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

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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. Ó2017 AACR. Introduction expression in AA and EA prostate cancer using large-scale expres- sion 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 Ó2017 American Association for Cancer Research. the specific loss of 4p16.3 described previously (22) and www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0669 Wang et al. 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.
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