The most common endocrine malignancies are referred to thyroid cancers across the globe. WDTCs, including PTC and FTC, allocated more than 90% of all thyroid malignancies (26). Despite a demonstrable preoperative diagnosis tool regarding thyroid nodules, namely fine-needle aspiration biopsy (FNAB), early diagnosis and prognosis, have yet to be improved using complementary approaches. Exploring molecular mechanisms of thyroid tumorigenesis may provide an exciting opportunity to advance our knowledge of cancer biology and lead to finding a novel and reliable diagnostic biomarker for distinct thyroid nodules.
DNA methylation changes are the most useful biomarkers for diagnosis, prognosis, and response predictions to treatment in the cancer era. The present study's essential findings were the methylation status of 15 CpG islands in TIMP3 promoter as a critical tumor suppressor gene and its significant association with BRAF V600E mutation in PTC nodules. Our findings demonstrated a significant increase in methylation degree at 5th (+ 3620) and 6th (+ 3625) CpG positions in the forward strand of TIMP3 promoter in PTC tissues compared to their corresponding ANT and benign tissues. Approximately 50% decrease in TIMP3 mRNA levels in PTC tumoral tissues compared to their corresponding ANT was found. Another relevant finding from our study was a significant correlation between TIMP3 expression and total methylation status in the ANT and tumoral tissues of PTC patients. Moreover, the mRNA levels in BRAF-positive cases were decreased, while complete methylation increased significantly. A literature review shows minimal investigations have evaluated the TIMP3 promoter methylation status in thyroid lesions.
In accordance with our results, Hoque et al. (2005) (18) evaluated the promoter methylation pattern of 11 genes and its relation to the BRAF V600E mutation in 23 PTC patients samples using the real-time quantitative methylation-specific PCR (QMSP) technique. They reported a significant increase in the PTC group's methylation degree of TIMP3 promoters compared to the non-cancer tissues. However, in contrast to our findings, they did not observe any significant correlation between BRAF T1796A mutation and TIMP3 methylation. Furthermore, Hu et al. (2006) (17) investigated the role of four tumor suppressor genes (TIMP3, SLC5A8, DAPK, RARb2) in 231 PTC tissue samples and human thyroid cancer cell lines to evaluate tumorigenesis by searching the association between methylation status of these genes and tumor aggressiveness with utilizing real-time QMSP method. They also assessed the relationship between the methylation status of these genes with BRAF T1796A mutation. Methylation of TIMP3 was associated with its silencing in thyroid cancer cell lines. A close relationship was observed between BRAF mutation and TIMP3 methylation. It is suggested that methylation of TIMP3 may be an important step in BRAF mutation-promoted PTC tumorigenesis and aggressiveness. A detailed examination of TIMP3 by Brait et al. (2012) (20) demonstrated 51% methylation in the promoter of TIMP3 in PTC tissues. Still, they showed that PTC tumor samples had overlapping frequencies of methylation for TIMP3 with benign and normal thyroid samples (51% tumors vs. 42% benign vs. 27% normal). Consequently, the synchronization of hyper-methylation of the gene promoter and decrease of its mRNA levels in our study and previous research support this hypothesis that there is an association between TIMP3 promoter methylation and PTC development. These results accord with our observations regarding TIMP3 promoter methylation.
We found a significant increase in methylation degree at 14th (+ 3686) and 15th (+ 3689) CpG positions in the forward strand of TIMP3 promoter in FTC lesions compared to their corresponding ANT and BENIGN tissues. Another relevant result from our study was a significant correlation between TIMP3 expression and total methylation status in the ANT and tumoral tissues of FTC patients. A study by Stephen et al. (2015) (19) investigated the promoter methylation status of seven genes, including CASP8, CDKN2A, DAPK1, ESR1, NIS, RASSF1, and TIMP3, related to thyroid cancers in a cohort of FTC in comparison with 26 Hurthle and 27 classic subtypes using QMSP method. TIMP3 was methylated (qMSP values > 0.0), but the methylation degrees were not statistically significant between different FTC subtypes. In the following, Stephen et al. (2018) (27) designed a multi-gene panel to examine 21 candidate genes’ promoter methylation status on formalin-fixed paraffin-embedded (FFPE) samples using the QMSP method in a retrospective cohort of 329 patients, including 71 normal thyroids, 83 benign nodules, 90 FTC and 85 PTC. They observed that TIMP3, accompanied by the other five genes (RARB2, SERPINB5, RASSF1, TPO, and TSHR), could differentiate PTC from normal thyroid, with the sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue; at the same time, they did not display the relation between TIMP3 methylation and FTC patients.
Recently, Su et al. (2019) (13) discussed the therapeutic role of TIMP3 and its potential as a direct target for cancer therapy, including its usefulness as a predictor of cancer progression. Among the TIMP family members, TIMP3 is outstanding; because only it can bind to the extracellular matrix (ECM) after secretion. Additionally, TIMP3 can block MMPs and a wide range of A Disintegrin and Metalloproteinase (ADAMs) and ADAM with thrombospondin motifs (ADAMTS). The evidence shows that the TIMP3 gene acts as a tumor suppressor gene by inducing apoptosis and inhibiting proliferation, angiogenesis, and metastasis (28, 29). Nevertheless, the TIMP3 mRNA level is downregulated by genetic and epigenetic alternations.
Since epigenetic changes are mitotically heritable alterations, they can act in concert with genetic variations and lead to tumorigenesis (30). The gene-silencing phenomenon takes in the internuclear transfer of signals, which is induced in wild-type nuclei and is maintained in progeny. In carcinogenesis, silencing the stable genes during the mitosis cell division causes silenced cell growth control and other genes (31). Epigenetic drugs that affect the most important mechanisms of epigenetic changes, DNA methylation, are of increasing interest to endocrinologists and oncologists alike. Clinical trials' results will determine the effect of epigenetic drugs used alone to treat advanced thyroid cancers (11). Therefore, complex communication between protein signaling pathways must be inhibited to affect thyroid cancer growth fundamentally. Considering the different methylated sites in the PTC and FTC patients, these CpG sites should be regarded more in epigenetic drugs, and more investigations are required on these regions.
To the best of our knowledge, this is the first study that determined the promoter methylation status in a wide range of CpG islands of TIMP3 in patients with thyroid nodules, especially FTC, using the direct bisulfite sequencing method. However, as for the limitations of this cross-sectional study, we had a small sample size to evaluate the probable constitutional changes in TIMP3 methylation. We did not have a follow-up study to investigate the prognostic role and/or recurrence to give more credibility to the results. The long-term diagnosis of TIMP3 methylation with BRAF mutation is not entirely understood due to the limited findings documented. The prognostic importance of TIMP3 methylation with BRAF mutation is recommended to be further evaluated based on long-term outcomes. Moreover, a multi-gene panel is required to differentiate between malignant and benign cases to obtain better and more accurate results in diagnosis and prognoses. Due to a lack of access to cytological specimens, it was impossible to investigate, and the study was performed only on surgically resected specimens.