CpG island status as an epigenetic alteration for NIS promoter in thyroid neoplasms; a cross-sectional study with a systematic review

Thyroid cancers are the most common malignancies and represent approximately 95% of all endocrine tumors. The global incidence of thyroid cancer was estimated to be 586,000 cases in 2020, and nearly 43,646 patients were presumed to die from thyroid cancer [1]. Thyroid cancer comprises several subtypes with remarkably different biological characteristics. Among these subtypes, papillary and follicular thyroid cancers (PTC and FTC) are referred to as well-differentiated thyroid cancers (WDTCs), accounting for more than 90% of all thyroid malignancies [2]. WDTCs typically are well managed and represent a favorable prognosis; however, sometimes, patients progress toward poorly differentiated and more aggressive subtypes [3].

Nowadays, environmental factors, alongside genetic alterations, play a pivotal role in developing thyroid cancers [4, 5]. Ecological factors in regulating gene expression are known as epigenetic alterations [6‐8]. Aberrant DNA methylation has been introduced as a major epigenetic event in tumorigenesis [9, 10]. Gene inactivation through hypermethylation of CpG islands located in promoter regions was described in many cancers, especially thyroid cancer, which correlated with progression and tumorigenesis [11].

Solute carrier family 5-member 5 (SLC5A5, also called NIS) gene is localized on chromosome 19 (19p13.11) and encodes an 80–90 kDa transmembrane glycoprotein that actively transports iodide from the bloodstream into the thyroid follicular cells [12, 13]. Indeed, thyroid-stimulating hormone (TSH) can regulate NIS protein activity. In this process, a Na+/K+-ATPase pump generates a sodium gradient, transporting two Na+ to an I− as the first step in the thyroid hormones biosynthesis [14]. The role of NIS in thyroid cancer diagnosis and treatment is important since NIS primarily mediates radioactive iodine (RAI) accumulation. Accordingly, the reduced NIS mRNA levels and/or impairment in NIS plasma membrane trafficking are well-demonstrated factors showing poor prognosis in thyroid cancer [15]. However, the association between NIS and thyroid cancer is complex and poorly understood [13].

Traditionally, it has been argued that CpG islands exist within the promoter of the NIS gene. In this regard, several studies have demonstrated that this island is a methylation target, which could have a vital role in DNA methylation, causing the reduction of NIS mRNA expression in thyroid carcinomas [12, 16‐20].

In molecular cancer medicine development history, epigenetics has been thought of as a critical factor. In essence, epigenetic alterations depend on different factors, including race, environmental changes, lifestyle, nutrition, and physical activity. As above-mentioned, there are several studies regarding NIS promoter’s methylation in thyroid cancers. However, no previous study has investigated changes in those factors in an Iranian population as epigenetic modifications are strongly linked with race. Therefore, according to the mentioned challenges, this study seeks to investigate the methylation degree of a large region of NIS promoter in a set of multinodular goiters (MNG) samples (as the benign samples), malignant thyroid tumors including PTC and FTC, and their surrounding non-tumor tissue samples to suggest a methylation region as a discriminatory biomarker for early diagnosis of thyroid nodules. In addition, a systematic review of the literature was completed to bring together studies regarding methylation of NIS gene promoter, to reach a general conclusion about the role of NIS promoter methylation in tumorigeneses, and to discuss with our results.

The current systematic review was designed according to the latest version of the PRISMA checklist for systematic review [22]. A literature search by using the terms including “Thyroid Neoplasm” OR "Thyroid Carcinoma" OR "Thyroid Cancer" AND "DNA Methylations” OR “hypermethylation" OR "CpG Island" AND “sodium iodide symporter” OR "SLC5A5" was conducted in PubMed/MEDLINE, Web of Science, and Scopus databases. The search was performed without restricting publication date. Only articles in English were evaluated. The search results were managed by EndNote version X7. Titles and abstracts were reviewed, and relevant studies were selected. Inclusion criteria were as follows: The study indicated methylation of the NIS gene promoter in differentiated thyroid cancer (PTC and FTC). Then the full-text versions of selected articles were retrieved. The unrelated studies, reviews, meta-analysis, whole-genome, and wide genome studies were excluded. However, the references of related reviews were checked to identify additional papers. The following characteristics were collected from included studies: authors, year of publication, sample size groups, methodology, methylation status, and potential clinical values.

Thyroid cancers are the most common endocrine malignancies, which comprise several subtypes; among them, PTC and FTC are referred to WDTCs, accounting for more than 90% of all thyroid malignancies [23]. It is well accepted that increased methylation may induce dedifferentiation and decrease the expression of thyroid tissue-specific genes. DNA methylations are heritable and reversible; it is speculated that target therapy may alter methylation status of these genes to achieve the goal of tumor treatment. In this regard, the present study was designed to determine the methylation status of NIS promotor in thyroid nodules.

NIS is a transmembrane glycoprotein mainly regulated by the TSH that mediates the active transport of iodide from the bloodstream into the follicular thyroid cells [24]. The CpG islands in the NIS gene promoter have been suggested as a methylation target in reducing gene expression [25]. This study showed that although the expression of NIS was significantly lower in PTC tumoral tissues compared to matched non-tumoral ones, the methylation status was not changed significantly in any of the CpG sites. Moreover, no significant differences were observed between the mRNA levels and methylation patterns in PTC tumoral lesions compared to the benign MNG group. Further, our results indicated that the mRNA level of NIS was significantly reduced in FTC tumoral tissues compared to both matched non-tumoral and benign MNG lesions. Besides, FTC tumoral tissues displayed significantly higher levels of NIS promoter methylation compared to non-tumoral and benign MNG tissues. These findings were consistent with the hyper-methylation of the 4th, 6th, and 7th CpG positions in the forward strand of the NIS promoter.

Our systematic research achieved nine studies that explored NIS gene promoter methylation status in normal and tumoral thyroid tissues and benign lesions (Table 3). Some studies emphasize an increase in NIS promoter methylation in malignant tissues, while others showed controversial results. In 1999, Venkataraman et al. [14] assessed the methylation of the NIS promoter in 100 bp upstream and downstream, extending to the first intron, of CpG-rich dinucleotides (named P, L, C regions) and its expression in PTC and FTC tumor tissues. Our results are in line with their findings, which showed that methylation in three regions was not associated with NIS loss in PTC tumors. A CpG rich segment of the coding region (region C) displayed heterogeneous methylation among PTC subjects without any correlation to hNIS mRNA expression. Similarly, FTC cases expressed NIS mRNA and had different methylation patterns between regions. Nevertheless, 5-azacytidine or sodium butyrate treatment of seven human thyroid carcinoma cell lines was found to restore NIS mRNA expression in four cell lines and iodide transport in two cell lines. In a study by Neuman et al. [26], a 42.8% (6/14) occurrence of NIS methylation was found in cold thyroid nodules (CTNs) without determining the type of thyroid cancer relative to non-methylation in their non-tumoral surrounding tissues. Consistent with our findings, they detected a significantly reduced NIS expression ranging from 3 to 68% compared to the corresponding surrounding tissues (ST) in 86% of the CTNs. Moreover, they did not observe significant correlation between NIS mRNA expression and the degree of methylation. In another study conducted by Smith et al. [27], increased methylation of NIS in 22% (7/13) of patients with PTC and 0% of the control group, including follicular adenomas (FA), goiters, and normal cases were observed. However, the author pointed out that the limited number of the study groups led to not concluding predictive value from the comparison. In our findings, methylation of the NIS gene was increased in all regions except the 5thCpG site in PTC tumoral tissues compared to adjacent normal tissues, while this elevation was not statistically significant. A pilot study by Stephen et al. in 2011 [28] showed NIS promoter was methylated in (7/13) of DTCs (without specifying PTC or FTC separately), (1/3) of hyperthyroid, and (1/5) of normal cases and suggested that aberrant methylation of NIS may be an early change in thyroid tumorigenesis regardless of cell type. By applying semi-quantitative (SQ) MSP, Galaro et al. [12] investigated the methylation levels of the NIS CpG island between nucleotide − 466 and + 246 from the ATG in fragmented regions. They showed that the 5′ upstream region of the CpG island had the highest methylation levels, decreasing toward the 3’ region (P1 < P2 < L < C, p < 0.05). Besides, similar degree of methylation was reported in PTC tumoral tissues (benign = 2.19 ± 0.50 AU, malignant = 1.92 ± 0.59 AU) and non-tumoral tissues (benign = 2.23 ± 0.58 AU, malignant = 2.20 ± 0.82 AU) and suggested that NIS promoter methylation is a common occurrence in benign and malignant tumors as well as in the surrounding tissues. In agreement with our result, no quantitative correlation was detected between methylation levels and mRNA expression in any of the groups. Later, Galaro et al. in 2014 [16] appraised two CpG islands; the first had been described by Venkataraman et al. [14], which was referred to as CpG-island1, and the second, the new NIS CpG-island2 with 14 CpG sites, comprised a sequence of 256 bp and located at − 2152/− 1887 relative to the ATG site. They found a novel distal enhancer regulating the mRNA expression of NIS through DNA methylation, then by using QBS-sequencing, indicating that the methylation degree of 66.1% in tumoral tissues (benign, 64.3%, and malignant, 69.6%), compared to 23.2% of none tumoral (NT) tissues (benign, 20.5%; malignant, 25.1%). In contrast to our result, a significant inverse correlation between NIS mRNA expression and the degree of CpG-island2 methylation in NT and T samples was observed. At the same year, Choi et al. (2014) [29], found more frequent NIS methylation, in six specific CpG sites in the promoter region (− 191 to + 73), in cases of BRAF V600E carrier PTC samples than surrounding normal (SN) tissues (normal 29% vs. cancer 58%). In particular, − 24 and − 26 CpG sites were highly methylated in BRAF V600E PTCs.

According to the literature, methylation status of NIS gene in FTC tumoral tissues compared to matched non-tumoral and benign tissues is limited. In 2015, Stephen et al. [30] investigated the promoter methylation status of NIS gene related to thyroid cancers in a cohort of FTC compared to 26 Hurthle and 27 classic subtypes using the QMSP method. NIS gene was methylated (QMSP values > 0.0), but inversely to our findings, the methylation degrees were not statistically significant between different subtypes. They also reported frequent methylation of NIS in normal thyroid samples, hyperthyroid nodules, and thyroid cancers, suggesting this is a signal of early thyroid tumorigenesis, regardless of cell type. Eventually, in the following, Stephen et al. in 2018 [31] found significant differences in methylation levels of NIS promoter between FTC and normal thyroid groups. These findings imply that the NIS promoter hyper-methylation may result in a reduction of its expression in FTC. In contrast to Stephen's [30] findings, we detected a significant decrease in NIS expression and an increase in promoter methylation status in FTC samples compared to their matched non-tumorous and benign samples, with no association between mRNA and methylation levels.

There are conflicting reports about NIS methylation status, mRNA level, and the correlation between these two events in limited studies with restricted sample sizes. Several reasons may contribute to the inconsistent results, including the use of different methods, study groups, and population, as well as the different CpG island regions used in the studies. Nonetheless, the present findings in PTC subjects seem to be consistent with Smith et al. [27], Galaro et al. [12], and Stephen et al. [31] reports, which found no significant differential methylation of NIS promoter in PTCs tumors. Due to the methods used, some studies did not specify the area investigated and results were just reported as methylated and non-methylated, consequently comparing the results is quite challenging.

According to our study, promoter methylation of the NIS is not a common event in PTC tumors, their matched non-tumor, and MNG tissues; consequently, promoter methylation of NIS could not be a possible mechanism leading to reducing its expression. Maybe other regulatory mechanisms that affect this gene’s expression during neoplastic transformations are involved. However, there are many other CpG sites in the NIS gene promoter and the lack of methylation difference in this region cannot be generalized to the overall percentage of promoter methylation. In fact, the study of methylation of all CpG promoter sites of this gene will provide valuable information. As well as, based on the literature, methylation status is heterogeneous in various CpG regions of NIS gene promoter; whether it is the upstream or downstream of the start codon (ATG) of the gene, subsequently, further research is needed in this area.

There are no distinct patterns of methylation linked with transcriptional failure. This shows that thyroid nodule methylation is heterogeneous. The absence of a distinct methylation pattern also implies the importance of other epigenetic processes, which may alter the production of NIS mRNA levels. We underlined the need for additional research to understand the epigenetic mechanisms underlying the reduction of NIS mRNA expression, which could lead to developing novel treatments to enhance radioiodine uptake in thyroid carcinoma cells. These investigations can also contribute to a better comprehension of how epigenetic events influence gene expression. Additionally, it is anticipated that the design of targeted demethylation medicines, which may play a role in the redifferentiation of thyroid carcinomas, will lead to a novel cancer therapeutic strategy.

DNA methylation as an epigenetic modification has improved the understanding of thyroid carcinogenesis. Much effort has been made to investigate the potential of drugs that block this form of epigenetic alteration to stimulate the re-expression of silenced genes in diverse malignancies. Lately, thyroid cancer management has been approached with more personalized medicine to prevent the over-diagnosis and over-treatment of tumors [32]. In addition, hypermethylation of thyroid-specific genes such as NIS is associated with the failure of clinical radioiodine treatment in thyroid cancer, one of the most common methods for treating DTCs [14]. A decrease in NIS expression will lead to a decrease in response to the treatment. Therefore, not only the investigations of methylation patterns in the NIS gene can be effective in choosing the type of treatment, but also in the prognosis of patients, it is expected that individuals with higher levels of methylation will respond less to iodine therapy, which ultimately leads to the design the effective drugs by reducing the methylation of this gene. There is little evidence of the effectiveness of demethylating agents in thyroid cancer. In the most studies, these drugs have been tested on cancer cell lines and have yielded promising results, however, have not yet been translated into clinical practice. But it may be used as a solo agent or combined with other drugs for proper therapy [33].

The strengths of the present study include this being the first study that determined the promoter methylation status in the selected region of NIS, a functional gene, in an Iranian population diagnosed with thyroid nodules. In order to obtain reliable results, and to avoid false-positive results from non-quantitative methods such as MSP, we evaluated NIS promoter methylation with a quantitative methodology. However, there were several limitations to this cross-sectional study, including the small sample size of patients and the non-achievable clinicopathological information. Time and budget constraints limited us from examining more CpG sites of this gene. According to the limited number of studies, further investigations with larger sample size and other sites that may be concerned with epigenetic regulation of NIS expression are required to elucidate the role of these mechanisms in the progression of thyroid nodules, leading to finding novel diagnostic biomarkers and improving the effectiveness of therapies.

The present study provides additional evidence concerning NIS promoter methylation in thyroid nodules. Although we did not find significant differences regarding NIS methylation in PTC patients, the NIS mRNA expression was substantially decreased in PTC cases. While the methylation of this gene was increased in tumoral tissues compared to matched non-tumoral tissues and MNG one. Intriguing results were found for FTC cases about the methylation pattern of the sodium-iodide symporter in CpGs islands. These differences in aberrantly methylated CpGs between PTC and FTC subjects reflect a potentially interesting findings and provide the following insights for future research. One hypothesis that warrants further review is that promoter regions are methylated more frequently in FTC than in PTC in our study. This hypothesis cannot be confirmed with our modest number of cases, but it does raise questions that can be investigated further. The investigations can also contribute to a better understanding of how epigenetic events influence gene expression. It is anticipated that the design of targeted demethylation medicines, which may play a role in the dedifferentiation of thyroid carcinomas, will lead to a novel cancer therapeutic strategy.