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Cancer Research 67, 9649, October 15, 2007. doi: 10.1158/0008-5472.CAN-06-4111
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Gene Amplification and Overexpression of PRDM14 in Breast Cancers

Noriko Nishikawa1,2, Minoru Toyota1,3,5, Hiromu Suzuki4, Toshio Honma2, Tomoko Fujikane1,2, Tousei Ohmura2, Toshihiko Nishidate2, Mutsumi Ohe-Toyota1, Reo Maruyama3, Tomoko Sonoda4, Yasushi Sasaki1, Takeshi Urano6, Kohzoh Imai3, Koichi Hirata2 and Takashi Tokino1

1 Department of Molecular Biology, Cancer Research Institute, 2 First Department of Surgery, 3 First Department of Internal Medicine, and 4 Department of Public Health, Sapporo Medical University, Sapporo, Japan; 5 PRESTO, Japan Science and Technology Corporation, Kawaguchi, Japan; and 6 Department of Biochemistry, Shimane University Faculty of Medicine, Izumo, Japan

Requests for reprints: Minoru Toyota, First Department of Internal Medicine, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo 060-8543, Japan. Phone: 81-11-611-2111, ext. 3211; Fax: 81-11-618-3313; E-mail: mtoyota{at}sapmed.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several genes that encode PR (PRDI-BF1 and RIZ) domain proteins (PRDM) have been linked to human cancers. To explore the role of the PR domain family genes in breast carcinogenesis, we examined the expression profiles of 16 members of the PRDM gene family in a panel of breast cancer cell lines and primary breast cancer specimens using semiquantitative real-time PCR. We found that PRDM14 mRNA is overexpressed in about two thirds of breast cancers; moreover, immunohistochemical analysis showed that expression of PRDM14 protein is also up-regulated. Analysis of the gene copy number revealed that PRDM14 is a target of gene amplification on chromosome 8q13, which is a region where gene amplification has frequently been detected in various human tumors. Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 by siRNA induced apoptosis in breast cancer cells and increased their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 may play an important role in the proliferation of breast cancer cells. That little or no expression of PRDM14 is seen in noncancerous tissues suggests that PRDM14 could be an ideal therapeutic target for the treatment of breast cancer. [Cancer Res 2007;67(20):9649–57]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the past few years, much progress has been made toward a better understanding of the molecular mechanisms involved in breast cancer. Among these are mechanisms by which gene expression is regulated, including the reversible modification of core histones through acetylation, phosphorylation, or methylation (1). The methylation of lysine residues on histone tails is catalyzed by enzymes containing a conserved Su(var)3-9, Enhancer-of-zeste and Trithorax (SET) domain. Such histone methyltransferases control epigenetic inheritance through transfer of methyl groups from the methyl donor S-adenosylmethionine to basic residues on histones (2). Methylation events driven by these enzymes have, in some cases, been implicated in gene silencing and carcinogenesis. For instance, up-regulated expression of one of the polycomb group genes, EZH2, which encodes a protein with H3-K27 histone methyltransferase activity, is associated with a high cell proliferation rate and aggressive breast cancers (3). The expression of SMYD3, which also contains a SET domain, also is up-regulated in human breast cancer (4), and the variable number of tandem repeats polymorphism in the SMYD3 gene promoter region is associated with an increased risk of breast cancer (5, 6).

The PRDI-BF1 and RIZ homologous (PR) domain proteins (PRDM) are a subclass of the SET domain proteins that was first noted in the homologous region shared by RIZ1/PRDM2 and BLIMP1/PRDM1 (7) and also show homology with SET domain proteins related to known histone methyltransferases (8, 9). Although the role of PRDMs in modulating transcription remains unclear, recent studies indicate that they do play a role in tumorigenesis. For instance, both RIZ1/PRDM2, which encodes a Rb-binding protein, and BLIMP1/PRDM1, which encodes a c-Myc transcription repressor and promotes B-lymphocyte maturation (7, 10–13), induce growth arrest and exhibit proapoptotic activities (12, 14). In addition, the MDS1-EVI1/PRDM3 gene is commonly mutated in myeloid leukemia (15), whereas the PMF1/PRDM4 gene is located at a tumor suppressor locus on 12q23-q24 (16), and its overexpression inhibits DNA synthesis (17). Finally, expression of both PRDM5 and RIZ1/PRDM2 is commonly silenced through CpG island promoter DNA methylation in several cancer types including breast, ovarian, and liver cancers (18, 19).

In the present study, we examined the expression profile of PR domain family genes in breast cancers. We found that among the 16 members, PRDM14 is frequently overexpressed and is amplified in breast cancer. Our data suggest that PRDM14 could harbor an oncogenic property and function as a novel molecular target in the treatment of these tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and specimens. Seven breast cancer cell lines (MCF7, MB231, MB435, MB436, MB468, SKBr-3, and T47D), 7 gastric cancer cell lines (MKN7, MKN28, MKN74, AZ521, KatoIII, JRST, and SNU638), and 15 ovarian cancer cell lines (SKOV-3, OVCA-3, PA-1, Caov-3, MH, KURA, AMOC-2, MCAS, KF, KFr, HTBOA, TOV-21G, SW626, TOV-112D, and OV-90) were obtained from the American Type Culture Collection or the Japanese Collection of Research Bioresources. All cell lines were cultured in appropriate medium supplemented with 10% fetal bovine serum and incubated under a 5% CO2 atmosphere at 37°C. In addition, 55 breast cancer specimens and 8 breast tissue samples from areas adjacent to tumors were obtained from Sapporo Medical University Hospital at surgery and stored at –80°C. In accordance with institutional guidelines, all patients gave informed consent before collection of the specimens. Genomic DNA was extracted using the phenol/chloroform method. Total RNA was extracted from cell lines using Trizol (Life Technologies, Inc.) according to the manufacturer's instructions.

Real-time PCR. The reverse transcriptase reaction was carried out with 5 µg of total RNA using a SuperScript II First-Strand Synthesis System (Invitrogen, Inc.) with random primers. Relative real-time PCR for detection of gene expression levels was carried out using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Primers and probes were purchased from Applied Biosystems. The reaction mixture (total volume 20 µL) contained ~10 ng of cDNA, primers, and probes at final concentrations of 300 and 200 nmol/L, respectively, as well as commercial reagents (Universal Master Mix, Applied Biosystems). Relative levels of gene expression were quantified using the {Delta}{Delta}Ct method, which results in a ratio of target gene expression to equally expressed housekeeping genes. For calibration, the ratio in a sample of normal breast tissue from an area adjacent to the tumor was determined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the housekeeping gene. The primer/probe sets used were as follows: PRDM1, Hs00153357; PRDM2, Hs00210612; PRDM3, Hs00602795; PRDM4, Hs00183764; PRDM5, Hs00218855; PRDM6, Hs01373000; PRDM7, Hs00364862; PRDM8, Hs00220274; PRDM9, Hs00360639; PRDM10, Hs00360651; PRDM11, Hs00220293; PRDM12, Hs00222080; PRDM13, Hs00222082; PRDM14, Hs00225842; PRDM15, Hs00411330; PRDM16, Hs00223161_A1; CEACAM6, Hs00366002_m1; CEACAM7, Hs00185152_m1; CCDC3, Hs00230222_m1; PEG10, Hs00248288_s1; POT1, Hs00209984_m1; GATA3, Hs00231122_m1; IGFBP7, Hs00266026_m1; ID4, SEMA3B, Hs00190328_m1.

Analysis of DNA copy numbers in breast cancers. Real-time PCR to determine genomic DNA copy numbers was carried out using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems); information on the PCR primers is available on request. The {Delta}{Delta}Ct method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate the copy numbers of five genes. In addition, the 2–{Delta}{Delta}Ct method of relative quantification (described in detail in ref. 20) was adapted to estimate the copy numbers of five genes. This method, which enabled us to estimate gene copy numbers in unknown samples, had two main prerequisites. The first was the existence of at least one calibrator consisting of template DNA with a known copy number of each of the studied genes. The second was the need to have a housekeeping gene whose copy number was the same in all samples. This enables normalization of the quantitative data. In this work, albumin was used as the calibrator, whereas GAPDH served as the housekeeping gene in all experiments. For quantitative PCR carried out with genomic DNA, we used a cutoff ratio of 2.2 to define genomic amplification.

Immunohistochemical and immunocytochemical analyses of PRDM14. Formalin-fixed, paraffin-embedded sections of human breast carcinomas in 10 mmol/L sodium citrate were heated in a microwave oven at maximum power for 15 min and then at a reduced power for 15 min to achieve antigen recovery. After they were blocked in a 5% solution of bovine serum albumin in PBS also containing 0.5% Tween 20, the sections were incubated with rabbit anti-human PRDM14 antibody (1:100 dilution; Abcam) overnight at 4°C. The sections were then developed using EnVision-Plus reagents (DAKO Corp.), with 3,3'-diaminobenzidine serving as the chromogen, and counterstained with hematoxylin, after which microscopic images were captured digitally. The results of PRDM14 immunostaining in breast cancers were scored independently by two pathologists (H.H. and M.S.) blinded to the protocol who concurred in the scoring.

For immunofluorescent staining of PRDM14, cells were fixed in 3.7% formalin solution for 10 min at 25°C, washed with PBS, and then incubated overnight with rabbit anti-human PRDM14 (ABGENT). The nuclei were stained with Vectashield mounting medium with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.).

Fluorescence in situ hybridization. Formalin-fixed paraffin-embedded tissues were used for fluorescence in situ hybridization (FISH) analysis. BAC clones (RP11-152C15) containing the genomic sequences of the 8q.13.3 amplicon were purchased from Invitrogen. BAC probe was labeled using the Nick Translation method, hybridized at 37°C for 16 h, and then incubated with anti-digoxigenin-Cy3. Nuclei were stained with DAPI. Signals were detected using a fluorescence microscope (Leica CW-4000).

Colony formation and cell growth assay. Cells (1 x 106) were transfected with 5 µg of pCMV-PRDM14 or pCMV-Tag2A expression vector using a Nucleofector Electroporation System (Amaxa). They were then plated in 60-mm culture dishes, selected for 14 days in medium containing G418, and stained with Giemsa. Experiments were carried out in triplicate and colonies were counted using NIH IMAGE software.

Anchorage-independent growth assays were done as previously described (21). MCF10A breast epithelial cells were transfected with mutant H-ras, PRDM14, or mutant Ha-ras + PRDM14. A layer of 1% agar in McCoy's medium was poured and allowed to solidify. Transfected cells (1 x 103) were suspended in a 0.36% bactoagar supplemented with McCoy's 5A medium and overlaid on the agar in triplicate wells. After 3 weeks, images of the colonies were captured with an Olympus IX71 microscope using the Metaview software (Universal Imaging Corp., Molecular Devices).

To assess cell growth, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out using SKBr-3 cells stably expressing PRDM14; cells transfected with pCMV-Tag2 and thus stably expressing Flag-tag served as a control. The cells (1 x 104) were plated in 96-well plates and incubated with 50 µmol/L cis-diammine-dichloroplatinum (CDDP), 50 µmol/L etoposide (VP-16), 1 mmol/L docetaxel, or 200 ng/mL Adriamycin. After 48 h, cell viability was assessed in MTT assays.

Gene expression profile in SKBr-3 cells stably expressing PRDM14. Microarray experiments were carried out using the Agilent Whole Human Oligo Microarray 41K platform (Agilent Technologies). Total RNA was extracted with RNAeasy (Qiagen) from two clones stably expressing PRDM14 and used as a template for synthesis of Cy5-labeled cDNA probes. Total RNA from SKBr-3 cells stably expressing pCMV-Tag2 vector also was labeled with Cy-3 and served as a control. The hybridized slides were scanned with a Microarray Scanner (Agilent Technologies).

Knockdown of PRDM14. For PRDM14 knockdown studies, 1 x 106 PA-1 cells plated in 60-mm plates were transfected using an Amaxa electroporation system (Amaxa). siRNA was designed by B-Bridge International, Inc. Three siRNA sequences that target PRDM14 were 5'-CCAGUGAAGUGAAGACCUATT-3' (siPRDM14-1F) and 5'-UAGGUCUUCACUUCACUGGTT-3' (siPRDM14-1R), 5'-GGACAAGGGCGAUAGGAAATT-3' (siPRDM14-2F) and 5'-UUUCCUAUCGCCCUUGUCCTT-3' (siPRDM14-2R), and 5'-GGGAAAAUCUUCUCAGAUCTT-3' (siPRDM14-3F) and 5'-GAUCUGAGAAGAUUUUCCCTT-3' (siPRDM14-3R). The sequences of the three control siRNA oligos used were 5'-ATCCGCGCGATAGTACGTA-3' (siRNA-control-1), 5'-TTACGCGTAGCGTAATACG-3' (siRNA-control-2), and 5'-TATTCGCGCGTATAGCGGT-3' (siRNA-control-3). Cells were harvested after 48 h and the RNA was isolated using an RNAEasy extraction kit (Qiagen). Cell growth was assessed by counting the numbers of cells. In some instances, the cells were treated with 50 µmol/L CDDP or 1 mmol/L docetaxel 48 h after transfection. After treatment with siRNA and/or drugs, the cells were harvested, fixed in 70% ethanol, incubated with 2 mg/mL RNase, and stained in 50 µg/mL propidium iodide solution. Approximately 5 x 104 stained cells were then analyzed with a Becton Dickinson FACScan flow cytometer.

Statistics. The statistical analysis was carried out using StatView software (SAS Institute, Inc.). Fisher's exact test (two-sided) was used to determine the association between PRDM14 methylation and clinicopathologic features. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression profile of PR domain family genes in breast cancers. We initially carried out quantitative real-time PCR analysis of PRDM1 to PRDM16 in a panel of primary breast cancers to characterize the expression profile of PR domain family genes (Fig. 1A and B ). We found that expression of PRDM5 was down-regulated in breast cancer, which is consistent with results previously reported (18), whereas we frequently noted overexpression of PRDM14. To examine the expression of PRDM14 in more detail, we determined the relative levels of PRDM expression in a panel of 55 primary breast cancer specimens and 7 cell lines (Fig. 2 ). We found that 3 of 4 (75.0%) ductal carcinoma in situ, 33 of 51 (64.7%) invasive tumors, and 4 of 7 (57.1%) breast cancer cell lines showed levels of PRDM14 that were >5-fold higher than in noncancerous breast tissues. Moreover, immunohistochemical analysis of PRDM14 protein expression with an anti-PRDM14 antibody (Supplementary Fig. S1) revealed strong staining within the cancers but not in noncancerous stromal and ductal cells (Fig. 3 ). We also found that expression of PRDM14 was often up-regulated in gastric and ovarian cancers (Fig. 2).


Figure 1
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  		Figure 1. Analysis of PR domain family gene expression in breast cancer. A, levels of expression of PRDM1-16 in breast cancer tissues relative to normal breast tissues were determined by real-time PCR. Y-axis, PRDM14 levels in breast cancer tissue normalized to normal breast tissue. Case numbers are shown below the column. B, expression profile of PRDM family genes in breast cancer. The expression level of each PRDM gene in the indicated samples is shown according to a pseudocolor gradient relative to the expression in normal breast epithelium.

 

Figure 2
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  		Figure 2. Quantitative analysis of PRDM14 expression in various tumors and normal tissues. Y-axis, levels of PRDM14 expression normalized to GAPDH. Tissue types are shown below the columns. Columns, mean of triplicates; bars, SE. DCIS, ductal carcinoma in situ.

 

Figure 3
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  		Figure 3. Representative results of immunohistochemical staining of PRDM14 in breast cancer tissues. Left, breast cancer specimens expressing high levels of PRDM14 mRNA (cases 97 and 96); right, specimens expressing low or negligible levels of PRDM14 mRNA (cases 15 and 106).

 
Correlation between overexpression and amplification of PRDM14. PRDM14 is located on chromosome 8q in region 8q13.3, where gene amplification has frequently been observed in human tumors including those of the breast (22, 23). Therefore, to learn whether overexpression of PRDM14 is associated with gene amplification, we used quantitative real-time PCR to determine the gene copy numbers in the region spanning 8q13-8q24 in 23 tumors expressing various levels of PRDM14 mRNA. We found that, indeed, the copy numbers of PRDM14 gene were relatively high in tumors that overexpressed PRDM14 mRNA (Fig. 4A and B ).


Figure 4
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  		Figure 4. Amplification of PRDM14 in breast cancers. A, relative copy numbers of five genes located around the PRDM14 locus and the relative expression levels of PRDM14. The loci examined are shown on the top. Expression of PRDM14 was significantly higher in tumors with gene amplification than in those without amplification (P = 0.014). B, FISH analysis. Validation of 8q13.3 amplification was carried out using FISH analysis in three representative specimens using a probe located at 8q13.3. C, expression profile of the 8q13.3 amplicon in breast cancers. Real-time PCR was carried out for eight genes located in the 8q13.3 region. The expression level of each gene in the indicated samples is shown according to a pseudocolor gradient relative to the expression in normal breast epithelium. bullet, amplification; {circ}, no amplification. Sample numbers are shown on the left.

 
To determine whether other genes in the 8q.13.3 amplicon are also up-regulated in tumors, we examined expression of eight genes in a panel of breast cancer specimens, some of which showed gene amplification whereas others did not (Fig. 4C). Only low levels of expression were detected for SLCO5A1, SULF1, TRAM1, LACTB2, XKR9, and EYA1 in breast cancers despite amplification. By contrast, expression of NCOA2 was high regardless of gene amplification, indicating that NCOA2 expression is regulated by molecular mechanisms other than amplification.

When we correlated expression of PRDM14 within the tumors with the clinical data obtained from these patients, we found no correlation between the levels of PRDM14 expression and any of the clinicopathologic characteristics of the patients, which likely reflects the small number of samples analyzed (data not shown).

Role of PRDM14 in cell growth and resistance to chemotherapeutic drugs. To obtain information about the function of PRDM14, we examined the effects of its overexpression in breast cancer cells. As shown in Fig. 5A , PRDM14 was localized in the nucleus, which is consistent with the fact that it has a PR domain and that it plays a role in gene regulation. To assess the effect of PRDM14 on cell growth, we carried out colony formation assays after transfecting MCF7 and SKBr-3 cells, which express only low levels of endogenous PRDM14, with pCMV-PRDM14 or pCMV-Tag2A. Following transfection, cells expressing PRDM14 showed markedly higher numbers of colonies than cells expressing Flag-tag (a result that was confirmed in three independent experiments; Fig. 5B). In addition, anchorage-independent growth assays using two independent clones stably expressing PRDM14 revealed that overexpression of PRDM14 enhances the ability of cells to grow without first attaching to a substrate (Supplementary Fig. S2). Apparently, overexpression of PRDM14 leads to increased breast cancer cell growth.


Figure 5
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  		Figure 5. Oncogenic activity of PRDM14. A, immunofluorescence analysis of PRDM14 in breast cancer cells. SKBr-3 cells were transfected with pCMV-Tag2A or pCMV-PRDM14 and then labeled with anti-PRDM14 antibody. The nucleus was stained with DAPI. B, overexpression of PRDM14 enhances colony growth of breast cancer cells. MCF7 and SKBr-3 cells were transfected with either pCMV-Tag2A (control plasmid encoding Flag-tag) or pCMV-PRDM14 and then selected by incubation with 0.7 mg/mL G418. After 14 d, plates were stained with Giemsa solution and the colonies were counted. C, ectopic expression of PRDM14 reduces the sensitivity of SKBr-3 cells to chemotherapeutic drugs. Two clones [SKBr-3-PRDM14-8 (P-8) and SKBr-3-PRDM14-9 (P-9)] showing stable expression of PRDM14 driven by the CMV promoter were established. The cells were then treated with 50 µmol/L CDDP, 50 µmol/L VP-16, 0.2 mg Adriamycin, or 1 µmol/L docetaxel for 48 h, after which cell viability was assessed by MTT assays. Columns, mean from three independent experiments; bars, SE; *, P < 0.01, versus control [SKBr-3-pCMV-Tag2A (V)]. D, real-time PCR analysis confirming the results of the microarray analysis. Expression of putative PRDM14 target genes was examined using cDNA from two clones stably expressing PRDM14 (P-8 and P-9). One clone stably expressing Flag-tag served as a control (V).

 
We then assessed the capacity of PRDM14 to mediate resistance to chemotherapeutic drugs by treating stable PRDM14 transfectants with CDDP, VP-16, Adriamycin, or docetaxel. As shown in Fig. 5C, cells stably expressing PRDM14 were significantly more resistant to the drugs than cells expressing Flag-tag.

Identification of genes regulated by PRDM14. Because PRDMs are involved in the regulation of gene transcription, we next used a microarray to examine the effect of overexpressing PRDM14 on gene expression and compiled a list of PRDM14-responsive genes. These genes were defined as exhibiting a >3-fold up-regulation or 2-fold down-regulation in the presence of PRDM14 (PRDM14-transfected SKBr-3/pCMV-Tag2–transfected SKBr-3 ratio). We found that overexpression of PRDM14 led to up-regulation of 116 genes and down-regulation of 107 genes in both the SK-P8 and SK-P9 clones (Supplementary Tables S1 and S2). These results were confirmed by real-time PCR, which showed that the expression levels of CEACAM6, CEACAM7, CCDC3, PEG10, POT1, and GATA3 were all significantly higher in cells stably expressing PRDM14 than in cells expressing pCMV-Tag2. Conversely, expression of IGFBP7, ID4, and SEMA3B was significantly down-regulated in SKBr-3 cells stably expressing PRDM14 (Fig. 5D).

Enhancement of Ha-ras–induced transformation of MCF10A cells by PRDM14. To assess the effect of overexpression of PRDM14 in untransformed cells, we evaluated the capacity of PRDM14 to induce transformation of MCF10A breast epithelial cells. As previously observed (21), transfection of MCF10A cells with activated H-ras induced cell proliferation and colony formation in soft agar. By contrast, introduction of PRDM14 into MCF10A cells did not induce proliferation, but it did enhance the growth induced by activated Ha-ras (Fig. 6A and B ). Thus, PRDM14 seems to act in concert with a growth signal to facilitate cellular transformation.


Figure 6
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  		Figure 6. Role of PRDM14 in cell transformation, growth, and apoptosis. A, PRDM14 enhanced Ha-ras–induced anchorage-independent growth of MCF10A cells. MCF10A cells were transfected with mutant Ha-ras, PRDM14, or mutant Ha-ras + PRDM14 and then plated in soft agar. After incubation for 3 wk, colonies were analyzed by Metaview software. B, numbers of colonies of Ha-ras, PRDM14, and Ha-ras + PRDM14 transfectants were counted. Columns, mean from three independent experiments; bars, SE; *, P < 0.05; **, P < 0.01. C, knockdown of PRDM14 by siRNA in the cancer cell lines. PA-1 and SK-P8 cells, which constitutively express high levels of PRDM14, were transfected with control or PRDM14-targeted siRNA (si-control and siPRDM14, respectively). After 48 h, the cells were harvested and expression of PRDM14 was assessed by quantitative real-time PCR. D, knockdown of PRDM14 suppresses cell growth. PA-1 and SK-P8 cells treated with either si-control or siPRDM14 were grown for 48 or 96 h, after which cell numbers were counted. Columns, mean from three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, versus si-control.

 
Down-regulation of PRDM14 suppresses cell growth. To further investigate the functional consequences of aberrant expression of PRDM14, we knocked down PRDM14 expression using siRNA in PA-1 cells, which constitutively express high levels of PRDM14. For this experiment, we first determined the optimal conditions for introduction of the siRNA using an Amaxa electroporation system with a vector encoding green fluorescent protein. By adjusting the conditions, we were ultimately able to express green fluorescent protein in ~80% of cells (data not shown). Under those optimized conditions, introduction of a mixture of three siRNA oligos targeting PRDM14 in PA-1 cells resulted in an 85% reduction in PRDM14 mRNA levels, as compared with cells transfected with control siRNA (Fig. 6C). When we then tested whether knocking down PRDM14 would affect cell growth, we found that cell numbers were significantly diminished by transfection with PRDM14 siRNA, as compared with transfection with control siRNA (Fig. 6D). Down-regulation of PRDM14 in SKBr-3 cells overexpressing the gene also reduced cell growth (Fig. 6D). Expression of two PRDM14 target genes, CEACAM6 and GATA3, was also down-regulated in PA-1 cells transfected with PRDM14 siRNA (Supplementary Fig. S3A). Conversely, expression of IGFBP7 was up-regulated when PRDM14 was knocked down. Finally, we examined the effect of knocking down PRDM14 on the sensitivity of cells to chemotherapeutic drugs. We found that PRDM14 knockdown induced apoptosis in PA-1 cells (Supplementary Fig. S3B) and also increased the susceptibility of cells to apoptosis induced by CDDP and docetaxel. The apoptotic effects of down-regulating PRDM14 and of the two chemotherapeutic drugs were additive, however, suggesting they act via independent pathways.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we analyzed the expression profiles of PR domain family genes in a panel of breast cancers. Our results indicate that expression of PRDMs in malignant breast tumors differs from that in normal breast tissues. For instance, we found that PRDM5 was down-regulated in breast tumors, which is consistent with the earlier finding that PRDM5 is methylated in breast cancers (18) and confirms that our high-throughput approach was reliable enough for valid characterization of the gene expression profile. In addition, we found that although there is little or no expression of PRDM14 in noncancerous tissues, it was frequently overexpressed in human breast cancer tissues and that this up-regulation is associated with gene amplification. Our results are consistent with the report by Hu et al. (24) showing that PRDM14 is expressed in breast cancer but not in normal breast epithelium. That overexpression of PRDM14 was observed in 75.0% of ductal carcinoma in situ and 64.7% of invasive breast tumors suggests that its amplification may occur at a relatively early stage of tumorigenesis. Moreover, our finding that overexpression of PRDM14 significantly increases the growth of breast cancer cells whereas knocking down PRDM14 reduces their growth and induces apoptosis suggests that PRDM14 could be a useful therapeutic target for treating primary breast cancers.

This comprehensive study, which included analyses of molecular genetics, gene transcription, and functional characterization of PRDM14, provides compelling evidence that amplification of PRDM14 is involved in breast carcinogenesis. Amplification of regions of chromosome 8q is frequently detected in malignant tumors, including tumors of the breast (25), which are often associated with amplification of the 8q24 region where c-myc is situated (26). Studies have suggested that several other candidate oncogenes are also located on chromosome 8q (27–29). The 8q13 region, where PRDM14 is located, is known to be amplified in breast cancer (22, 23). Given that there is often a correlation between copy number and expression level, it is not surprising that breast cancer tissue in which we detected increased copy numbers of PRDM14 gene also showed significantly higher levels of the transcript (P = 0.014). On the other hand, we noted several cases in which the PRDM14 copy number was increased, but the level of its expression was not, implying the presence of other oncogenes within the 8q13 amplicon. Conversely, we detected high levels of PRDM14 mRNA in several cases despite the fact that PRDM14 copy number was not increased, implying gene amplification is not the only cause of PRDM14 overexpression and that further study will be necessary.

We also found several genes situated near PRDM14, including SLCO5A1, SULF1, TRAM1, LACTB2, XKR9, and EYA1, which were not up-regulated in a subset of tumors showing 8q13-8q24 amplification. In addition, our finding that expression of NCOA2 was high regardless of gene amplification indicates that NCOA2 expression is regulated by molecular mechanisms other than gene amplification. And given that NCOA2 is involved in estrogen receptor–mediated gene transcription, it would be interesting to know whether up-regulation of both PRDM14 and NCOA2 is a significant factor in the development and progression of breast cancer.

The molecular mechanism by which PRDM14 enhances cell growth remains unknown. PRDM14 contains a PR domain that is likely a derivative of SET domain, which is involved in methylation of lysine residues on the histone tail, and affects chromatin structure and gene expression (1). To date, two histone methyltransferases, EZH2 and SMYD3, have been shown to have oncogenic properties (30, 31). It is noteworthy in that regard that our microarray analysis revealed that in SKBr-3 cells stably expressing PRDM14, there was up-regulated expression of a variety of genes known to be involved in breast cancer, including carcinoembryonic antigen family genes such as CEACAM6 (32) and CEACAM7 (33); POT1, a component of telomerase that binds to the single-stranded form of human telomeric DNA and facilitates telomerase activity by disrupting the G-quadruplex (34); GATA3, a transcription activator highly expressed in breast cancer (35); and PEG10, an imprinted gene up-regulated by c-myc and associated with cell proliferation (36). Moreover, a number of genes associated with growth suppression and differentiation (37–39) were down-regulated by PRDM14. Taken together, our findings indicate that up-regulated levels of PRDM14 in malignant breast tumors enhance tumor cell growth and provide them with a survival advantage. The mechanism by which this is achieved remains to be determined, however. Clarification of the role played by PRDM14 in the regulation of gene expression should shed light on whether or not the genes up-regulated or down-regulated in SKBr-3 cells overexpressing PRDM14 are direct targets of PRDM14.

In summary, we have shown that PRDM14 is frequently overexpressed in breast cancers and that its overexpression is often associated with gene amplification. Ectopic expression of PRDM14 enhances cell growth and reduces the susceptibility of tumor cells to chemotherapeutic drugs. That there is little or no expression of PRDM14 in normal tissues suggests that inhibition of PRDM14 activity could represent a novel approach to the treatment of breast cancer.


   Acknowledgments
 
Grant support: Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (M. Toyota, K. Imai, and T. Tokino), Grants-in-Aid for Scientific Research (S) from Japan Society for Promotion of Science (K. Imai), and a Grant-in-Aid for the Third-term Cancer Control Strategy, and Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan (M. Toyota).

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.

We thank Dr. William F. Goldman for editing the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 11/ 7/06. Revised 6/ 1/07. Accepted 8/18/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhang Y, Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 2001;15:2343–60.[Free Full Text]
  2. Bannister AJ, Kouzarides T. Reversing histone methylation. Nature 2005;436:1103–6.[CrossRef][Medline]
  3. Bachmann IM, Halvorsen OJ, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 2006;24:268–73.[Abstract/Free Full Text]
  4. Hamamoto R, Silva FP, Tsuge M, et al. Enhanced SMYD3 expression is essential for the growth of breast cancer cells. Cancer Sci 2006;97:113–8.[CrossRef][Medline]
  5. Frank B, Hemminki K, Wappenschmidt B, et al. Variable number of tandem repeats polymorphism in the SMYD3 promoter region and the risk of familial breast cancer. Int J Cancer 2006;118:2917–8.[CrossRef][Medline]
  6. Tsuge M, Hamamoto R, Silva FP, et al. A variable number of tandem repeats polymorphism in an E2F-1 binding element in the 5' flanking region of SMYD3 is a risk factor for human cancers. Nat Genet 2005;37:1104–7.[CrossRef][Medline]
  7. Keller AD, Maniatis T. Identification and characterization of a novel repressor of ß-interferon gene expression. Genes Dev 1991;5:868–79.[Abstract/Free Full Text]
  8. Schultz J, Copley RR, Doerks T, Ponting CP, Bork P. SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res 2000;28:231–4.[Abstract/Free Full Text]
  9. Rea S, Eisenhaber F, O'Carroll D, et al. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 2000;406:593–9.[CrossRef][Medline]
  10. Buyse IM, Shao G, Huang S. The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein. Proc Natl Acad Sci U S A 1995;92:4467–71.[Abstract/Free Full Text]
  11. Huang S. Blimp-1 is the murine homolog of the human transcriptional repressor PRDI-BF1. Cell 1994;78:9.[CrossRef][Medline]
  12. Lin Y, Wong K, Calame K. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science 1997;276:596–9.[Abstract/Free Full Text]
  13. Turner CA, Jr., Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 1994;77:297–306.[CrossRef][Medline]
  14. He L, Yu JX, Liu L, et al. RIZ1, but not the alternative RIZ2 product of the same gene, is underexpressed in breast cancer, and forced RIZ1 expression causes G2-M cell cycle arrest and/or apoptosis. Cancer Res 1998;58:4238–44.[Abstract/Free Full Text]
  15. Fears S, Mathieu C, Zeleznik-Le N, et al. Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family. Proc Natl Acad Sci U S A 1996;93:1642–7.[Abstract/Free Full Text]
  16. Yang XH, Huang S. PFM1 (PRDM4), a new member of the PR-domain family, maps to a tumor suppressor locus on human chromosome 12q23-24.1. Genomics 1999;61:319–25.[CrossRef][Medline]
  17. Chittka A, Chao MV. Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor. Proc Natl Acad Sci U S A 1999;96:10705–10.[Abstract/Free Full Text]
  18. Deng Q, Huang S. PRDM5 is silenced in human cancers and has growth suppressive activities. Oncogene 2004;23:4903–10.[CrossRef][Medline]
  19. Du Y, Carling T, Fang W, et al. Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily. Cancer Res 2001;61:8094–9.[Abstract/Free Full Text]
  20. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–{Delta}{Delta}C(T)) method. Methods 2001;25:402–8.[CrossRef][Medline]
  21. Martinez-Lacaci I, Kannan S, De Santis M, et al. RAS transformation causes sustained activation of epidermal growth factor receptor and elevation of mitogen-activated protein kinase in human mammary epithelial cells. Int J Cancer 2000;88:44–52.[CrossRef][Medline]
  22. Fejzo MS, Godfrey T, Chen C, Waldman F, Gray JW. Molecular cytogenetic analysis of consistent abnormalities at 8q12-22 in breast cancer. Genes Chromosomes Cancer 1998;22:105–13.[CrossRef][Medline]
  23. Forozan F, Mahlamaki EH, Monni O, et al. Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data. Cancer Res 2000;60:4519–25.[Abstract/Free Full Text]
  24. Hu M, Yao J, Cai L, et al. Distinct epigenetic changes in the stromal cells of breast cancers. Nat Genet 2005;37:899–905.[CrossRef][Medline]
  25. Struski S, Doco-Fenzy M, Cornillet-Lefebvre P. Compilation of published comparative genomic hybridization studies. Cancer Genet Cytogenet 2002;135:63–90.[CrossRef][Medline]
  26. Afify A, Bland KI, Mark HF. Fluorescent in situ hybridization assessment of chromosome 8 copy number in breast cancer. Breast Cancer Res Treat 1996;38:201–8.[CrossRef][Medline]
  27. Clancy JL, Henderson MJ, Russell AJ, et al. EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer. Oncogene 2003;22:5070–81.[CrossRef][Medline]
  28. Ehlers JP, Worley L, Onken MD, Harbour JW. DDEF1 is located in an amplified region of chromosome 8q and is overexpressed in uveal melanoma. Clin Cancer Res 2005;11:3609–13.[Abstract/Free Full Text]
  29. Tsuneizumi M, Emi M, Nagai H, et al. Overrepresentation of the EBAG9 gene at 8q23 associated with early-stage breast cancers. Clin Cancer Res 2001;7:3526–32.[Abstract/Free Full Text]
  30. Hamamoto R, Furukawa Y, Morita M, et al. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol 2004;6:731–40.[CrossRef][Medline]
  31. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002;419:624–9.[CrossRef][Medline]
  32. Poola I, Shokrani B, Bhatnagar R, et al. Expression of carcinoembryonic antigen cell adhesion molecule 6 oncoprotein in atypical ductal hyperplastic tissues is associated with the development of invasive breast cancer. Clin Cancer Res 2006;12:4773–83.[Abstract/Free Full Text]
  33. Ohlsson L, Hammarstrom ML, Israelsson A, et al. Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT-PCR. Br J Cancer 2006;95:218–25.[CrossRef][Medline]
  34. Loayza D, De Lange T. POT1 as a terminal transducer of TRF1 telomere length control. Nature 2003;423:1013–8.[CrossRef][Medline]
  35. Hoch RV, Thompson DA, Baker RJ, Weigel RJ. GATA-3 is expressed in association with estrogen receptor in breast cancer. Int J Cancer 1999;84:122–8.[CrossRef][Medline]
  36. Li CM, Margolin AA, Salas M, et al. PEG10 is a c-MYC target gene in cancer cells. Cancer Res 2006;66:665–72.[Abstract/Free Full Text]
  37. Burger AM, Zhang X, Li H, et al. Down-regulation of T1A12/mac25, a novel insulin-like growth factor binding protein related gene, is associated with disease progression in breast carcinomas. Oncogene 1998;16:2459–67.[CrossRef][Medline]
  38. Castro-Rivera E, Ran S, Thorpe P, Minna JD. Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, whereas VEGF165 antagonizes this effect. Proc Natl Acad Sci U S A 2004;101:11432–7.[Abstract/Free Full Text]
  39. Yu L, Liu C, Vandeusen J, et al. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. Nat Genet 2005;37:265–74.[CrossRef][Medline]



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