Abstract
Background/Aim: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan. Materials and Methods: Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined. Results: Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%). Conclusion: In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.
- Renal cell carcinoma
- von Hippel-Lindau gene
- tumor suppressor gene
- methylation sensitive multiplex ligation-dependent probe amplification analysis
- promoter methylation
Kidney cancer affects about 300,000 people worldwide and is responsible for 129,000 deaths annually (1). The global age-standardized incidence rate is 4 per 100,000 people per year (2). Moreover, age standardized incidence rate increased from 3.35/100,000 individuals in 2002 to 5.09/100,000 individuals in 2012 in Taiwan (3). Renal cell carcinoma (RCC) is the most common type in adults, accounting for around 90% of all kidney cancers (4). The incidence rates have increased over time in most populations, but mortality rates have levelled off or decreased since the 1990s (5). Based on the 2016 WHO classification, the major subtypes are clear cell, papillary, and chromophobe, which comprise 65-70%, 15-20%, and 5-7% of all RCCs, respectively (6). Clear cell RCC accounts for most kidney cancer-related deaths and is characterized by cells with clear cytoplasm (7).
The genetic feature most closely associated with sporadic clear cell RCC is loss or mutation of the von Hippel-Lindau (VHL) tumor suppressor gene (8-10). However, inactivation of VHL alone is not sufficient to cause RCC (11, 12). Other genes are likely to be important for its development, including PBRM1 (29-41% of tumor samples), SETD2 (8-12%), BAP1 (6-10%), KDM5C (4-7%), and MTOR (5-6%) (5). Epigenetic inactivation of tumor suppressor genes by methylation of promoter region CpG dinucleotides has also been implicated in the pathogenesis of RCC (13, 14). Early studies have demonstrated that VHL, CDKN2A/p16INK4a, and RASSF1A tumor suppressor genes are frequently inactivated by methylation in clear cell RCC (14, 15). More recent studies have demonstrated tumor-specific promoter methylation of BNC1, PDLIM4, RPRM, CST6, SFRP1, GREM1, COL14A1, and COL15A1 genes in more than 30% of RCCs (13).
The genetic aspects of RCC have received little attention in Taiwan. Acquired cystic disease-associated RCC has been reported to be associated with frequent abnormalities on chromosome 3 (16). Yano et al. noted that the CpG islands of connexin 32 gene are methylated in RCCs of hemodialysis patients (17). The aim of this study was to elucidate the possible etiological role of molecular pathogenesis in sporadic RCCs in Taiwan. A total of 15 patients with RCC were screened for mutations in the VHL gene and methylation of 24 tumor suppressor genes. Mutations were identified by PCR and Sanger sequencing. Methylation was determined by methylation sensitive multiplex ligation-dependent probe amplification (MS-MLPA) analysis.
Materials and Methods
Study subjects. Fifteen paraffin-embedded tumor and normal tissue samples (Cases 1 to 15, 8 males and 7 females) were provided by the Tumor Tissue Bank of Koo Foundation Sun Yat-Sen Cancer Center which is funded by the National Science and Technology Program for Pharmaceuticals and Biotechnology (#NSC89-2323-B-368-001). The study procedures were approved by the Institutional Review Board of Chung Shan Medical University Hospital (reference number CS2-03052). All procedures that involved human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
DNA extraction. Genomic DNA was extracted from the sections with the QIAamp Tissue Kit (Qiagen GmbH., Hilden, Germany), according to the manufacturer’s instructions and finally dissolved in 100 μl of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). DNA concentration of each sample was measured using NanoDrop UV-VIS Spectrophotometer.
Polymerase chain reaction (PCR) and direct sequencing. The three exons of the VHL gene were amplified in 7 fragments using published primers and protocols (18). PCR products were purified using QIAquick PCR Purification kits (Qiagen GmbH., Hilden, Germany). The purified PCR products were sequenced via the cycle sequencing method with fluorescently labelled dideoxy chain terminators from the ABI Prism kit (Applied Biosystems, Taipei, Taiwan, ROC) in an ABI Model 377 automated DNA sequencer, according to the distributor’s protocol. The sequencing primers were the same as those for the preceding PCRs. When a mutation was detected, the nucleotide sequence was confirmed on both strands.
Copy number and methylation analyses. MS-MLPA analysis was performed using Salsa MS-MLPA kit ME001-C2 Tumor suppressor-1 (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer’s instructions. Samples were then subjected to capillary electrophoresis on an ABI PRISM 3130XL (Applied Biosystems). Twenty-six MS-MLPA probes were used to detect the methylation status of promoter regions of 24 different tumor suppressor genes by HhaI digestion (Table I). MLPA results were analyzed using GeneMarker version 3.2.1 (SoftGenetics, LLC, State College, PA, USA) to determine copy numbers and methylation status of the HhaI sites. For copy number, each sample peak area was divided by the nearest control peak areas. Relative copy number was obtained by comparing this ratio with that of a control sample (19). The internal methylation ratio was calculated by comparison of the HhaI digested aliquot with the paired undigested aliquot from each sample with intra-sample data normalization according to the manufacturer’s instructions (20). Methylation, compared to normal reference, was assessed by comparing the probe methylation percentages obtained for the test sample with the percentages of the 5 normal reference samples. Copy number ratio of 1.0 and methylation ratio of 0 were expected in most genes in normal reference. If so, the methylation compared to normal reference was unlimited (∞). If methylation ratios of the test sample and normal reference samples were appropriate, methylation compared to normal reference was around 1.0.
Results
VHL gene inactivation: mutation and promoter methylation. The DNA sequences of VHL gene were determined via direct sequencing. Four mutations were identified in the exon region of VHL gene in the DNA samples prepared from paraffin-embedded tumor specimens (Table II). Among them were three missense mutations. Valine was substituted for glycine via heterozygous mutation at codon 155 (V155L) in exon 3, 677T>G in case 1 (Figure 1A); asparagine was substituted for serine via heterozygous mutation at codon 141 (N141S) in exon 2, 635A>G in case 5 (Figure 1B); and glutamate was substituted for aspartate via heterozygous mutation at codon 52 (E52D) in exon 1, 369G>T in case 9 (Figure 1D). There was one silent mutation, with no change in amino acid sequence, via heterozygous mutation at codon 33 in exon 1, 312G>A in case 7 (Figure 1C).
MLPA results were analyzed to determine copy numbers and methylation status of the HhaI sites in the promoter region of VHL gene located in chromosome 3p25.3 (Table II). In case 3, methylation ratios were unlimited (∞) in both normal and tumor tissue DNA compared to average normal reference (Table II, Figure 2A and B). This indicated that the VHL gene is inactivated by methylation of its promoter in both germline and somatic DNA. Copy number ratio of 0 was detected in tumor somatic DNA from case 13 indicating that the VHL probe failed to hybridize with its promoter region due to a small deletion (Table II, Figure 2E and F). In addition, partial inactivation of VHL gene was identified due to copy number ratio decreases in cases 11, 12, and 15 in both normal and tumor tissue DNA compared to average normal reference (Table II).
Copy number and methylation analyses. MS-MLPA analysis was performed with DNA from case 2 to case 15 using Salsa MS-MLPA kit ME001-C2 tumor suppressor-1. Increases in copy number ratio of CADM1 were found in all RCCs. The copy number ratios were 1.38, 1.40, 1.20, 1.29, 1.27, 1.37, 1.42, 1.26, 1.40, 1.30, 1.58, 1.43, 1.54 and 1.22, respectively. For case 13, in addition to the VHL gene, copy number ratio of 0 was detected for the FHIT gene indicating a small deletion (Figure 2E and F).
Methylation of APC (100%) was found in all RCCs (Figure 2, Table III). The second most commonly methylated gene was CDKN2B (92.9%). Only case 11 was found to be unmethylated. Methylation of CASP8 (not in case 11 or 13), MLH1_167 (not in case 2 or 11), and KLLN (not in case 13 or 14) was found in 12 out of 14 (85.7%) RCCs (Table III). Methylation of RASSF1_382 (not in case 2, 4, 6, or 11), CDH13 (not in case 8, 11, 13, or 14), and CDKN2A (not in cases 11 to 14) was found in 10 out of 14 (71.4%) RCCs. Frequencies of 9 genes with medium level of methylation were ATM 64.3%, RASSF1_328 57.1%, CD44 50.0%, TP73 42.9%, RARB, ESR1, and BRCA1 35.7%, TIMP3 and GSTP1 28.6%. Moreover, methylation of CDKN1B (case 12), BRCA2 (case 12), and CADM1 (case 2) was identified in only one (7.1%) case of RCC. Methylation of CHFR was identified in only two RCCs, case 3 and case 8. Twenty-one out of 26 MS-MLPA probes showed somatic DNA methylation only. CDKN2B, MLH1_167, CDH13, RASSF1_328 and RARB demonstrated germline DNA methylation (data not shown). Somatic DNA methylation means that methylation is found in RCC tissue only, not in their corresponding normal tissues. Four of the 24 genes (FHIT, MLH1_463, DAPK1, and HIC1) did not show detectable promoter region methylation (Table III).
Patient characteristics in relation to methylations status of tumor suppressor genes. Age (≥50, <50), clear cell type RCC (yes/no), and tumor stage (early, stage I and II; late, stage III to IV) are dichotomous variables based on Moore’s work (21). Pathological stage is an important determinant of survival. We found a novel and interesting correlation between methylation of the CHFR gene promoter and late stage. No other gene associations were found between promoter methylation and age, or clear cell type RCC. RCC incidence is higher in men than in women (5). However, there was no significant difference in the numbers of males and females in this study (8 males and 7 females).
Discussion
For clear cell RCC, chromosome 3p deletion occurs in at least 95% of the cases and duplication, but not deletion of chromosome 5q, in approximately 30-40% of the cases (22). It has been suggested that the VHL tumor suppressor gene is a major gatekeeper gene for clear cell RCC (23). Approximately 50%-80% of sporadic RCCs are shown to have mutations of the VHL gene in Western countries (24, 25). In this study, which was conducted in Taiwan, the frequency of VHL mutation events for sporadic RCCs was only 20% (3/15), which is much lower than that in Western countries. Our results showed promoter hypermethylation in 1 of 15 (6.6%) tumors. This ratio is also lower than that of a previous study in which silencing of the VHL gene by DNA methylation occurred in about 20% of RCCs (10, 24). Recently, it has been reported in the Cancer Genome Atlas that 7% of clear cell RCCs showed epigenetic silencing at VHL (7, 26). The discrepancy may be attributed to ethnic effects. However, further studies using larger samples are recommended to verify our results. In the present study, both FHIT and VHL deletions were found in case 13 (age ≥50, stage II). A previous study has suggested that FHIT deletion is an early event and VHL deletion as an early and/or late event in RCC (27).
Dulaimi et al. reported that the frequencies of hypermethylation in 100 kidney tumors were RASSF1A (45%), APC (14%), RARB2 (12%), CDKN2A/p16INK4a (10%), and VHL (8%) (14). Morris et al. noted that RCCs are most frequently methylated at DAPK (24%), not at RARB2 (0%), CDKN2A/p16INK4a (0%) or CDH13 (3%) (28). However, these results were not verified by this study as RCCs were found to be most frequently methylated at APC (100%), CDH13 (71.4%, 10/14), CDKN2A (71.4%, 10/14), RARB2 (35.7%, 5/10), and VHL (7.14%, 1/14), not at DAPK1 (0%). Based on the results of this study, frequencies of promoter methylation in RASSF1A, 71.4% (10/14) for RASSF1_382 on MV location 03-050.353347, and 57.1% (8/14) for RASSF1_328 on MV location 03-050.353298 were much higher than in previous studies in which RASSF1A promoter methylation was detected in 56% and 40% of primary clear cell RCCs by Yoon et al. and Tokinaga et al., respectively (29, 30). In this study, neither germline nor somatic DNA methylations in DAPK1 were identified, which is inconsistent with the findings of a previous study (31). The reasons for this discrepancy are unclear but may be related to the sensitivity of the methods used. With older molecular methods based on radio-labeled primers and polyacrylamide gel electrophoresis, small minor bands may be missed or mistaken. Capillary gel electrophoresis with fluorescence detection allows for the analysis of methylation status with high sensitivity. Dulaimi et al. also noted that RASSF1A methylation is significantly associated with high-grade tumors (14). Recent studies have highlighted that 16% of RCC cases have loss of CDKN2A through mutation, deletion, or promoter hypermethylation (7, 32).
Although there were differential methylation patterns of the 24 tumor suppressor genes among the 14 RCCs, at least two (mean=10.7) genes were methylated in each tumor sample. In this study, all RCCs showed methylation of APC specific to RCC, not in normal tissues, which did not change with age. APC gene encodes a 312-kDa protein that acts as an antagonist of the Wnt signaling pathway (33). Deregulation of Wnt signaling pathway through APC deficiency or loss of heterozygosity has recently been implicated in human RCC (34-36). Aberrant methylation of the APC gene promoter has been reported not only in colon (37), but also in breast and lung carcinomas (38). The accumulation of a variety of genetic aberrations is necessary for the initiation and progression of RCCs (39). These results indicated that methylation of APC is a universal pathognomonic event in tumorigenesis of RCC and can be a candidate diagnostic and therapeutic biomarker in liquid biopsy as it is found early in the process of carcinogenesis.
In addition to APC methylation, there were a variety of other genetic aberrations. CDKN2B gene methylation was observed in all RCCs, except for case 11. CDKN2B gene on 9p21.3 encodes the p15INK4B protein that binds to and inhibits activation of CDK4 or CDK6 (40). Germline mutations in CDKN2B have been identified as a novel cause of familial RCC (41). CASP8 gene encodes Caspase-8 that is an apoptosis-related cysteine peptidase (42). Methylation at CASP8 has been demonstrated in 16% of RCCs (28). MLH1 gene encodes proteins that detect and repair DNA mismatches (43). Expression of mismatch repair MLH1 proteins has been shown to be reduced in 83.7% (118/141) of sporadic RCCs (44). KLLN gene encodes the protein killin, which is a p53-regulated nuclear inhibitor of DNA synthesis (45). Bennett el al. found germline methylation in 23/41 (56%) RCC patients and somatic methylation in 19/20 (95%) patients with advanced RCC (46). These results indicated that methylation of APC, CDKN2B, CASP8, MLH1_167, and KLLN is important in the tumorigenesis of RCC, and may have diagnostic, clinical, and therapeutic significance.
In conclusion, inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, V155G in case 1, N141S in case 5, and E52D in case 9, promoter methylation in case 3, and small deletion in case 13 (Figure 3A and B). RCCs were most frequently methylated at APC (100%, 14/14), CDKN2B (92.9%, 13/14), CASP8, MLH1_167, and KLLN (85.7.4%, 12/14), but not at FHIT, MLH1_463, DAPK1, and HIC1 (0%) (Figure 3C). The rate of VHL inactivation and promoter methylation profile for RCCs in the Taiwanese population differ from those in Western populations. This may be attributed to ethnic effects. However, larger sample size is required to confirm these findings. Moreover, methylation of APC may be a universal pathognomonic event in tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.
Acknowledgements
Tissue samples were provided by the Tumor Tissue Bank of Koo Foundation Sun Yat-Sen Cancer Center which is funded by the National Science and Technology Program for Pharmaceuticals and Biotechnology (#NSC89-2323-B-368-001). The authors would like to thank GenePhile Bioscience Laboratory of Ko’s Obstetrics and Gynecology Clinic for the help with the acquisition of data.
Footnotes
Authors’ Contributions
Yen-Chein Lai: designed the experiments, performed the experiments, interpreted the results, and drafted the manuscript. Wen-Chung Wang: designed the experiments, interpreted the results, and made critical revisions to the manuscript. All authors have read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in regard to this study.
- Received June 23, 2021.
- Revision received July 20, 2021.
- Accepted July 21, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.