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

Author Manuscript Published OnlineFirst on June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0669 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RGS12 is a novel tumor suppressor gene in African American prostate cancer that represses AKT and MNX1 expression

Yongquan Wang*1,2, Jianghua Wang* 2, Li Zhang2,3, Omer Faruk Karatas#2, Longjiang Shao2 , Yiqun Zhang4, Patricia Castro2, Chad J. Creighton4,5 and Michael Ittmann2

1Department of Urology, Southwest Hospital, Third Military Medical University Chongqing, China; 2Dept. of Pathology & Immunology, Baylor College of Medicine and Michael E. DeBakey Dept. of Veterans Affairs Medical Center; 3Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China; 4Dan L. Duncan Cancer Comprehensive Cancer Center Division of Biostatistics; 5Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

*These authors contributed equally to this project as co-first authors

# Current address: Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum, Turkey.

Running title: RGS12 in African American prostate cancer

Keywords: prostate cancer, RGS12, MNX1, AKT, African American

Financial support: This work was supported grants from the Department of Defense Prostate Cancer Research Program (W81XWH-12-1-0046 MI); the National Cancer Institute supporting the Dan L. Duncan Cancer Center (P30 CA125123) Human Tissue Acquisition and Pathology and Genomic and RNA Profiling Shared Resources; the Prostate Cancer Foundation (MI) and by the use of the facilities of the Michael E. DeBakey VAMC.

Conflicts of interest: The authors declare no potential conflicts of interest

Word count: 4484

Figures: 7

Correspondence:

Michael Ittmann MD/PhD Department of Pathology and Immunology Baylor College of Medicine One Baylor Plaza Houston, TX 77030 Tele: (713) 798-6196 Fax: (713) 798-5838 E-mail: [email protected]

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0669 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

African American (AA) men exhibit a relatively high incidence and mortality due to prostate cancer (PCa) even after adjustment for socioeconomic factors, but the biological basis for this disparity is unclear. Here we identify a novel region on chromosome 4p16.3 that is lost selectively in AA PCa. The negative regulator of G-protein signaling RGS12 was defined as the target of 4p16.3 deletions, although it has not been implicated previously as a tumor suppressor gene. RGS12 transcript levels were relatively reduced in AA PCa and PCa cell lines showed decreased RGS12 expression relative to benign prostate epithelial cells. Notably, RGS12 exhibited potent tumor suppressor activity in PCa and prostate epithelial cell lines in vitro and in vivo. We found that RGS12 expression correlated negatively with the oncogene MNX1 and regulated its expression in vitro and in vivo. Further, MNX1 was regulated by AKT activity and

RGS12 expression decreased total and activated AKT levels. Our findings identify RGS12 is a candidate tumor suppressor gene in AA PCa which acts by decreasing expression of AKT and

MNX1, establishing a novel oncogenic axis in this disparate disease setting.

INTRODUCTION

African American (AA) men have a significantly higher incidence of prostate cancer

(PCa) compared to European American (EA) men(1) and are twice as likely to die from PCa compared to EA men. The biological basis for this difference in PCa mortality is unclear. Since

AA men account for a significant fraction of all PCa related deaths in in the US, it is important to understand the basis for this higher mortality in order to optimize prevention and treatment strategies for this higher risk group of men.

There have been a number of studies comparing PCa tissues from AA and EA men.

Several studies have compared gene expression in AA and EA PCa using large scale expression microarrays (2-5) including a study from our group (6). A number of studies have focused on a smaller set of preselected genes (7-9). All of these studies indicate that there is differential gene expression between AA and EA PCa. The TMPRSS2/ERG fusion gene is much less frequent in AA PCa based on studies of DNA, RNA and protein (8-16). Elevated SPINK1 expression appears to be more common in AA PCa (8,9,17-19). Among other genes upregulated in AA PCa, inflammatory genes are prominent (2,4,7). We have recently identified

MNX1 as an oncogene that is expressed at significantly higher levels in AA PCa compared to

EA PCa (6). We further demonstrated that MNX1 is regulated by AKT and androgen receptor activity and upregulates lipid synthesis, which has been linked to aggressive disease (20,21) and thus MNX1 may contribute to disease aggressiveness in AA PCa.

We have published (22) a study of allelic loss and gain in 20 AA PCas using Affymetrix

500k SNP arrays to define regions of recurrent copy number gain and loss in localized PCa and compared the pattern of copy number alterations (CNAs) to that of a similar cohort of EA men

(23). We found multiple cytobands with a statistically higher frequency of CNAs in our AA cohort over the EA cohort. The only unique CNA identified in this initial analysis that had not been previously linked to PCa was loss of chromosome 4p16.3.

We have now extended our original CNA studies to a new set of 40 highly tumor- enriched primary PCas and matched benign prostate tissues from AA men using high resolution

Affymetrix 6.0 SNP arrays and expression array analysis using RNAs from the same tissues.

We have confirmed the specific loss of 4p16.3 described previously (22) and identified a novel tumor suppressor gene, RGS12 at this locus that shows significantly decreased expression in

AA PCa but not EA EA PCa. Both in vitro and in vivo data show that RGS12 is a tumor suppressor gene, as would be predicted from its known ability to negatively regulate pro- oncogenic signal transduction. Furthermore, we have found that loss of RGS12 increases expression of MNX1 at least in part by regulating AKT protein levels. Our findings establish a novel oncogenic axis in AA PCa.

MATERIALS AND METHODS

Prostate and prostate cancer tissue. Tissue samples were obtained from the Human Tissue

Acquisition and Pathology Core of the Dan L. Duncan Cancer Center and were collected from fresh radical prostatectomy specimens after obtaining informed consent under a Baylor College of Medicine Institutional Review Board approved protocol and as such followed the principals of the Declaration of Helsinki and the Belmont Report. Cancer tissues include at least 70% tumor tissue and benign tissues were free of cancer on pathological examination. DNAs and RNAs were extracted using a Qiagen DNA/RNA mini kit following the manufacturer’s protocol.

Affymetrix 6.0 SNP and Agilent 60K expression arrays. DNAs from AA PCa tissues and matched benign tissue were analyzed using Affymetrix 6.0 SNP arrays by the Albert Einstein

College of Medicine Genomics Core. SNP array data were processed using the crlmm package in Bioconductor, with the preprocessing steps for copy number estimation as follows: (1) quantile normalization of the raw intensities (quantile normalizing the SNPs and nonpolymorphic markers separately), (2) genotyping, (3) for total copy number, translating the normalized

intensities to an estimate of raw copy number by adding the allele-specific summaries. For each of the 1M SNP probes, each tumor profile was centered on the paired normal, in order to generate tumor:normal ratios. Tumor:normal logged values were averaged by gene, and each profile was centered on the median of log ratios across all genes. For heat map presentation, gene-level tumor:normal values were further collapsed into cytobands. When combining datasets from multiple studies, values for each dataset were binned as gain or loss or no change, using a similar approach to that of our previous study (22). For the Lapointe dataset, the standard deviation (SD) of the tumor profile with the smallest SD across cytobands was used as the reference for defining gain or loss events within each cytoband; cytobands with average values greater than +3SD were called as gain, and cytoband values less than −3 SD were called as loss. Gene-level copy alterations for the Taylor dataset were previously binned in that study, with average cytoband log2 ratio>0.6 or <-0.6 being called here as gain or loss, respectively. For our own SNP array datasets (present study and Castro et al. (22)), a log2 of

0.2 was used as the cutoff (similar to that of Castro et al. (22)). Expression array analysis has been described previously (6). GEO accession number pending.

Quantitative real-time PCR (Q-RT-PCR). Gene expression levels were tested using quantitative real-time PCR (Q-RT-PCR) on an Applied Biosystem (StepOne, Lifetechnologies).

Total RNAs were extracted using the RNasy kit (Qiagen). cDNAs were synthesized as described previously. TagMan probes used are listed in Supplementary Table 1. Differences in mRNA levels were analyzed using the ΔΔCt method normalized to β-actin expression. Each measurement point was repeated at least in triplicate.

Cell culture. Human immortalized normal prostate epithelial cell line PNT1A and PCa cell lines

LNCaP, DU145 and PC3 were all maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). LAPC4 cells were cultured in RPMI-1640 medium with 10% FBS supplemented with 10nM R1881 (Sigma). VCaP and 293T cells were

maintained in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) with 10% FBS. All cell culture medium contained 1x Antibiotic-Antimycotic (Gibco). PNT1A cells were obtained from the European Type Culture Collection. PNT1A with myristoylated AKT and controls have been described previously (24). All other cell lines were obtained from the American Type Culture

Collection. Cell were obtained between 2001 and 2012, expanded, frozen and stored as stocks in liquid nitrogen. All cell lines are authenticated by STR analysis at MD Anderson Cancer

Center Characterized Cell Line Core Facility. Cells are tested monthly for mycoplasma contamination.

Stable knockdown of RGS12. LNCaP cells with stable knockdown of RGS12 were produced by utilizing RGS12-shRNAs in lentiviral vector pGFP-C-shLenti (Origene). Four unique human shRNAs (A-D) for RGS12 constructs in lentiviral GFP vector were purchased from Origene (Cat

# TL302015). Another 3 unique shRNA-RGS12 constructs (V3LHS_310594; 310595 and

310599) were purchased from the Baylor College of Medicine C-Bass core. Lentiviruses carrying these stable shRNAs were produced in 293T cells using Lenti-vpak Packaging kit

(Origene) following manufacture’s instruction. LNCaP and PNT1A cells were infected by these viruses and were selected with 0.5ug/ml puromycin (Sigma)

Plasmid construction and transfection. Primers used to amplify three human RGS12 isoforms (GenBank accession NM_198229, NM_002926, and NM_198227) are listed in Table

2. Three RGS12 isoforms were cloned into pcDNA3.1/ V5-His-TOPO vector (Invitrogen,

Carlsbad, CA, USA) containing CMV promoter. Constructs were sequenced and confirmed their accuracies before transfection into cells. The transfection was performed using Fugene 6 reagent (Promega, USA) and transfected cells were selected and maintained in media containing 200ug/ml G418.

Cell proliferation assay. 5X103 cells were plated in each well of 96-well plates. Proliferation was determined using the Cell Counting Kit-8 Cell Proliferation Assay kit (Dojindo Molecular

Technologies) as described by the manufacturer. The absorbance was read at 450 nm with

VERSAmax Tunable microplate reader.

Matrigel Invasion Assays. 2.5 × 104 cells were plated in the top chamber of Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). The cells on the apical side of each insert were then scraped off after 24h. The wells were washed with PBS, fixed with 100% methanol and stained with DAPI. After staining, membranes were removed from the insert and mounted on slides, and the invading cells were counted under the Nikon Eclipse TE2000-U microscope. Matrigel assays were performed in triplicate.

Soft agar growth assay. 6-well plates with 0.5% base agar layer mixed with 1Xculture media plus 10% FBS were prepared before the seeding of cells. PNT1A cells (5 × 104) with stable knockdown of RGS12 or vector controls were plated in 0.35% top agar layer each agar dishes.

Cell colonies were counted after incubation at 37°C in an incubator for three weeks and staining with 1mg/ml of idonitrotetrazolium chloride for 8h This experiment was repeated twice.

Western Blot. Total cellular protein lysate was prepared as described previously. Anti-β-actin was obtained from Sigma-Aldrich (dilution 1:5000). Anti-MNX1 was purchased from Origene

(Cat# TA337035) and used at a dilution of 1:3000. Anti-RGS12 was purchased from Santa Cruz

(sc-514173). Antibodies to total AKT and phospho-AKT-T308 and S473 were from Cell

Signaling. Western blotting procedures were described previously (6).

Mouse Xenograft Studies. All procedures were approved by the Baylor College of Medicine

Institutional Animal Use and Care Committee. Experiments were carried out on 8-10 week old male SCID mice. Tumor xenografts were established by subcutaneous injection over each flank in 50ul volume mixed with 50ul Matrigel (BD Bioscience). Tumors were harvested 8 weeks after inoculation and the tumor weights were recorded. Tumor tissues were snap frozen for further mRNA and protein expression studies.

Statistical analysis. Numerical values from two groups were compared by Mann-Whitney test, with p<.05 considered significant. For more than two groups, ANOVA was used followed by pairwise comparison to controls, which were considered significant if p<.05.

RESULTS

Copy number alterations in AA PCa. We have extended our original CNA studies of AA PCa

(22) to a new set of 40 highly tumor-enriched primary PCas and matched benign prostate tissues from AA men using high resolution Affymetrix 6.0 SNP arrays (906K SNPs). We combined this new data, our published data (Castro et al (22)) and the predominantly EA data sets of Lapointe et al (23) and Taylor et al (25) (total AA: n=89; EA: n=169) for analysis. Data organized by race is shown in Supplementary Figure 1. Cluster analysis of this data revealed that the vast majority of AA PCas clustered in two major groups (Figure 1A, right), indicating that

AA and EA PCa have different patterns of CNAs than EA PCa. The AA cluster to the far right shows a distinct pattern of CNAs. A smaller cluster of AA PCas clusters adjacent to the metastatic samples and has significant similarities to CNAs in these samples.

We then compared frequencies of CNAs at all cytobands between AA and EA patients after excluding metastatic samples and samples without data on race. A total of 32 cytobands showed significantly higher loss or gain (p<.01, one-sided Fisher’s exact test) in AA PCa when compared to EA PCa (Figure 1B and Supplementary Table 3). Of note, we confirmed the specific loss of 4p16.3 described previously (p<.001). It is well known that hereditary cancer loci often show somatic alterations as well, so it is noteworthy that 6 of the 32 cytobands we identified have been implicated in hereditary AA PCa by linkage analysis, including 8q24(26-

28),11q13(28), 12q24(29), 14q32(30), 17p11(31) and 17q21(32). We then carried out a cluster analysis of the cytobands that were significantly different between AA and EA PCa. Most of the

AA PCas cluster into four groups in this analysis as indicated in Figure 1B. Group A has no losses in these cytobands. Group B shows multiple gains and some losses while Group D

shows more focal gains. Group C shows loss in multiple cytobands preferentially lost in AA

PCa. Most of the AA PCa cases with loss of 4p16.3 cluster in this group. Of note, Group C clusters adjacent to the metastatic PCas, which are predominantly derived from EA men, and the metastatic cases also show loss at 4p16.3, indicating that loss of 4p16.3 is likely to be associated with aggressive disease in primary PCa.

Identification of a novel tumor suppressor in AA PCa. We have also carried out expression array analysis using RNAs from the same cancers used for CNA analysis (6). We identified a total of 4341 probes altered in PCa Vs benign (p<.01) in AA PCa and the overall quality of the data has been confirmed as described previously (6). To identify the potential tumor suppressor on 4p16.3 that was preferentially lost in AA PCa, we systematically examined expression of genes on 4p16.3 in AA PCa in the expression array data. We found that two genes that are adjacent on 4p16.3 (HTT and RGS12) which both show downregulation of mRNA in AA PCas.

Detailed examination of deletions showed that losses are more concentrated in RGS12 and

CNAs correlate with expression levels for RGS12 (r=.59, p=.016) but not HTT (Supplementary

Table 4).

RGS12 has three alternatively spliced protein coding isoforms as shown in

Supplementary Figure 2. The proteins encoded by isoforms 1 and 2 are almost identical and encode full length proteins. Isoform 3 lacks the amino terminal PTB and PDZ domains. Because of the extensive overlap of the three isoforms we were not able to measure the mRNA levels of all 3 isoforms individually. We were able to analyze total RGS12 using a probe from the common region of all three isoforms, total isoforms 1+2, total isoforms1+3 and isoform 3 alone.

Q-RT-PCR analysis using a TaqMan probe which detects all three RGS12 isoforms showed significantly decreased RGS12 expression in AA PCa (Fig 2A; p<.001, Mann-Whitney). Analysis using primers detecting both isoforms 1 + 2 or isoform 3 only also showed significantly decreased expression levels in AA PCas compared to benign prostate tissues (Fig 2B and 2C;

both p<.001; Mann-Whitney). However, no loss was seen in EA PCa using primers detecting all three isoforms (Figure 2D). Scatter plots of this data are shown in Supplementary Figure 3.

Overall the CNA and expression analysis data show that there is loss of RGS12 alleles and/or gene expression in AA PCa that is not seen in EA PCa..

We then compared expression of RGS12 in PNT1A cells, an immortalized normal prostate epithelial cell line to LNCaP, VCaP, LAPC4, DU145 and PC3 PCa cell lines. As shown in Figure 2E, all 5 PCa cell lines showed decreased RGS12 relative to PNT1A. The differences were highly statistically significant (p<.001; ANOVA) in all PCa cell line except DU145 where the decrease was relatively small (p<.05, ANOVA). Analysis of isoform expression in the same cell lines revealed that isoform 1+2 and isoform 1+3 expression was decreased in all PCa cell lines except DU145 (Fig 2F). Isoform 3 had relatively low expression in PNT1A compared to isoforms

1 and 2 and while there was a trend for lower isoform 3 expression in the PCa cell lines, this was not statistically significant.

Biological effects of RGS12 knockdown in vitro. In order to study RGS12’s potential tumor suppressor function, we knocked down RGS12 expression in PNT1A cells with three different

RGS12-shRNAs using a lentiviral vector (pGFP-C-shLenti). After stable selection, knockdown of

RGS12 in each group was confirmed using a TaqMan probe which detects all three isoforms

(Figure 3A). Knockdown of RGS12 significantly increased cell proliferation (p<.001 at 4 days,

ANOVA) in all three groups compared to scrambled control (Fig 3B). We also tested colony formation in soft agar, a hallmark of transformation. PNT1A cells are not fully transformed and form only rare small colonies in this assay. As shown in Figure 3C, knockdown of RGS12 markedly increased colony formation in soft agar (p<.001, ANOVA). The overall colony sizes in

RGS12 knockdown lines were also much larger compared to controls (Fig 3D). As a positive control, we used PNT1A cells expressing Huntington interacting protein-1 which formed colonies in soft agar (data not shown) as we have shown previously (33). We also knocked

down RGS12 in LNCaP using four different shRNAs (Fig 3E). As in PNT1A, proliferation was significantly increased with all four shRNAs (Fig 3F).

We then cloned all three major isoforms of RGS12 from LNCaP cells into pCDH-CMV-

MCS-EF1-Neo lentiviral vector. As seen in Figure 4A, V5 antibody detected all three bands with correct estimated sizes using lysate from 293T cells following transient transfection of the three isoforms. Using an anti-RGS12 antibody, we were able to confirm the overexpression of different RGS12 isoforms, although based on the relative band intensity in anti-V5 and anti-

RGS12 Westerns the affinity of the anti-RGS12 antibody for isoform 3 was higher than for isoforms 1 and 2. After stable transfection of all three isoforms into LNCaP cells, we confirmed significantly increased RGS12 expression at RNA level in LNCaP cells (Fig 4B). Overexpression of each isoform significantly decreased cell proliferation (Fig 4C). Isoform 3 had a lower effect on growth compared with the other two isoforms (p<.05 Vs Iso1 or Iso2, ANOVA).

Overexpression of each isoform dramatically inhibited cell invasion (Fig 4D), and again isoform

3 showed a weaker effect on invasion (p<.01 Vs Iso1 or Iso2, ANOVA)

Biological effects of RGS12 expression in vivo. To evaluate tumor suppressive activities in vivo we carried out xenograft experiments in SCID mice. In the first experiment, two groups of mice (10 mice /group) were injected subcutaneously with LNCaP with vector or LNCaP-shD respectively. Tumor growth was monitored twice weekly. At end of 5 weeks, mice were euthanized and primary tumors were excised, weighed, and a portion of the tumor was frozen in liquid nitrogen for molecular analysis and another portion fixed and paraffin-embedded. The difference of tumor weight between shD group and controls was statistically significant (Fig 5A,

P<0.05, Mann-Whitney), with higher tumor weights in tumors with RGS12 knockdown. In the second experiment, we used LNCaP cells overexpressing RGS12 isoform 2 or 3 or control cells with scrambled vector. Tumor weights were significantly decreased in both isoform expressing

groups compared to control cells (Fig 5B, P<.001, Mann-Whitney). Surprisingly, isoform 3 tumors were smaller than isoform 2 expressing tumors given that isoform 3 appeared to be less tumor suppressive than isoform 2 in vitro. Analysis of RGS12 mRNA expression in the final tumors revealed that isoform 3 tumors had approximately 4-fold higher levels of RGS12 compared to isoform 2 tumors (data not shown). This implies there may have been preferential growth of tumor cells with lower RGS12 knockdown during the in vivo growth, and this effect was more profound in the isoform 2 expressing cells than the isoform 3 expressing cells. Finally, since PNT1A cells with RGS12 knockdown formed colonies in soft agar, we injected PNT1A- shD or shE cells or control PNT1A cells into mice. After 8 weeks, there were no tumors found in the PNT1A control group while obvious tumor masses were seen in both groups with RGS12 knockdown. The average weight of tumors collected were 68 mg and 84 mg in the shD and shE groups, respectively (Fig 5C). To confirm that the tumors were from PNT1A cells, we used SV40

T-antigen immunohistochemistry, since PNT1A cells were originally immortalized with SV40 large T-antigen. As shown in Figure 5D, tumor cells were positive for SV40 T-antigen. Overall our data shows that RGS12 is tumor suppressor gene in vivo for PCa and prostate epithelial cells.

RGS12 represses expression of MNX1, an AKT regulated oncogene. We have recently shown that MNX1 is an oncogenic transcription factor whose expression is preferentially increased in AA PCa. Examination of the gene expression data in our AA PCas (6) revealed a significant negative correlation between MNX1 and RGS12 mRNA expression in cancer tissues

(-.278; p=.028, Spearman). We saw a similar negative correlation (-.230; p=.01, Spearman) in the dataset of Grasso et al (34).This data is from a predominantly EA cohort but has a mixture of localized (59 cases) and metastatic PCa (35 cases), unlike our data which is all from localized disease. These correlations are shown in Supplementary Figure 4. We hypothesized that RGS12 may repress MNX1 expression.

We examined expression of MNX1 in the LNCaP cell lines with overexpression of

RGS12 isoforms 1-3. As seen in Figure 6A, MNX1 protein expression was completely lost in isoform 1 expressing cells and markedly diminished in isoform 2 and 3 expressing cells.

Conversely, knockdown of RGS12 markedly increased MNX1 expression in LNCaP cells in vitro

(Fig 6B). Similar increased levels of MNX1 protein were seen in xenograft tumors from mice with RGS12 knockdown (Fig 6C). Examination of MNX1 in LNCaP xenografts expressing

RGS12 isoform 3 or vector controls significant downregulation of MNX1 protein (Fig 6D). Thus

RGS12 significantly decreases MNX1 protein expression.

We have shown previously using inhibitors of the PI3K (LY294002) and AKT (AZD5363) that MNX1 is strongly regulated by AKT activity (6). We have now confirmed this observation using PNT1A cells expressing myristoylated AKT. As shown in Figure 7A these cells show increased levels of total and phosphorylated AKT compared to vector controls and also show increased MNX1 protein levels. We then examined the impact of RGS12 expression on AKT activity. As shown in Figure 7B, isoform 1 almost completely abolishes AKT protein expression as well as expression of phosphorylated AKT. Expression of isoforms 2 and 3 resulted in lesser decreases in total and phosphorylated AKT. Knockdown of RGS12 in PNT1A cells showed increased in S473 phosphorylated AKT (Supplementary Figure 5). Of note, PNT1A are PTEN wild type.

Examination of AKT mRNA in cells overexpressing RGS12 showed increased mRNA that was statistically significant for isoforms 1 and 2 (Fig 7C). This strongly suggests that

RGS12 regulates AKT post-transcriptionally, with a feedback upregulation of AKT mRNA.

Consistent with this, we observed a positive correlation between RGS12 and AKT mRNA levels in our expression microarray data (.338, p=.009) (Supplementary Figure 6).

DISCUSSION

In this report, we have shown that RGS12 is preferentially deleted in AA compared to EA

PCa and there is significantly lower RGS12 mRNA expression in PCa compared to benign tissues in AA but not EA PCa. We observed a significant correlation of genomic deletion and decreased expression (r=.59, p=.016) in AA PCa, but at this level of correlation it is likely that other factors may also impact RGS12 mRNA expression in AA PCa in addition to genomic deletion. Such factors will require further studies to elucidate.

Our in vitro and in vivo data shows that RGS12 is a tumor suppressor gene. Of note, decreased RGS12 is by itself capable of fully transforming immortalized normal prostate epithelial cells such that they form large colonies in soft agar and tumors in SCID mice, indicating that it has a strong tumor suppressive effect in this context. It can also impact tumorigenesis and transformation related cellular phenotypes in vitro and in vivo in fully transformed PCa cells.

RGS12 has not been previously implicated as a tumor suppressor gene. A SNP in

RGS12 has been shown to be associated with overall survival in lung cancer(35) but the mechanism for this association is unknown. RGS12 is a negative regulator of G-protein signaling which 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 pathogenesis of PCa (37-42) although the exact targets of RGS12 are not clear. RGS12 has been shown to interact with the IL-8 receptor(43). It also contains a phospho-tyrosine binding domain and has been shown to interact with MAPK/ERK and PI-3-kinase signaling in various contexts (44-46) and thus may act as link between G-protein coupled signaling and other signaling pathways(36).

We have shown previously that MNX1 is an oncogenic transcription factor whose expression is markedly increased in AA PCa and to a much lesser extent in EA PCa(6).

Remarkably, examination of gene expression data in our AA PCas revealed a significant negative correlation between MNX1 and RGS12 mRNA expression in AA PCa tissues. Both knockdown and overexpression studies confirm that RGS12 can strongly inhibit expression of

MNX1. AKT activity strongly regulates MNX1 expression based on our published data (6) and analysis of PNT1A cells expressing myristoylated AKT confirms this observation. Our data indicates that RGS12 significantly negatively regulates AKT protein levels and activity. It should be noted that levels of phosphorylated AKT were roughly proportional to AKT protein levels, indicating that activation of 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 post-transcriptional since AKT mRNA is actually increased by RGS12 overexpression.

The regulation of AKT protein levels has not been as intensively 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 post-transcriptional mechanisms potentially impacting AKT protein levels. AKT can be phosphorylated at threonine-450 and this phosphorylation impacts 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 PCa and is associated with aggressive disease (48). Interactions with heat shock proteins can also increase

AKT stability. On the other hand, ubiquitin-mediated proteolysis or degradation by caspases of

AKT has also been described(47). Additional studies are needed to understand the mechanism by which AKT protein levels are controlled by RGS12.

Our data indicates that decreased RGS12 enhances transformed phenotypes at least in part via increasing expression of MNX1, establishing a novel oncogenic axis in AA PCa. MNX1 enhances lipid synthesis, which has been shown to be associated with disease aggressiveness

(20,21). The increased MNX1 resulting 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 impact other AKT targets as well. Whether decreased RGS12 also impacts 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 PCa.

Our current and previous copy number alteration studies in AA PCa have shown that there are significant quantitative and qualitative differences in CNAs between AA and EA PCa.

Of the 32 cytobands with CNAs identified as being more common in AA PCa, 6 coincide with a region linked to familial AA PCA. However, the majority of CNAs we have identified have not been previously linked to AA PCa. While we have focused on 4p16.3 in these studies, other areas of CNA that are more frequently present in AA PCa identified in our studies may also harbor novel tumor suppressors or oncogenes relevant to AA PCa. Future studies will hopefully identify additional genes that impact initiation and/or progression in AA PCa and provide new insights into the optimal strategies for prevention and treatment of PCa in AA men.

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FIGURE LEGENDS

Figure 1. Copy number alteration in African American prostate cancer

A. Cluster analysis of copy number alterations in AA and EA PCa. 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 while 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 PCa identified using SNP arrays of

DNAs from 40 highly tumor enriched tissues and matched benign tissues, our prior analysis of

20 AA PCas (Castro) and two primarily EA datasets (Taylor and Lapointe). Cytobands that were more commonly altered in AA PCa (p<.01, one-sided Fisher’s exact test) were identified. Yellow is gain and blue is loss. Most of the AA PCas 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 while Group D shows more focal gains. Note that both group B and C cluster adjacent to blocks of metastatic cancers. Losses at 4p16.3 were concentrated in Group C.

Figure 2. RGS12 expression in prostate cancer.

A-C. Relative RGS12 mRNA expression in by Q-RT-PCR in benign prostate and PCa from AA men. A. All isoforms B. Isoforms 1 and 2. C. Isoform 3. Mean +/- standard deviation (SD) is shown. *** p<.001, Mann-Whitney. D. Relative RGS12 mRNA expression in by Q-RT-PCR in benign prostate and PCa from EA men, all isoforms. E. Relative RGS12 expression (all isoforms) in PCa cell lines and the benign prostate epithelial cell line PNT1A. Mean +/- SD; * p<.05; *** p<.001; ANOVA pairwise comparisons versus PNT1A. F. RGS12 expression using

probes recognizing specific isoforms 1 and 2, 1 and 3 or 3 only are shown. Means +/- SD; * p<.05; *** p<.001; ANOVA pairwise comparisons versus same probe in PNT1A

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<.05; ** p<.001; *** p<.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<.001; ANOVA.

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 since 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<.001; ANOVA pairwise versus vector control.

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<.05, Mann Whitney. B. Final tumor weights LNCaP vector controls and LNCaP with expressing RGS12 isoform 2 or isoform 3. Mean +/- SD. *** p<.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.

Figure 6. RGS12 represses MNX1 expression

A. Western blot with anti-MNX1 antibody of lysates from LNCaP cells and cell lines expressing

RGS12 isoforms 1-3 or vector control. Actin is 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 is 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.

Figure 7. RGS12 represses AKT protein expression. A. Western blots of cell lysates from

PNT1A cells expressing myristoylated AKT (m-AKT) or vector controls (Vec) for MNX1, phosphorylated AKT (S473 and T308) or total AKT. Actin is a loading control. B. Western blots of lysates from LNCaP cell lines expressing RGS12 isoforms 1-3, control LNCaP and vector controls for MNX1, total AKT and phosphorylated AKT (T308). Actin is a loading control. C. AKT mRNA in LNCaP cells expressing RGS12 isoforms 1-3 and vector controls. Mean +/-SD. * p<.05, ANOVA.

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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