Methylation of tumour suppressor genes associated with thyroid cancer

1. Introduction

Thyroid cancer, the most common malignant endocrine disease (more than 95% cases worldwide) [1], that can affect both genders, although it is most common in women, three to four times higher than in men. Though thyroid cancer can occur at any age, most tumours are diagnosed after the age of sixty [2].

Thyroid neoplasia represents a highly heterogeneous entity that possesses different cytological, histological and genetic characteristics. Starting with benign lesions – follicular adenoma (FA), to the well differentiated carcinomas, such as papillary thyroid carcinoma (PTC) in 85–90% of thyroid cancer cases, or follicular thyroid carcinoma (FTC) in 5–10% of all thyroid cancer cases, to poorly/undifferentiated thyroid carcinoma and anaplastic thyroid carcinoma (ATC) in 2% of thyroid cancers, or medullary carcinoma (MTC) in about 5% of thyroid cases, most primary thyroid cancers are originated from thyroid follicular cells [2].

The most prevalent type of thyroid neoplasia is papillary thyroid cancer (PTC). The survival rate is higher compared with other cancers. Prognosis for nodules less than 20 mm in diameter is excellent, but a clinically significant thyroid nodule may develop in 5% to 10% of the general population during their lifetime. 99% of patients with nodules smaller than 20 mm will be alive for 20 years, in contrast with FTC (10–20% of will be alive for 10 years), MTC (25% of patients with will be alive for 10 years) and ATC (90% of patients with will be alive for 5 years) [3].

Thyroid cancer is a multifactorial disease, the patients presenting a genetic susceptibility, but more evidences showed the involvement of epigenetic deregulated mechanisms in thyroid oncogenesis [4]. The well-known point mutations and rearrangements found in thyroid cancer tend to affect the effector genes of the MAPK pathway, such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), the RAS family and the protooncogene RET. These alterations have been shown to be subtype specific. Almost all tumours presenting RAS mutations have a follicular pattern of growth (FA, FTC or PTC follicular variant [fvPTC]). Mutations in BRAF gene and rearrangements in the RET gene are characteristic of PTC and poor prognosis [5, 6, 7]. RAS mutations have diagnostic but not prognostic value.

DNA methylation is one of the most important epigenetic modifications along with histone modifications and silencing mediated by RNA interference [8]. The epigenetic mechanisms are deregulated in cancer and the hallmark for cancer cells is represented by hypermethylation of tumour-suppressor gene and hypomethylation of repetitive DNA sequences and/or oncogenes. DNA methylation is important for transcriptional regulation, being essential for the normal cellular development. In chromosomal regions of tumour-associated genes, the epigenetic modifications can change important associated regulatory mechanisms for pathogenic malignant transformation. With DNA methylation, a methyl group is added to the fifth carbon of the cytosine residue in a CpG dinucleotide. CpG dinucleotides’ rich regions (CpG islands) are usually located in the 50-flanking gene promoter areas. Gene promoter methylation, particularly near the transcription start site, is associated with chromatin remodelling, which affects gene silencing [9, 10].

Tumour suppressor gene inactivation by promoter hypermethylation is thought to be important in carcinogenesis. Thus, measuring DNA methylation in a genome-wide manner would be valuable for studying mechanisms of epigenetic control involved in gene expression regulation. For personalized therapy, molecularly-defined tumour subgroup identification appears promising. For example, gene expression panels associated with breast cancer are now clinically used to provide individualized therapy. These panels, which depict carcinogen exposure-associated differences in individual tumours, include DNA hypermethylation involved in gene silencing as well as DNA hypomethylation, which is associated with oncogene activation and genomic instability [11, 12].

It has been observed that methylation of the thyroid cancer genes, especially methylation of tumour suppressor genes, can be correlated with clinical parameters, risk factors and the prognosis of the disease. Despite the efforts, this mechanism is not completely known.

Recent studies focused on the global methylation pattern in thyroid cancer. Ceolin et al. proved differences between the global MTC and PTC methylation level, therefore, the overall methylation profile of DNA can be correlated with thyroid cancer histology [13].

Lee et al. reported decreased levels of MAPK signal-inhibiting gene SERPINA5 in papillary thyroid cancer, due to DNA methylation [14, 15].

The aim of this study is to evaluate the degree of promoter methylation for a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with thyroid oncogenesis.

2. Materials and methods

Sample collection. Biopsy samples ( n = 114), tumour and adjacent tissue were obtained from 57 patients who, undergo surgical resection. The samples originated from patients with thyroid neoplasms: thyroid adenomas (age range: 37–68 years median: 55), papillary thyroid cancer (age range: 23–79 years median: 47.5) and papillary thyroid cancer follicular variant (age range: 17–63 years median: 34). For nucleic acids preservation all sample were collected in a sterile tube with RNA later (Thermo Fisher Scientific, Massachusetts, USA). Tumours were immediately stored at - 80 ∘ C. Blood samples from patients diagnosed with thyroid neoplasms ( n = 57) and from 20 subjects without thyroid case history (as controls) were collected in EDTA tubes. Informed consent was obtained from each subject prior to the study enrolment.

All the samples were provided with patient’s agreement and the study protocol was approved by the Ethic Committee.

DNA isolation. DNA was isolated from thyroid tissue samples and blood using High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Mannheim, Germany). DNA samples were stored at - 20 ∘ . The DNA concentration and purity of all samples were evaluated with NanoDrop spectrophotometer (NanoDropTechnologies, Montchanin, DE, USA).

BRAF V600E mutation was detected using PCR-RFLP analysis. PCR primer sequences used were: forward primer: 5’-GCT TGC TCT GAT AGG AAA ATG AG-3’; reverse primer: 5’-GAT AGA CAG CAG CAT CTC AGG-3’. PCR reaction mix contains: 100 ng DNA, 1.5 U/ μ l Taq polymerase, 10 pmol/ μ l primer, 0.2 mM dNTP, 1.5 mM MgCl 2 , buffer up to 20 μ l reaction volume (Promega, Madison, WI, USA). Amplification protocol: 5 min 95 ∘ C, followed by 40 X (95 ∘ C, 30 sec, 58 ∘ C, 30 sec, 72 ∘ C 30 sec) and final elongation step 72 ∘ C 30 sec. Enzyme restriction: 20U TspRI in 20 μ l reaction volume. Incubation for 16 h, 65 ∘ C. Identification of restriction fragments: electrophoresis in agarose gel 2%, TBE 1x, using DNA ladder 50 bp DNA step Ladder (Promega). Restriction products: wildtype 3 fragments – 117 bp, 87 bp, 33 b; mutant homozygote, 2 fragments – 204 bp, 33 bp, mutant heteozygote, 4 fragments – 204 bp, 117 bp, 87 bp, 33 bp.

RET split detection was performed using FISH technique. The protocol is specific for formalin-fixed paraffin-embedded (FFPE) tissue sections and consists of successive stages of dewaxing, rehydration, deproteinization and pretreatment followed by in situ hybridization with cDNA probes double labelled with fluorochromes (Zytovision GmbH – Germany). The two ends of the gene were labelled with different fluorochromes, green and orange, respectively. If the gene is intact, one see one single yellow spot. If the gene is splitted there were two distinct green and orange spots on the image. 3 μ m sections displayed on the slide were fixed by exposure for 16 hours at 50 ∘ C on histological thermostatic plate, then gradually dewaxed using xylene. After dewaxing the sections were rehydrated by successive baths of 100%, 90%, 70% ethanol and deionized water, respectively. The next step was an enzymatic deproteinization at 98 ∘ C for the nuclear membrane permeabilization, then hybridization with cDNA double labelled probes, overnight (16–18 hours). After hybridization, the samples were washed and counterstained with DAPI.

RNA isolation. Isolation of total RNA was performed using Trizol technology (Invitrogen) according to manufacturer’s instructions. 50 mg of tissue was disrupted using a homogenizer and K1 cells were deposited after centrifugation and 1 ml Trizol reagent was added. Isolated RNA quality check was done by reading the concentration and purity NanoDrop spectrophotometer and Experion.

Human tumour suppressor genes DNA methylation PCR array, signature panel

The Human Tumour Suppressor Genes EpiTect Methyl II Signature PCR Array profiles the methylation status of 22 tumour suppressor gene promoters whose hypermethylation has been reported in the literature to occur frequently in a variety of cancers and tumours. Profiling tumour samples with these arrays may help correlate CpG island methylation status with biological phenotypes. The results may provide insights into the molecular mechanisms and biological pathways behind oncogenesis, as well as cancer pathology. The protocol is based on restriction enzyme digestion, followed by real-time PCR, for the analyse of the 22 different tumour suppressor genes methylation status.

Apoptosis: BRCA1, CDKN2A, DAPK1, GSTP1, MGMT (AGT), PTEN, RUNX3, TIMP3, TP73, VHL.

Cell adhesion: APC, CDH1, CDH13, CDKN2A.

Cell cycle: APC, BRCA1, CDKN2A, FHIT, MLH1, NEUROG1, PTEN, RASSF1, RUNX3, TP73, VHL.

DNA damage and repair: APC, BRCA1, MGMT (AGT), MLH1, TP73.

Signal transduction: APC, BRCA1, CDH13, ESR1, PTEN, RASSF1, SOCS1, WIF1.

Transcription factors: BRCA1, CDH1, ESR1, NEUROG1, RARB, RUNX3, TP73, VHL.

Other: PDLIM4 (RIL).

Unmethylated C residues conversion was realized with bisulphite treatment using EpiTect Bisulfite kit (Qiagen, Valencia, California, USA). 700 ng/ μ l of each isolated DNAs (tumours and normal adjacent tissue) were bisulphite treated along with positive (CpGenome Universal Methylated DNA) and negative (CpGenome Universal Unmethylated DNA) controls (Millipore, Billerica, MA, USA).

Primers In this purpose the promoter sequence was retrieved from EMBL database (https://www.ebi.ac.uk/ research/publications/embl-nucleotide-sequence-data base-1). The methylation primers were selected using MethPrimer (http://www.urogene.org/methprimer/) which use an CpG islands prediction algorithm. By default a CpG island is defined as a 200 bp DNA sequence with a GC content $>$ 50% (Li and Dahiya, 2002). The primers distinguish between methylated and unmethylated DNA after bisulphite treatment of the template (Table 1) and were synthesized by Invitrogen Corporation (Carlsbad, California, USA). On the other hand, the primers are usually placed in the vicinity of the TSS, because the methylation in the region around the TSS (Transcription Start Site) must have a profound effect on the gene transcription [16].

WIF1 gene promoter presents 3 CpG islands (199 bp, 255 bp, 357 bp) and the transcription is on the minus strand. The TSS is situated at - 238 nt, the primers being located around - 533 nt, a critical region for transcriptional silencing according to Licchesi et al. [17]. PDLIM4 gene promoter presented one CpG island of 314 bp, the primers were situated upstream of TSS + 294 nt, this region being found hypermethylated in colon cancer [18]. TP73 presents 5 CpG islands (357 bp, 382 bp, 176 bp, 560 bp, 822 bp), the primers were selected downstream of TSS around-800nt, the region found also hypermethylated in chondrosarcoma [19].

Direct Q-MSP of Genomic DNA is a quantitative method for evaluating the degree of sample methylation.

Standard curves were designed using positive controls (fully methylated) and negative controls (fully unmethylated). Positive and negative controls were diluted in order to obtain a serial dilution: 10 pg, 100 pg, 1 ng and 10 ng according to Absolute Quantitation Using Standard Curve Getting Started Guide (Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System, Foster City, CA, USA). All DNA samples were diluted to a final concentration of 10 ng/ μ l. 25 μ l FastStart Universal SYBR Green Master (ROX) (Roche Molecular Biochemicals, Mannheim, Germany) 0.3 μ M primers and 5 μ l DNA were used. Direct Q-MSP was performed in 50 μ l final volume and the parameters we used were: 95 ∘ C–10 min (1 cycle), and 40 cycles at 95 ∘ C for 15 sec and 60 sec at the specific annealing temperature.

qPCR experiments were performed in duplicate ( Δ Ct ≈ 1.2 between replicates) and mean values were used for calculations. In order to establish the quality of DNA samples we used ACTB gene as reference [20]. All umethylated samples for methylation presented a Ct for ACTB below 35, excluding false negative results.

Methylation percentage calculation method was done according to Fackler et al. [20]. The concentration of unmethylated and methylated DNA for each patient sample was extrapolated using the standard curves. The percentage of methylation was calculated according to the formula: % M = 100 × [ng methylated gene A/(ng methylated gene A + unmethylated gene A)], where total target gene DNA was taken as the sum of U + M.

Cell culture treatment. In order to evaluate the role of methylation in gene expression suppression, K1 cell line culture (derived from a papillary carcinoma primary thyroid) was cultured for 24 hours in DMEM-Ham’s F12 medium supplemented with 2 mM glutamine and 10% foetal calf serum (FCS) to allow cell adherence to a minimum of 50%. Then the cells were treated with 5-azacytidine (AzaC) a DNA demethylating agent in a concentration of 5 μ M and 10 μ M for 24 h and 48 h. This treatment can free CpG islands promoters from methylation [21].

Apoptotic process evaluation by flow cytometry. Changes that occur in the plasma membranes are part of the early events in apoptosis. Apoptotic cells present phosphatidylserine translocated into the inner face of the plasma membrane allowing external annexin family members (proteins that bind calcium dependent phospholipids) to attach. Following the characteristic changes occurring during the apoptotic process, the test used allows a double staining: annexin V labelled with FITC (fluorescein isothiocyanate) which binds to phosphatidylserine exposed to the extracellular environment and PI (propidium iodide) – a dye used for the evaluation viability is tied intracellular DNA. For staining of cells an apoptosis kit was used (BD Bioscience, NJ, USA). More than 1 × 10 5 cells suspended in 100 μ l binding buffer were added 5 μ l of Annexin V/FITC and 5 μ l PI solution, incubating the mixture for 15 minutes in the dark. In each tube were added 400 μ L of Annexin V binding buffer and the sample was collected using FACSCantoII flow cytometer. Data was analysed using Diva 6.2 software (Becton Dickinson) and shown as two-colour dot plot with FITC-Annexin V (green fluorescence, X axis) vs. PI (red fluorescence, Y axis) [22].

4. Discussion

Along with various molecular alterations found in thyroid neoplasia (mutations and/or gene rearrangements) that were described (RET germline mutation, and chromosomal rearrangements resulting in the fusion gene PAX8/PPAR γ [24], epigenetic modifications are also common in thyroid tumours. Deciphering the genetic and epigenetic alterations that are involved in thyroid cancer, represents an essential step in understanding the disease pathogenesis, and help to improve the diagnosis methods for an individual appropriate disease management.

Like other human tumours, aberrant methylation of tumour suppressor genes is also a characteristic for thyroid tumours. Some reports mention the involvement in thyroid tumorigenesis of aberrant silencing trough promoter methylation of PTEN [25], RASSF1A [26, 27], tissue inhibitor of metalloprotei- nase-3 (TIMP3), SLC5 A8, death-associated protein kinase (DAPK) and retinoic acid receptor β 2 (RAR β 2) [28, 29] tumour suppressor genes.

Our study confirms the precedent reports regarding the gene promoter methylation of RARB, RASSF1, PTEN in thyroid cancer. According to gene panel studied we found new factors characteristic to thyroid tumours: methylation of TP73, WIF1 and PDLIM4 tumour suppressor genes, which can be a contributor to thyroid neoplasia.

The TP73 gene is a member of TP53 family involved in controlling cellular proliferation, differentiation and cell death, which includes also the TP63 gene [30]. TP73 role compensates for the loss of p53 function, and is rarely mutated compared to TP53 [31]. One interesting fact is that TP73 gene transcription is controlled by two different promoters (P1 and P2), leading to p73 protein isoforms with opposite functions, one containing (TA) or lacking ( Δ N) the transactivation domain. While TAp73 possesses a pro-apoptotic tumour suppressor protein function, Δ Np73 acts as an anti-apoptotic oncoprotein [32, 33]. It was observed that Δ Np73 isoform expression increases in tumours which presented P1 promoter hypermethylated [34].

Hypermethylation of the TP73 gene promoter was also found in a variety of solid tumours such as squamous cell lung cancer [35], gastric carcinoma [36] and cervical cancer [37].

The current study showed that the methylation of TP73 gene promoter is significantly higher in tumour tissue compared to normal tissue ( 0.0001), and a borderline significance ( p = 0.05) was found when the different neoplasias are compared. The highest methylation values were characteristic for PTC samples. The expression level of TP73 gene without discrimination between isoforms is higher in tumour samples vs. normal, but no differences were observed between the thyroid neoplasia samples. Traces of TP73 methylation were detected in blood of patients with PTC and fvPTC, the amount being significant ( p = 0.0148) compared to normal, having a good potential for diagnosis and disease management.

The WNT inhibitory factor-1 (WIF-1) gene is a secreted antagonist that binds to WNT proteins to suppress WNT/ β -catenin signalling, which is known to be involved in oncogenesis process [38, 39].

WIF-1 gene expression was found to be down-regulated through promoter hypermethylation, geno- mic loss, or genomic rearrangement in many human neoplasia, including lung, colon, breast, bladder, kidney, prostate and salivary gland tumours [40, 41, 42, 43, 44, 45, 46].

We showed that WIF1 is hypermethylated in tumour samples, the percentage of methylation being significantly higher ( 0.0001). The WIF1 methylation values were increased also in thyroid adenoma samples, indicating this factor could contribute in early stages of oncogenesis. The median values of WIF1 gene were lower in the thyroid neoplasm group. WIF1 gene promoter presented higher methylation percentage values in blood samples than controls.

PDLIM4 (reversion-induced LIM gene-RIL) was a potential tumour suppressor gene involved in cell growth regulation and modulates actin stress fibre dynamics through its association with alpha-actin [47]. Moreover, it has been shown that PDLIM4 promoter methylation could be the main mechanism to suppress its expression in prostate cancer tissues [48]. Other report showed also an association of PDLIM4 promoter methylation with breast cancer [49]. In colon cancer hypermethylation of PDLIM4 suppresses activation of Src (a regulatory protein that functions in several fundamental processes, including cell differentiation, proliferation, migration, and survival) [50]. PDLIM4 promoter is hypermethylated in tumour samples, especially in PTC samples. Follicular adenomas presented higher PDLIM4 methylation levels, suggesting the possibility to use this factor as a marker for diagnosis as long as significant methylation levels were quantified from blood of patients with thyroid neoplasms. Lowest PDLIM4 expression values were found in fvPTC samples.

Detection of methylation levels of the tested genes in blood could represent a potential method to evaluate the patient status. Even if PDLIM4 and TP73 genes expression decreases gradually from benign neoplasia to malign status, and WIF1 presented the lowest value in PTC samples; evaluation of gene expression in blood does not provides the same informational value.

We indicated also that the treatment with demethylating agents as 5-AzaC could restore the gene function as the expression increases significantly and the promoter becomes active. The increment was notable especially for TP73 gene, but also for PDLIM4 and WIF1 gene.

Among the tested genes we noted four more important genes NEUROG1, ESR1, RUNX3, MLH1, which presented the promoter with higher methylation values in tumour samples compared to normal.

The genetic tests for mutations in thyroid cancer are innapropiate due to the low incidence of mutations in thyroid oncogenesis [51] and no specifc inactivating mutations have been identifed in well-differentiated thyroid cancers [52, 53, 54]. However, BRAF V600E mutation is present in papillary thyroid cancer and is associated with an aggressive tumor phenotype and higher risk of recurrent and persistent disease [55].

Various studies suggested the involvement of BRAF V660E mutation in aberrant methylation processes of certain genes in colon cancer [56, 57] and in thyroid cancer [10, 29]. A high-throughput study demonstrated the effect of BRAF V600E signalling on aberrant methylation that may have a profound impact on cell functions and behaviors of PTC [58]. We found also a significant correlation between Braf V600E mutation presence and TIMP3 gene promoter methylation, but also with MGMT, NEUROG1, RAR-beta gene promoter methylation.

RET/PTC rearrangements were associated with a more aggressive phenotype, a larger tumor size, and it was shown to influence the methylome [59, 60]. The present study showed a significant correlation between RET/PTC rearrangement and CDH13, RARB genes promoter methylation. A correlation was also observed for this translocation with ESR1, NEUROG1 and SOCS1 gene promoter methylation.

5. Conclusions

The hypermethylation of tumour suppressor promoter is a hallmark of cancer and by inhibiting the genes expression, it alters the cell signalling pathways. Therefore, a test that uses as a method for methylation quantification of tumour suppressor genes is suitable for diagnosis and prognosis of thyroid cancer.