A Promising Marker of Hormone Refractory Metastatic Prostate Cancer

$A Promising Marker of Hormone Refractory Metastatic Prostate Cancer$

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

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 Kit (Roche Applied Science). Automated in situ the tissue microarray to serve as a negative control. A second hybridization was done on the Discovery System (Ventana array containing predominantly metastatic cases was also stained Medical Systems, Tucson, AZ). After deparaffinization, slides and scored. were soaked in 2Â SSC for 5 minutes and digested with Scoring of Reg IV Expression. Reg IV expression was proteinase K (Life Technologies, at a final concentration of determined using a validated scoring method (7, 14–16) where 10 Ag/mL) for 30 minutes at 37jC. Sense and antisense staining was evaluated for intensity. Benign epithelial glands and riboprobes were diluted at 1:100 (1 Ag of probe/mL) in prostate cancer cells were scored for Reg IV staining intensity on hybridization solution (50% deionized formamide, 10% a four-tiered system ranging from negative to strong expression. polyethylene glycol, 0.3 mol/L NaCl, 10 mmol Tris (pH 8.0), A score of 1 was negative, a score of 2 was considered low 1 mmol EDTA, Denhardt’s solution 1Â, yeast tRNA 500 Ag/mL, expression, a score of 3 indicated moderate expression, and a 50 mmol DTT). The hybridization was done for 6 hours at score of 4 correlated with strong expression. Slides were read 65jC with 100 AL of hybridization solution. After hybridiza- independently by two pathologists (M. Rubin and M. Loda) with tion, slides were washed twice at 70jC for 6 minutes in 1.0Â >90% interobserver agreement. SSC. The hybridization was followed by a 30-minute Construction and Production of Lentivirus Expressing incubation with a biotinylated anti-digoxigenin (Sigma Bio- Reg IV-Myc.His. A Myc.His-tagged Reg IV construct was Sciences, St. Louis, MO), followed by alkaline phosphatase– PCR-amplified from a pcDNA Reg IV-myc.his vector and conjugated streptavidin for 16 minutes. Visualization procedure inserted into the lentiviral vector chemokine/chemokine was done in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl receptor through restriction sites of EcoRI and NheI (17). phosphate (Ventana mapBlue) for 5 hours and finally in Lentivirus stocks were generated by calcium phosphate hematoxylin counterstain. mediated transfection of 293T cells. The titer of the virus Recombinant Reg IV Expression and Immunoprecipi- was checked with 293T cells using chemokine/chemokine tation. 293T cells were transiently transfected with a myc-his receptor-enhanced green fluorescent protein as a positive tagged Reg IV expression vector by calcium phosphate control and indicator. precipitation for 48 hours. Cell labeling and immunoprecipita- LNCaP and LAPC-9 Prostate Xenograft Models. tion was done as described (12). Briefly, cells were labeled LNCaP or LNCaP-Reg IV.myc.his cells (5 Â 106) were mixed with 500 ACi of trans-35S label (ICN) in methionine and lysine- with an equal volume of Matrigel and inoculated into severe free DMEM (Invitrogen) containing 5% of dialyzed fetal combined immunodeficiency mice s.c. In the case of LAPC-9, bovine serum for 4 hours. Cell lysates and conditioned medium explanted tumor is digested with Pronase and cultured in were incubated with 3 Ag of anti-myc monoclonal antibody 10% fetal bovine serum-RPMI 1640 for 16 hours. Cells are then

transduced by chemokine/chemokine receptor lentivirus alone or RESULTS lentivirus RegIV.myc.mis at a multiplicity of infection of 5 for Cloning and Characterization of Reg IV. The LAPC-9 2 hours. Cells are then washed with culture media, mixed with xenograft was established from metastatic prostate tumors and Matrigel, and inoculated back to severe combined immunode- progresses in vivo from androgen dependence to independence. ficiency mice (1 Â 106 cells/mice). In order to identify novel candidate markers of prostate cancer ELISA Detection of Serum RegIV.myc.his. Twenty-five progression, RNAs from paired androgen-dependent and andro- microliters of mouse serum was mixed with 75 mL of PBS and gen-independent LAPC-9 tumors were labeled and hybridized to incubated in Ni-NTA HisSorb strips (Qiagen) for 3 hours. The 24,000 spot cDNA arrays with common reference RNA. Two strips were washed with PBS-0.1% Tween 20 and then incubated hundred and four clones representing 101 named genes and 59 with 1:5,000 diluted anti-Myc monoclonal antibody (Invitrogen) expressed sequences tags showed expression variation of at least for 45 minutes. The 1:10,000 diluted anti-mouse IgG antibodies 4-fold between the androgen-dependent and androgen-indepen- conjugated with alkaline phosphatase (Promega) were then dent samples. Out of the 101 named genes, 75 have been incubated for 45 minutes. After washing, the color was characterized functionally to some degree and most can be developed using one-step nitroblue tetrazolium/5-bromo- categorized into six biological processes according to Gene 4-chloro-3-indolyl phosphate (Pierce) for 20 minutes and read Ontology annotations: cell-cell signaling/signal transduction, cell at 450 nm. adhesion/motility, structural molecule/cytoskeleton, immune Statistics. The Pearson v2 statistic was used to test response, cell proliferation/cell cycle, and metabolism (Table 1). whether the rows (e.g., tumor type) and columns (e.g., Reg Eleven androgen-responsive genes, including well-known an- expression) of a table were independent. To test for differences drogen targets such as klk3 (prostate-specific antigen), were of Reg expression across different tumor types, we also used the identified by comparison to the expression profiles of 567 Kruskal-Wallis test, which is a nonparametric multigroup androgen-regulated transcripts we had identified previously comparison tests. Statistical analyses were done using the freely (Table 1). With one exception (WWP1), all of the genes normally available R software (http://www.r-project.org). up-regulated by androgens showed decreased expression in the

Table 1 Genes highly differentially expressed in LAPC-9 androgen-dependent and androgen-independent tumors Up-regulated in androgen-independent tumors Down-regulated in androgen-independent tumors Cell-cell signaling/signal transduction WWP1 (19.2), APOB (10.9), CX3CR1*(9.5), AGTR1 (13.7), ADRB1 (13.1), LIM (9.5), GDF1 (7.9), RGS1*(6.8), EDNRB (6.3), MME (5.8) FGF12 (5.0) ABCA5*(4.7) RGS5 (5.9) RAB32 (5.8) ANXA1*(4.8) Cell adhesion/junction/motility CDH2 (24.3), CSPG2 (11.2) CX3CR1*(9.5) DSC (19.1) PCDH16 (6.8) CLDN8*(6.5) ANXA1*(4.8) Structure molecule/cytoskeleton CAV1 (11.8), NEFL (9.6), SLIT1 (5.8), ANK3 (9.2), CLDN8*(6.5) MME*(5.8) BIN3 (5.6) ABLIM1 (5.2) SGCD (4.8) PTPN4 (5.4) Immune response APOE (11.0), RGS1*(6.8), IGLL1 (5.4) ULBP2 (14.7), DPP4 (14.2), TRG@(13.3), HLA-C (5.0) ANXA1*(4.8) IL1R1 (10.4) SEMA3C*(5.4) PLA2G7 (4.9) Cell proliferation/cell cycle PTN (23.3), DNAJA2 (14.7) SEMA3C*(5.4) ABCA5*(4.7) Electron transport CYP4B1 (8.2) MAOA (8.7) STEAP2 (5.5) CYP1B (4.7) Other functions SLC7A5 (15.5), GSTM2 (15.1), RBP5 (13.4), KLK4 (26.2), RTN4R (15.9), KLK3 (10.2), LDHB (10.7), SERPING1 (10.4), HOOK1 (8.0), PPP3R2 (10.1), CXADR (8.3), LPL (6.2), BDNF (8.0), SCUBE2 (7.5), COL2A1 (6.8), NUCB2 (5.6), TMEPA1 (5.5), PTPN4 (5.4), PRESS16 (6.4), MAN1A1 (6.0), NRXN1 (5.6), KLK2 (5.2), CPNE1 (5.2), CA1 (5.2) GSTM4 (5.4), SIAT9 (5.3), PROX1 (5.0) TRIM29 (5.1) SLC36A1 (5.0) SOX17 (5.0) CUGBP2 (4.7) Genes with unknown function REG4 (19.5), NSE1 (13.9), LAMA3 (12.8), SLC15A2 (15.3), NEDD4L (10.9), EPS8L3 (9.7), SFRP1 (9.4), SPG2 (8.0), MSP (6.2), BMPR1B (9.3), BCMP11 (8.4), USP43 (8.0), BGN (5.9), NAALAD2 (5.5) SATB1 (5.1) DNAH11 (6.6), CDC6 (6.3), INPP4B (6.2), SIAH1 (5.1) PSK-1 (5.7), ABCC4 (5.6), PTK2 (5.6), MBNL1 (5.4), ChGn (5.0), CYP4Z1 (4.9) ATSV (4.7) MGC4544 (4.7) Androgen responsive WWP1 (19.2) KLK4 (26.2), NEDD4L (10.9), IL1R1 (10.4), KLK3 (10.2), CDC6 (6.3), ABCC4 (5.6), NUCB2 (5.6), TMEPA1 (5.5) KLK2 (5.2) ABCA5 (4.7)

androgen-independent tumor growing in the castrated animal, androgen-dependent ones (data not shown). These results show confirming that androgens modulate their expression in vivo. that two expressed sequences tags related to the Reg gene family We focused our attention on uncharacterized genes that are reproducibly up-regulated during androgen-independent were differentially expressed between the androgen-dependent progression of LAPC-9. and androgen-independent samples. Two of the most highly up- A full-length cDNA was obtained by 5V and 3V rapid regulated transcripts in the androgen-independent tumor have amplification of cDNA ends PCR, sequenced, and found to be extensive homology to the Reg family of secreted C-type lectins, identical to Reg IV, a newly described member of the Reg gene a family of proteins normally expressed in the upper gastroin- family. Reg IV has an open reading frame of 474 bp, predicting testinal tract and believed to play important roles in response to a peptide of 158 amino acids with an NH2-terminal signal tissue injury, islet cell regeneration, and tumorigenesis. On sequence of 22 amino acids. It is 39% similar to Reg I and Reg Northern blot, a single 1.2 kb band was present in multiple III, the other two members of this gene family in humans (9). independently derived hormone refractory LAPC-9 tumors, but A multiple tissue Northern blot was probed and showed that not in the paired parental androgen-dependent LAPC-9 tumors Reg IV expression is restricted to the gastrointestinal tract, most or other xenografts (Fig. 1A). The microarray result was prominently the colon (Fig. 1B). Expression was also also confirmed by quantitative PCR, which showed an average seen in pancreas and small intestine (duodenum and jejunum) 70-fold increase in expression of these expressed sequences tags on a 76-tissue dot blot, suggesting that there may be in androgen-independent LAPC-9 tumors compared with interindividual variations in the level and location of Reg IV expression (data not shown). No expression was seen in prostate on either blot. Digital Northern analysis using the Cancer Genome Anatomy Project (NIH) database confirmed this normal tissue distribution, and also showed that Reg IV expressed sequences tags were present in several prostate, gastric, and colon cancers (UniGene cluster Hs. 105484), suggesting that Reg IV expression may be expressed more broadly in prostate cancer and not limited to LAPC-9. Reg IV Encodes a Secreted Protein of f20 kDa and Is Detectable in Serum of Tumor-Bearing Animals. Reg IV is predicted to be a secreted protein based on the presence of a putative signal sequence and on its homology to Reg I and III (9). To confirm this prediction, we transiently expressed a myc- tagged Reg IV cDNA construct in 293T cells and harvested the cell pellets and conditioned media. As shown in Fig. 1C, the majority of Reg IV protein was found in the culture medium, consistent with the conclusion that Reg IV is a secreted protein. A single band of f20 kDa was identified, again consistent with the predicted molecular weight of Reg IV. In order to determine if secreted Reg IV can be detected in the serum of prostate cancer-bearing mice, LNCaP and LAPC-9 prostate cancer cells were stably transduced with lentivirus constructs expressing myc.his-tagged human Reg IV. Expression of tagged Reg IV was confirmed by Western blot and then tumors were established s.c. in severe combined immunodeficiency mice. Non-Reg IV expressing tumors were also established as controls. Once tumors reached an average size of 1 cm, serum was obtained and the mice were sacrificed. An ELISA assay was developed to detect the presence of the myc.his-tagged protein as described in MATERIALS AND METHODS. Control sera were used to normalize for background signal. The sera from animals containing his.myc.Reg IV-positive LNCaP and LAPC-9 tumors were positive, whereas Fig. 1 Reg IV expression in prostate cancer xenografts and in normal all control animals were negative (ELISA data not shown). tissues and Reg IV secretion. Northern analysis of Reg IV in prostate These results suggest that Reg IV is secreted and that it is xenografts (A), showing overexpression in two hormone refractory (androgen-independent) sublines of LAPC-9, and multiple normal tissues released into and is detectable in serum. (B), with notable expression in the colon. Expression was also seen in Reg IV Is Expressed by High-Risk Tumors Treated with pancreas and small bowel in a multiple tissue dot blot (not shown here). Neoadjuvant Hormone Ablation Therapy. Reg IV was C, myc-tagged Reg IV cDNA was transiently transfected into 293 T cells identified in hormone refractory LAPC-9 sublines, suggesting and recovered from the conditioned media with an anti-Myc antibody. A that Reg IV might be involved in hormone refractory prostate control antibody did not identify this band, indicating that it is Reg IV. Likewise, a Myc antibody did not pull down a specific protein from a cancer progression. In order to test this hypothesis prelimi- vector-only transfectant control (data not shown). narily, sense and antisense Reg IV probes were generated and

hybridized to four radical prostatectomy specimens obtained specimens expressed Reg IV, whereas 44.6% (25/56) of primary from patients with high-risk (high grade and locally advanced) tumors and 62.5% (40/64) of metastatic tumors stained positively tumors treated with neoadjuvant hormone ablation therapy for (Table 2). These differences (between normal and primary 3 to 8 months. All four cases had residual disease, which tumors, and between primary tumors and metastases) were stained specifically with the antisense Reg IV probe, but not statistically significant (P = 0.00000038 and one-sided P = 0.038, the control sense probe. No staining was seen in residual respectively) and show that the prevalence of Reg IV expression adjacent normal tissue (Fig. 2A). These results show that Reg increases as prostate cancers progress. IV is expressed in residual hormone refractory prostate cancer. We also evaluated the relative level of Reg IV expression The pretreatment sample for these patients was not available in benign, localized, and metastatic tumors. As shown in Fig. 3, to test the hypothesis that Reg IV expression was induced by the overall intensity of Reg IV staining increased from benign androgen ablation. to clinically localized to metastatic prostate cancer, with a To determine if Reg IV expression is androgen-regulated, median staining intensity of 1.0, 1.7, and 2.5, respectively LAPC-4 and LNCaP cell lines were grown in the absence of (Kruskal-Wallis test; P < 0.001; note that a score of 1 means androgen and assayed for Reg IV expression. No Reg IV no detectable expression). Whereas a majority of positive induction was seen after androgen starvation in tissue culture or localized tumors expressed only weak levels of Reg IV, a in hormone refractory variants of these cell lines in vivo majority of positive metastatic tumors stained strongly (Table 2). (Fig. 1A), suggesting that the Reg IVexpression seen in hormone The increase in Reg IV staining intensity between benign refractory LAPC-9 tumors and in tumors treated with neo- prostate tissue and localized prostate cancer was statistically adjuvant hormone ablation is not regulated simply by the significant (P = 0.00000016). Likewise, metastatic prostate removal of androgen. cancer had statistically higher expression of Reg IV than Reg IV Is Strongly Expressed by a Majority of Metastatic localized prostate cancer (Kruskal-Wallis test; P < 0.00033). Prostate Cancers. In order to study Reg IV expression further, These differences show that the level of Reg IV expression a tissue array spanning the gamut of prostate histology (n =211 increases as prostate cancers progress, particularly in metastatic tissue microarray elements) was evaluated by RNA in situ cancer. hybridization (Fig. 2B). The percentage of samples staining We also asked whether Reg IV expression is associated with positive for Reg IV increased from benign to clinically localized tumor grade in localized tumors. As shown in Fig. 4, Reg IV to metastatic prostate cancer. None of the 48 evaluable benign expression was significantly more intense among high grade

Fig. 2 In situ expression analysis of Reg IV expression. A, the antisense probe (right) shows Reg IV (brown) expression in an androgen-independent tumor but not in the adjacent normal tissue to the left of the tumor. The sense control (left), is negative; B, progression of Reg IV expression. Top left (a) is normal prostate, which is not staining; top right (b), negative Gleason 6 cancer; bottom left (c) is a Gleason 9/10 primary tumor staining strongly for Reg IV (note intense purple color); bottom right (d), strongly staining lymph node metastasis.

Table 2 Distribution of Reg IV expression on a prostate cancer androgen, because androgen starvation of both LAPC-4 and tissue array LNCaP prostate cancer cell lines in tissue culture did not result None Weak Moderate Strong in Reg IV expression. Nor did androgen-independent sublines of Benign 48 0 0 0 LNCaP or LAPC-4 express Reg IV in vivo. Additional studies Primary 31 (55.4%) 15 (26.7%) 8 (14%) 2 (3.6%) will be needed to understand the regulation of Reg IV in prostate Metastatic 24 (37.5%) 5 (7.8%) 11 (17%) 24 (37.5%) cancer. The biological role of Reg IV in prostate cancer progression is not known. Reg proteins have been associated with tumors (i.e., Gleason 7-10) than in low grade ones (i.e., Gleason proliferation and regeneration, cell survival, resistance to 5-6; Mann-Whitney test, P = 0.03). There was no association of apoptosis, and cell adhesion. Hartupee et al. (9) reported that Reg IV expression with recurrence or survival. Reg IV is highly expressed in ulcerative colitis and hypothesized To confirm the high-intensity expression in metastatic that it might be related to the high rate of colon cancer in prostate cancer, we also evaluated an array containing 259 individuals with this disease. Violette et al. (18) found a metastases obtained from 24 patients who died of hormone- consistent relationship between Reg IV expression and chemo- refractory metastatic prostate cancer. The mean staining intensity therapy resistance in colon cancer cell lines. They found that Reg in autopsy cases was 3.2, similar to that in the ‘‘progression’’ IV is expressed in five of seven chemoresistant lines, but is array. Benign prostate tissue on this array was negative. Among absent from all chemosensitive lines. Importantly, they noted that positive tumors, almost all cells stained positive, again similar to Reg IV is expressed by LS513, a cell line that survives but does the progression array. These results confirm that as prostate not proliferate in the presence of chemotherapy, suggesting that cancer progresses, there is increasing expression of Reg IV. Reg IV may be a survival factor rather than a mitogen. Similarly, Expression is highest in hormone refractory metastatic tumors. recent studies have shown that Reg Ia is a signaling intermediate in a survival pathway in motoneurons (19). The hypothesis that Reg IV might play a role in cell survival is consistent with its DISCUSSION expression in hormone-refractory prostate cancer. The associa- The two seminal events in the natural history of prostate tion of Reg IV expression with chemotherapy resistance is also cancer are metastasis and progression to androgen independence. consistent with the fact that a majority of patients with lethal The ability to predict at diagnosis the clinical course of an prostate cancer metastases on our tissue array received individual tumor is currently suboptimal. Thirty percent of chemotherapy during their clinical course. clinically localized tumors recur after local therapy and a subset Reg IV is the second gastrointestinal secreted protein that of these go on to metastasize and kill their host. The association we have identified in prostate cancer. Intestinal trefoil factor of Reg IV expression with androgen independence and (ITF/TFF3) was initially identified in prostate cancer arrays and metastasis raises the possibility that expression of Reg IV may has since been reported to be expressed by f40% of localized correlate with the risk of progression to hormone refractory prostate cancers and a higher percentage of metastases (20–22). metastasis. Expression of the Reg IV homologues Reg 1a and Trefoil factors are known to play an important role in intestinal PAP has been reported to predict for reduced survival from colon protection and restitution, a process in which mucosal continuity cancer (10). Indeed, we found that increasing Reg IV expression did correlate with higher grade primary tumors, suggesting that Reg IV expression may have prognostic utility in primary tumors. However, there was no association with recurrence in this initial small series. Analysis of Reg IV expression in a larger patient cohort with long-term follow-up will be necessary to determine its relationship to recurrence and prostate cancer survival. None of the patients with localized tumors in our database went on to die from prostate cancer. Because Reg IV is secreted, it might also be useful as a serum marker to identify patients with metastasis or at risk to develop metastases. This possibility is supported by the ability to detect Reg IV in the serum of tumor-bearing animals. Antibodies against Reg IV are currently being generated to assess Reg IV protein expression in tissue samples and to measure circulating Reg IV levels in normal and cancer patients. An important issue will be to determine if Reg IV expression in the gastrointestinal tract interferes with the detection of Reg IV from tumor tissue. Reg IV was cloned from a hormone refractory xenograft and is expressed by both androgen-resistant local tumors and metastases. It is not known whether Reg IV expression is related Fig. 3 Reg IV expression in normal, primary, and metastatic prostate cancer. The mean expression score F SD for Reg IV expression is shown specifically to androgen independence and/or metastasis because for normal prostate, primary prostate cancer, and metastatic prostate all of the metastases were obtained from hormone refractory cancer. The results summarize a prostate cancer tissue array representing patients. Reg IV expression does not seem to be regulated by the gamut of prostate tissues.

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Zhennan Gu, Mark A. Rubin, Yu Yang, et al.

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