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] 1 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. 2 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. 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. 3 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. 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 4 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. 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).