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By Linda L. Xu, MD, and Shiv Srivastava, PhD
Prostate cancer (cap) is the most common malignancy and the second leading cause of cancer mortality in American men.1 Outward symptoms are not always apparent; when untreated, CaP may spread, or metastasize, to other parts of the body or vital organs. When this happens, the patient has fewer treatment options than he would have if the disease had been discovered earlier.
Serum prostate-specific antigen (PSA) and other diagnostic tools have been very successful in the early detection of CaP.1 However, the wide spectrum of biologic behavior exhibited by prostatic neoplasms poses a difficult problem in predicting the clinical course for the individual patient.2 Traditional prognostic markers such as grade, clinical stage, and pretreatment PSA have limited prognostic value for individual men.3 Molecular studies have shown a significant heterogeneity between multiple cancer foci present in a cancerous prostate gland. These studies also have documented that metastatic lesions can arise from cancer foci other than dominant tumors. Approximately 50-60% of patients treated with radical prostatectomy for localized CaP have microscopic disease that is not organ-confined, and a significant portion of these patients relapse.4 Therefore, identification and characterization of genetic alterations defining CaP onset and progression are crucial to understanding the biology and clinical course of disease.
PSA has been used clinically as a biomarker for CaP diagnosis and post-treatment follow-up due to its specific expression in prostate epithelial cells. Over the past 10 years, the PSA test has revolutionized the early detection of CaP; organ-confined disease can be cured effectively by surgical intervention or radiation treatment. Since the introduction of the PSA test, there has been a sharp decline in the incidence of metastatic CaP. However, the PSA blood test is not always entirely accurate, and it is not CaP-specific. PSA testing may identify men with CaP, but often PSA is elevated in men with benign prostate hyperplasia, prostatitis (inflammation of the prostate), and other non-malignant, non-life threatening prostate disorders. Current figures on this common diagnostic test show that about 25% of men with CaP will have normal PSA levels and more than one-half of men with higher PSA levels may be cancer-free.
Discovery of Prostate-Specific Genes
The discovery of additional prostate-specific genes has resulted in enthusiasm for evaluating their potential utility in the diagnosis and disease progression of CaP. HK2, a member of kallikrein gene family, currently is being evaluated for its role in CaP.5 Prostate-specific membrane antigen (PSMA) is a membrane-bound glycoprotein that is expressed in prostate and few other tissues. Expression of PSMA is increased in CaP, particularly in hormone-refractory disease. PSMA has been exploited as a marker for tumor detection and treatment by immunoscintiscanning with the 111indium-labeled, anti-PSMA monoclonal antibody 7E11.C5. Increased concentrations of 7E11.C5-reactive antigen are present in the serum of CaP patients compared with healthy individuals. Also, hematogenous circulating CaP cells are detectable with reverse transcriptase-polymerase chain reaction analysis.6
Current strategies for defining CaP-specific genetic alterations include positional cloning of candidate genes from chromosome loci frequently altered in CaP and comparison of the global gene expression profiles in cancer cells and corresponding normal cells by differential display (DD), serial analysis of gene expression (SAGE), and cDNA microarrays.7-9 The database of cDNA sequence libraries from defined tissues or cells also have provided impetus for identifying unique expression patterns in specific target tissues. These gene discovery approaches recently have led to the identification of several new prostate-specific/abundant genes, such as NKX3.1, prostase, prostate stem cell antigen (PSCA), TMPRSS2, STEAP, PDEF, PART-1, HOXB13, DD3, PCGEM1, PMEPA1, and PSGR, all of which exhibit diverse characteristics.10-20 Of special interest is the identification of prostate-specific expression markers, which not only are prostate-specific but also show elevated expression in CaP. Furthermore, several laboratories, including ours, are addressing the potential utility of new prostate-specific molecules as biomarkers of CaP onset and progression and as cancer vaccine targets.
Our laboratory has employed DD, SAGE, DNA microarrays, and electronic subtraction of cDNA sequence libraries to study CaP-associated gene expression alterations.19-21 These techniques led us to identify new prostate-specific genes that are overexpressed in CaP, such as PCGEM1 and PSGR, novel androgen-
regulated prostate-specific or abundant genes, such as PMEPA1, and other promising candidates.19-22 Here we briefly review our first observations on isolation and characterization of PSGR, a seven-transmembrane G-protein coupled receptor.21
PSGR was identified as a prostate tissue-specific cDNA during a search of the expressed sequence tag database at Human Genome Sciences. Analysis of the 1,474 bp PSGR cDNA sequence revealed an open reading frame (ORF) of 963 bp nucleotides encoding a 320 amino acid protein with a predicted molecular mass of 35.4 kDa. The PSGR ORF revealed intriguing homology (~ 50% identity and ~ 70% similarity) to the G-protein coupled odorant receptor (OR) family. A protein motif search using ProfileScan indicated the existence of seven transmembrane domains between amino acid residues 22 and 293 that are characteristic of G-protein coupled receptors. ORs belong to a superfamily of G-protein coupled receptors (GPCRs) that are transmembrane proteins mediating cellular responses to diverse extracellular stimuli, including light, neurotransmitters, hormones, and odorants.23 GPCRs are the largest gene family known to exist in a given animal genome. Through selective ligand binding, the GPCRs discriminate between multiple signals. GPCRs amplify and transduce the information inherent in ligand binding to the cell interior by interacting with the heterotrimeric G-proteins. The ligand-bound GPCR activates the G-protein complex, which in turn modulates a number of effector proteins such as adenylyl cyclase, phospholipase C-beta, G-protein-gated calcium and potassium channels, and membrane proximal components of the MAP kinase pathway. ORs are localized in nasal epithelium and are highly selective in expression. However, OR-like genes have been detected in testis, and their functions are not well understood. It has been suggested that OR-like genes may serve a chemosensory role in sperm chemotaxis during fertilization. Prostate-specific expression of PSGR, suggests an as yet undiscovered function in the prostate. Also of note is the overexpression of PSGR in about two-thirds of CaP specimens analyzed.
The distribution of PSGR mRNA in 50 different normal human adult and fetal tissues examined by Northern blot and slot blot analyses showed that PSGR expression was detected only in prostate tissue. In situ RNA hybridization analysis of PSGR expression in prostate tissues revealed that PSGR expression was localized predominantly to epithelial cells of the gland. Comparison of PSGR expression in normal and tumor tissues by laser capture microdissection-derived normal/tumor cell RNAs, using semiquantitative or real time PCR assays and RNA in situ hybridization, revealed overexpression of PSGR in 62% (32/52) of the tumor specimens.
We have demonstrated that prostatic epithelial cells restrict expression of PSGR and tumor cells exhibit significantly increased expression of this putative seven-transmembrane G-protein coupled receptor. However, it is not yet clear how PSGR overexpression may play a role in the process of tumorigenesis. G-protein coupled receptor may play important roles in cell signaling and cell proliferation. Therefore, future experiments will focus on biologic function of PSGR in tumorigenesis of CaP and its potential utility as a CaP biomarker. Membrane localization of PSGR makes PSGR protein an attractive target for immunotherapy approaches and antibody-based imaging of metastatic CaP. It also will be very important to determine the signals transduced by PSGR in the prostate gland and future studies will address these issues. Using the well-studied prostate-specific gene (PSA or PSMA) paradigm, further studies of PSGR, focusing on basic research and preclinical models, hold promise in evaluating the utility of this novel prostate-specific membrane protein in CaP diagnosis, prognosis, and therapy. (Dr. Xu is Staff Scientist and Dr. Srivastava is Professor and Scientific Director, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Rockville, MD.)
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