Vol. 11, 2237–2243, March 15, 2005 Clinical Cancer Research 2237 Reg IV: A Promising Marker of Hormone Refractory Metastatic Prostate Cancer Zhennan Gu,2 Mark A. Rubin,4 Yu Yang,4 growing tumors that may not impact an individual’s natural life Samuel E. Deprimo,5 Hongjuan Zhao,5 span, although others are struck by rapidly progressive, metastatic tumors. Prostate-specific antigen screening is limited by a lack of Steven Horvath,1 James D. Brooks,5 4 2,3 specificity and an inability to predict which patients are at risk to Massimo Loda, and Robert E. Reiter develop hormone refractory metastatic disease. Recent studies Departments of 1Statistics and 2Urology, and the 3Molecular Biology advocating a lower prostate-specific antigen threshold for Institute, Geffen School of Medicine at University of California at diagnosis may increase the number of prostate cancer diagnoses Los Angeles, Los Angeles, California; 4Department of Pathology, Dana-Farber Cancer Institute, Harvard School of Medicine, Boston, and further complicate the identification of patients with indolent Massachusetts; and 5Department of Urology, Stanford University versus aggressive cancers (1). New serum and tissue markers that School of Medicine, Stanford, California correlate with clinical outcome or identify patients with potentially aggressive disease are urgently needed (2). Recent expression profiling studies suggest that expression ABSTRACT signatures for metastatic versus nonmetastatic tumors may reside The diagnosis and management of prostate cancer is in the primary tumor (2–4). Additional features that predispose hampered by the absence of markers capable of identifying tumors to metastasize to specific organs may also be present at patients with metastatic disease. In order to identify potential some frequency in the primary tumor (5). These recent new markers for prostate cancer, we compared gene observations suggest that novel markers of premetastatic or expression signatures of matched androgen-dependent and prehormone refractory prostate cancer may be identified in early hormone refractory prostate cancer xenografts. One candi- stage disease. These markers may also play a role in the biology date gene overexpressed in a hormone refractory xenograft of metastatic or hormone refractory prostate cancer progression. was homologous to the regenerating protein gene family, a Recent examples of genes present in primary tumors that group of secreted proteins expressed in the gastrointestinal correlate with outcome and play a role in the biology of prostate tract and overexpressed in inflammatory bowel disease and cancer progression include EZH2 and LIM kinase (6, 7). cancer. This gene, Reg IV, was confirmed to be differentially However, neither of these two genes is secreted. expressed in the LAPC-9 hormone refractory xenograft. In order to identify new candidate serum or tissue markers Consistent with its up-regulation in a hormone refractory of hormone refractory prostate cancer, we compared gene xenograft, it is expressed in several prostate tumors after expression profiles of paired hormone-dependent and hormone neoadjuvant hormone ablation therapy. As predicted by its refractory prostate cancer xenografts. The LAPC-9 xenograft sequence homology, it is secreted from transiently transfected was established from an osteoblastic bone metastasis and cells. It is also expressed strongly in a majority of hormone progresses from androgen dependence to independence follow- refractory metastases represented on two high-density tissue ing castration in immunodeficient mice (8). It has been used microarrays. In comparison, it is not expressed by any previously to identify candidate therapeutic targets in prostate f normal prostate specimens and only at low levels in f40% of cancer. Differentially expressed genes were validated and then primary tumors. These data support Reg IV as a candidate examined for sequence homology to secreted or cell surface marker for hormone refractory metastatic prostate cancer. proteins. We report here on the identification, characterization, and initial validation of one such candidate gene, Reg IV, a new INTRODUCTION member of the regenerating family of secreted C-lectin proteins Prostate cancer is the most common malignancy and the (9). Reg proteins are normally expressed in the gastrointestinal second leading cause of cancer-related death in American men. tract and are induced in inflammatory bowel disease and some Prostate cancer is a biologically and clinically heterogeneous gastrointestinal malignancies. Their pleiotropic functions include disease. A majority of men with this malignancy harbor slow- promoting tissue regeneration, proliferation, and resistance to apoptosis (10). We show that Reg IV encodes a secreted protein, which is not expressed in the normal prostate. Reg IV is expressed at low levels in a subset of primary tumors and is Received 2/24/04; revised 10/26/04; accepted 11/19/04. moderately or highly expressed in a majority of hormone Grant support: Department of Defense grant PC 001588 and American refractory and metastatic tumors. These results suggest that Reg Cancer Society grant (R. Reiter). IV may be a potential marker of prostate cancer metastasis The costs of publication of this article were defrayed in part by the or hormone refractory growth. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MATERIALS AND METHODS Requests for reprints: Robert E. Reiter, Department of Urology, 66-128 Microarray Analysis of Gene Expression. Tumor UCLA Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. Phone: 310-794-7224; Fax: 310-206-5343; samples from a matched pair of androgen-dependent and E-mail: [email protected]. -independent LAPC-9 xenografts were grown and prepared as D2005 American Association for Cancer Research. described previously (8). Total RNA was isolated by using Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. 2238 Reg IV in Prostate Cancer Ultraspec RNA isolation systems (Biotecx). mRNA was (9E10), and 20 mL of protein G-Sepharose CL-4B (Amersham) purified using Oligotex mRNA Midi Kit (Qiagen). Two for 2 hours at 4jC. Samples were washed and boiled in SDS micrograms of mRNA was reverse-transcribed, and cDNA sample buffer for 5 minutes and separated on 12.5% SDS- was then labeled with Cy-5. Labeled tumor cDNA was PAGE. The gel was treated with Amplify (Amersham) before combined with a Cy-3-labeled common reference RNA derived being dried and autoradiographed. from 11 different cell lines and hybridized to cDNA micro- Northern Blot Analysis of Gene Expression. RNAs arrays containing 22,648 elements representing 17,083 genes, were extracted as described above. Ten micrograms of RNA was as reported previously (11). The slides were scanned with a separated on a 1.2% agarose denaturing gel, transferred to GenePix microarray scanner (Axon Instruments) and were nitrocellulose filters, and hybridized with RT-PCR-prepared analyzed with Genepix software. Spots of insufficient quality DNA fragments of Reg IV (Genbank AI732541). Probes were were excluded from analysis by visual inspection. Data files labeled with a 32PdCTP by random priming using the random were entered into the Stanford Microarray Database, where spot primer labeling system (Amersham) and hybridization was intensity was correlated with gene identification. Only features carried out at 62jCin6Â SSC overnight, followed by washing with a signal intensity >50% above background in either Cy5 with 2Â SSC-0.1% SDS and 0.2Â SSC-0.1% SDS at 62jC. For or Cy3 channel and whose expression varied at least 4-fold multiple tissue Northern analysis, the hybridization was done as between the paired samples were retrieved from the Stanford described by the manufacturer (Clontech). Microarray Database. Detailed descriptions of array manufac- Case Selection for Tissue Microarray. In order to ture, hybridization protocols, and data analysis are available at evaluate Reg IV, we used a prostate cancer progression tissue http://cmgm.Stanford.EDU/pbrown. microarray. This tissue microarray is composed of benign Construction of myc-His-Tagged Reg IV Expression prostate tissue, localized prostate cancer, and hormone refractory Vector. The Reg IV coding sequence was subcloned into the metastatic prostate cancer. These cases came from well-fixed multiple cloning site of pcDNA3.1/myc-His expression vector radical prostatectomy specimens from the University of (Invitrogen) at the BamHI and EcoRI sites. The reading frame Michigan (Ann Arbor, MI), the University Hospital Ulm (Ulm, was confirmed by sequencing. Germany), and the rapid autopsy program from the University of RNA Probes and In situ Hybridization. A 399 bp Michigan Specialized Program of Research Excellence in DNA fragment from the 3V-untranslated region of Reg IV Prostate Cancer (13). All samples were collected with prior (Genbank AI732541) was inserted into the pCR2.1 vector Institutional Review Board approval at each respective institu- (Invitrogen) in both sense and antisense orientations under the tion. This tissue microarray was composed of classic acinar control of the T7 promoter. Plasmids were linearized and prostate cancers and areas demonstrating foamy gland features digoxigenin-labeled riboprobes were generated using the DIG from the same cases. Benign tissue samples were also placed in RNA Labeling