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[Cancer Research 66, 4055-4064, April 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Detection of Recurrent Copy Number Loss at Yp11.2 Involving TSPY Gene Cluster in Prostate Cancer Using Array-Based Comparative Genomic Hybridization

Sapna Vijayakumar1, Devon C. Hall1, Xavier T. Reveles1, Dean A. Troyer2, Ian M. Thompson3, Dawn Garcia1, RuiHua Xiang1, Robin J. Leach1,4, Teresa L. Johnson-Pais4 and Susan L. Naylor1

Departments of 1 Cellular and Structural Biology, 2 Pathology, 3 Urology, and 4 Pediatrics, University of Texas Health Science Center, San Antonio, Texas

Requests for reprints: Susan L. Naylor, Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: 210-567-3842; Fax: 210-567-6781; E-mail: naylor{at}uthscsa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the second leading cause of cancer deaths among American men. The loss of Y chromosome has been frequently observed in primary prostate cancer as well as other types of cancer. Earlier, we showed that introduction of the human Y chromosome suppresses the in vivo tumorigenicity of the prostate cancer cell line PC-3. To further characterize the Y chromosome, we have developed a high-density bacterial artificial chromosome (BAC) microarray containing 178 BAC clones from the human Y chromosome. BAC microarray was used for array comparative genomic hybridization on prostate cancer samples and cell lines. The most prominent observation on prostate cancer specimens was a deletion at Yp11.2 containing the TSPY tandem gene array. Out of 36 primary prostate tumors analyzed, 16 (44.4%) samples exhibited loss of TSPY gene copies. Notably, we observed association between the number of TSPY copies in the blood and the incidence of prostate cancer. Moreover, PC-3 hybrids with an intact Yp11.2 did not grow tumors in nude mice, whereas PC-3 hybrids with a deletion at Yp11.2 grew tumors in nude mice. (Cancer Res 2006; 66(8): 4055-64)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Various cytogenetic studies conducted on prostate cancer specimens have identified a wide variety of genetic abnormalities, including gain or loss of whole or portions of chromosomes (1). Techniques like chromosome banding, fluorescence in situ hybridization (FISH), loss of heterozygosity, and comparative genomic hybridization (CGH) have been used to detect genetic alterations on cancer chromosomes. Additionally, the genetic aberrations can also be studied using microarray-based CGH (array CGH), which provides greater resolution (up to 30 kb; ref. 2) compared with 10-Mb resolution of conventional CGH. Array CGH can be done using various platforms, such as cDNA arrays (3), oligonucleotide arrays (4), and bacterial artificial chromosome (BAC) microarrays, to isolate submicroscopic genetic aberrations. Using array CGH, single clone variations were observed in cell lines established from the bone marrow of patients with prostate cancer (5). Many of these copy number changes were not evident by multicolor FISH or by conventional CGH. Furthermore, a study on prostate cancer cell lines using array CGH reported several new genetic alterations affecting the expression levels of various genes present in the affected regions (6). These observations show the potential of array CGH in discovering candidate tumor suppressor genes and/or oncogenes.

Using prior knowledge concerning a specific chromosomal aberration, custom arrays are fabricated and used for narrowing the minimal critical region of chromosomal deletion or duplication (7) and also in identifying the candidate gene involved in a specific cancer type (8). In prostate cancer, genome-wide screening using array CGH reported chromosomal losses to be more abundant than chromosomal gains (9). Previous studies have shown that the loss of the Y chromosome is a frequent genetic anomaly in prostate cancer (10). In the human prostate cancer cell line PC-3, the introduction of a Y chromosome (11) suppressed tumor formation in vivo, indicating the functional significance of the Y chromosome in prostate tumorigenesis. At present, there is limited information available about any regional losses on the Y chromosome in cancer (12). The whole genome arrays previously used in the analyses of chromosomal aberrations in prostate cancer (9, 12, 13) did not contain a high-resolution array (approximate resolution of 1 Mb) for the Y chromosome. Our present study is aimed at identifying the submicroscopic deletions or gains on the Y chromosome that are unique to prostate tumor. We have constructed a tiling path resolution BAC microarray for the human Y chromosome and used it to analyze prostate tumors, prostate cancer cell lines, and WBC. The results from these experiments showed a common deletion of Yp11.2 in prostate tumors, a tumorigenic PC-3 microcell hybrid, and prostate cancer cell line 22Rv1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary Tumor Samples
All studies on human tissue samples were conducted as per the regulation of the Institutional Review Board at the University of Texas Health Science Center at San Antonio. The tumor samples were from patients who had undergone prostatectomies at the Audie Murphy Veterans Administration Hospital, San Antonio, TX.

DNA Extraction
Paraffin-embedded tissue. For each individual tumor, DNA was extracted from four 10-µm tissue sections. The tumor region on each slide was identified using the corresponding H&E-stained slide, examined by a pathologist. The adjacent normal tissue from each slide was first removed using a sterile blade. The slides containing the remaining tumor tissue were immersed in xylene and incubated for 10 minutes at room temperature. The deparaffinization was repeated with fresh xylene for 10 minutes at room temperature. The slides were air-dried for 1 to 2 minutes and later washed twice in 100% ethanol at room temperature for 10 minutes each. After brief air-drying, the tumor section was scraped with a sterile needle and transferred to a sterile Eppendorf tube. The tissue was left in a sterile environment for an additional 10 minutes for complete drying. Before adding Proteinase K (Roche Diagnostics, Indianapolis, IN), the dried tissue was incubated in 1 mL of lysis buffer [10 mmol/L Tris-HCl (pH 8), 0.5% SDS, and 20 µg/mL RNase] at 65°C for 15 minutes and vortexed for 3 seconds. After the addition of Proteinase K (20 mg/mL) at a final concentration of 0.6 µg/µL, the tissue was incubated at 50°C in a water bath overnight. Proteinase K was added every 12 to 14 hours until the tissue was completely digested. The duration of digestion varied from 24 to 36 hours. Once the digestion was completed, a phenol/chloroform method of DNA extraction was used. Twenty micrograms of glycogen (Roche Diagnostics) was added to enhance the precipitation of DNA. The DNA pellet was washed once in ice-cold 75% ethanol and was resuspended in 50 to 100 µL of sterile water.

Blood. The blood samples were collected from patients registered with the San Antonio Center of Biomarkers of Risk for Prostate Cancer. Genomic DNA from blood samples was extracted using QIAamp DNA Blood Maxi kit (Qiagen, Valencia, CA). The DNAs were quantified spectrometrically and distributed as aliquots of 10 ng/µL in 96-well plates and stored at –20°C.

DNA from prostate cancer cell lines. The standard phenol/chloroform method was used for extracting genomic DNA from all cell lines used in the study.

Tissue Culture
Human prostate cancer cell lines, including 22Rv1, DU145, LNCaP, and PC-3, were purchased from the American Type Culture Collection (Manassas, VA) and grown in the suggested media and conditions. PC-3 hybrids were grown as previously described (11).

Array CGH
Slide manufacturing. From the Y chromosome tiling path (National Center for Biotechnology Information browser, 2002), 198 clones were selected and PCR verified using known sequence-tagged site markers or custom-made primers from the database sequence information. The BAC clones were purchased either from BACPAC Resources (Oakland, CA) or Research Genetics (Carlsbad, CA). DNA was extracted from 300-mL overnight cultures using a Plasmid Maxi kit (Qiagen) according to the manufacturer's protocol. The BAC DNAs were spotted in duplicate on microarray slides by Spectral Genomics (Houston, TX). The slide format was based on the company's Constitutional Chip array that allows experiments involving dye reversal to be conducted on the same slide. Several control clones preselected by Spectral Genomics were also added to the slide for data normalization.

Labeling and hybridization. All experiments were conducted in low-light conditions; 1.5 µg of either tumor DNA or normal male DNA (Promega, San Luis Obispo, CA) was diluted in 300 µL of sterile water and sonicated to yield a fragment size of 300 to 3 kb. Labeling of the fragmented DNA with Cy5 and Cy3 dyes (Perkin-Elmer, Wellesley, MA) was done according to the protocol recommended by Spectral Genomics. Hybridization was done in a microarray hybridization chamber (Fisher Scientific, Pittsburgh, PA) for 18 to 20 hours at 37°C in a hybridization oven with a rocking platform.

Washing and scanning. The slides were dipped in a Coplin jar containing 2x SSC (pH 7.4) at room temperature and agitated to remove the coverslips. The slides were then washed once in each of the following buffers: wash 1 (2x SSC, 50% deionized formamide; EM Science, Gibbstown, NJ; pH 7.4 for 20 minutes), wash 2 (2x SSC, 0.1% Igepal; Sigma, St. Louis, MO; pH 7.4 for 20 minutes), and wash 3 [0.2x SSC (pH 7.4) for 10 minutes], each at 42°C in a water bath with constant agitation. After the third wash, the slides were dipped in MilliQ water briefly and quickly dried using N2 gas at 40 p.s.i. The dried slides were immediately scanned using the GenePix4000A scanner (Axon Instruments, Union City, CA), and images were saved as single digital files.

Data processing. The scanned images were loaded onto SpectralWare, version 2.1 (Spectral Genomics). Data normalization was done by the software using the control points that contain a mixture of several BAC clones from the human genome, excluding the chromosomes 18, X, and Y. Further analyses of the images from the reverse labeling experiments were conducted using the SpectralWare.

Real-time Quantitative PCR
The primer and probe pairs were designed using the software Primer Express 2 (Applied Biosystems, Foster City, CA). DNA copy number variation: TSPY, 5'-AGAGCGTCCCTGGCTTCT-3' (forward) and 5'-GCCCATCGGTCACTTACACT-3' (reverse); albumin, 5'-AGGGTAAAGAGTCGTCGATATGCT-3' (forward; ref. 14) and 5'-CAATCTCAACCCACTGTCAGCTA-3' (reverse).

The probe (Applied Biosystems) sequences were TSPY, 5'-FAM-ATGTTGTATCCTTCTCAGTGTTTCTTCGG-NFQ-3' and albumin, 5'-VIC-CAAACGCATCCATTCTACCAACTTGAGCAT-TAMRA-3' (14).

PCR reactions were conducted for TSPY and albumin in 384-well plates using the 7900HT Sequence Detection System (Applied Biosystems) following the protocol recommended by the manufacturer. The reaction consisted of Taqman Universal PCR Master mix (Applied Biosystems) and TSPY (400 pg of DNA, 900 nmol/L of each primers, 200 nmol/L probe) and albumin (20 ng DNA, 300 nmol/L of each primers and 200 nmol/L probe). The PCR conditions used were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles each of 95°C for 15 seconds and 60°C for 1 minute. The results were analyzed by SDS software (Applied Biosystems). The standard curve method of quantification was used to calculate the relative TSPY copy number in the samples.

Quantification of mRNA levels. The following primers and probe were used to measure the mRNA expression of TSPY and CYorf16: TSPY, 5'-GGCTTCTGGGCCAATGTT-3' (forward) and 5'-TGCAGAGATGAACAGGATGC-3' (reverse); CYorf16, 5'-GTTTGGCTGAACATGACTGC-3' (forward) and 5'-TGAAGCTGGAGGGAGAGGTT-3' (reverse). Taqman probes were TSPY, 5'-FAM-CTCCAGCTGACCATGTAGCTCAGCA-TAMRA-3' and CYorf16, 5'-FAM-CACTTGACTGCAGCCTGGATGACACAG-TAMRA-3'.

Total RNA from frozen sections of prostate tumor samples and prostate cancer cell lines was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total RNA samples from normal human testis and prostate tissues (Clontech, Mountain View, CA) were used as controls. Taqman Reverse Transcription kit (Applied Biosystems) was used to synthesize the first-strand cDNA from these RNAs. Each reaction consisted of Taqman Universal PCR Master mix (Applied Biosystems), 900 nmol/L of TSPY or CYorf16 primers, and 200 nmol/L TSPY or CYorf16 probe. TATA-box binding protein expression assay (Applied Biosystems) was used for the endogenous control. Approximately 60 ng of cDNA was used in each reaction. The PCR cycling conditions were the same as those to detect DNA copy number.

Statistical analyses. The {chi}2 test was used to analyze the distribution of the copies of TSPY in the samples. The difference in the distribution of samples in the cancer and control groups was tested using a test for proportions. The Cochran-Amitage trend test was used to find any trend between copies of TSPY and cancer incidence. The statistical software containing these tests was from Software and Services (Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Quality testing of the array. Out of 198 BAC clones tested by PCR, 178 clones were selected from Ypter-Yqter to be processed for microarray preparation. The remaining 20 clones either tested negative using custom-made primers based on publicly available sequences for the corresponding clones, or the clones mostly contained repeats as detected by RepeatMasker (Institute for Systems Biology, Seattle, WA). There were 46 clones from the X chromosome, 165 clones from chromosome 18, and 20 clones selected from the remaining autosomes, making a total of 409 clones printed in duplicate on the array. The sensitivity of the custom-fabricated array to detect Y chromosome–specific clones was tested by comparing the hybridization of DNA from male and female samples with the microarray slides (Fig. 1 ). In the male/female experiment, 28 of 62 clones on Yp and 11 of 116 on Yq exhibited equal signal intensities in both male and female DNA, implying homology to other autosomes (Table 1 ). The remaining 139 clones showed greater hybridization signals from the male DNA. All clones on the X chromosome exhibited a higher copy number in female DNA, reflecting the dosage difference of the X chromosome in the two sexes. As expected, clones on the autosomes did not show any difference in male/female comparison (data not shown).


Figure 1
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  		Figure 1. The log2 ratio plots from a hybridization comparing normal male and normal female blood genomic DNA. X axis, base position (Mb) of the BAC clones; Y axis, Cy5/Cy3 log2 ratio. Blue curve, log 2 ratio obtained from Cy5-labeled female DNA and Cy3-labeled male DNA; red curve, ratio from the dye swap experiment. The Cy5/Cy3 ratio is a mean value obtained from duplicate clones spotted on the microarray slide. An amplified chromosomal region is identified when the blue plot (test, Cy5/reference, Cy3) is above the baseline (set at value 1), and the corresponding clone on the red plot (reference, Cy5/test, Cy3) is below the base line. A deletion on the other hand will have red plot above the baseline and blue plot below the baseline. A, ratio plot for Y chromosome. The straight lines between base positions 10 and 20 Mb indicate the centromeric region, and the lines after 30 Mb base positions depict the heterochromatin block on Yq. The region between positions 5.9 and 27.5 Mb, where red plot lies above the baseline, represents the Y chromosome–specific region as female DNA shows copy loss in the region. The BAC clones between positions 2.9 and 5.8 on Yp, where the two plots overlap, share homology to the X chromosome. B, ratio plot for X chromosome. The hemizygosity of the X chromosome in male DNA is clearly evident from the blue curve, which is above the baseline for all clones on the X chromosome.

 

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  		Table 1. BAC clones on the human Y chromosome sharing high homology to other chromosomes

 
Array CGH on prostate tumors. We have developed a high-resolution microarray for human Y chromosome and used it to further dissect the submicroscopic changes occurring on the Y chromosome in prostate tumors. The tumor DNA used in the analyses were extracted from frozen tissue, microdissected paraffin-embedded tissue sections, or prostate cancer cell lines. We obtained comparable array CGH results for DNA extracted from frozen tissue versus paraffin-embedded tissue (data not shown). A total of 36 primary prostate tumors with varying grades and Gleason scores were analyzed by array CGH (Table 2 ).


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  		Table 2. Prostate tumors with different pathologic grades showing various copy number changes on the human Y chromosome

 
The most common pattern of Y chromosome alteration (16 of 36 tumors) in prostate tumors is depicted in Fig. 2A (ii) . All data for these tumors are presented in Table 2. We also observed whole chromosome loss and loss of entire Yq in prostate tumor samples by array CGH (Table 2). DNA copy number changes were observed at multiple locations along the Y chromosome in prostate tumors. However, the most predominant copy number imbalance was the deletion of a ~700-kb region on proximal Yp. This segment of the chromosome is represented on the array by three BACs (RP11-441G8, RP11-344D2, and RP11-370N2). The public database reports only one functional gene, the testis-specific protein on Y (TSPY), a multicopy gene cluster, at this chromosomal region. Furthermore, a multicopy transcription unit cluster CYorf16 is also transcribed from the antisense strand of the TSPY gene cluster. The only other frequent deletion observed on Yp was more distal and often overlapped with clones RP11-218E11, RP11-492C2, and RP11-573O23. Remarkably, two copies of TSPY gene are also located in this region. An interstitial deletion proximal to the heterochromatin block on Yq was also seen in addition to Yp11.2 deletions in prostate tumors. Among the four prostate cancer cell lines tested (22Rv1, DU145, LNCaP, and PC-3), only 22Rv1 showed the 700-kb interstitial deletion at Yp11.2 and the Yq deletions. PC-3 showed complete loss of the Y chromosome. The other two cell lines did not show any obvious aberrations of the Y chromosome.


Figure 2
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  		Figure 2. A, distinct regions on the Y chromosome show copy number variation in prostate tumor. i, prostate tumor sample with a near intact Y chromosome. ii and iii, prostate tumor samples with deletions at various locations along the Y chromosome. A deletion corresponding to the red peak coinciding with a region on proximal Yp11.2 in the TSPY gene cluster. Amplification was seen between base positions 2.7 and 8.7 Mb on Yp and 13.5 and 21.9 Mb on Yq. This was the most common type of Y chromosome seen in prostate tumor (clone RP11-57J19 in the pseudoautosomal region on Yq has been removed from analysis in the last panel). B, compared with a tumorigenic PC-3 hybrid (ii), a nontumorigenic PC-3 hybrid (i) contained a near intact Yp. Prostate tumors and tumorigenic PC-3 hybrid shared similar Yq deletions, implying that these regions are not necessary for tumor suppression.

 
Array CGH on PC-3 hybrids containing the introduced human Y chromosome. In a previous study, we showed that an introduced Y chromosome suppressed tumor formation in PC-3, a human prostate cancer cell line lacking cytogenetically detectable Y chromosome, in nude mice (11). We compared one hybrid subclone (2-2C12E), which formed aggressive tumors in vivo, with its parental nontumorigenic PC-3 hybrid cell line 2-2C12. The array data showed that similar deletions were observed on Yq in both the tumorigenic and nontumorigenic PC-3 hybrids (Fig. 2B). However, the Yp11.2 deletions involving BACs RP11-441G8, RP11-344D2, and RP11-370N2 were seen only in the tumorigenic hybrid and not in the nontumorigenic hybrid. Furthermore, we did not observe any deletion of Yp by array CGH in another nontumorigenic PC-3 hybrid (data not shown). Because both tumorigenic and nontumorigenic PC-3 hybrids carried similar deletions on Yq, it implies that these deletions might not be as critical as the Yp11.2 deletion during prostate tumorigenesis.

Real-time quantitative PCR. Because four BACs (RP11-71C4, RP11-344D2, RP11-441G8, and RP11-370N2) constitute the multicopy TSPY gene array, we sought to analyze the deletion at Yp11.2 by determining the copy number of the TSPY gene. TSPY copy number in the tumors ranged from 0 to 41 (Fig. 3 ). The copy number data obtained using array CGH and quantitative PCR agreed with each other in most of the tumors (Table 3 ). An additional tumor (N36) showed TSPY copy loss by PCR. Tumors N29, N30, N31, and N37 showed greater copy loss of TSPY by quantitative PCR. Because the TSPY array is repeated within four BACs (RP11-441G8, RP11-344D2, RP11-71C4, and RP11-370N2), we were not able to show the microdeletion at Yp11.2 in the tumors by FISH using these BACs as probes (data not shown).


Figure 3
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  		Figure 3. The distribution of TSPY copy number in prostate tumors and blood samples. Real-time quantitative PCR was conducted to analyze for copies of TSPY, a multicopy gene cluster present at Yp11.2 where frequent copy number loss was observed in prostate tumors by array-CGH. A, TSPY copy range in prostate tumors. B, TSPY copy distribution in the blood samples of men with prostate cancer ({square}) and without prostate cancer ({diamondsuit}).

 

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  		Table 3. Correlation of the loss at Yp11.2 and the status of TSPY in prostate tumor

 
Irrespective of the TSPY copy number, TSPY mRNA was at low levels in prostate tumors (Table 3). Among the cell lines, TSPY was detected in 22Rv1 and LNCaP, whereas DU145 and PC-3 did not express TSPY. The relative expression level of TSPY mRNA in normal prostate was 100-fold lower than normal testis. The transcription unit CYorf16 was expressed at high levels in the normal testis sample, whereas it was not expressed in the normal prostate. We also analyzed mRNA expression of TSPY in nontumorigenic and tumorigenic PC-3 hybrids. In the tumorigenic PC-3 hybrid 2-2C12E, the mRNA expression of TSPY was ~10-fold lower compared with its nontumorigenic PC-3 hybrid 2-2C12. We analyzed blood samples from people with and without prostate cancer for TSPY copy number by quantitative real-time PCR. In the control population, 167 Caucasians and 151 Hispanic men were tested for TPSY copy number variation in WBC. The experimental group included 171 Caucasians and 112 Hispanic men who had prostate cancer. Because only a few prostate tumor samples were available from these men, a study comparing matched blood and prostate tumors could not be conducted. The TSPY copy number for all blood samples predominantly ranged between 20 and 30 copies (Fig. 3B). Based on these data, we have divided the prostate tumor and blood samples into three categories: <20 copies, 20 to 30 copies, and >30 copies of TSPY. For the tumor samples, information on race/ethnicity was not available. In the blood samples from men with prostate cancer, 29% of the samples had <20 TSPY copies, whereas only 12% of men without prostate cancer showed <20 TSPY copies (Fig. 4E ). Statistically significant overall association was present between TSPY copies in the blood and cancer incidence for the age group 50 to 70 years in Caucasian ({chi}2 = 11.03, P < 0.004) and Hispanic ({chi}2 = 8.06, P < 0.018) men. In the age group 71 to 90 years, the association between TSPY copies and prostate cancer incidence was less significant ({chi}2 = 3.31, P < 0.19 and {chi}2 = 3.05, P < 0.21 for Caucasian and Hispanic men, respectively). The difference in the proportion of samples between cancer cases and the normal control was highly significant in the group with <20 copies of TSPY (Fig. 4). In addition, statistically significant association of cancer incidence with decreasing number of TSPY copies was observed in both Caucasian (z = 1.63, P < 0.05 for age group 50 to 70 and z = 1.73, P < 0.04 for age group >70) and Hispanic (z = 2.23, P < 0.01 for age group 50 to 70 and z = 1.48, P < 0.07 for age group >70) men.


Figure 4
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  		Figure 4. Blood samples from Caucasian and Hispanic men show loss of TSPY gene copies. A, proportion of blood samples from Caucasian men in the age group of 50 to 70 with various TSPY copies. Empty columns, Caucasian men without prostate cancer; black columns, Caucasian men with prostate cancer. B, proportion of blood samples from Hispanic men in the age group of 50 to 70 with various TSPY copies. Empty columns, Hispanic men without prostate cancer; black columns, Hispanic men with prostate cancer. C, proportion of blood samples from Caucasian men >70 years old with various TSPY copies. Empty columns, Caucasian men without prostate cancer; black columns, Caucasian men with prostate cancer. D, proportion of blood samples from Hispanic men >70 years old with various TSPY copies. Empty columns, Hispanic men without prostate cancer; black columns, Hispanic men with prostate cancer. E, loss of TSPY copies is seen in prostate tumor and blood samples. On an average, the prostate tumor and blood samples contained 20 to 30 TSPY copies. The samples are classified into three groups: <20 copies, 20 to 30 copies, and >30 copies. A high proportion of prostate tumor samples showed <20 TSPY copies. *, statistically significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutations of the classic tumor suppressor gene p53 are rare in the primary prostate tumors and might not be the critical step involved in the progression to metastasis (15). Studies thus far regarding the genetic status of retinoblastoma in prostate cancer can not unequivocally resolve the issue concerning its involvement in the genesis of prostate cancer (16–18). Among the tumor suppressor genes, the expression of PTEN located on chromosome 10q22-q24 has been reported to be affected at high frequency in prostate cancer (19, 20). Several candidate hereditary prostate cancer genes have been identified (21), but none of these genes seem to be inactivated or lost in the majority of prostate cancers. The loss of Y chromosome is a frequent event in prostate cancer as well as in other types of cancer (10, 22–25). The percentage of whole Y chromosome loss has varied from study to study (10-53% of the prostate cancer cases analyzed; refs. 10, 26–28). It is not known, however, at which stage during prostate tumorigenesis the loss of Y chromosome occurs and how the genes on the Y chromosome contribute to tumorigenesis (29).

Using array CGH and real-time quantitative PCR, we have identified a novel and frequent deletion at Yp11.2 in prostate tumors. In a separate study, we found high incidence of TSPY copy loss in the blood of men who had prostate cancer compared with men who did not have prostate cancer. Previous reports have suggested that the average copy number of TSPY in a normal population is about 27 to 40 copies (30, 31). In our study, TSPY copy number distribution in prostate tumor and blood samples averaged around 20 to 30 copies. In both Caucasian and Hispanic men with <20 TSPY copies in blood, the incidence of prostate cancer was higher compared with rest of the samples. Moreover, the proportion of samples with >30 copies of TSPY was comparatively higher in the normal control relative to the cancer cases. This further suggests that fewer copies of TSPY and not increased copies of TSPY are associated with the pathologic condition. Because we observed a similar trend in both young and old men, we ruled out the possibility that the extreme loss of TSPY copies observed in prostate tumor is a random event induced due to aging. Whether the loss of TSPY copy number in the WBC of normal people indicates a risk factor for developing prostate cancer is at present unknown.

A previous study on prostate cancer described loss of BPY1, BPY2, SMCY, and RMBY on Yq (32). Our array CGH analyses on prostate tumors also showed frequent deletions on the distal Yq, removing copies of these multicopy genes on Yq. Our data and the earlier report (32) on prostate cancer imply that genomic instability is present on Yq. Because identical deletions on Yq were present in the nontumorigenic PC-3 hybrid and tumorigenic PC-3 hybrids, these deletions may not have a direct effect on prostate tumorigenesis.

Recent discoveries of large-scale copy number variation identified by microarray technology pinpoint the importance of the genomic architecture that facilitates duplications and deletions at highly homogeneous regions on the chromosomes (33, 34). In several genetic diseases, nonallelic homologous recombination has been implicated as the causative mechanism for genetic rearrangements, leading to gene inversion, duplication, or deletions (35). These types of recombination can be mediated through tandemly repeated genes as seen in {alpha}-thalassemia and ß-thalassemia gene clusters (36).

Unique low copy repeat sequences present at the pericentromeric regions have been shown to undergo homologous recombination, leading to genomic imbalance causing severe phenotypes (37, 38). We speculate that the TSPY gene cluster located close to the centromere on Yp has the potential to undergo genomic rearrangement through homologous recombination. Each TSPY gene is contained within a 20.4-kb repeat tandemly repeated into one long array at Yp11.2 (31). There is >99% sequence similarity between these repeats. Because these are direct repeats, recombination between the TSPY repeat units can lead to deletion or duplication. The genomic architecture of the Y chromosome in humans as well as nonhuman primates is marked by long palindromes whose arms share 99.940% to 99.997% identities at the nucleotide level (39). It was shown that some of these palindromes on Yq mediate nonallelic homologous recombination, resulting in massive deletions of >1 Mb in size and leading to reproductive failure in men (40). Because these deletions on Yq do not result in the complete loss of any of the multicopy genes located within the region, the gene dosage may contribute to the pathologic condition (41). Whether the TSPY gene cluster also imparts such gene dosage effects in prostate tumorigenesis is not clear. In our study, we rarely detected the expression of TSPY in tumors. The reason for the lack of TSPY expression in those tumors that do not show any significant copy loss is at present unknown. It has been reported that in high-grade melanomas, TSPY undergoes epigenetic silencing (42).

Due to its homology to suppressor of variegation, enhancer of zeste and trithorax (SET) domain, TSPY has been proposed to function as an oncogene in gonadoblastoma (43). In contrast, a recent report suggested that the expression of TSPY is lost during tumor progression in melanoma (42). Our study also shows preferential loss of TSPY in prostate tumor samples and lower TSPY expression than was found by Lau et al. (43). Whether TSPY genes function in a tissue context manner is not clear. Additionally, the loss of TSPY may be a later event during prostate tumorigenesis. At this time, we do not know whether the loss of TSPY coincides with advanced stages of prostate cancer. In our study, prostate samples analyzed were Gleason ≥5. There are no published reports of any kind supporting the functional role of TSPY. It was shown that TSPY undergoes cryptic exon splicing and intron skipping to generate a heterogeneous population of mRNA in both testis and prostate cells (43). Apart from an alternative splicing mechanism that can generate proteins with varied functions, epigenetic silencing regulates the expression of TSPY (44). Although TSPY is the only functional gene located at Yp11.2, a tandem array of transcription unit CYorf16 is transcribed from the antisense strand. In our study, we did not find CYorf16 to be transcribed in prostate tissue.

It is not known whether the preferential loss of TSPY copies seen in prostate tumors is the initiator for prostate tumorigenesis or is the result of a prior genomic defect that occurred elsewhere in the genome. Increased expression of wild-type RAD51, a key player in the double-strand break repair pathway that is the initial step involved in homologous recombination, has been observed in several cancer cell lines, including prostate cancer cells (45). Although elevated levels of other proteins involved in double-strand break repair, such as RAD52, RAD54, and XRCC3, are seen in prostate cancer cell lines (46), their status in prostate tumors is mostly unknown. Furthermore, chromosome 8q, a region often amplified in prostate cancer (47) contains RAD54B.

In conclusion, we have identified a novel recurrent deletion at Yp11.2, involving the TSPY gene cluster, in prostate tumors. Furthermore, the loss of copies of TSPY in WBC is associated with an increased incidence of prostate cancer. The genomic architecture of this region is unique in that it contains the largest tandem repeat array known in the entire human genome. Even more interesting is the fact that both strands are transcribed giving rise to two different groups of transcripts. Moreover, TSPY, the better-studied gene of the two, undergoes tissue-specific alternative splicing, yielding potentially a wide variety of proteins. The genomic structure of the region possibly marks it as a "hotspot" for rearrangements, leading preferentially to deletions in prostate tumorigenesis. Further studies will provide a better insight about whether these genetic rearrangements lead only to deletion of copies of TSPY gene in prostate cancer, or it also generate novel hybrid genes within the cluster, as is the case in opsin gene rearrangements, leading to red-green color blindness (48).


   Acknowledgments
 
Grant support: Department of Defense grants DAMD17-99-1-9469 (S.L. Naylor) and DAMD17-2-1-0044 (T.L. Johnson-Pais), National Cancer Institute's Early Detection Network grants U01 CA86402 and U01 CA84986, San Antonio Cancer Institute grant P30 CA54174, Cancer Center Council, and American Cancer Society grant TURSG-03-152-01-CCE (R.J. Leach).

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 10/21/05. Revised 1/11/06. Accepted 2/10/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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