Mol Biol Rep (2014) 41:6125–6131 DOI 10.1007/s11033-014-3491-0

Expression profile of MAGI2 as a novel biomarker in combination with major deregulated in prostate cancer

Reza Mahdian • Vahideh Nodouzi • Mojgan Asgari • Mitra Rezaie • Javad Alizadeh • Behzad Yousefi • Hossein Shahrokh • Maryam Abolhasani • Mohamadreza Nowroozi

Received: 9 September 2013 / Accepted: 17 June 2014 / Published online: 2 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Complex molecular changes that occur during CI 0.002–12.08]). The expression of PTEN, NKX3.1, prostate cancer (PCa) progression have been described SPINK1, DD3, and AMACR genes was significantly recently. Whole genome sequencing of primary PCa sam- deregulated in prostate tumor samples (p range ples has identified recurrent gene deletions and rearrange- 0.000–0.044). A significant correlation was observed ments in PCa. Specifically, these molecular events disrupt between MAGI2 and NKX3.1 expression in tumor samples the gene loci of phosphatase and tensin homolog (PTEN) (p = 0.006). Furthermore, the inclusion of MAGI2 in the and membrane-associated inverted-2 gene panel improved the accuracy for discrimination (MAGI2). In the present study, we analyzed the expression between PCa and BPH samples with the sensitivity and profile of MAGI2 gene in a cohort of clinical PCa (n = 45) specificity of 0.88 [CI 0.76–0.95] and 0.83 [CI 0.68–0.92], and benign prostatic hyperplasia (BPH) samples (n = 36) respectively. The data presented here suggest that MAGI2 as well as three PCa cell lines. We also studied the gene can be considered as a novel component of of PCa-related genes, including PTEN, NKX3.1, signatures for the detection of PCa. SPINK1, DD3, AMACR, ERG, and TMPRSS2-ERG fusion in the same samples. The expression of MAGI2 mRNA was Keywords MAGI2 Á PTEN Á Prostate cancer Á Gene significantly down-regulated in PC3, LNCaP and DU-145 expression Á BPH PCa cell lines (p = 0.000), and also in clinical tumor samples (Relative expression = 0.307, p = 0.002, [95 % Introduction

R. Mahdian (&) Á V. Nodouzi Á J. Alizadeh Prostate cancer (PCa) is the second most common cause of Molecular Medicine Department, Biotechnology Research male cancer deaths in the United States. However, the full Center, Pasteur Institute of Iran, Tehran 1316943551, Iran e-mail: [email protected] range of molecular changes associated with PCa develop- ment has not been characterized yet. Nearly half of all PCa M. Asgari Á M. Abolhasani samples harbor various gene deletions and rearrangements. Oncopathology Research Center, Iran University of Medical These gene rearrangements may affect the expression of Science (IUMS), Tehran, Iran the corresponding mRNAs and proteins [1, 2]. Many PCa M. Asgari Á H. Shahrokh Á M. Abolhasani cases can be characterized by the status of E26 transfor- Hasheminejad Clinical Research Developing Center (HCRDC), mation-specific and/or phosphatase and tensin homolog Iran University of Medical Science (IUMS), Tehran, Iran (PTEN) genes. Nevertheless, more genomic events may M. Rezaie occur in smaller subsets of PCa cases. Recently, two Flowcytometry Department, Medical Diagnosis and Reference reports in the Journal ‘‘Nature’’ have shown complex Laboratories of Iranian Blood Transfusion, Tehran, Iran molecular changes occur during PCa progression [3, 4]. Berger et al [3] completed whole genome sequencing of B. Yousefi Á M. Nowroozi Department of Urology, Uro-oncology Research Center seven primary PCa samples which identified novel somatic (UORC), Imam Khomeini Hospital, Tehran, Iran genomic events in PCa [3]. They identified rearrangements 123 6126 Mol Biol Rep (2014) 41:6125–6131 that disrupted the PTEN and membrane-associated guan- Tissue samples were obtained via open radical prosta- ylate kinase inverted-2 (MAGI2) gene loci [3]. tectomy. Each tissue sample was sectioned into two rep- MAGI2, known as synaptic scaffolding molecule, licates. One replicate was examined by a pathologist for the belongs to membrane-associated guanylate kinases super- detection of malignant changes and the determination of family. MAGI2 acts as a scaffold protein in assembling and Gleason score. The other replicate was microdissected to anchoring cellular signaling proteins, such as Atrophin-1, obtain PCa tumor tissue and its matched normal tissue. The glutamate receptors, neuroligins-1, b1-adrenergic receptor, tissue samples were instantly immersed in RNAlater b-catenin, and PTEN [5]. As a scaffold protein for PTEN, solution (Qiagen, Germany) and kept at room temperature MAGI2 interacts with the C-terminus of PTEN through its for 24 h. Then, the samples were transferred into liquid PDZ domain and enables PTEN to convert PIP3 into PIP2 nitrogen containers for long term storage. To rule out the [6–8]. Moreover, MAGI2 enhances PTEN activity via effect of tumor grade variation on the result of the gene decreasing its protein degradation [5–7]. MAGI2 gene rear- expression analysis, only tumor samples with intermediate rangement has been shown in the genome of a melanoma cell Gleason score (Mean ± SEM; 6.8 ± 0.1, Median = 7) line, another cancer type in which PTEN loss is prevalent [3]. were included in the study. The samples were assigned as Therefore, deregulation of MAGI2 expression either via tumor (T, n = 45) harboring at least 80 % tumor cell somatic genomic events or post-transcriptional regulation content, tumor adjacent normal tissue (N, n = 33), or BPH (e.g. by micro-RNAs) can affect PTEN activity [9]. This fact (n = 36). Mean plasma level of prostate specific antigen suggests the involvement of the phosphatidylinositol (PSA) was 17.82 ± 3.71 ng/ml and 7.71 ± 1.28 ng/ml 3-kinase pathway as a driver of prostate carcinogenesis and a (Mean ± SEM) in PCa and BPH group, respectively. potential therapeutic target. Mutations in PTEN and MAGI2 within the genome of PCa Total RNA extraction and cDNA synthesis cells appear to be mutually exclusive. MAGI2 was found to be recurrently affected by a copy-neutral rearrangement in Total RNA containing small RNAs (e.g. miRNAs) was PCa [3]. This status makes MAGI2 gene rearrangement extracted and purified from tissue samples (50 mg) using invisible to detection methods other than whole genome miRNeasyÒ Mini kit (Qiagen, Germany) according to the sequencing or fluorescent in situ hybridization (FISH). kit instruction. High quality RNA samples (A260/ Alternatively, gene expression analysis can indirectly detect 280 [ 1.8) were used as templates for cDNA synthesis. disruptive genomic events that have affected the mRNA Fifteen samples were excluded from the study due to the expression [10, 11]. In the present study, for the first time, we low quality of their RNAs. cDNA synthesis was performed analyzed the expression profile of MAGI2 mRNA in a cohort using oligo(dT) or random hexamers (ProtoscriptÒ kit, of clinical PCa samples as well as PCa cell lines. We also New England BioLabs, USA) to convert mRNA or non- studied the expression of other PCa-related genes, including coding RNAs, respectively. PTEN, NKX3.1, SPINK1, DD3, AMACR, ERG, and TMPRSS2-ERG fusion in the same clinical samples. Development of quantitative TaqMan probe real-time PCR assays

Patients and methods Exon-exon junction spanning primers and TaqMan probes were designed using Primer ExpressÒ V.3 software Tissue sample collection (Applied Biosystems, USA) and verified to be specific for their targets by BLAST analysis (Table 1). MAGI2, PTEN, PCa and BPH patients were referred to the urology NKX3.1, ERG, AMACR, SPINK1, DD3, and TMPRSS2- department at Uro-oncology Research Center (UORC) at ERG gene fusion were assigned as target genes. GAPDH Imam Khomeini Hospital or Hasheminejad Clinical and PSA genes were used to normalize the gene expression Research Development Center (Tehran, Iran) from June variations as described previously [12–14]. According to 2011 to February 2013. All the patients were new cases the preliminary experiments, b-actin mRNA was selected with no medical history of surgery or chemotherapy. The as the most stable housekeeping gene in the PCa cell lines median age of the patients was 63 years ranged from 47 to and used to normalize the gene expression levels in the cell 75. The patients were examined by an expert urologist and line experiments. evaluated according to the standard imaging procedures The primers of TMPRSS2-ERG gene fusion were and laboratory analyses for PCa. Each patient contributed designed to amplify the most common form of the rear- to the study signed a written informed consent approved by rangement occurred by joining of exon 1 of TMPRSS2 and the Ethics Committee of UORC and Pasteur Institute of exon 4 of ERG gene. To determine the dynamic range and Iran. amplification efficiency of each target, specific PCR 123 Mol Biol Rep (2014) 41:6125–6131 6127

Table 1 The sequence of the primers and TaqMan probes used in the gene expression analysis Gene Forward primer Reverse primer TaqMan Probe

PSA TCTGCGGCGGTGTTCTGG GCTGTGGCTGACCTGAAATAC TGTGCCGACCCAGCAAGATCAC DD3 GGTGGGAAGGACCTGATGA GGGCGAGGCTCATCGAT AGAAATGCCCGGCCGCCATC AMACR GCTGAATCTCCTGGCTGACT TGCTGTTCCTTCCACCATATTT TGACCGCACACGCACTGGCAAG SPINK1 AGGTAACAGGCATCTTTCTT CCCACAGACAGGGTCATATAT TGGCCTCTCTTCCCAGGGAGTCA ERG GACGACTTCCAGAGGCTCAC GACGACTTCCAGAGGCTCACC GACGACTTCCAGAGGCTCACC TMPRSS2-ERG AGTAGGCGCGAGCTAAGCA AGTAGGCGCGAGCTAAGCAG AGTAGGCGCGAGCTAAGCAG PTEN CACACGACGGGAAGACAAG CCTCTGGTCCTGGTATGAAGA AGTTCCCTCAGCCGTTACCTGTG MAGI2 TGTGAGAAGAAAGGTGCTAT CGTGGTTGCTGTTGGTGTAGG AAGTCCAGGCTCTGTATCCACC NKX3.1 CCAGAGCCAGAGCCAGAGG TCCAACAGATAAGACCCCAA CTCGGTCTCTGCCAGCGTCTCGG GAPDH ACACCCACTCCTCCACCTTT TCCACCACCCTGTTGCTGTAG TGGCATTGCCCTCAACGACCAC b-actin CAGAGCCTCGCCTTTGCC CACGATGGAGGGGAAGACG ACACCCGCCGCCAGCTCACCA

Table 2 The relative expression of the PCa-related genes in the clinical tumor samples Gene Type PCR Efficiency Expression Std. error 95 % CI. p value Result

GAPDH REF 0.91 1.000 PTEN TRG 0.94 0.186 0.035–1.06 0.005–10.56 0.000 DOWN NKX3.1 TRG 0.89 0.003 0.000–0.26 0.000–0.28 0.000 DOWN ERG TRG 0.93 0.371 0.006–30.46 0.000–27.05 0.254 AMACR TRG 0.92 6.918 0.124–44.54 0.000–23.55 0.031 UP SPINK1 TRG 0.96 5.130 0.088–14.06 0.003–24.19 0.044 UP DD3 TRG 0.91 104.24 1.815–603.14 0.066–595.86 0.000 UP MAGI2 TRG 0.99 0.307 0.047–3.02 0.002–12.08 0.002 DOWN PSA TRG 0.90 1.139 1.000–1.64 0.216–1.94 0.128 The expression ratio of each gene in tumor group was compared to their expression in the BPH group. The expression of the target genes (TRG) was normalized to the reference genes GAPDH (REF) and PSA. p values less than 0.05 were considered statistically significant for the data analysis products were purified, sequenced and ligated into TA- The comparison between mean gene expression levels in vector plasmids (Thermo Scientific, USA). The recombi- PCa and BPH group was performed using Relative nant plasmids were serially diluted and used as templates Expression Software Tool (RESTÓ 2009, Qiagen, Ger- for the generation of Real-time PCR standard curves. many) [17]. RESTÓ software compares two or more

treatment groups or conditions with data points (CT)in Data analysis sample or control group for multiple reference and target genes [17]. Chi square analysis (SPSS software, v 16.0) Relative mRNA expression in each particular tumor tissue was performed to evaluate the correlation between gene and its adjacent normal tissue was normalized to the geo- expression variations in the study groups. In comparison metrical mean of the CT values determined for GAPDH and and correlation analyses of the gene expression data, PSA genes as described previously [12–14]. Three BPH p values \ 0.05 were considered statistically significant. samples were used as external controls in each experiment. Gene expression ratio in each sample was determined rel- ative to the mean DCT value of the BPH samples included Results in the experiments [15]. Gene expression variations with more than two-fold change were considered significant. Validation of quantitative real-time PCR assays Pooled RNA sample from three PBH cases was used as calibrator sample (Relative expression ratio = 1) for the The Real-time PCR experiments were set up using PC3, gene expression analysis in the PCa cell lines [16]. LNCaP, and DU-145 PCa cell lines. The specificity of each

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PCR amplification reaction was verified by agarose gel MAGI2 expression was significantly down-regulated electrophoresis and sequencing of the amplified fragments in PCa cell lines through the experiments. The accuracy of the quantitative assays was also verified by the generation of standard The quantitative expression analysis of MAGI2 in PCa cell curves, which were drawn using template plasmids con- lines showed significant (p = 0.000) down-regulation of taining the desired amplicons. The slope and R2 coefficient the gene expression in PC3, LNCaP, and DU-145 cell lines. of the standard curves were determined for each gene. The The expression of MAGI2 mRNA was almost lost in PC3 PCR efficiency of the studied genes is indicated in Table 2. and LNCaP cell lines (Fig. 1). PTEN mRNA was not detectable in androgen-independent PC3 cells which har- bor homologous deletions of the PTEN gene. The PTEN expression profiles observed in PC3, LNCaP, and DU-145 PSA mRNA expression cells were consistent with the previously published data A [18].

to BPH) MAGI2 expression was down-regulated in clinical tumor sample cohort ∗ ∗ The expression of MAGI2 was down-regulated in tumor (Normalized

RELATIVE EXPRESSION sample group (p = 0.002) suggesting probable involve- ment of MAGI2 in PCa tumorigenesis (Table 2 and Fig. 2). MAGI2 expression was down-regulated in 72 % of the MAGI-2 mRNA expression tumor samples but remained unchanged (18 %) or up- B regulated (10 %) in the other cases. The mean expression of MAGI2 gene in tumor tissues relative to PBH samples was 0.307 (S.E. 0.047–3.02, [95 % CI 0.002–12.08]). Chi ∗ ∗ ∗ square analysis of the gene expression results showed a significant correlation between MAGI2 and NKX3.1 mRNA expression in tumor samples (p = 0.006). (Normalized to BPH) RELATIVE EXPRESSION The inclusion of MAGI2 in the gene expression profile increased the assay accuracy PTEN mRNA expression The landscape of the data achieved by the quantitative gene C expression assay on each patient tumor sample is illustrated in Fig. 2. NKX3.1 gene was down-regulated in the most tumor tissue samples (Mean relative expression = 0.003, [95 % CI 0.00–0.28], p = 0.000). NKX3.1 expression was ∗ almost lost in most of tumor samples and was also sig-

(Normalized to BPH) nificantly decreased in tumor adjacent normal tissues RELATIVE EXPRESSION (Mean relative expression = 0.302, [95 % CI 0.011–5.272], p = 0.001). The expression of PTEN, SPINK1, DD3, and AMACR genes was also deregulated in Fig. 1 Expression profile of MAGI2 and PTEN mRNA in prostate prostate tumor samples compared to the BPH group cancer cell lines. PC3, LNCaP, and DU-145 PCa cell lines were cultured in RMPI-1640 medium containing 10 % FBS. Total RNA (p range 0.000–0.044, Table 2 and Fig. 3). However, was extracted from 106 cells and was converted to cDNA using a despite different gene expression levels of these genes in commercial kit. The relative expression of MAGI2 and PTEN tumor tissue (T) vs. their matched pathologically normal normalized to b-actin gene expression was determined by quantitative tissue (N) in each PCa patient (Fig. 2), there was no sta- TaqMan real-time PCR assay. The mean expression level of the analyzed genes in three BPH samples (calibrator sample) and three tistically significant difference between the mean expres- prostate cancer samples (positive control) are also indicated. a. PSA sion ratios of these genes in T versus N group. mRNA expression was detectable only in LNCap cells, PCa and BPH TMPRSS2-ERG gene fusion was detected in 39 % (14/33) samples. b. MAGI2 was significantly down-regulated in all the cell of PCa tumor samples. Interestingly, this gene fusion was lines and PCa sample compared to PBH control samples (p = 0.000). c. PTEN mRNA was not detectable in PC3 cell line which harbor present in all PCa samples obtained from the patients with homozygous PTEN gene deletion. (*p \ 0.01) distant metastasis (n = 4). The expression of SPINK1 was 123 Mol Biol Rep (2014) 41:6125–6131 6129

Fig. 2 The landscape of the PCa-related gene expression in the (N) is indicated relative to the mean expression of those genes in the clinical prostate cancer tissue samples. The color intensity of each cell PBH group. The samples with undetectable levels of the target genes of the diagram demonstrates the gene expression changes as: up- are indicated by blank cells ( ). The status of TMPRSS2-ERG gene regulated ( , fold change[2), unchanged ( , 0.5\fold change\2) fusion is indicated as either positive ( ) or negative ( ). The data and down-regulated ( , fold change \0.5). The expression ratio of are indicated for 33 out of 45 PCa patients for whom both tumor and the target genes in tumor tissue (T) and its matched normal tissue matched normal tissue samples was available inversely correlated to the presence of the TMPRSS2-ERG during PCa progression [3, 4]. Various gene deletions and fusion discriminating a subset of TMPRSS2-ERG negative rearrangements in the genome of PCa cells have been tumors. Collectively, the gene expression profile could dis- described by Berger et al. [3]. These gene mutations and tinguish between PCa (n = 45) and BPH samples (n = 36) rearrangements eventually affect gene expression at either with the sensitivity and specificity of 0.88 [CI 0.76–0.95] and mRNA or protein level. In this study, we examined MAGI2 0.83 [CI 0.68–0.92], respectively. The exclusion of MAGI2 gene expression in clinical PCa samples. Interestingly, we from the gene panel decreased the accuracy of the assay for observed that MAGI2 expression was significantly down- discrimination between PCa and BPH cases (with the sen- regulated in primary PCa tissue as well as in PCa cell lines. sitivity and specificity of 0.78 [CI 0.63–0.87] and 0.69 [CI Decreased MAGI2 expression would negatively affect 0.53–0.82], respectively) (Table 3). PTEN function and enhance the activation of Akt. This was shown in a study on breast cancer by Sachdeva et al. [9]. They showed that the suppression of MAGI2 expression by Discussion miR-101 reduces PTEN activity and Akt activation. However, the genomic rearrangements are likely to be the Recent findings about the genomic aberrations in PCa have main cause of MAGI2 down regulation in PCa, because revealed the complexity of molecular changes that occur miR-101 has been reported to be down-regulated in PCa

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Table 3 The sensitivity and specificity of the gene expression profile MAGI2 at mRNA level in tumor samples further confirmed for the discrimination between prostate cancer and BPH samples that molecular events at genomic level (i.e. rearrange- Assay statistical MAGI2 included MAGI2 excluded ments) but not post-transcriptional regulations (i.e. trans- measures lational repression) might be involved in PCa. In our study on PCa cell lines harboring different Sensitivity 0.88 [CI 0.76–0.95] 0.78 [CI 0.63–0.87] genomic alterations in PTEN gene alleles, there was a Specificity 0.83 [CI 0.68–0.92] 0.69 [CI 0.53–0.82] significant decrease in the expression of MAGI2 while Positive 5.33 [CI 2.55–11.15] 2.54 [CI 1.5–4.26] likelihood ratio PTEN gene expression was lost only in PC3 cell line. In Negative 0.13 [CI 0.05–0.30] 0.32 [CI 0.17–0.57] fact, despite the unchanged expression of PTEN, the likelihood ratio expression of MAGI2 was significantly down-regulated in Diagnostic odds 40.00 [CI 11.14–143.54] 7.95 [CI 2.93–21.58] the LNCaP and DU-145 cells, indicating that distinct ratio molecular events might be involved in each gene alteration. The tumor samples (n = 45) and BPH samples (n = 36) were ana- Also, whole genome sequencing of primary PCa showed lyzed in parallel by pathologic assessment (the standard method) and that the genomic events affecting these two genes may quantitative TaqMan Real-time PCR assay for the gene expression occur in a mutually exclusive manner [20]. signature, including MAGI2, PTEN, NKX3.1, AMACR, SPINK1, DD3, Our investigation demonstrates a significant correlation and TMPRSS2-ERG gene fusion (the alternative method). The assay statistical measures were calculated either when MAGI2 was included between the expression profile of MAGI2 and NKX3.1 in or excluded from the gene panel. The inclusion of MAGI2 in the gene clinical primary PCa samples. This correlation may be panel enhanced the assay accuracy for the discrimination between mediated via the reduced PTEN protein function as PTEN prostate cancer and BPH samples. The statistical analysis was per- controls the activity of NKX3.1 through regulation of its formed with Wilson score method (Elie et al. BMC Medical Research Methodology 2008, 8:7) expression [21]. PTEN function loss correlates with the decreased expression of NKX3.1 and PCa progression in both mice and humans [22]. In the present study, the down regulation of MAGI2 was 103 not correlated to PTEN mRNA expression in the PCa sam- 102 ples. This may further confirm that different genomic events cause distinct chromosomal aberrations in each gene. The 101 discovery of MAGI2 genomic rearrangements in PCa pro- 100 poses that interrogating both the PTEN and MAGI2 loci might improve prognostication and patient stratification for 10-1

(Relative to BPH) clinical trials of PI3 kinase pathway inhibitors [3, 7]. 10-2

Gene expression ratio However, the genomic alteration in MAGI2 (i.e. balanced rearrangements) are undetectable by conventional 10-3 molecular methods other than whole genome sequencing or PTEN NKX3.1 ERG AMACR SPINK1 DD3 MAGI2 SHARPIN PSA FISH analysis. Here, we showed that the analysis of MAGI2 gene expression, as previously established for other PCa Fig. 3 Box-plot charts demonstrating median expression level of the PCa-related genes in the clinical PCa samples. The quantitative biomarkers [10, 13, 23, 24], may reflect the genomic defects TaqMan probe Real-time PCR assay was performed for the gene and improve the molecular assays for PCa diagnosis. expression analysis in PCa and BPH samples. The CT values were transferred to gene expression analysis software (RESTÓ 2009). The Acknowledgments This work was funded by Pasteur Institute of gene expression data are shown relative to the BPH group. The Iran (Grant No. 562). The authors would like to thank Ms. M. Saffari statistical analysis results are illustrated as box-plot graph for each at Iranian Biomedical Journal editorial office for her contribution in gene. Dashed line in each box represents the median value of the data. the final editing of the manuscript. MAGI2, PTEN, and NKX3.1 genes were significantly down-regulated, whereas DD3, SPINK1, and AMACR genes were up-regulated in tumor tissue samples References

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