Ca

Figure 1. Matrix metalloproteinases groups.

Matrix metalloproteinases (MMPs) can be classified into eight distinct groups by their domain structure, five of which are secreted and three of which are membrane-type MMPs (MT-MMPs). Secreted MMPs: The minimal-domain MMPs contain an N-terminal signal sequence (Pre) that directs them to the endoplasmic reticulum, a propeptide (Pro) with a zinc-interacting thiol (SH) group (from cysteine) that maintains them as inactive zymogens and a catalytic domain with a zinc-binding site (Zn). In addition to the domains that are found in the minimal domain MMPs, the simple hemopexin domain MMPs have a hemopexin-like domain—that is connected to the catalytic domain by a hinge (H), which mediates interactions with tissue inhibitors of metalloproteinases, cell-surface molecules and proteolytic substrates. The first and the last of the four repeats in the hemopexin-like domain are linked by a disulphide bond. The gelatin-binding MMPs contain three inserts that resemble collagen-binding type II repeats of fibronectin (Fi) and is responsible for the specific binding to gelatins and collagens. The furin-activated secreted MMPs contain a recognition motif for intracellular furin-like serine proteinases (Fu) between their propeptide and catalytic domains that allows intracellular activation by these proteinases. This motif is also found in the vitronectin-like insert (Vn) MMPs and the membrane-type MMPs (MT-MMPs). MT-MMPs: MT-MMPs include transmembrane MMPs that have a C-terminal, single-span transmembrane domain (TM) and a very short cytoplasmic domain (Cy), and the glycosylphosphatidylinositol (GPI)-anchored MMPs. MMP-23 represents a third type of membrane-linked MMP. It has an N-terminal signal anchor (SA) that targets it to the cell membrane, and so is a type II transmembrane MMP. MMP-23 is also characterized by its unique cysteine array (CA) and immunoglobulin (Ig)-like domains instead of the hemopexin domain. Adapted from Ref. 7.

sequences. Two important elements for transcriptional regulation are an AP-1 binding site for AP-1 transcription factors which comprise of members of the FOS and JUN family of transcription factors, and a PEA-3 element that binds ETS transcription factors. The AP-1 site, located approximately 70 bp upstream from the transcriptional start site, has been considered to play an important role in the transcriptional activation of the MMP promoters, whereas interaction between AP-1 and PEA-3 site is necessary for basal transcription and trans-activation by cytokines and growth factors. The DNA binding and trans-activation of both AP-1 and ETS transcription factors are regulated by mitogen-activated protein kinases (MAPKs) (12). Interestingly, AP-1 site is not present in the promoter region of MMP-2, a critical metalloproteinase involved in cancer metastasis, and MMP-14, which is involved in the activation of MMP-2 (13). Another transcriptional control of MMP expression is the presence of naturally occurring sequence variation or single nucleotide polymorphisms (SNPs) in the promoters of MMP genes (14). These genetic polymorphisms have been shown to have allele-specific effects on the MMP promoter activities, e.g. an insertion of a guanine at position -1607 in the MMP-1 gene promoter creates the core sequence (5'-GGA-3') of a binding site for the ETS transcription factors. The 2G allele has a higher transcriptional activity in melanoma cells and is associated with more invasive tumors (15).

All MMPs are synthesized as prepro-enzymes. Most MMPs are secreted as inactive, latent pro-MMPs, with the exception of MT-MMPs, which are membrane bound and localize at the cell surface. Since MMP activation occurs after secretion into the extracellular milieu, an important control point for MMP activity is the proteolytic cleavage of pro-MMPs. It has been demonstrated that serine proteases such as trypsin, plasmin, or urokinase initiate activation of MMPs from the zymogen form (16). Some MMPs can also activate other members of the family. A good example is the activation of pro-MMP-2 at the cell surface by MMP-14 and TIMP-2 (17): TIMP-2 binds MMP-14 at its amino terminus and pro-MMP-2 at its carboxyl terminus, which allows an adjacent, non-inhibited MMP-14 to cleave the bound pro-MMP-2. MMP-14 does not fully activate MMP-2 and another already activated MMP-2 is required to remove a residual portion of the MMP-2 propeptide (18). Pro-MMP2 can also be activated by MMP-15 through TIMP-2 independent mechanism (19). Although most MMPs are activated outside the cells by serine proteases or other activated MMPs, MMP-11, MMP-28, and the MT-MMPs can also be activated by intracellular furin-like serine proteases before they reach the cell surface (20).

A final and important control point of MMP activity is the inhibition of activated enzymes by endogenous inhibitors. The main inhibitor of MMPs in tissue fluids is a-macroglobulin, an abundant plasma protein (21). a-Macroglobulin binds to MMPs and the MMP/cx-macroglobulin complex then binds to a scavenger receptor and is irreversibly cleared by endocytosis. In a similar way to thrombospondin-2 forms a complex with MMP-2 and facilitates scavenger-receptor-mediated endocytosis and clearance (22). By contrast, thrombospondin-1 binds to pro-MMP-2 and -9 and directly inhibits their activation (23-24). Curiously, thrombospondin-1 has also been reported to increase MMP-2 and -9 activation (25).

Another group of endogenous MMP inhibitors are TIMP family of inhibitors. At present, four structurally related members have been characterized (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) with 40-50% sequence identity at the ammo acid levels (26). TIMPs are small, low molecular weight proteins (20-30 kDa). They differ in tissue-specific expression and ability to inhibit various MMPs. They reversibly inhibit active MMPs with relatively low selectivity by occupying the catalytic domain of activated enzymes (27-28). The TIMP/MMP complex is a tight binding, non-covalent complexes with a stoichiometric 1:1 molar ratio. Unlike TIMP-1, TIMP-2, and TIMP-4, which are secreted in soluble form, TIMP-3 has a unique association with ECM. Studies with Timp-2-deficient mice indicate that the dominant physiological function of TIMP-2 is activation of MMP-2 (29). Apart from inhibiting MMPs, TIMP-3 has been shown to promote apoptosis whereas TIMP-1 is active in blocking apoptosis and overexpression of TIMP-2 protect cancer cells from apoptosis (30-32).

MMPs AND TIMPs IN THYROID CANCER PROGRESSION AND METASTASIS

The expression and activity of MMPs are increased in many types of human cancer, and this correlates with advanced tumor stage, increased invasion and metastasis, and shortened survival. Many studies show a negative association between MMPs activity and prognosis (7). MMP-2 and MMP-9 are of particular importance in tumor cell invasion, because they degrade type IV collagen, the main structural component of the basement membrane. Tumor cells expressing high levels of these enzymes are highly metastatic. Cancer cells are not the only source of MMPs. Stromal cells are also participated in the production of MMPs (20). MMPs that are secreted by stomal cells can still be recruited to the cancer cell membrane, e.g. MMP-2 mRNA is expressed by stromal cells of human breast cancers, whereas MMP-2 protein is found on both stromal and cencer cell membranes (33). It has been shown that cancer cells can stimulate tumor stromal cells to produce MMPs in a paracrine fashion through secretion of cytokines, growth factors, and EMMPRIN (extracellular matrix metalloproteinase inducer). EMMPRIN is an intrinsic plasma membrane glycoprotein produced in high amounts by cancer cells, which stimulates local fibroblasts to synthesize MMPs (34). Tumor cell interactions with fibroblasts via EMMPRIN leads to fibroblast-induced local degradation of basement membrane and ECM components, thus facilitating tumor cell invasion. It has been shown that MMP-9 production in tumor infiltrating macrophages play a critical role in angiogenesis and progressive growth of human ovarian tumors in mice (35). Stromal cells and their products have been reported to even cause tumorigenic transformation of adjacent epithelial cells (36).

Earlier studies have shown that invasion by cultured human follicular thyroid carcinoma is correlated with increased production of beta 1 integrins and MMPs (37). Correlation between MMPs and ECM degradation is further demonstrated by the study of plasmin activation system in metastatic follicular thyroid carcinoma cell lines (38). As mentioned earlier, plasmin is a serine protease involved in the activation of MMPs. Plasmin is generated from plasminogen by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). UPA-mediated plasmino-gen activation is an important pathway in tumor invasion and can be inactivated by plasminogen activator inhibitors (PAI-1 and -2) (39). Decreased activity of PAI-1 is associated with greater ECM degradation in follicular thyroid carcinoma cell lines (38).

Overexpression of MMP-2, and MMP-9 has been found in thyroid carcinomas and is correlated with large tumor size, high intrathyroid invasion, presence of lymph node metastasis, and advanced disease stage (40). A more comprehensive study of MMPs profile involving seven secreted MMPs (MMP-1, -2, -3, -7, -8, -9, and -13) and three membrane-bound MMPs (MMP-14, -15, and-16) demonstrates that the major MMPs produced in papillary thyroid carcinomas are MMP-2 and MMP-14 (41). The pro-MMP-2 activation and the expression of MMP-14, known to activate pro-MMP-2 at the cell surface, are considerably higher in carcinomas with lymph node metastasis than those without metastasis. MMP-15 expression is confined to 26% of cases. MMP-2, MMP-14, and MMP-15 are immunostained in both carcinoma and stromal cells (41). In a separate study, increased MMP-2 expression is found in follicular and anaplastic thyroid carcinomas, but not in follicular adenomas (42). Interestingly, MMP-2 mRNA expression is restricted to fibroblasts in the stroma adjacent or close to invading tumor cells (42). MMP-1 expression is significantly greater among follicular and papillary thyroid carcinomas compared to benign lesions. However, there is no relationship between MMP-1 expression and invasion, metastasis, or disease recurrence (43). Both carcinoma and stromal cells have been shown to express MMP-1 (43-44). A recent cDNA and tissue microarray study shows that MMP-11 is up-regulated in 67% of papillary thyroid carcinoma tissues (45).

Both TIMP-1 and TIMP-2 expression are increased in thyroid carcinomas, and are correlated with large tumor size and advanced disease stage (40,46), which seem to be contradictory to the role of TIMPs as inhibitors of tumor cell invasion and metastasis. Further study shows stronger TIMP-1 immunostaining in the stromal cells surrounding the tumor, suggesting that the high levels of TIMP-1 transcripts in advanced stage of thyroid carcinoma are likely represent a stroma response to tumor cell invasion. Overexpression of TIMP-1 by gene transfer has resulted in a significant suppression of invasive potential of NPA cells, a poorly differentiated thyroid carcinoma cell line (46). Reduced TIMP-1 expression has been shown in recurrent papillary thyroid carcinoma when compared to non-recurrent carcinomas (43). Apparently, tumor invasion is not dependent on the absolute levels of TIMPs or MMPs. It is the balance between TIMPs and MMPs that determines the potential of thyroid tumor invasion and metastasis. Indeed, the molar ratio of total amounts of MMPs:TIMPs is significantly higher in the thyroid carcinoma samples than in the adenoma and normal samples (41).

Many MMP genes are transcribed at low or undetectable levels in normal thyrocytes. Analysis of MMPs and TIMPs expression in vitro demonstrates that MMP-1, -2, -9, -14, and TIMP-1, -2, -3 mRNA are present in normal thyrocytes, malignant thyroid cells and thyroid-derived fibroblasts. The basal levels of MMP-1, -9, and -14 are much lower in thyrocytes than in malignant thyroid cells and thyroid-derived fibroblasts, whereas high basal levels of MMP-2, TIMP-1, -2, and -3 are found in all three cell types without striking difference (47-48). IL-1 can upregulate MMP-l and MMP-9 mRNA in all the cell types through activating nuclear factor of and has no significant effect on TIMPs, MMP-2, and MMP-14. also acting via

NF-/cB passway, can stimulate MMP-9 mRNA expression in malignant thyroid cells and thyroid-derived fibroblasts. EGF, acting via protein tyrosine kinase, can only stimulate MMP-1 expression in malignant cells (49). Phorbol—myristate acetate (PMA, an active phorbol ester) can induce MMP-1, MMP-9 and TIMP-1 mRNA in all the cell types, MMP-14 in malignant thyroid cells and thyroid-derived fibroblasts (47-49). Since PMA, acting via protein kinase C (PKC), can induce c-jun and c-fos gene expression in human thyroid cells, and their gene products are AP1 transcriptional factors (50), it is likely that PKC is involved in the induction ofMMP transcription. Although thyroid-stimulating hormone (TSH) has no significant effect on the basal MMP-1, or TIMP-1 mRNA levels, it can cause a dose-dependent inhibition in PMA or EGF-induced MMP-1 mRNA in malignant cells, and PMA-induced MMP-1 and TIMP-1 mRNA in benign thyroid cells. The repressive action of TSH on MMP-1 mRNA can be mimicked by the forkolin and 8-bromo-cAMP, and can be abrogated by a protein kinase A (PKA) inhibitor, H-89, suggesting that it is PKA-mediated (49). MMP-11, -13, and -18 genes are thyroid hormone responsive genes. Although they have not been shown to be involved in thyroid cancer, they have distinct functions during frog embrogenesis (51).

Several studies have shown that high serum levels of MMP-2, MMP-9, and TIMP-1 are associated with tumor invasion and poor survival in several types of cancer (5254). Thus, they may be used as prognostic markers in cancer patients. Higher levels of MMP-2 and TIMP-2 are detected by ELISA in peripheral blood of thyroid cancer patients when compared to normal control, and increased blood levels of MMP-3 and MMP-9 appear to be associated with medullary thyroid cancer (55). It remains to be determined whether serum levels of MMPs and TIMPs can be used as diagnostic or prognostic markers for thyroid carcinoma.

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