Functional and Epigenetic Characterization of the KRT19 Gene in ...

DNA AND CELL BIOLOGY Volume 30, Number 2, 2011 ª Mary Ann Liebert, Inc. Pp. 85–90 DOI: 10.1089/dna.2010.1108

Functional and Epigenetic Characterization of the KRT19 Gene in Renal Cell Neoplasms Filipa Paiva,1,2 Sara Duarte-Pereira,1,2 Vera Lućia Costa,1,2 Joaõ Ramalho-Carvalho,1,2 Patrıćia Patrıćio,1,2 Franclim Ricardo Ribeiro,2,3 Francisco Lobo,4 Jorge Oliveira,4 Carmen Jerońimo,1,2,6 and Rui Henrique1,5,6

The KRT19 gene encodes cytokeratin 19, an element of the cytoskeleton whose expression is frequently altered in renal cell carcinoma (RCC). Epigenetic phenomena, such as promoter methylation, may be a regulatory mechanism of expression of this gene. The aim of this study was to assess the epigenetic regulation of the KRT19 gene using epigenetic-modulating drugs, through the evaluation of methylation and expression status of the promoter region of KRT19 in 6 renal carcinoma cell lines and 112 primary renal tumors (52 clear cell RCC, 22 papillary RCC, 22 chromophobe cell RCC, and 16 oncocytomas). The diagnostic and prognostic value of KRT19 methylation levels in RCC was also evaluated. In cell lines 769-P, A498, and Caki-1, KRT19 re-expression was observed after treatment with 5-aza-20 deoxycytidine and trichostatin A. Conversely, a decrease in promoter methylation levels was apparent for the same cell lines. In primary renal tumors, KRT19 promoter methylation frequency was low (20.5% of cases). Although chromophobe cell RCC showed the lowest frequency compared with the remaining subtypes, this difference did not reach statistical significance. Moreover, no correlation between KRT19 methylation and expression was apparent in tumor samples and no significant correlations with clinicopathological parameters were observed. KRT19 methylation is not a frequent feature of primary RCC and oncocytomas, nor is it associated with clinicopathological parameters. Although we found evidence that KRT19 gene expression is epigenetically regulated in cell lines, this finding was not translated to primary tumors, suggesting the intervention of other genetic mechanisms for in vivo regulation of the KRT19 gene.

Renal cell tumors (RCTs), the most common types of kidney neoplasms (85%), are mainly comprised of clear cell renal cell carcinoma (ccRCC), papillary RCC (pRCC), chromophobe cell RCC (chrRCC), and oncocytoma, a benign neoplasm, along with other less common epithelial tumors (Baldewijns et al., 2008). The genetic features of the main subtypes of RCT are well characterized and allowed for the simultaneous histomorphological and genetic definition of each tumor type in the Heidelberg classification (Baldewijns et al., 2008). However, the epigenetic characterization of RCTs has been much less extensively documented (Costa et al., 2007; Morris et al., 2008). Indeed, epigenetic alterations, mainly aberrant promoter methylation, have been recognized as an alternative mechanism for tumor suppressor gene silencing, as it prevents the access of transcription activators to DNA (Illingworth and Bird, 2009). The KRT19 gene, located at 17q21.2, encodes for the protein cytokeratin (CK) 19 (Bader et al., 1988). In normal kidney

Introduction

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idney cancer is a clinically, morphologically, and genomically heterogeneous disease that represents 4% of all adult malignancies worldwide, with 57,760 estimated new cases and 12,980 estimated deaths for 2009 in the United States, for both genders ( Jemal et al., 2009). In terms of incidence, kidney and renal pelvis tumors occupy the seventh (5%) and sixth (3%) position among all noncutaneous tumors for men and women, respectively, representing 5% of all cancer-related mortality in men and 3% in women ( Jemal et al., 2009). In Europe, for 2008, the estimated adjusted incidence of kidney cancer was 14.2 and 6.1 cases per 100,000 habitants for men and women, respectively (Ferlay et al., 2010). In the same period, the mortality rate was 6.1 per 100,000 habitants for men and 2.4 per 100,000 habitants for women (Ferlay et al., 2010). 1

Cancer Epigenetics Group, Research Center of the Portuguese Oncology Institute—Porto, Porto, Portugal. Department of Genetics, Portuguese Oncology Institute—Porto, Porto, Portugal. 3 Cancer Genetics Group, Research Center of the Portuguese Oncology Institute—Porto, Porto, Portugal. Departments of 4Urology and 5Pathology, Portuguese Oncology Institute—Porto, Porto, Portugal. 6 Department of Pathology and Molecular Immunology, ICBAS—Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal. 2

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86 tissue, CK19 is expressed by distal tubules and collecting duct cells, although it is absent in proximal tubules (Cao et al., 2000). CK19 shows a restricted expression in ccRCC (10%– 15%), suggesting that these ccRCCs may represent a specific subtype originating from the collecting duct system or with neo-expression of CK19 (Mertz et al., 2008). The sequence analysis of the KRT19 gene revealed a GC-rich region spanning 1282 bp, *50 bp upstream of the translational start site (Morris et al., 2008). Remarkably, KRT19 promoter methylation was found in RCC cell lines (40%) as well as in primary RCCs (38%), but not in normal renal tissue from patients without cancer. Further, KRT19 promoter methylation was rarely (14%, 3/22) detected in adjacent normal tissue to primary RCC (Morris et al., 2008). Thus, an epigenetic regulation of the KRT19 gene was hypothesized, and aberrant KRT19 promoter methylation might serve as a specific renal cell cancer biomarker. Thus, we sought to extend those previous observations, assessing the functional and epigenetic characterization of the KRT19 gene in all the main RCT subtypes. For accomplishing that goal, we first verified the epigenetic regulation of the KRT19 gene using epigenetic-modulating drugs in renal cancer cell lines and then characterized quantitatively the methylation status of the promoter region of KRT19 in a relatively large series of RCT and normal kidney tissue samples. Materials and Methods RCC cell line culture conditions and treatment with 5-aza-2 0 deoxycytidine and trichostatin A RCC cell lines, from American Type Culture Collection, 769-P, 786-O, Caki-1, Caki-2, A498, and ACHN (all are ccRCC, except 769-P, which is derived from pRCC), were plated in 75 cm2 flasks and routinely maintained in RPMI 1640 Medium þ GlutaMAX (Invitrogen GIBCO) (769-P, 786-O), Mc Coy’s 5A Medium Modified (Invitrogen GIBCO) (Caki-1 and Caki-2), and Eagle’s Minimum Essential Medium (Invitrogen GIBCO) (A498 and ACHN) supplemented with 10% fetal bovine serum at 378C, 5% CO2, and 1% penicillin/streptomycin (Invitrogen, GIBCO) for isolation of DNA and RNA. For pharmacological treatment, the cells were split to a confluence of 25% in a T-75 flask 12–24 h before treatment. Then, cells were treated at days 1 and 4 with 1 or 5 mM 5aza-20 deoxycytidine (DAC) (Sigma) from 100 mM 50% acetic acid dissolved stock. In another flask, 1 mM of DAC and 0.5 mM of trichostatin A (TSA) from 5 mM 100% ethanoldissolved stock (Sigma) were added. Cells that were not exposed to DAC or TSA, were used as control (Mock). The medium and the drugs were changed every 24 h. On day 5, cells were harvested by trypsinization and processed for DNA and RNA extraction. Patients, sample collection, and DNA extraction One hundred twelve patients with RCT (52 ccRCC, 22 pRCC, 22 chrRCC, and 16 oncocytomas) who were consecutively treated with partial or radical nephrectomy at the Portuguese Oncology Institute, Porto, Portugal, between 2001 and 2005 were selected for this study, after informed consent. Tumor tissues were collected, snap-frozen immediately after surgical resection, and stored at 808C. Standard pathological evalua-

PAIVA ET AL. tion was performed in formalin-fixed, paraffin-embedded tissue. Hematoxylin-eosin stained slides were examined by a pathologist to determine the histologic subtype, nuclear grade, and pathologic stage. Normal renal tissue was procured from two kidneys, not involved by neoplastic or inflammatory disease, and removed during surgical procedures for extrarenal malignancy resection. Relevant clinical data were collected from patient’s clinical records and these studies were approved by the institutional review board. Genomic DNA was extracted from tissues using a standard technique of an overnight digestion with proteinase K (20 mg/mL) in the presence of 10% SDS at 558C, followed by phenol-chloroform extraction and precipitation with 100% ethanol (Pearson and Stirling, 2003). All samples were analyzed in a spectrophotometer NanoDrop ND-1000 (NanoDrop Technologies) to assess the concentration and quality of the DNA extracted. A ratio of 1.8 was accepted as pure for DNA. Bisulfite treatment and quantitative methylation-specific polymerase chain reaction Sodium bisulfite conversion was performed as previously described (Clark et al., 1994). Briefly, 2 mg of genomic DNA was denatured in 0.3 M NaOH for 20 min at 508C. The denatured DNA was diluted in 450 mL of a freshly prepared bisulfite solution, and the mixture was incubated at 708C for 3 h in the dark. After incubation, the resulting bisulfiteconverted DNA was desalted and purified through a column by using Wizard DNA purification resin (Wizard DNA Clean-Up System; Promega). The eluted DNA was denatured in 0.3 M NaOH, precipitated with 100% ethanol, resuspended, and stored at 808C. The chemically modified DNA was amplified by fluorescence-based real-time methylation-specific polymerase chain reaction (PCR), using SYBR Green technology (PerkinElmer Corp.). The primers used for the target gene (KRT19; GenBank accession no. NG_012285.1) and the internal reference gene (b-actin and ACTB) were, respectively, 50 -TTC GCG CGT TTA GTA TTT AC-30 (sense), 50 -TTA ACT TCT CGT TAC CCG C-30 (antisense), 50 -TGG TGA TGG AGG AGG TTT AGT AAG T-30 (sense), and 50 - AAC CAA TAA AAC CTA CTC CTC CCT TAA-30 (antisense). Serial dilutions of fully methylated DNA (CpGenome Universal Methylated DNA; Millipore) were used to perform the calibration curve to quantify the amount of fully methylated alleles in each reaction. The amplifications were carried out in 96-well plates on a 7500 Real-time PCR system (Applied Biosystems). Fluorogenic PCRs were carried out in a reaction volume of 20 mL consisting of 2 mL of modified DNA, 0.5 mL of 10 mM primers sense and antisense, 10 mL of Power SYBR Green PCR Master Mix (Applied Biosystems), and 7 mL of diethyl pyrocarbonate-treated (DEPC) water. The samples were run in triplicate and multiple water blanks were used as controls for contamination (negative control). PCR was performed according to the following conditions: 508C for 2 min, 958C for 10 min followed by 40 cycles of 958C for 15 s, and 608C for 1 min. Then, a dissociation–curve analysis was performed using the following conditions: 958C for 15 s, 608C for 20 s, and 958C for 30 s. To determine the relative levels of methylated promoter DNA in each sample, the values of the target gene were compared with the values of the internal

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Table 1. KRT19 Promoter Methylation Levels (KRT19/ACTB1000) and mRNA Relative Expression Levels (KRT19/HPRT11000) in Renal Cell Lines Before and After Treatment with Epigenetic-Modulating Drugs Cell lines

Methylation levels mRNA expression levels

Mock 1 mM DAC 5 mM DAC Mock 1 mM DAC 5 mM DAC 0.5 mM TSA

768-O

Caki-1

Caki-2

A498

ACHN

769-P

1.9 nd nd 0.2 nd nd nd

100.4 1333.3 0 9.6 43.2 40.3 26.9

12.2 12.3 0 395.6 395.6 316.5 316.5

318.1 210.4 95.3 0.1 67.7 85.6 173.0

0 nd nd 3451.3 nd nd nd

116.0 189.3 0 8.8 154.0 69.5 1872.6

All cells were harvested at day 5. DAC, 5-aza-20 deoxycytidine; nd, not determined; TSA, trichostatin A.

reference gene to obtain a ratio that was then multiplied by 1000 for easier tabulation (KRT19/ACTB1000). RNA extraction and quantitative reverse transcriptase PCR Total RNA was extracted from each sample using the RNeasy Mini Kit (Qiagen) and it was reverse-transcribed using the Revert Aid H Minus First Strand cDNA synthesis Kit (Fermentas), with random hexamers as reaction primers, according to the manufacter’s instructions. The KRT19 (GenBank accession no. NG_012285.1) primers used were 50 CTT CCG AAC CAA GTT TGA GAC-30 (sense) and 50 -CTT CAG TCC GGC TGG TGA AC-30 (antisense), which amplify a 338 bp product. The hypoxanthine phosphoribosyltransferase 1 gene (HPRT1; GenBank no. NM_000194) was adopted as endogenous control for RNA normalization. HPRT1 sense and antisense primers were 50 -TGA CAC TGG CAA AAC AAT GCA GAC TT-30 and 50 -TTC GTG GGG TCC TTT TCA CCA GCA A-30 , respectively, which amplify a 101 bp product (Primer 3, Rozen and Skaletsky, 2000). All amplifications were carried out in 96-well plates on a 7500 Real-time PCR System (Applied Biosystems), at 508C for 2 min, 958C for 10 min, followed by 40 cycles of 958C for 15 s, and 648C for 1 min (annealing temperature). Each well included 2 mL cDNA, 0.5 mL of 10 mM primers sense and antisense, 10 mL of Power SYBR Green PCR Master Mix (Applied Biosystems), and 7 mL of DEPC-treated water. Serial dilutions of this cDNA were used for constructing the calibration curves on each plate. Each plate included multiple negative controls, as well as serial dilutions of a positive control used to make the standard curve. To determine the relative levels of KRT19 expression, the values obtained by quantitative reverse transcriptase PCR (mean quantity) were divided by the respective values of the internal reference gene (HPRT1). The ratio generated, which constitutes an index of the percentage of input copies of cDNA at the primer, was then multiplied by 1000 for easier tabulation (expression level ¼ target gene/reference gene1000).

ables (Furhman grade, pTNM stage, age, and gender) and molecular variables was assessed using the Kruskall–Wallis test (for analysis of variance) and the Mann–Whitney U-test (for post-hoc and pairwise comparisons) with Bonferroni’s correction when appropriate. The chi-square and Fischer’s exact test were used for the comparison of the frequency of KRT19 methylation among tumor subtypes. All tests were two sided and the level of significance was set at p < 0.05. All analyses were performed using SPSS version 15.0 (SPSS). Results KRT19 methylation and expression levels in RCC cell lines All RCC cell lines analyzed displayed KRT19 promoter methylation except for ACHN (Table 1). Although the values varied widely among the cell lines, methylation and

Statistical analysis The normalized expression values obtained for renal cell lines and primary RCT were compared with normalized methylation values using the nonparametric Spearman coefficient test. The association between clinicopathological vari-

FIG. 1. Correlation of KRT19 promoter methylation levels (KRT19/ACTB1000) and expression levels (KRT19/ HPRT11000) among renal cell lines.

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Table 2. Clinical and Pathological Characteristics of Patient Population Clinicopathological features Patients, n Gender, n (%) Male Female Age median Histologic subtype, n (%) ccRCC pRCC chrRCC Oncocytoma Pathologic stage, n (%) T1 T2 T3 T4 Furhman grade, n (%) 1 2 3 4

112 65 (58) 47 (42) 61 52 22 22 16

(46.5) (19.6) (19.6) (14.3)

53 21 21 1

(55.2) (21.9) (21.9) (1)

3 28 46 19

(3.1) (29.2) (47.9) (19.8)

ccRCC, clear cell renal cell carcinoma; chrRCC, chromophobe cell renal cell carcinoma; pRCC, papillary renal cell carcinoma; RCC, renal cell carcinoma.

expression levels appeared to be follow inverse trends, except for 786-O, which showed both low KRT19 gene methylation and mRNA expression levels. Indeed, a statistically significant inverse correlation was found between KRT19 promoter methylation levels and mRNA relative expression levels (r ¼ 1, p < 0.001) for Caki-1, Caki-2, A498, ACHN, and 769-P cell lines (Fig. 1).

Table 3. KRT19 Methylation and Expression Frequencies in Renal Tumor Subtypes Renal subtypes ccRCC pRCC chrRCC Oncocytoma All subtypes

Methylation frequencies 17.3% 27.3% 9.1% 37.5% 20.5%

(9/52) (6/22) (2/22) (6/16) (23/112)

Expression frequencies 76.5% 66.7% 50% 33.3% 66.7%

(13/17) (4/6) (2/4) (1/3) (20/30)

the clinical or pathological parameters (age, gender, pathological stage, and nuclear grade). KRT19 methylation and expression levels in tissue samples The frequencies of KRT19 promoter methylation and expression among the four RCT subtypes are depicted in Table 3, and the distribution of methylation levels is graphically displayed in Figure 2. To categorize the promoter methylation status, a cutoff value of 9.5 was used. This value was chosen considering the highest methylation level detected in normal kidney tissue. Although the prevalence of KRT19 methylation was higher in oncocytomas, it did not differ significantly among the four tumor subtypes. However, a trend was apparent for oncocytomas to display more frequent KRT19 hypermethylation than chrRCC (Bonferronicorrected p ¼ 0.014). Likewise, no statistically significant differences were disclosed for KRT19 mRNA expression frequencies, either. According to Spearman’s test, no correlation was found between promoter methylation and expression levels in tumor samples.

Pharmacological unmasking of KRT19 Considering the results described in the preceding section, pharmacological unmasking of KRT19 was performed in cell lines 769-P, Caki-1, Caki-2, and A498 but not in cell lines 786-O and ACHN because they showed only vestigial or absent KRT19 promoter methylation. Globally, treatment with 5 mm DAC was the most effective in achieving promoter demethylation, whereas exposure to 1 mm DAC provided inconsistent results. Except for Caki-2, KRT19 promoter demethylation resulted in increased mRNA expression. However, Caki-2 had the lowest methylation levels to start with. The addition of TSA caused an increase in the mRNA expression levels for 769-P and A498, but not for the remaining cell lines (Table 1). Correlation of methylation data with clinical and pathological data The relevant clinical and pathological characteristics of the patients included in this study are summarized in Table 2. As expected, men were more frequently found to have RCT than women and ccRCC was the most frequent subtype. Most cases of RCC were confined to the organ and were classified as nuclear grade 2 or 3. No statistically significant association was found between KRT19 promoter methylation levels and

FIG. 2. Distribution of KRT19 promoter methylation (KRT19/ACTB1000) levels among clear cell renal cell carcinoma (ccRCC), papillary RCC (pRCC), chromophobe cell RCC (chrRCC), and oncocytomas.

KRT19 EXPRESSION REGULATION IN RENAL CELL NEOPLASMS Discussion There is accumulating evidence that epigenetic alterations constitute an important alternative mechanism for the inactivation of cancer-related genes. These alterations have been implicated in the pathogenesis of many human cancers, including RCC. Hence, this study was designed to expand the current knowledge on epigenetic-mediated gene alterations in renal tumorigenesis. The target gene of this study was the KRT19 gene, which has been previously reported to be silenced by promoter methylation in RCC cell lines and in some primary RCCs. Among a restricted number of candidate epigenetically inactivated tumor suppressor genes in RCC, KRT19 was found to be methylated in several RCC cell lines (including Caki-1) and tissues (39.2% of ccRCC and 33.3% of pRCC). Moreover, KRT19 mRNA re-expression was apparent following cell line exposure to 5 mm DAC (Morris et al., 2008). However, that study did not investigate KRT19 promoter methylation in all major subtypes of RCTs nor did it quantitatively characterize promoter methylation status or mRNA expression. Therefore, we first determined KRT19 promoter methylation status in six RCC cell lines using quantitative methylationspecific PCR, a highly sensitive and specific method for CpG methylation analysis. Similarly to Morris and coworkers (2008), KRT19 promoter methylation was detected in RCC cell line Caki-1 (as well as in 769-P, Caki-2, and A498), but not in ACHN. Moreover, 786-O showed very low methylation levels, which is also in accordance with the aforementioned report (Morris et al., 2008). Remarkably, expression levels inversely correlated with promoter methylation levels, further supporting an epigenetic-mediated regulation of the KRT19 gene. Then, we tested that hypothesis through exposure of cell lines to epigenetic-modulating drugs. In this phase of the study, 786-O and ACHN were not tested owing to the low or absent promoter methylation levels. It became clear from those experiments that the highest DAC concentration (5 mm) was more effective in inducing promoter demethylation and consequent gene re-expression. The highest mRNA re-expression levels were observed when cell lines were simultaneously exposed to both 1 mm DAC and TSA. Thus, a synergistic effect between CpG methylation and histone deacetylation is suggested, as previously demonstrated in other tumors (Kang et al., 2004; Zhang et al., 2005). These findings reinforce the intricate connection between DNA methylation and histone modifications in gene regulation. However, detailed characterization of the histone modification status (namely by means of chromatin immunoprecipitation) at the KRT19 promoter is required to validate this hypothesis. Based on the results of the RCC cell lines, we further analyzed a total of 112 RCTs, comprising the four most frequent histologic subtypes (including oncocytoma, a benign neoplasm), thus extending the findings of Morris and coworkers (2008). Although no statistically significant differences in the frequency of KRT19 promoter methylation were apparent among RCT subtypes, a striking trend was noticeable for oncocytomas and chrRCC. This is an interesting finding as oncocytomas and chrRCC derive from the same segment of the nephron (Polascik et al., 2002). Indeed, in a previous quantitative promoter methylation profiling

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study from our group, no significant differences between oncocytomas and chrRCC were apparent, thus limiting a potential use of methylation markers for urine-based differential detection of RCT (Costa et al., 2007). It is possible that the present lack of statistical significance of KRT19 promoter methylation frequency between oncocytomas and chrRCC might be due to the low sampling number, thus requiring further investigation in a larger case series. Contrarily to RCC cell lines, no significant correlation was observed between KRT19 promoter methylation and mRNA expression levels in primary RCTs. Indeed, except for one oncocytoma, all tested RCTs showing KRT19 promoter methylation displayed mRNA expression. This finding may be due to the low levels of KRT19 promoter methylation detected in RCTs, as depicted in Figure 2. Because dense promoter CpG methylation is required for effective gene silencing, the methylation status of the KRT19 gene in primary tumors might not be sufficient to efficiently impair gene expression. Moreover, genetic alterations might play a significant role in KRT19 gene expression in RCTs. Indeed, whereas deletion of chromosome 17 (to which the KRT19 gene is mapped) is frequent in chrRCC, chromosome 17 trisomy is characteristic of pRCC (Teixeira and Heim, 2005). Thus, simultaneous cytogenetic characterization is needed to better understand the role of genetic and epigeneticmediated KRT19 altered expression in RCTs. Cancer cell lines are not fully representative of their parent primary tumor types as their adaptation to their specific conditions introduces additional genetic and epigenetic alterations in an already complex genome. Thus, the discrepancy of methylation results between RCC cell lines and primary tumors observed in our study might be explained by the acquisition of novel alterations by cancer cell lines in the course of their immortalization process. However, cancer cell lines remain a valuable research tool owing to the ability to perform functional studies, which are very difficult or even impossible to perform in primary tumor cells, although additional validation in vivo is required. Conclusion KRT19 promoter methylation is frequent in RCC cell lines and inversely correlates with mRNA expression, thus suggesting epigenetic regulation of this gene in RCC. This result was further supported by pharmacological unmasking of the KRT19 gene, which was maximal with the combined use of a demethylating agent and an HDAC inhibitor. Although KRT19 promoter methylation was detected in all subtypes of renal cell primary tumors, the frequency is low and no significant differences were apparent among the four subtypes. Moreover, and contrarily to cell lines, KRT19 promoter methylation did not correlate with gene expression. Thus, additional genetic and/or epigenetic mechanisms might be involved in the KRT19 gene altered expression in renal carcinogenesis. Acknowledgments V.L.C. is supported by a grant from FCT—Fundaçaõ para a Cieˆncia e Tecnologia (SFRH/BD/23374/2005). This study was funded by research grants from the Research Center of Portuguese Oncology Institute—Porto (CI-IPOP 4-2008), and from the Comissaõ de Fomento da Investigaçaõ

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Address correspondence to: Rui Henrique, M.D., Ph.D. Department of Pathology Portuguese Oncology Institute—Porto Rua Dr. Antońio Bernardino de Almeida Porto 4200-072 Portugal E-mail: [email protected] Received for publication July 12, 2010; received in revised form July 30, 2010; accepted July 30, 2010.