling in physiology and cancer. Biochim Xp11 in African Americans. Prostate 2010;70:270–5. Biophys Acta 2013;1833:1732–44. 29. Ledet EM, Sartor O, Rayford W, Bailey-Wilson JE, Mandal DM. Sugges- 40. Rodriguez M, Luo W, Weng J, Zeng L, Yi Z, Siwko S, et al. PSGR promotes tive evidence of linkage identified at chromosomes 12q24 and 2p16 in prostatic intraepithelial neoplasia and prostate cancer xenograft growth African American prostate cancer families from Louisiana. Prostate through NF-kappaB. Oncogenesis 2014;3:e114. 2012;72:938–47. 41. Cai Y, Wang J, Ren C, Ittmann M. Frequent heterogeneous missense 30. Ledet EM, Hu X, Sartor O, Rayford W, Li M, Mandal D. Characterization of mutations of GGAP2 in prostate cancer: implications for tumor biology, germline copy number variation in high-risk African American families clonality and mutation analysis. PLoS One 2012;7:e32708. with prostate cancer. Prostate 2013;73:614–23. 42. Cai Y, Wang J, Li R, Ayala G, Ittmann M, Liu M. GGAP2/PIKE-a directly 31. Baffoe-Bonnie AB, Kittles RA, Gillanders E, Ou L, George A, Robbins C, et al. activates both the Akt and nuclear factor-kappaB pathways and promotes Genome-wide linkage of 77 families from the African American Hereditary prostate cancer progression. Cancer Res 2009;69:819–27. Prostate Cancer Study (AAHPC). Prostate 2007;67:22–31. 43. Snow BE, Hall RA, Krumins AM, Brothers GM, Bouchard D, Brothers CA, 32. Haiman CA, Chen GK, Blot WJ, Strom SS, Berndt SI, Kittles RA, et al. et al. GTPase activating specificity of RGS12 and binding specificity of an Genome-wide association study of prostate cancer in men of African alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain. J Biol Chem ancestry identifies a susceptibility locus at 17q21. Nat Genet 2011; 1998;273:17749–55. 43:570–3. 44. Willard MD, Willard FS, Li X, Cappell SD, Snider WD, Siderovski DP. 33. Wang J, Yu W, Cai Y, Ren C, Ittmann MM. Altered fibroblast growth factor Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor- receptor 4 stability promotes prostate cancer progression. Neoplasia mediated differentiation. EMBO J 2007;26:2029–40. 2008;10:847–56. 45. Huang J, Nalli AD, Mahavadi S, Kumar DP, Murthy KS. Inhibition of 34. Grasso CS, Wu YM, Robinson DR, Cao X, Dhanasekaran SM, Khan AP, et al. Galphai activity by Gbetagamma is mediated by PI 3-kinase-gamma- and The mutational landscape of lethal castration-resistant prostate cancer. cSrc-dependent tyrosine phosphorylation of Galphai and recruitment of Nature 2012;487:239–43. RGS12. Am J Physiol Gastrointest Liver Physiol 2014;306:G802–10. 35. Dai J, Gu J, Lu C, Lin J, Stewart D, Chang D, et al. Genetic variations in the 46. Sambi BS, Hains MD, Waters CM, Connell MC, Willard FS, Kimple AJ, et al. regulator of G-protein signaling genes are associated with survival in late- The effect of RGS12 on PDGFbeta receptor signalling to p42/p44 mitogen stage non-small cell lung cancer. PLoS One 2011;6:e21120. activated protein kinase in mammalian cells. Cell Signal 2006;18:971–81. 36. Siderovski DP, Willard FS. The GAPs, GEFs, and GDIs of heterotrimeric G- 47. Liao Y, Hung MC. Physiological regulation of Akt activity and stability. Am protein alpha subunits. Int J Biol Sci 2005;1:51–66. J Transl Res 2010;2:19–42. 37. Bagnato A, Loizidou M, Pflug BR, Curwen J, Growcott J. Role of the 48. Ayala G, Wang D, Wulf G, Frolov A, Li R, Sowadski J, et al. The prolyl endothelin axis and its antagonists in the treatment of cancer. Br J isomerase Pin1 is a novel prognostic marker in human prostate cancer. Pharmacol 2011;163:220–33. Cancer Res 2003;63:6244–51.

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RGS12 Is a Novel Tumor-Suppressor Gene in African American Prostate Cancer That Represses AKT and MNX1 Expression

Yongquan Wang, Jianghua Wang, Li Zhang, et al.

Cancer Res Published OnlineFirst June 13, 2017.

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