Reg IV: A Promising Marker of Hormone Refractory Metastatic Prostate Cancer

INTRODUCTION

Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in American men. Prostate cancer is a biologically and clinically heterogeneous disease. A majority of men with this malignancy harbor slow-growing tumors that may not impact an individual's natural life span, although others are struck by rapidly progressive, metastatic tumors. Prostate-specific antigen screening is limited by a lack of specificity and an inability to predict which patients are at risk to develop hormone refractory metastatic disease. Recent studies advocating a lower prostate-specific antigen threshold for diagnosis may increase the number of prostate cancer diagnoses and further complicate the identification of patients with indolent versus aggressive cancers (1). New serum and tissue markers that correlate with clinical outcome or identify patients with potentially aggressive disease are urgently needed (2).

Recent expression profiling studies suggest that expression signatures for metastatic versus nonmetastatic tumors may reside in the primary tumor (2–4). Additional features that predispose tumors to metastasize to specific organs may also be present at some frequency in the primary tumor (5). These recent observations suggest that novel markers of premetastatic or prehormone refractory prostate cancer may be identified in early stage disease. These markers may also play a role in the biology of metastatic or hormone refractory prostate cancer progression. Recent examples of genes present in primary tumors that correlate with outcome and play a role in the biology of prostate cancer progression include EZH2 and LIM kinase (6, 7). However, neither of these two genes is secreted.

In order to identify new candidate serum or tissue markers of hormone refractory prostate cancer, we compared gene expression profiles of paired hormone-dependent and hormone refractory prostate cancer xenografts. The LAPC-9 xenograft was established from an osteoblastic bone metastasis and progresses from androgen dependence to independence following castration in immunodeficient mice (8). It has been used previously to identify candidate therapeutic targets in prostate cancer. Differentially expressed genes were validated and then examined for sequence homology to secreted or cell surface proteins. We report here on the identification, characterization, and initial validation of one such candidate gene, Reg IV, a new member of the regenerating family of secreted C-lectin proteins (9). Reg proteins are normally expressed in the gastrointestinal tract and are induced in inflammatory bowel disease and some gastrointestinal malignancies. Their pleiotropic functions include 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 moderately or highly expressed in a majority of hormone refractory and metastatic tumors. These results suggest that Reg IV may be a potential marker of prostate cancer metastasis or hormone refractory growth.

MATERIALS AND METHODS

Microarray Analysis of Gene Expression. Tumor samples from a matched pair of androgen-dependent and -independent LAPC-9 xenografts were grown and prepared as described previously (8). Total RNA was isolated by using Ultraspec RNA isolation systems (Biotecx). mRNA was purified using Oligotex mRNA Midi Kit (Qiagen). Two micrograms of mRNA was reverse-transcribed, and cDNA was then labeled with Cy-5. Labeled tumor cDNA was combined with a Cy-3-labeled common reference RNA derived from 11 different cell lines and hybridized to cDNA microarrays containing 22,648 elements representing 17,083 genes, as reported previously (11). The slides were scanned with a GenePix microarray scanner (Axon Instruments) and were analyzed with Genepix software. Spots of insufficient quality were excluded from analysis by visual inspection. Data files were entered into the Stanford Microarray Database, where spot intensity was correlated with gene identification. Only features with a signal intensity >50% above background in either Cy5 or Cy3 channel and whose expression varied at least 4-fold between the paired samples were retrieved from the Stanford Microarray Database. Detailed descriptions of array manufacture, hybridization protocols, and data analysis are available at http://cmgm.Stanford.EDU/pbrown.

Construction of myc-His-Tagged Reg IV Expression Vector. The Reg IV coding sequence was subcloned into the multiple cloning site of pcDNA3.1/myc-His expression vector (Invitrogen) at the BamHI and EcoRI sites. The reading frame was confirmed by sequencing.

RNA Probes and In situ Hybridization. A 399 bp DNA fragment from the 3′-untranslated region of Reg IV (Genbank AI732541) was inserted into the pCR2.1 vector (Invitrogen) in both sense and antisense orientations under the control of the T7 promoter. Plasmids were linearized and digoxigenin-labeled riboprobes were generated using the DIG RNA Labeling Kit (Roche Applied Science). Automated in situ hybridization was done on the Discovery System (Ventana Medical Systems, Tucson, AZ). After deparaffinization, slides were soaked in 2× SSC for 5 minutes and digested with proteinase K (Life Technologies, at a final concentration of 10 μg/mL) for 30 minutes at 37°C. Sense and antisense riboprobes were diluted at 1:100 (1 μg of probe/mL) in hybridization solution (50% deionized formamide, 10% polyethylene glycol, 0.3 mol/L NaCl, 10 mmol Tris (pH 8.0), 1 mmol EDTA, Denhardt's solution 1×, yeast tRNA 500 μg/mL, 50 mmol DTT). The hybridization was done for 6 hours at 65°C with 100 μL of hybridization solution. After hybridization, slides were washed twice at 70°C for 6 minutes in 1.0× SSC. The hybridization was followed by a 30-minute incubation with a biotinylated anti-digoxigenin (Sigma BioSciences, St. Louis, MO), followed by alkaline phosphatase–conjugated streptavidin for 16 minutes. Visualization procedure was done in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Ventana mapBlue) for 5 hours and finally in hematoxylin counterstain.

Recombinant Reg IV Expression and Immunoprecipitation. 293T cells were transiently transfected with a myc-his tagged Reg IV expression vector by calcium phosphate precipitation for 48 hours. Cell labeling and immunoprecipitation was done as described (12). Briefly, cells were labeled with 500 μCi of trans-³⁵S label (ICN) in methionine and lysine-free DMEM (Invitrogen) containing 5% of dialyzed fetal bovine serum for 4 hours. Cell lysates and conditioned medium were incubated with 3 μg of anti-myc monoclonal antibody (9E10), and 20 mL of protein G-Sepharose CL-4B (Amersham) for 2 hours at 4°C. Samples were washed and boiled in SDS sample buffer for 5 minutes and separated on 12.5% SDS-PAGE. The gel was treated with Amplify (Amersham) before being dried and autoradiographed.

Northern Blot Analysis of Gene Expression. RNAs were extracted as described above. Ten micrograms of RNA was separated on a 1.2% agarose denaturing gel, transferred to nitrocellulose filters, and hybridized with RT-PCR-prepared DNA fragments of Reg IV (Genbank AI732541). Probes were labeled with α ³²PdCTP by random priming using the random primer labeling system (Amersham) and hybridization was carried out at 62°C in 6× SSC overnight, followed by washing with 2× SSC-0.1% SDS and 0.2× SSC-0.1% SDS at 62°C. For multiple tissue Northern analysis, the hybridization was done as described by the manufacturer (Clontech).

Case Selection for Tissue Microarray. In order to evaluate Reg IV, we used a prostate cancer progression tissue microarray. This tissue microarray is composed of benign prostate tissue, localized prostate cancer, and hormone refractory metastatic prostate cancer. These cases came from well-fixed radical prostatectomy specimens from the University of Michigan (Ann Arbor, MI), the University Hospital Ulm (Ulm, Germany), and the rapid autopsy program from the University of Michigan Specialized Program of Research Excellence in Prostate Cancer (13). All samples were collected with prior Institutional Review Board approval at each respective institution. This tissue microarray was composed of classic acinar prostate cancers and areas demonstrating foamy gland features from the same cases. Benign tissue samples were also placed in the tissue microarray to serve as a negative control. A second array containing predominantly metastatic cases was also stained and scored.

Scoring of Reg IV Expression. Reg IV expression was determined using a validated scoring method (7, 14–16) where staining was evaluated for intensity. Benign epithelial glands and prostate cancer cells were scored for Reg IV staining intensity on a four-tiered system ranging from negative to strong expression. A score of 1 was negative, a score of 2 was considered low expression, a score of 3 indicated moderate expression, and a score of 4 correlated with strong expression. Slides were read independently by two pathologists (M. Rubin and M. Loda) with >90% interobserver agreement.

Construction and Production of Lentivirus Expressing Reg IV-Myc.His. A Myc.His-tagged Reg IV construct was PCR-amplified from a pcDNA Reg IV-myc.his vector and inserted into the lentiviral vector chemokine/chemokine receptor through restriction sites of EcoRI and NheI (17). Lentivirus stocks were generated by calcium phosphate mediated transfection of 293T cells. The titer of the virus was checked with 293T cells using chemokine/chemokine receptor-enhanced green fluorescent protein as a positive control and indicator.

LNCaP and LAPC-9 Prostate Xenograft Models. LNCaP or LNCaP-Reg IV.myc.his cells (5 × 10⁶) were mixed with an equal volume of Matrigel and inoculated into severe combined immunodeficiency mice s.c. In the case of LAPC-9, explanted tumor is digested with Pronase and cultured in 10% fetal bovine serum-RPMI 1640 for 16 hours. Cells are then transduced by chemokine/chemokine receptor lentivirus alone or lentivirus RegIV.myc.mis at a multiplicity of infection of 5 for 2 hours. Cells are then washed with culture media, mixed with Matrigel, and inoculated back to severe combined immunodeficiency mice (1 × 10⁶ cells/mice).

ELISA Detection of Serum RegIV.myc.his. Twenty-five microliters of mouse serum was mixed with 75 mL of PBS and incubated in Ni-NTA HisSorb strips (Qiagen) for 3 hours. The strips were washed with PBS-0.1% Tween 20 and then incubated with 1:5,000 diluted anti-Myc monoclonal antibody (Invitrogen) for 45 minutes. The 1:10,000 diluted anti-mouse IgG antibodies conjugated with alkaline phosphatase (Promega) were then incubated for 45 minutes. After washing, the color was developed using one-step nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Pierce) for 20 minutes and read at 450 nm.

Statistics. The Pearson χ² statistic was used to test whether the rows (e.g., tumor type) and columns (e.g., Reg expression) of a table were independent. To test for differences of Reg expression across different tumor types, we also used the Kruskal-Wallis test, which is a nonparametric multigroup comparison tests. Statistical analyses were done using the freely available R software (http://www.r-project.org).

RESULTS

Cloning and Characterization of Reg IV. The LAPC-9 xenograft was established from metastatic prostate tumors and progresses in vivo from androgen dependence to independence. In order to identify novel candidate markers of prostate cancer progression, RNAs from paired androgen-dependent and androgen-independent LAPC-9 tumors were labeled and hybridized to 24,000 spot cDNA arrays with common reference RNA. Two hundred and four clones representing 101 named genes and 59 expressed sequences tags showed expression variation of at least 4-fold between the androgen-dependent and androgen-independent samples. Out of the 101 named genes, 75 have been characterized functionally to some degree and most can be categorized into six biological processes according to Gene Ontology annotations: cell-cell signaling/signal transduction, cell adhesion/motility, structural molecule/cytoskeleton, immune response, cell proliferation/cell cycle, and metabolism (Table 1). Eleven androgen-responsive genes, including well-known androgen targets such as klk3 (prostate-specific antigen), were identified by comparison to the expression profiles of 567 androgen-regulated transcripts we had identified previously (Table 1). With one exception (WWP1), all of the genes normally up-regulated by androgens showed decreased expression in the androgen-independent tumor growing in the castrated animal, confirming that androgens modulate their expression in vivo.

We focused our attention on uncharacterized genes that were differentially expressed between the androgen-dependent and androgen-independent samples. Two of the most highly up-regulated transcripts in the androgen-independent tumor have extensive homology to the Reg family of secreted C-type lectins, a family of proteins normally expressed in the upper gastrointestinal tract and believed to play important roles in response to tissue injury, islet cell regeneration, and tumorigenesis. On Northern blot, a single 1.2 kb band was present in multiple independently derived hormone refractory LAPC-9 tumors, but not in the paired parental androgen-dependent LAPC-9 tumors or other xenografts (Fig. 1A). The microarray result was also confirmed by quantitative PCR, which showed an average 70-fold increase in expression of these expressed sequences tags in androgen-independent LAPC-9 tumors compared with androgen-dependent ones (data not shown). These results show that two expressed sequences tags related to the Reg gene family are reproducibly up-regulated during androgen-independent progression of LAPC-9.

A full-length cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends PCR, sequenced, and found to be identical to Reg IV, a newly described member of the Reg gene family. Reg IV has an open reading frame of 474 bp, predicting a peptide of 158 amino acids with an NH₂-terminal signal sequence of 22 amino acids. It is 39% similar to Reg I and Reg III, the other two members of this gene family in humans (9).

A multiple tissue Northern blot was probed and showed that Reg IV expression is restricted to the gastrointestinal tract, most prominently the colon (Fig. 1B). Expression was also seen in pancreas and small intestine (duodenum and jejunum) on a 76-tissue dot blot, suggesting that there may be 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 ∼20 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 ∼20 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 all control animals were negative (ELISA data not shown). These results suggest that Reg IV is secreted and that it is released into and is detectable in serum.

Reg IV Is Expressed by High-Risk Tumors Treated with Neoadjuvant Hormone Ablation Therapy. Reg IV was identified in hormone refractory LAPC-9 sublines, suggesting that Reg IV might be involved in hormone refractory prostate cancer progression. In order to test this hypothesis preliminarily, sense and antisense Reg IV probes were generated and hybridized to four radical prostatectomy specimens obtained from patients with high-risk (high grade and locally advanced) tumors treated with neoadjuvant hormone ablation therapy for 3 to 8 months. All four cases had residual disease, which stained specifically with the antisense Reg IV probe, but not the control sense probe. No staining was seen in residual adjacent normal tissue (Fig. 2A). These results show that Reg IV is expressed in residual hormone refractory prostate cancer. The pretreatment sample for these patients was not available to test the hypothesis that Reg IV expression was induced by androgen ablation.

To determine if Reg IV expression is androgen-regulated, LAPC-4 and LNCaP cell lines were grown in the absence of androgen and assayed for Reg IV expression. No Reg IV induction was seen after androgen starvation in tissue culture or in hormone refractory variants of these cell lines in vivo (Fig. 1A), suggesting that the Reg IV expression seen in hormone refractory LAPC-9 tumors and in tumors treated with neoadjuvant hormone ablation is not regulated simply by the removal of androgen.

Reg IV Is Strongly Expressed by a Majority of Metastatic Prostate Cancers. In order to study Reg IV expression further, a tissue array spanning the gamut of prostate histology (n = 211 tissue microarray elements) was evaluated by RNA in situ hybridization (Fig. 2B). The percentage of samples staining positive for Reg IV increased from benign to clinically localized to metastatic prostate cancer. None of the 48 evaluable benign specimens expressed Reg IV, whereas 44.6% (25/56) of primary tumors and 62.5% (40/64) of metastatic tumors stained positively (Table 2). These differences (between normal and primary tumors, and between primary tumors and metastases) were statistically significant (P = 0.00000038 and one-sided P = 0.038, respectively) and show that the prevalence of Reg IV expression increases as prostate cancers progress.

We also evaluated the relative level of Reg IV expression in benign, localized, and metastatic tumors. As shown in Fig. 3, the overall intensity of Reg IV staining increased from benign to clinically localized to metastatic prostate cancer, with a median staining intensity of 1.0, 1.7, and 2.5, respectively (Kruskal-Wallis test; P < 0.001; note that a score of 1 means no detectable expression). Whereas a majority of positive localized tumors expressed only weak levels of Reg IV, a majority of positive metastatic tumors stained strongly (Table 2). The increase in Reg IV staining intensity between benign prostate tissue and localized prostate cancer was statistically significant (P = 0.00000016). Likewise, metastatic prostate cancer had statistically higher expression of Reg IV than localized prostate cancer (Kruskal-Wallis test; P < 0.00033). These differences show that the level of Reg IV expression increases as prostate cancers progress, particularly in metastatic cancer.

We also asked whether Reg IV expression is associated with tumor grade in localized tumors. As shown in Fig. 4, Reg IV expression was significantly more intense among high grade tumors (i.e., Gleason 7-10) than in low grade ones (i.e., Gleason 5-6; Mann-Whitney test, P = 0.03). There was no association of Reg IV expression with recurrence or survival.

To confirm the high-intensity expression in metastatic prostate cancer, we also evaluated an array containing 259 metastases obtained from 24 patients who died of hormone-refractory metastatic prostate cancer. The mean staining intensity in autopsy cases was 3.2, similar to that in the “progression” array. Benign prostate tissue on this array was negative. Among positive tumors, almost all cells stained positive, again similar to the progression array. These results confirm that as prostate cancer progresses, there is increasing expression of Reg IV. Expression is highest in hormone refractory metastatic tumors.

DISCUSSION

The two seminal events in the natural history of prostate cancer are metastasis and progression to androgen independence. The ability to predict at diagnosis the clinical course of an individual tumor is currently suboptimal. Thirty percent of clinically localized tumors recur after local therapy and a subset of these go on to metastasize and kill their host. The association of Reg IV expression with androgen independence and metastasis raises the possibility that expression of Reg IV may correlate with the risk of progression to hormone refractory metastasis. Expression of the Reg IV homologues Reg 1α and PAP has been reported to predict for reduced survival from colon 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 specifically to androgen independence and/or metastasis because all of the metastases were obtained from hormone refractory patients. Reg IV expression does not seem to be regulated by androgen, because androgen starvation of both LAPC-4 and LNCaP prostate cancer cell lines in tissue culture did not result in Reg IV expression. Nor did androgen-independent sublines of LNCaP or LAPC-4 express Reg IV in vivo. Additional studies will be needed to understand the regulation of Reg IV in prostate cancer.

The biological role of Reg IV in prostate cancer progression is not known. Reg proteins have been associated with proliferation and regeneration, cell survival, resistance to apoptosis, and cell adhesion. Hartupee et al. (9) reported that Reg IV is highly expressed in ulcerative colitis and hypothesized that it might be related to the high rate of colon cancer in individuals with this disease. Violette et al. (18) found a consistent relationship between Reg IV expression and chemotherapy resistance in colon cancer cell lines. They found that Reg IV is expressed in five of seven chemoresistant lines, but is absent from all chemosensitive lines. Importantly, they noted that Reg IV is expressed by LS513, a cell line that survives but does not proliferate in the presence of chemotherapy, suggesting that Reg IV may be a survival factor rather than a mitogen. Similarly, recent studies have shown that Reg Iα 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 expression in hormone-refractory prostate cancer. The association of Reg IV expression with chemotherapy resistance is also consistent with the fact that a majority of patients with lethal prostate cancer metastases on our tissue array received chemotherapy during their clinical course.

Reg IV is the second gastrointestinal secreted protein that we have identified in prostate cancer. Intestinal trefoil factor (ITF/TFF3) was initially identified in prostate cancer arrays and has since been reported to be expressed by ∼40% of localized prostate cancers and a higher percentage of metastases (20–22). Trefoil factors are known to play an important role in intestinal protection and restitution, a process in which mucosal continuity is re-established following tissue injury, whereas Reg proteins are believed to play a role in tissue regeneration (23). Both Reg and trefoil proteins are overexpressed in inflammatory bowel disease. Both are also overexpressed in malignancy. TFF3, for example, is an adverse prognostic factor in gastric cancer (23). It will be important to understand the reasons why several related gastrointestinal proteins are expressed in prostate cancer, their regulation and their functions as paracrine or autocrine factors. Reg IV, TFF3, and their receptors (a receptor for Reg 1 was recently identified) might also be useful therapeutic targets for the management of prostate cancer (24).