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Archivos Españoles de Urología ISSN: 0004-0614 [email protected] Editorial Iniestares S.A. España

van der Heijden, Antoine G. THE ROLE OF METHYLATION IN UROLOGICAL TUMOURS Archivos Españoles de Urología, vol. 66, núm. 5, junio, 2013, pp. 432-439 Editorial Iniestares S.A. Madrid, España

Available in: http://www.redalyc.org/articulo.oa?id=181031087005

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative PERSPECTIVAS ACTUALES DE LA BIOLOGÍA MOLECULAR APLICADA A LA URO-ONCOLOGÍA Arch. Esp. Urol. 2013; 66 (5): 432-439

THE ROLE OF METHYLATION IN UROLOGICAL TUMOURS

Antoine G. van der Heijden

Department of Uorology. Radboud University Nijmegen Medical Centre. Nijmegen. The Netherlands.

Summary.- Alterations in DNA methylation have been Resumen.- Las alteraciones de la metilación del ADN described in human for more than thirty years se han descrito en el cáncer humano desde hace más now. Since the last decade DNA methylation gets more de 30 años. Desde la última década la metilación del and more important in cancer research. In this review the ADN gana más y más importancia en investigación del different alterations of DNA methylation are discussed in cáncer. En esta revisión se discuten las alteraciones de testicular germ cell tumours, Wilms’ tumours, renal cell la metilación del ADN en los tumores testiculares de carcinomas, urothelial cell carcinomas of the bladder células germinales, tumores de Wilms, carcinomas de and of the . Eventually, the células renales, carcinomas de células uroteliales de potential use in diagnostics, prognostics and therapy are vejiga y adenocarcinomas prostáticos. Finalmente se discussed. discute su uso potencial en diagnóstico, pronóstico y tratamiento.

Keywords: DNA methylation. Testis. Kidney. Palabras clave: ADN metilación. Riñón. Veji- Bladder. Prostate. Cancer. Wilm’s tumour. Biología ga. Próstata. Cáncer. Tumor de Wilms. Molecular molecular. biology.

DNA Methylation

Epigenetics is defined as the cellular information, other than the DNA sequence itself, which is heritable during cell division (1). Almost all cancer cells have multiple epigenetic abnormalities, which combine with genetic changes to affect many cellular processes, including cell proliferation and invasion, by silencing tumour-suppressor . Epigenetic alterations in cancer cells include DNA methylation, CORRESPONDENCIA histone modification and RNA interference. However, the most studied epigenetic alteration is DNA methylation that can occur at the cytosine that @A.G. van der Heijden MD PhD precedes the in a CpG dinucleotide. While Department of CpG dinucleotides are generally underrepresented in the human genome sequence, the regions Radboud University Nijmegen Medical Centre of around half of the human genes contain CpG- Geert Grooteplein 10 rich areas termed CpG islands that are generally 6525 GA Nijmegen (The Netherlands) unmethylated in normal cells (2).

[email protected] Three different types of DNA methylation have been observed in cancer, global hypomethylation, localized hypermethylation and loss of imprinting (3). 433 THE ROLE OF METHYLATION IN UROLOGICAL TUMOURS

Hypomethylation represents a decrease of the overall the embryonal carcinoma is undifferentiated and methylcytosine content compared to normal tissue cells may differentiate along an embryonal lineage and is often more pronounced in advanced stages into teratomas, containing tissues from all three germ of malignant disease (4). Methylcytosine levels can layers, or they may differentiate along extra-embryonal be substantially diminished, often to less than 50% lineages into yolk sac tumours or choriocarcinomas of the normal level. To date, there are few reports (11). In general, DNA methylation seems to increase on hypomethylation of human associated with differentiation. with activation by overexpression (5). In contrast with hypomethylation, hypermethylation is not a Smiraglia and co-workers demonstrated genome-wide event, but it is found at specific sites significant epigenetic differences between seminomas (6). Hypermethylation has often been observed in the and non-seminomas by restriction landmark genomic promoter regions of many tumour suppressor genes scanning (12). Seminomas showed almost no CpG and is correlated with decreased expression. island methylation, in contrast to nonseminomas that Since hypermethylation is concentrated at the promoter show CpG island methylation at a level similar to region it often does not become evident when overall other solid tumours. Furthermore, they demonstrated a DNA methylation is being measured. It has to be higher level of general hypomethylation in seminomas studied on a gene-by-gene basis (7). Hypermethylation compared to nonseminomas. The same year the is considered a key mechanism involved in silencing group of Smith-Sørensen endorsed the significantly of cancer-associated genes and occurs at genes with higher methylation frequency found in nonseminomas diverse functions related to tumorigenesis and tumour compared to seminomas, respectively 67% and 24%; progression and thereby hypermethylation needs to P=0.0003 (13). Furthermore, they discovered a high be considered as an alternative to gene mutation and frequency of MGMT promoter methylation (32/69) deletion. in patients with TGCT. Their conclusion was MGMT contributes to the development of nonseminoma The third alteration related to DNA TGCT. DNA repair genes MGMT and RASSF1A methylation is loss of imprinting (LOI). LOI includes became potential target genes also due to the partial or complete extinction of the difference studies of Koul and Honorio (14,15). Subsequently, between the expression levels of the maternal and several studies on novel hypermethylated target paternal alleles at imprinted loci, which results in an genes have been published (16-18). PRSS21, increased or decreased overall expression. In other SCGB3A1, HIC1, BRCA1, HOXA9, APC, FHIT, words, imprinted genes display a characteristic HOXB5, CDH13 and CDH1 were presented as novel parent-of-origin-specific DNA methylation pattern target genes hypermethylated at high frequencies that results in only a single allele being expressed among nonseminomas. Nevertheless, only a limited (either the paternal or maternal allele) (8). Since number of tumour suppressor genes showed to be the differences in the expression of imprinted alleles inactivated by DNA promoter hypermethylation in are associated with differential methylation, LOI is more than a minor percentage of TGCTs, including associated with alterations in DNA methylation. LOI MGMT, SCGB3A1, RASSF1A, HIC1, and PRSS21. In has been observed most often in tumours with an addition, imprinting defects, DNA hypomethylation, embryonic phenotype (9) and has been implicated in and the presence of the unmethylated XIST gene are the progression of several tumours (10). often observed in TGCTs.

In this review the different alterations of DNA Aberrant DNA methylation has the potential methylation in urological tumours are discussed and to improve current TGCT diagnostics. The PRSS21 eventually followed by the potential use in diagnostics, gene can be a useful biomarker. Initially PRSS21 prognostics and therapy. showed to be hypermethylated in 8 out of 8 TGCTs (16). Nevertheless, these findings have not been validated in a larger cohort of patients since. DNA Testicular germ cell tumour methylation might also serve as a prognostic marker for treatment response. Cell line research suggests Testicular germ cell tumour (TGCT) is the most an association between differentiation, global DNA common malignancy among young males and can methylation status and response to be classified into two main histological subgroups: (19). The degree of methylation might explain the seminomas and non-seminomas. Seminomas are chemotherapy sensitivity of TGCT. But what is the generally undifferentiated, and morphologically underlying mechanism for this? Normally primordial they resemble the precursor stage intratubular germ germ cells and embryonic stem cells have an open cell neoplasia. Non-seminomas, on the other hand, chromatin structure that provides a good DNA access. include several histological subtypes. Among these, If TGCT carry a similar chromatic structure, this might 434 A. G. van der Heijden explain the extreme chemotherapy sensitivity; the state markers are being studied to offer patients with low of the chromatin might determine how efficiently DNA risk of relapse a therapy that minimizes the potential targeting drugs can access the DNA. Subsequently, late toxicities, without decreasing the survival rates. increased methylation results in a more condensed Maschietto and co-workers found five genes, TSPAN3, chromatin structure and a decreased chemo sensitivity NCOA6, MPP2, CDO1 and MCM2, which were (20). Koul and co-workers showed a more frequent down regulated in patients with relapse (25). promoter hypermethylation of RASSF1a and HIC1 among chemo resistant tumours (17). These findings Recently, methylation of the RASSF1A did not reach statistical significance due to the limited promoter was found predictive for poor outcome in number of patients but suggest that the promoter Wilms’ tumours (26). Methylation was more frequent methylation status may serve as prognostic marker for in tumours of older compared to younger children, chemo sensitivity. and in advanced-stage compared to early stage tumours. However, multivariate analysis could not confirm the prognostic significance of RASSF1A Wilms’ tumour methylation, possibly because of the small number of advanced stage tumours examined. Nevertheless, Wilms’ tumour or nephroblastoma is a variety the methylation status of RASSF1A might be a novel of that typically occurs in children. biomarker to predict outcome of patients with a Wilms’ tumours are characterized by proliferation Wilms’ tumour. of immature renal cells, mesenchymal nephroblasts, which are hampered in their ability to differentiate into tubular epithelial cells. Furthermore, the growing tumour compresses the normal kidney parenchyma. Genetically, Wilms’ tumours are heterogeneous. Renal cell carcinoma (RCC) is the most The best-known tumour suppressor gene involved common of the adult kidney. Compared to is the WT1 gene, which encodes a transcriptional four decades ago, there has been a five-fold increase repressor, expressed in maturing nephroblasts. WT1 in the incidence of, and a two-fold increase in mortality mutations are observed in approximately 20% of from, RCC (27). The disease is histopathologically Wilms’ tumours (21). heterogeneous, comprising several subtypes of which the most common (75-80%) is the clear cell subtype. A further important change in many Wilms’ Papillary (10–15%), chromophobe (5%) and other tumours is loss of imprinting (LOI) resulting in aberrant more rare forms such as collecting duct carcinoma expression of the imprinted allele in cancer cells (<1%) comprise the remainder (28). (22). LOI at the IGF2/ locus on chromosome 11p15.5 is associated with an overexpression of The most frequent form of familial RCC IGF2, to stimulate proliferation and enhance survival occurs in individuals with inherited von-Hippel Lindau of mesenchymal and epithelial cells, and diminished (VHL) syndrome. The identification of the VHL tumour expression of H19 with a potential tumour suppressor suppressor gene located on chromosome 3p led to function. More recently, a somatic deletion targeting a the finding that, in addition to inactivation by point previously uncharacterized gene on the X chromosome mutation (50-80% of the clear cell RCCs) or deletion, was discovered. This gene called WTX is inactivated the VHL gene also showed allelic inactivation by in approximately one-third of Wilms’ tumours (23). In hypermethylation of the promoter region associated contrast to biallelic inactivation of autosomal tumour- with transcriptional silencing in 10-15% of familial suppressor genes, WTX is inactivated by a monoallelic and sporadic RCC (29). VHL inactivation occurs only “single-hit” event targeting the single X chromosome in clear cell tumours and similarly, methylation of VHL in tumours from males and the active X chromosome has been found only in clear cell RCC. Since VHL in tumours from females. inactivation is the initiating event in familial clear cell renal tumours, it is likely an early event in sporadic Despite the efforts to identify a marker clear cell renal tumorigenesis. useful for prognosis, tumour stage remains the most significant prognostic variable widely used in treatment Fiona and co-workers studied DNA methylation regimens, separating Wilms’ tumours in two distinct profiling in both VHL-related and unrelated RCC subgroups: patients with either low or intermediate and have shown that the RASSF1, TWIST1, PITX2, risk of relapse. Currently, the only molecular analysis CDH13, HS3ST2, TAL1, WT1, MMP2, DCC, ICA1 for this stratification is based on genomic observation. and TUSC3 genes are more frequently methylated in Genomic losses of both 1p and 16q are considered RCC patients with wild-type VHL than in RCC patients adverse risk factors (24). Additional molecular with mutant VHL (30). These findings demonstrate 435 THE ROLE OF METHYLATION IN UROLOGICAL TUMOURS different patterns of CpG methylation in VHL and non- diagnosis and prognosis of RCC, and in the longer VHL clear cell RCC. term may enable more individualized treatments.

Recently, Girgis and co-workers conducted an integrated analysis of copy number, gene expression, Urothelial cell carcinoma of the bladder protein expression, and methylation changes in clear cell RCC (31). They identified genes located in peak In the western world is the copy-number aberrations with concordant changes in fourth most common cancer in men and the eighth methylation; hypomethylated in copy-number gains most common in women (38). The most important such as STC2 and CCND1 or hypermethylated in histological subtype of bladder cancer is urothelial deletions such as CLCNKB, VHL and CDKN2A/2B. cell carcinoma. It accounts for 90% of all bladder Furthermore, they found that CA-IX expression is tumours. The remaining 10% consists of squamous significantly increased in CA-IX hypomethylated cell carcinoma and . Since these tumours and these patients revealed to have a better tumours are in direct contact with urine, methylation prognosis then the non-hypomethylated. Moreover, studies can focus on urine instead of tissue. Hoque the copy number loss of CA-IX in conduction with and co-workers studied nine genes (APC, ARF, CDH1, methylation status synergistically worsens patient GSTP1, MGMT, CDKN2A, RARbeta2, RASSF1A, survival among CA-IX non-hypomethylated patients. and TIMP3) in urine and tissue samples of 15 bladder cancer patients and 25 controls (39). The methylation An extensive study on 11 RCC cell lines pattern in urine matched that in the primary tumour. identified eight genes (BNC1, PDLIM4, RPRM, CST6, Four genes (CDKN2A, ARF, MGMT, and GSTP1) SFRP1, GREM1, COL14A1 and COL15A1) that were displayed 100% specificity. Results were validated on frequently (>30%) methylated in primary RCC (32). 175 urine samples, which displayed methylation in at Hypermethylation and re-expression of BNC1, CST6, least one of these genes (sensitivity 69%; specificity RPRM and SFRP1, suppressed the growth of RCC cell 100%). lines. Subsequently, in tissue samples, methylation of BNC1 and COL14A1 was associated with a To identify primary bladder cancer in poorer prognosis independent of tumour size, stage urine samples Renard and co-workers performed a or grade. Another cell line study reported UCHL1 as methylation-specific polymerase chain reaction (MSP) hypermethylated in around a third of primary RCC assay on DNA from bladder tissue (40). Genes with (33). Hypermethylation of UCHL1 is also correlated 100% specificity were retained for subsequent MSP with a poor prognosis in RCC patients (34). analysis on DNA extracted from urine samples. MSP assays performed on 466 urine samples identified With respect to Wnt antagonist family genes TWIST1 and NID2 (sensitivity 90%; specificity 93%) in RCCs, DNA hypermethylation has been observed in for the primary detection of bladder cancer. SFRP1, SFRP2, SFRP5, WNT, WIF1 and DKK3 genes (35). SFRP1 showed to be a significant independent In another study on urine samples, DNA predictor of RCC. The multigene methylation score methylation of 10 genes, discovered by microarray showed a sensitivity of 79% and a specificity of 76% analysis, were analysed by MSP (41). A panel of 5 as diagnostic biomarker. Furthermore, the methylation genes (MYO3A, CA10, NKX6-2, DBC1, and SOX11 score could significantly distinguish grade, T stage or PENK) had 85% sensitivity and 95% specificity and overall survival, which makes Wnt antagonist for detection of bladder cancer (area under curve = genes an excellent epigenetic biomarker panel for 0.939). Analysing the data by cancer invasiveness detection, staging and prognosis of RCC. showed a detection rate of 81% in non-muscle invasive bladder cancer and 90% in muscle invasive disease. A mass spectrometry analysis of a set of genes For the detection of bladder cancer Costa and co- down-regulated in RCC compared to normal renal workers identified novel methylation markers by gene tissue in RCC identified besides SFRP1 also SCNN1B, expression microarray analysis of bladder cancer cell SYT6, DACH1, TFAP2A and MT1G hypermethylated lines and tissue samples (42). A panel comprising in RCC (36). GDF15, TMEFF2, and VIM correctly identified bladder cancer in tissue with 100% sensitivity and specificity. A first generation methylation array analysis In urine samples, the panel achieved a sensitivity of of RCC identified many genes (33 out of 42) not 94%, a specificity of 100% and an area under the previously reported to be methylated in >20% of curve of 0.975. primary RCC patients (37). Further characterization Another study of Costa focused on a set of of these genes and their pathways may lead to the biomarkers to identify bladder, renal and prostate development of potential epigenetic biomarkers for cancer simultaneously (43). After gene expression 436 A. G. van der Heijden microarray analysis, PCDH17 and TCF21 were by a fast and aggressive grow with a high chance of selected for further validation. Methylation levels of metastasizing. There are two challenges in prostate both genes were quantified in 12 cancer cell lines and cancer, early detection and discriminating these 318 tissue samples. PCDH17 and TCF21 methylation aggressive from indolent tumours. levels provided a sensitivity rate of 92% for bladder cancer, 67% for renal cell cancer and 96% for The best single risk stratification tool prostate cancer. Methylation levels were differentially described so far, Gleason grading, has several expressed for all tumour types compared to cancer limitations. Gleason grading is consistent and highly free controls (p < 0.001), providing 83% sensitivity correlated with outcome, but it is observer dependent and 100% specificity for cancer detection. Although and the chance remains that part of the prostate is not in urine samples the sensitivity was decreased sampled correctly. For this reason there is an urgent considerably to 60%, 32% and 26% for bladder, need for new prostate cancer biomarkers. renal, and prostate tumours, respectively (39% overall), absolute specificity was retained. Obviously, The most studied gene affected by de additional efforts are required to increase the assay’s novo methylation during prostatic is sensitivity. GSTP1, which encodes an enzyme responsible for detoxifying electrophiles and oxidants, including Reinert and co-workers performed a gene those that threaten cell and genome damage (45). expression microarray analysis in 56 tissue samples Hypermethylation of GSTP1 transcriptional regulatory (44). Independent validation was conducted in 63 sequences has been consistently detected as an early samples by a PCR-based method and bisulphite event in prostate carcinogenesis in more than 90% sequencing. Subsequently, methylation levels in 174 of prostate in more than 50 independent urine specimens were quantified. A panel of four analyses. genes, ZNF154, POU4F2, HOXA9, and EOMES (P < 0.0001), achieved 84% sensitivity and 96% specificity In addition to GSTP1, more than 40 genes for early detection of bladder cancer. Furthermore, have been reported to be targets of epigenetic gene they identified a marker for disease progression TBX4 silencing in prostate cancers (46). Yegnasubramanian (P < 0.04). and co-workers reported that although hypermethylation (>85%) of GSTP1, APC, RASSF1a, COX2, and MDR1 Currently, we are investigating a panel could be detected both in localized and in metastatic of 20 previously identified methylation markers prostate cancers, hypermethylation of ERα, hMLH1, by pyrosequencing analysis. Nine hundred urine and p14/INK4a tended to appear only later during samples obtained from patients from four different disease progression (47). countries are being analysed. Patients with proven bladder cancer of all stages and grades, patients Jerónimo and co-workers found that with a history of bladder cancer or controls without methylation levels of GSTP1, APC, RASSF1A, and (a history of) bladder cancer are included. One of CRBP1, differed significantly between prostate the objectives is to verify whether methylation analysis carcinoma and high grade PIN, and/or high grade can be used in the follow up of patients with non- PIN or benign prostate hyperplasia (P < 0.0001) (48). muscle invasive bladder cancer in order to decrease The combined use of GSTP1 and APC demonstrated the number of needed in the follow up. a sensitivity of 98.3% for prostate carcinoma, with Results of this by the Dutch Cancer Society supported 100% specificity. validation study will be published soon. Hypomethylation is less studied in prostate cancer, but it also occurs during prostatic Adenocarcinoma of the prostate carcinogenesis. Analyses of 5-meC content in genomic DNA have revealed hypomethylation mostly In Europe and the of America in association with disease progression; metastatic prostate cancer is the most common type of cancer in prostate cancer cases express low 5-meC levels (49). men. One man in six will eventually develop prostate More recently, Cho and co-workers found LINE-1 cancer during lifetime, and up to 20% will die of this hypomethylation in 53% of all prostate cancer cases, disease. Although prostate cancer can be found early in 67% of cases with metastases but in through PSA testing, this analysis is not very accurate. only 8% of cases without lymph node metastases A part of the prostate cancer patients’ progress (50). relatively slow and often these patients die with prostate cancer rather than of prostate cancer. The Hoque and co-workers looked at the feasibility other part of prostate cancer patients is characterized of using urine samples of prostate cancer patients (51). 437 THE ROLE OF METHYLATION IN UROLOGICAL TUMOURS

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