The role of matrix metalloproteinase-9 as a prognostic biomarker in papillary thyroid cancer

Well-differentiated thyroid carcinomas are classified into papillary and follicular thyroid carcinoma (PTC and FTC), and are the most frequent endocrine malignancy, consisting over 70% of the cases [1]. The estimated incidence of thyroid cancer is > 3 fold higher in women [2].

Environmental, genetic and hormonal factors are the most important triggers in the etiology and increased prevalence of human cancers [1], with genetic mutations playing the most important role in tumorigenesis. Numerous oncogenes and rearrangements with different frequencies and properties have been implicated in the pathogenesis of PTCs, mostly involving the abnormal activation of RAS-RAF-MEK- mitogen-activated protein kinase (MAPK) pathway [3]. Of these, activating mutation in the B isoform of the Raf kinase gene, results in a valine to glutamic acid substitution at amino acid 600 (BRAF V600E) which occurs in PTC and PTC-derived anaplastic thyroid cancer (ATC) [4]. Its prevalence is highly variable, ranging from 29 to 83% among different reports [5]. In the previous study, its prevalence was reported as 40% in a sample of Tehran’s population [6]. In some studies, this mutation was correlated with advanced tumor or aggressive phenotypes; whereas, no such association was found in other studies [5, 7‐10]. Moreover, it was found to be associated with larger tumor size and lymph node metastasis (LNM) in the Tehranian population [6].

Modifications in matrix metalloproteinases (MMPs) also play a main role in various human cancers, and can be involved in the differentiation, morphogenesis and tissue remodeling during angiogenesis, tumor invasion and metastasis [11]. One of the most significant MMPs is MMP-9, also known as 92-kDa gelatinase B type IV collagenase. Due to its complex regulation activity, it is involved in the pathogenesis of many diseases [12]. Some reports showed that the BRAF V600E can induce MMPs [13, 14]; a positive correlation has been reported between BRAF V600E and MMP-9 and MMP-2 expressions, both being correlated with tumor extrathyroid extension [15].

Evaluating the role of MMP-9 along with BRAF mutation can be useful in predicting the outcome of many cancers, especially PTC. Therefore, first, we tested the hypothesis that whether BRAF V600E mutation has an association with MMP-9 activity or not? We then studied the clinical relevance of MMP-9, in terms of invasion and metastasis among PTC patients also evaluating the predictive and prognostic role of MMP-9 levels.

In the present study, the MMP-9 was significantly overexpressed in tumoral compared to adjacent non-tumoral tissues, and the protein levels showed a significant increase. In addition, the MMP-9 expression in PTC patients was significantly elevated in comparison to MNG participants, also with a significant increase in its protein levels. The MMP-9 mRNA levels in patients aged ≥45 years, with higher TNM stages and lymphovascular invasion was also significantly increased; its protein levels were upregulated as well. No significant association was found between MMP-9 mRNA and protein levels with BRAF V600E mutation and other clinicopathological features. After adjustment for age and gender, logistic regression showed that the odds of MMP-9 expression in the PTC group is about 1.2 times the MNG group; in higher TNM stages, it is 1.83 times the lower stages; and in patients with lymphovascular invasion it is 3.97 times those without invasion.

Although the MMP-9 regulation is not related to transformation of the normal cells into tumoral, it plays an essential role in degrading of type IV collagen (the major component of the basement membrane), thus promoting tumor invasion. Neutrophils, macrophages, or even transformed cells, secrete MMP-9. The extreme secretion of MMP-9 damages tissues in the absence or lack of their inhibitors. In carcinomas, this damage by MMP-9 leads to metastasis and invasion, which are serious challenges.

There are limited studies on the relation between MMP-9 expression and clinicopathological features in PTC. However, in line with our results, Buergy et al. (using the ELISA method) showed that MMP-1 and MMP-9 expressions significantly increase in aggressive FTC or advanced clinical PTC (with extrathyroid invasion, lymph-node metastasis, and high degree of tumor infiltration) compared to adenomas [18].

Although recent studies reported that MMP-9 upregulation is related to BRAF V600E mutation [15], our results showed no significant association between BRAF status and MMP-9 activity in our sample set; this finding is in line with previous reports from other groups in ATC cell lines and colorectal carcinoma [19, 20]. Actually, not all MMP genes have an AP-1 site in their promoter region, regulated by the BRAF-dependent ERK pathway. Other transcription factors, such as STAT, NF-kB and ETS, modulate the regulation of each MMP gene differently [21]. Briefly, BRAF may be involved in the development of tumor via other mechanisms, independent from the MMPs pathway. Furthermore, it seems that the combination of MMP/TIMP at the leading margin of each tumor may be able to create a fine-tuned microenvironment, regardless of MMP expression or baseline oncogenic BRAF mutation. Therefore, further studies should focus on the leading margin of tumors and consider the complete MMP/TIMP system to gain a more detailed insight into the biology of extracellular matrix (ECM) degradation and tumor cell invasion, as a prerequisite for PTC metastasis. Moreover, these contradictory reports may be due to different types of PTC.

In accordance to our results, Huang et al. evaluated mRNA and protein levels of HMGB1, MMP-9 and VEGF-C in 58 PTC, 20 adenomas, 25 MNG and 10 normal participants, using the IHC method; they found that protein levels of MMP-9, VEGF-C, and HUMGB1 were increased in PTC patients. However, in contrast, they demonstrated a positive association with tumor size and LNM, and found no associations with age and gender [22]. Marečko et al. reported a positive correlation between active MMP-9 and LNM, extrathyroidal invasion and degree of tumor infiltration in 120 PTC patients, using IHC and gel zymography. In addition, this correlation was found with the age of the patient, but not with tumor size or gender [23]. Similar to our findings, Kumar et al. assessed MMP-9 activity in some human cancers, including thyroid, breast and colorectal carcinomas; they revealed that this activity was associated with neoplasm growth, invasion and metastasis. Moreover, in thyroid cancer it increased from 33% in stage III to 75% in stage IVA, indicating that MMP-9 was expressed more in advanced stages of malignant diseases [24]. He et al. evaluated MMP-9 and MMP-2 levels in 41 PTC serum samples using ELISA, before and after Radiofrequency Ablation (RFA). They observed that the levels of these two MMPs reduced after RFA; also, they showed that age, degree of calcification, regularity of shape and diameter, and number of foci were effective independent risk factors for the prognosis after RFA [25]. Zhang et al. evaluated the diagnostic values of ultrasounds such as conventional ultrasound (US), contrast enhanced ultrasound (CEUS) and MMP-9 regulation in predicting the cervical LNM in 156 PTC patients, using IHC. They recognized a significant difference in MMP-9 between PTCs with and without cervical LNM; suggesting that combining conventional US, CEUS features and MMP-9 may be useful in predicting the cervical LNM of PTC [26].

Our results indicated that MMP-9 regulation could be used as a potential biomarker to differentiate PTC from MNG, with 70% diagnostic precision; thus, it is suggested that using this gene as a potential diagnostic biomarker for PTC could be more helpful. For further validation of MMP-9’s diagnostic value in the preoperative evaluation of thyroid nodules, analyzing the proposed gene in “intermediate” or “suspicious” FNA cases is required.

Since the pathogenesis of thyroid cancer involves a wide range of molecular disorders occurring throughout life, numerous types of molecular mechanisms in the formation of thyroid tumors have been investigated, including the role of MMPs [27]. MMPs are zinc-ion dependent endopetidases, digesting the underlying membrane and ECM. They take part in a variety of physiological and pathological processes, such as wound healing, angiogenesis, chronic inflammatory disease and tumor growth including metastasis, invasion, and progression [28]. The mechanism by which MMPs contribute to invasion via proteolytic destruction of the ECM (a central event of tumor invasion) is regulating the dynamic interactions between ECM–cell and cell–cell, during migration. Overregulation of MMP-9 has been associated with epithelial to mesenchymal morphological transition [29].

Various efforts have been made to develop new therapies that can control stimulation of MMPs expression in cancerous tissues to block the growth of cancer invasion and metastasis [30, 31]; these findings provide further evidence for MMPs as a suitable target for molecular therapies in aggressive thyroid cancer patients.

Several studies have demonstrated MMPs being involved in the growth and development of thyroid cancer, while there is no agreement on the precise immunolocalization of MMPs, either in stromal cells or tumor cells, or in both. Tumor cell invasion through proteolytic enzymes results in the destruction of cells surrounding the ECM [13, 27, 30]. However, it appears that MMPs are important enzymes involved in local attack and metastatic PTC cancer, being reported in several studies related to different members of the MMP family, including MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, MMP-14 and MMP-15 [13, 32]. The cDNA microarray assessment showed that MMP-2 expression had over two folds increase in PTC tissues compared to benign thyroid tissues. In particular, previous studies have reported an increase in MMP-2 and MMP-9’s mRNA and protein levels in a variety of thyroid cancer cells [30, 33]. Other studies reported that the expression of MMP-2, along with MMP-9, TIMP-1 and TIMP-2, is associated with thyroid tumor invasion and metastasis [13, 34]; these investigations indicate the major role of MMPs expression and the necessity for understanding the mechanism and effectiveness of different factors in thyroid cancer treatment.

Of the current study’s limitations was that we had no information on the follow-up of patients, to evaluate the correlation between MMP-9 expression and persistence of the disease or mortality. We had no access to FNA samples of the studied patients, to perform MMP-9 expression analysis for the diagnosis of PTC.

In conclusion, our findings revealed that the MMP-9 mRNA and protein levels had no association with BRAF V600E mutation in Iranian PTC patients. However, these levels were associated with age, TNM stages and lymphovascular invasion, which were defined as malignant factors. Moreover, elevated levels were observed in PTC patients compared to MNG participants. Therefore, it could be concluded that MMP-9 levels may be a potential biomarker to distinguish PTC from MNG patients.