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[Cancer Research 65, 2130-2138, March 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Functional Loss of the {gamma}-Catenin Gene through Epigenetic and Genetic Pathways in Human Prostate Cancer

Hiroaki Shiina2, Julia E. Breault1, William W. Basset1, Hideki Enokida1, Shinji Urakami1, Long-Cheng Li1, Steven T. Okino1, Masao Deguchi1, Masanori Kaneuchi1, Masaharu Terashima3, Tatsuaki Yoneda2, Kazushi Shigeno2, Peter R. Carroll1, Mikio Igawa2 and Rajvir Dahiya1

1 Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California and Departments of 2 Urology and 3 Biochemistry and Molecular Medicine, Shimane University School of Medicine, Izumo, Japan

Requests for reprints: Rajvir Dahiya, Urology Research Center (112F), University of California San Francisco and Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. Phone: 415-750-6964; Fax: 415-750-6639; E-mail: rdahiya{at}urol.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{gamma}-Catenin is a cell adhesion molecule and a candidate mediator of Wnt signal transduction. We hypothesized that impaired regulation of {gamma}-catenin through genetic and epigenetic pathways is associated with the pathogenesis of prostate cancer. To test this hypothesis, cytosine-phosphate-guanine methylation, loss of heterozygosity (LOH), and mutation status of the {gamma}-catenin gene were analyzed in cultured prostate cancer cell lines, 180 localized prostate cancers, 69 benign prostatic hyperplasias, and 11 hormone refractory prostate cancers (HRPC). In prostate cancer cell lines (DuPro, LNCaP, ND-1, and PC3), {gamma}-catenin mRNA transcripts were increased after 5-aza-2'-deoxycytidine treatment. In localized prostate cancer, {gamma}-catenin expression was lower but prevalence of {gamma}-catenin methylation was higher compared with benign prostatic hyperplasia. However, {gamma}-catenin methylation did not correlate with Gleason sum, pT category, or capsular penetration. Among localized prostate cancers with positive {gamma}-catenin methylation, the presence of LOH at chromosome 17q21 was closely related to down-regulation of {gamma}-catenin mRNA expression. The {gamma}-catenin mutations were not found in localized prostate cancers, whereas six mutations were found in five HRPCs within or close to the GSK-3ß consensus motif phosphorylation site, among which four HRPCs showed strong nuclear {gamma}-catenin accumulation. In these four HRPCs, Bcl-2 expression was increased, whereas the target of the Wnt signal, c-myc, was only expressed in one HRPC. Therefore, although epigenetic {gamma}-catenin methylation is an early event in the development of prostate cancer, simultaneous events of epigenetic cytosine-phosphate-guanine methylation and genetic LOH may be responsible for functional loss of {gamma}-catenin. The {gamma}-catenin mutation related to Bcl-2 overexpression has a significant effect on the pathogenesis of HRPC. This is the first report to characterize the epigenetic and genetic regulation of {gamma}-catenin in human prostate cancer.

Key Words: {gamma}-catenin • methylation • human prostate cancer • mutation • LOH and apoptosis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the most frequently diagnosed malignant disease and the second leading cause of cancer death in men in the United States (1–3). Understanding the genetic and epigenetic pathways involved in the pathogenesis of prostate cancer is essential so that better strategies for diagnosis and treatment of prostate cancer can be designed. In this regard, the functional loss of {gamma}-catenin results in the disruption of intercellular interaction among tumor cells and allows them to invade into surrounding tissues, thus representing worse biological potential (4). The {gamma}-catenin is a cytoplasmic protein and is structurally related to ß-catenin with similar functions, such as cell adhesion and signal transduction (5). Both ß- and {gamma}-catenins play a pivotal role in maintaining cellular polarity and serve as cell-to-cell attachment molecules through direct interaction with the cytoplasmic domain of E-cadherin (6, 7). In cancer tissue, escape from the cell adhesion system is an important mechanism allowing tumor cells to invade and/or metastasize. In signal transduction, both ß- and {gamma}-catenins are important constituents of the Wnt signaling pathway associated with cell differentiation (8). These catenins are involved in diverse processes during development and malignant transformation (9). The ß-catenin protein found usually in the lateral cell membrane partially reflects the function of cell adhesion, whereas that found in the cytoplasm and nucleus functions as a mediator of Wnt signal transduction (10). Posttranslational stabilization of the ß-catenin protein affects the Wnt signal to the nucleus with the aid of transcription factor T cell factor/lymphocyte enhancer factor 1 (10), leading to the activation of c-myc (11) and cyclin D1 (12), which are involved in the regulation of cell growth. Although different from the well-documented role of ß-catenin in Wnt signaling, the functional role of {gamma}-catenin remains unclear.

Prior studies have shown that hypermethylation of normally unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (CpG islands) located in the promoter region of genes is involved in transcriptional silencing (13). In several tumor suppressor genes, epigenetic CpG hypermethylation of the promoter is an alternative mechanism to genetic events, such as loss of heterozygosity (LOH), deletions, or mutations responsible for inactivating gene function (14–16). In subsets of thyroid (17) and lung cancers (18), {gamma}-catenin expression in cancer tissue has been decreased or lost. In these cancers, one of the mechanisms underlying down-regulation of {gamma}-catenin is CpG hypermethylation of the {gamma}-catenin promoter (17, 18). In prostate cancer, however, the epigenetic and genetic regulation of {gamma}-catenin has not been investigated.

The present study was designed to test the hypothesis that impaired regulation of {gamma}-catenin is involved in the processes of initiation, progression, and metastasis of prostate cancer. We analyzed CpG promoter methylation, mutation of the promoter and NH2-terminal region, and LOH status of the {gamma}-catenin gene in pathologic prostate samples comprising 180 localized prostate cancer, 69 benign prostatic hyperplasia (BPH), and 11 hormone refractory prostate cancers (HRPC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Samples. One hundred and eighty localized prostate cancers (from radical prostatectomy) and 69 pathologically proven BPHs (from transurethral resection) and 11 HRPCs (from transurethral resection) were obtained at Shimane University Hospital (Izumo, Japan). The pathologic findings of prostate cancer samples were determined by the General Rule for Clinical and Pathological Studies on Prostate Cancer by Japanese Urological Association and Japanese Society of Pathology (19), which is essentially based on the WHO criteria and the Gleason pattern (20). The 180 localized prostate cancers comprised 120 cases of pT2, 57 cases of pT3, and 3 cases of pT4 disease. Our routine diagnostic strategy for prostate cancer included serum prostate-specific antigen level, transrectal ultrasonography, color Doppler ultrasonography, and magnetic resonance imaging, which enabled us to detect the localization of prostate cancer before radical prostatectomy (21). We obtained control prostates from radical prostatectomized samples according to the following criteria: (a) prostate cancer presented as a single cancer focus, (b) preoperative prostate cancer localization identical to postoperative pathologic findings, and (c) prostate cancer localized to one side of the prostate. Finally, we selected and used 70 samples as control prostate, which did not contain any cancer areas. Written informed consent was obtained from all patients.

Tissue Preparation. All of the BPH samples and half of each prostate cancer tissue were fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin wax. Five-micrometer-thick sections were used for H&E staining for histologic evaluation and microdissection was done as previously described (22). The remaining half of each prostate cancer sample was immediately frozen and stored at –80°C for RNA extraction. Microscopically dissected samples were analyzed for methylation, LOH, and mutation. In BPH samples, high-grade prostate intraepithelial neoplasia and cancer were ruled out by microscopic analysis.

Cell Culture. Human prostate cancer cell lines DuPro, LNCaP, and PC3 were obtained from American Type Culture Collection (Rockville, MD). Human prostate cancer cell line (ND1) was developed in our laboratory (23). The media used in this study for DuPro, LNCaP, ND1, and PC3 was RPMI 1640 supplemented with 10% FCS.

Nucleic Acid Extraction. Genomic DNA from all prostate samples was extracted using a QIAamp Tissue kit (Qiagen, Valencia, CA) after microdissection (22) and was precipitated with ethanol. Genomic DNA of cell line samples was extracted with DNAzol reagent (Invitrogen Life Technologies, San Diego, CA). Total RNA was extracted with TRI reagent (Molecular Research Center, Cincinnati, OH). The RNA pellet obtained after isopropanol and ethanol precipitation was dried, resuspended in 25 µL RNase-free water, and stored in aliquots at –80°C until reverse transcribed. The concentrations of DNA and RNA were determined with a spectrophotometer, and their integrity was checked by gel electrophoresis.

cDNA Preparation and Differential Reverse Transcription-PCR of {gamma}-Catenin. One microgram RNA was added to 0.5 µg oligodeoxythymidilic acid primer (Promega, Madison, WI) in a final volume of 25 µL. The samples were placed at 55°C for 5 minutes and then cooled on ice. The primer RNA mixture was then combined with 0.25 unit of Avian myeloblastosis virus reverse transcriptase (Promega) and 0.5 unit of RNase inhibitor. The reverse transcription reaction was carried out at 45°C for 45 minutes. The cDNA was then incubated at 95°C for 5 minutes to inactivate the reverse transcriptase. Samples were stored at –20°C until used.

The cDNA samples (2 µL) were diluted into 20 µL solution containing 50 mmol/L of deoxynucleotide triphosphate, 0.5 units of RedTaq polymerase, and PCR reaction buffer provided by the manufacturer (Sigma, St. Louis, MO). For differential reverse transcription-PCR (RT-PCR) with {gamma}-catenin and GAPDH, different sets of s-GAPDH primers and l-GAPDH primers were used with prostate cancer cell lines and clinical samples, respectively. For cell line samples, the primer concentration was 150 nmol/L each. For clinical samples, the l-GAPDH-S and l-GAPDH-AS primer concentration was 100 nmol/L, whereas {gamma}-catenin RT-S and RT-AS primer concentrations were 200 nmol/L. The annealing temperature was 55°C for differential RT-PCR reactions, with 26 cycles for cell line samples and 32 cycles for clinical samples. The primer sequences are shown in Table 1. The PCR products were electrophoresed on 1.5% agarose gels, the expression level of the genes was evaluated by ImageJ software (http://rsb.info.nih.gov/ij), and the areas under the curves were calculated and analyzed. Expression level of {gamma}-catenin was quantified relative to GAPDH expression level and expressed as arbitrary units. For semiquantitative analysis of the amplified products, a suitable number of PCR cycles for {gamma}-catenin and GAPDH was determined so that it was within the exponential phase. For this purpose, we did RT-PCR using five diluted specimens (1:1, 1:2, 1:4, 1:8, and 1:16) from the same sample and calculated the absorbance of each band. At 32 cycles, the logistically converted densities of each band from the same sample were on the same regression line and are considered within the exponential phase.


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  		Table 1. Primer sequence

 
5-Aza-2'-Deoxycytidine Treatment. To screen for epigenetic alterations in the {gamma}-catenin gene, cells (DUPro, LNCaP, ND-1, and PC-3) were treated with 5-aza-2'-deoxycytidine (5-aza-dC, 5 µmol/L) in duplicate. The cultured cells were harvested after 4 days of 5-aza-dC treatment. Using cDNA, the difference in the expression level of {gamma}-catenin mRNA transcripts before and after 5-aza-dC treatment was analyzed by differential RT-PCR.

Methylation Analysis. Genomic DNA (100 ng) was modified with sodium bisulfite using a commercial kit (Invitrogen Life Technologies, San Diego, CA). Based on the functional promoter sequence (17), methylation-specific and unmethylation-specific primers were designed using our MethPrimer (http://itsa.ucsf.edu/~urolab/methprimer). The regions amplified by these primers have 16 CpG sites and their relationship to the CpG sites are shown in Fig. 1A. For methylation-specific PCR (MSP), a second round of nested PCR (MSP-A, MSP-B, and USP) was done using the universal PCR product amplified by Uni-S and Uni-AS primers as a template. The first universal primer sets have no CpG sites in either the forward or reverse primer. The PCR products MSP-A, MSP-B, and USP correspond to those obtained by the primer sets GM1-S and GM2-AS, GM2-S and GM1-AS, and {gamma}U-S and {gamma}U-AS, respectively. For semiquantitative analysis, a preliminary suitable number of PCR cycles for each MSP-A, MSP-B, and USP were carried out to determine the linear range of the reaction. To ensure this, at least one initial PCR was done using 32 cycles each for MSP-A and MSP-B and 30 cycles for USP. Then, a suitable PCR cycle was chosen for each sample. In each assay, absence of DNA template served as negative control. The primer sequences are shown in Table 1. The MSP-A, MSP-B, and USP products were analyzed by 2% agarose gel electrophoresis.



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  		Figure 1. Primer design and expression of {gamma}-catenin in human prostate samples. A, schematic representation of the location of CpG sites and primers within the functional promoter of the {gamma}-catenin gene. The first universal PCR was done using the Uni-S and Uni-AS primers. These universal primers do not cover any CpG sites within the primer sequence. MSP-A and MSP-B methylation was determined by the primer sets GM1-S and GM2-AS and GM2-S and GM1-AS, respectively, using the first universal PCR product as a template. USP was determined by the primer sets {gamma}U-S and {gamma}U-AS. B, {gamma}-catenin expression evaluated by immunostaining in human prostate samples. Immunostaining of {gamma}-catenin in BPH tissue (x200, hematoxylin counterstained) showed strong membrane expression at the lateral border of each cell. On the other hand, {gamma}-catenin expression in prostate cancer tissue (x200, hematoxylin counterstained) showed weaker membrane staining compared with the BPH sample. The differences in {gamma}-catenin immunostaining between BPH and prostate cancer reached statistical significance (P < 0.01). Protein expression was graded according to the proportion of positive (i.e., uniformly positive, >30% of positive cells) and negative (0-30% of positive cells) cells. C, differential RT-PCR results are shown. Relative expression of {gamma}-catenin to GAPDH mRNA was determined by RT-PCR. ImageJ software was used to determine the area under the curve and (GC, {gamma}-catenin)/(G3, GAPDH) ratios were calculated as described.

 
In localized prostate cancer samples with positive MSP-A or MSP-B, each relative methylation ratio was determined after MSP-A, MSP-B, or USP product was electrophoresed on nondenaturing 12% polyacrylamide gels. The area under the curve corresponding to each band was calculated using ImageJ software (http://rsb.info.nih.gov/ij) and the relative MSP-A and MSP-B ratios were determined (Fig. 3A).



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  		Figure 3. Relative MSP-A and MSP-B ratios and clinical samples. A, the relative methylation level of MSP-A (MSP-A ratio) was determined by [A / (A + U)] x 100%, where A and U are the areas under the curve corresponding to the MSP-A and USP band intensity. The MSP-B ratio was also determined by the same procedure. B, relationship of the relative MSP-B ratio with Gleason sum. C, relationship of the relative MSP-B ratio with pathologic stage. D, relationship between the relative MSP-B ratio and capsular penetration (Cap.).

 
Bisulfite DNA Sequencing Analysis. One microliter bisulfite-modified DNA was amplified using a pair of universal primers (Uni-S and Uni-AS) in a total volume of 20 µL. Direct bisulfite DNA sequencing of the PCR products using either forward UniG1-S primer or reverse PanGm-AS primer was done according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).

Mutation Analysis. We focused on the NH2-terminal regulatory region of {gamma}-catenin, where the consensus motif for the GSK-3ß phosphorylation site is present (5), and did mutational analysis using the primer sets {gamma}-S and {gamma}-AS. The primer sequences are shown in Table 1. Mutational screening by single-strand conformational polymorphism was done using a nonradioactive approach as described in our laboratory (24). If single-strand conformational polymorphism gels showed aberrant band migration, direct DNA sequencing was done to confirm the mutation using either {gamma}-nest-S or {gamma}-nest-AS primer (Table 1).

LOH Analysis. Genomic DNAs from microdissected prostate cancer and nonprostate cancer areas were separately amplified by PCR using two polymorphic markers mapping to the chromosome 17q21 region (D17S1787 and D17S1818). The primer sequence and PCR condition are shown in Table 1. PCR products were analyzed using 10% denaturing polyacrylamide gels with GelaStar staining (BMA, Rockland, ME). The intensity of each band was analyzed using ImageJ software (http://rsb.info.nih.gov/ij), and LOH was considered if there was a >30% reduction in the allelic ratio of tumor sample compared with control sample (25).

Immunostaining. Immunostaining of {gamma}-catenin, Bcl-2, and c-myc was done on 5-µm-thick consecutive sections obtained from paraffin-embedded materials, using mouse monoclonal antibody for {gamma}-catenin (1:500; Transduction Laboratories, Lexington, KY), Bcl-2 (clone 124, 1:80, DAKO, Carpinteria, CA), and c-myc (clone 9E10, 1:50, DAKO). The slides were prepared with antigen retrieval using citrate buffer (10 mmol/L, pH 6.0) before incubation of primary antibody. In negative controls, the primary antibody was replaced with nonimmune serum. 3,3'-Diaminobenzidine (Sigma-Aldrich) was used as the chromogen and counterstaining was done using hematoxylin. Protein expression was graded according to the proportion of positive (i.e., uniformly positive, >30% of positive cells) and negative (0-30% of positive cells) cells. ImageJ software was used to quantify immunohistochemical staining. Ninety-eight localized prostate cancer samples and 69 BPH samples were evaluated for {gamma}-catenin immunostaining.

Statistical Analysis. Differences in {gamma}-catenin expression between benign and malignant prostate was analyzed using {chi}2 test for immunostaining and Student's t test for RT-PCR, respectively. Relationship of the expression level of {gamma}-catenin transcripts with methylation status was analyzed by Pearson's correlation. The relationship of clinicopathologic findings with the methylation status of {gamma}-catenin was done using ANOVA test. P < 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of {gamma}-Catenin in Human Prostate. Typical {gamma}-catenin immunostaining is shown in Fig. 1B. In BPH specimens, membranous expression of {gamma}-catenin was strong, whereas in localized prostate cancer specimens weak {gamma}-catenin immunostaining was frequently observed (P < 0.01; Table 2). Likewise, expression of {gamma}-catenin mRNA transcript was significantly lower in localized prostate cancer than in control prostates (P < 0.01; Fig. 1C).


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  		Table 2. Summary of methylation and immunostaining results in prostate cancer and BPH

 
Methylation Status and {gamma}-Catenin Messenger RNA Expression in Prostate Cancer Cell Lines and Clinical Samples. As shown in Fig. 2A, before 5-aza-dC treatment, all prostate cancer cell lines showed USP bands, whereas strong MSP-B bands were observed in DuPro, ND1, and PC3 cells and a weak MSP-B band was found in LNCaP cell line. After 5-aza-dC treatment, the MSP-B band completely disappeared in all prostate cancer cell lines. Although before 5-aza-dC treatment low levels of {gamma}-catenin mRNA transcript was found in these prostate cancer cell lines, {gamma}-catenin mRNA transcripts were markedly increased in DuPro, ND1, and PC3 after 5-aza-dC treatment. Typical MSP and USP bands in clinical prostate samples are shown in Fig. 2B. The prevalence of MSP-A in prostate cancer and BPH was 43.3% and 31.9%, respectively, whereas that of MSP-B was 33.3% and 15.9%, respectively (Table 2). The majority of both BPH and localized prostate cancer samples showed USP bands, although the intensity of the USP band was strong in BPH and weak in localized prostate cancer. As shown in Fig. 2C, bisulfite DNA sequencing of localized prostate cancer samples showed coexistence of completely and partially methylated CpG sites with some CpG sites being unmethylated. Even in the same prostate cancer samples, the extent of methylation was different from one CpG site to the others.



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  		Figure 2. {gamma}-Catenin methylation in prostate cancer cell lines and clinical samples. A, in DuPro, PC3, and ND1 prostate cancer cell lines, mRNA transcripts of {gamma}-catenin were significantly increased after 5-aza-dC treatment. These alterations in levels of mRNA transcript were accompanied by a complete loss of MSP-B bands after 5-aza-dC treatment. In all prostate cancer cell lines, {gamma}-catenin mRNA was expressed and USP bands were present before 5-aza-dC treatment although the levels were different among cell lines. B, in clinical samples, all prostate cancer samples had strong MSP bands and BPH samples had strong USP bands. Approximately 40-50% of the BPH samples showed strong USP and weak MSP bands, suggesting partial methylation in these samples. In 80-90% of the prostate cancer samples, there were strong MSP bands and weak USP bands suggesting that most of the prostate cancer samples were highly but not completely methylated. C, methylation status of the {gamma}-catenin promoter analyzed by bisulfite DNA sequencing in clinical prostate cancer samples is shown. In sample 1, there was complete CpG methylation at sites +47 and +92, whereas the +59 and +87 CpG sites were completely unmethylated. The remaining CpG sites (+54, +56, +71, and +82) were partially methylated. In prostate cancer sample 2, the CpG sites at +47, +54, +59, +76, and +92 were completely methylated. In the same prostate cancer samples, the extent of methylation was different from one CpG site to the other. The number attributed to CpG sites is the sequence number from the transcription start site. Complete methylation (CM), partial methylation (PM), and unmethylation (UM).

 
{gamma}-Catenin Methylation Ratio in BPH and Localized Prostate Cancer. The relative methylation ratios (MSP-A and MSP-B ratios) were calculated in the individual samples (Fig. 3A). Either the MSP-A or MSP-B ratio was significantly higher in localized prostate cancer than in BPH (P < 0.01 each). However, each MSP ratio was not significantly associated with Gleason sum, pT category, or capsular penetration in localized prostate cancer (Fig. 3B-D). In the series of {gamma}-catenin methylation (+) localized prostate cancer, an inverse correlation was found between {gamma}-catenin mRNA expression and either MSP-A ratio (r = –0.233, P = 0.052) or MSP-B ratio (r = –0.329, P < 0.01). In the seven localized prostate cancer samples exhibiting both LOH and a positive MSP-B band, an inverse correlation between the methylation MSP-B ratio and {gamma}-catenin expression was pronounced and significant (r = –0.791, P < 0.05; Fig. 4A). There was no significant difference in {gamma}-catenin mRNA transcript level between LOH (+) and LOH (–) localized prostate cancer samples. For example, in case A, where there was no LOH and no methylation (negative MSP-B), the expression of {gamma}-catenin was strong (Fig. 4B). On the other hand, in case B, where LOH was negative and methylation was complete, expression of {gamma}-catenin was lost (Fig. 4C). In case C, where LOH was present and methylation partial, the expression of {gamma}-catenin was significantly reduced (Fig. 4D).



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  		Figure 4. Effects of LOH and methylation on expression of {gamma}-catenin in prostate cancer tissues. A, in the seven prostate cancer samples with both MSP-B (+) and LOH (+), there was a strong inverse correlation of MSP-B ratio with {gamma}-catenin expression (r = –0.79, P < 0.05; AU, arbitrary units). B, in case A, the MSP-A and MSP-B ratios are 44.3% and 0%, respectively, and relative {gamma}-catenin expression was 5.3 arbitrary units. Membrane expression of {gamma}-catenin was reduced but not lost. C, in case B, with no LOH, {gamma}-catenin expression was low owing to the complete methylation of the {gamma}-catenin promoter (no presence of USP band). D, in case C with LOH, {gamma}-catenin expression was significantly decreased although MSP-A and MSP-B ratios indicated incomplete methylation (MSP-A = 46.5% and MSP-B = 48.3%).

 
{gamma}-Catenin Regulation and Its Effect in HRPC Samples. Because the MSP-B methylation status is closely associated with expression of {gamma}-catenin mRNA transcripts, an MSP-B assay was done in 11 HRPC samples. As shown in Fig. 5A, 5 of 11 HRPC samples had MSP-B bands and all samples had USP bands. Typical bisulfite DNA sequencing obtained from HRPC sample 2 revealed that all CpG sites were unmethylated. On the other hand, as shown in Fig. 5B, five of the HRPC samples had six mutations (five missense mutations and one nonsense mutation) at the NH2-terminal regulatory region of the {gamma}-catenin gene. These mutations were located within or close to the putative consensus motif for GSK-3ß phosphorylation. Nuclear {gamma}-catenin staining was found in 4 of 11 HRPC samples (samples 4, 7, 10, and 11). All of these samples had missense mutations. HRPC sample 10 showed nuclear accumulation of {gamma}-catenin in the focal area. This sample also had both methylation (MSP-B) and missense mutations of the {gamma}-catenin gene. Reduced expression of {gamma}-catenin was found in the remaining four MSP-B-positive HRPCs. Nonsense mutation was observed in HRPC sample 6, where loss of {gamma}-catenin expression was restricted to only small foci (data not shown). In the HRPC tissue (sample 10), foci with nuclear {gamma}-catenin accumulation were always accompanied by both increased Bcl-2 expression and strong nuclear c-myc expression. In four of these HRPC samples except one (sample 10), Bcl-2 expression was increased but nuclear expression of c-myc was not found. These data are summarized in Fig. 5C. There were no mutations detected in localized prostate cancer samples, BPH samples, and prostate cancer cell lines.



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  		Figure 5. Epigenetic and genetic regulation of {gamma}-catenin in HRPC samples. A, methylation status of {gamma}-catenin and typical bisulfite DNA sequencing in HRPC. Five of eleven HRPC samples showed MSP-B bands, whereas all samples showed USP bands. Typical bisulfite DNA sequencing from HRPC sample 2 revealed that all CpG sites were unmethylated. The prevalence of methylation in HRPC samples is lower than in primary prostate cancer samples. B, expression of {gamma}-catenin protein and mutation in HRPC samples. In HRPC sample 4, a missense mutation (GAC -> GGC) with an amino acid change from ASP to Gly was found. Strong nuclear accumulation of {gamma}-catenin was observed. In HRPC 10, there were two different missense mutations (GGC -> GTC and AGC -> AAC), and the corresponding amino acid changes were from Gly to Val and from Ser to Asn, respectively. In this case, strong nuclear accumulation of {gamma}-catenin was focally observed (arrowhead) with reduced {gamma}-catenin expression in cancer cells (*) adjacent to the {gamma}-catenin accumulated foci. This sample was also positive for MSP-B. In HRPC 11, a missense mutation (GTG -> GCG) with an amino acid change from Val to Ala was found. Strong {gamma}-catenin expression was observed in nuclei (arrowhead) as well as cytoplasm. Six different mutations were found in five HRPC samples. Four mutations affected the putative consensus motif of the GSK-3ß phosphorylation site located at the NH2 terminal of the {gamma}-catenin gene. The locations affected by the two remaining mutations were outside but close to the GSK-3ß phosphorylation site. Underline, putative consensus motif of the GSK-3ß phosphorylation site; parenthesis, amino acid changes caused by the mutations. C, summary of {gamma}-catenin methylation and mutation in relation to Bcl-2 and c-myc immunostaining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanism underlying dysregulation of {gamma}-catenin in malignant cells is not fully understood. Recent publication on lung cancer suggests that CpG island methylation is one of the mechanisms contributing to the loss of {gamma}-catenin expression (18). However, such studies are lacking in prostate cancer. In the current study, after 5-aza-dC treatment, the expression of {gamma}-catenin mRNA transcripts was significantly increased in DuPro, LNCaP, ND-1, and PC-3 prostate cancer cell lines. Based on these results, we hypothesized that CpG hypermethylation of the {gamma}-catenin promoter might be one of the mechanisms responsible for its down-regulation.

As shown in Fig. 2A, the changes of {gamma}-catenin mRNA transcripts in prostate cancer cell lines coincided with the lack of the MSP bands. Before 5-aza-dC treatment, {gamma}-catenin expression and USP bands were simultaneously observed in these prostate cancer cell lines. Likewise, although {gamma}-catenin expression (protein and mRNA) was significantly lower in localized prostate cancer compared with BPH, both types of samples frequently exhibited USP and MSP bands (Fig. 2B). This data suggest that {gamma}-catenin expression is partially lost in prostate cancer. We further confirmed our methylation data using the sodium bisulfite method developed in our laboratory (26). Also, when the sequencing data is examined (Fig. 2C), the CpG sites in a sample do not always have the same percentage of methylation, suggesting that CpG sites are partially methylated. Thus, our data indicate that partially methylated CpG sites, as reflected by the simultaneous presence of MSP and USP bands, are a common characteristic of the {gamma}-catenin gene in human prostate cancer. In this study, we calculated the MSP-A and MSP-B ratio, which reflects the balance between methylated and unmethylated CpG sites, to evaluate the methylation status of the {gamma}-catenin promoter in individual prostate cancer samples. As shown in Fig. 3B-D, although the MSP-A and MSP-B ratios were significantly higher in localized prostate cancer than in BPH, {gamma}-catenin methylation was not related to Gleason sum, pT category, or capsular penetration. These findings suggest that CpG methylation of the functional {gamma}-catenin promoter is associated with its down-regulation and may be a very early event during the pathogenesis of prostate cancer.

The {gamma}-catenin gene locus on chromosome 17q21 is frequently a site for genetic events, such as LOH in prostate cancer (22). Recent studies have shown that some tumor suppressor genes are inactivated through biallelic events, such as LOH (25, 27). Therefore, we hypothesized that in addition to partial methylation, other mechanisms might be responsible for the significant reduction of {gamma}-catenin expression in prostate cancer tissue. We found that seven localized prostate cancer samples harbored both LOH at chromosome 17q21 and positive MSP-B methylation. In these samples, there was a significant inverse correlation of relative MSP-B ratios with {gamma}-catenin mRNA expression (Fig. 4A). This inverse correlation was less evident in the rest of the MSP-B-positive localized prostate cancer samples. For example, {gamma}-catenin expression was not reduced in LOH (–) and MSP-B (–) prostate cancer tissue (Fig. 4B). However, in prostate cancers that were LOH (+) and had partial methylation (coexistence of USP with MSP-A and MSP-B; Fig. 4C), or in prostate cancers with complete methylation (no USP but strong MSP-A and MSP-B; Fig. 4D), {gamma}-catenin expression levels were markedly reduced. These findings suggest that {gamma}-catenin expression is regulated by CpG promoter hypermethylation and genetic LOH in prostate cancer. At present, there are no reports on {gamma}-catenin inactivation in prostate cancer.

{gamma}-Catenin is very closely related to ß-catenin in amino acid sequence (5) and also has a GSK-3ß phosphorylation consensus motif at the NH2 terminal (5, 28). In localized prostate cancers, we found no mutations within the NH2-terminal regulatory region of {gamma}-catenin gene. On the other hand, in five HRPCs, six different mutations (five missense mutations and one nonsense mutation) were identified at the NH2-terminal regulatory region. As for {gamma}-catenin promoter methylation, there were positive MSP-B bands in five HRPC samples that were different from those samples harboring {gamma}-catenin mutations except for HRPC sample 10. The functional role of {gamma}-catenin in signal transduction is not fully understood. One gastric cancer cell line harboring a {gamma}-catenin mutation at the GSK-3ß phosphorylation site showed only a slight increase in TCF4-mediated transcriptional activation, compared with cell lines carrying mutation of ß-catenin or APC (29). This result indicates that the posttranslational effect of {gamma}-catenin mutation on the activation of the Wnt signal transduction may be minimal and potentially different from that caused by ß-catenin mutation. In the present study, four HRPC samples harboring {gamma}-catenin mutations exhibited strong nuclear staining of {gamma}-catenin with a concomitant increase in Bcl-2 expression. In contrast, c-myc, which is the target of Wnt signaling pathway, was not expressed in three of these four HRPCs. A recent study has shown that overexpression of {gamma}-catenin regulates cell growth through induction of the antiapoptotic factor Bcl-2 using human squamous carcinoma cells SCC9 (30). In agreement with this report, we also found that nuclear accumulation of {gamma}-catenin was associated with increased expression of Bcl-2 protein in HRPCs. Considering that (a) progression of prostate cancer is associated with inhibition of apoptosis and (b) Bcl-2 overexpression is a frequent event in advanced prostate cancer including HRPC (31), our results suggest that {gamma}-catenin mutation seems to be related to inhibition of apoptosis but not necessarily to cell proliferation through c-myc overexpression in HRPC.

This is the first report on the inactivation of the {gamma}-catenin gene through genetic and epigenetic pathways in prostate cancer. We conclude that CpG hypermethylation of {gamma}-catenin is an early event in the development of prostate cancer and that {gamma}-catenin expression is regulated by both CpG methylation and LOH in this disease. Our data further indicates that {gamma}-catenin mutation is related to inhibition of apoptosis, which might be one of the mechanisms underlying the development and/or progression of HRPC.


   Acknowledgments
 
Grant support: NIH grants RO1CA101844, RO1AG21418, and T32DK07790; VA Merit Review; and REAP award.

The costs of publication of this article were defrayed in part by the 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.

Received 9/18/04. Revised 12/ 6/04. Accepted 12/30/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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