Enhanced activation of matrix metalloproteinase-9 correlates with the degree of papillary thyroid carcinoma infiltration

Abstract

Aim: To determine whether matrix metalloproteinase-9 (MMP-9) may be a useful adjunctive tool for predicting unfavorable biological behavior of papillary thyroid carcinoma (PTC) by evaluating the expression profile and proteolytic activity of MMP-9 in PTC by different techniques and correlating the findings with clinicopathological prognostic factors.

Methods: Immunohistochemical localization of MMP-9 was analyzed with antibodies specific for either total or active MMP-9. Activation ratios of MMP-9 were calculated by quantifying gel zymography bands. Enzymatic activity of MMP-9 was localized by in situ zymography after inhibiting MMP-2 activity.

Results: Immunostaining of total and active MMP-9 was observed in tumor tissue and occasionally in non-neoplastic epithelium. Only active MMP-9 was significantly associated with extrathyroid invasion, lymph-node metastasis, and the degree of tumor infiltration (P<0.001, P=0.004, and P<0.001, respectively). Gelatin zymography revealed a correlation between the MMP-9 activation ratio and nodal involvement, extrathyroid invasion, and the degree of tumor infiltration. In situ zymography showed that gelatinases exerted their activity in tumor parenchymal and stromal cells. Moreover, after application of MMP-2 inhibitor, the remaining gelatinase activity, corresponding to MMP-9, was highest in cancers with the most advanced degree of tumor infiltration.

Conclusions: This is the first report suggesting that the evaluation of active MMP-9 by immunohistochemistry and determination of its activation ratio by gelatin zymography may be a useful adjunct to the known clinicopathological factors in predicting tumor behavior. Most important, in situ zimography with an MMP-2 inhibitor for the first time demonstrated a strong impact of MMP-9 activity on the degree of tumor infiltration during PTC progression.

Figure 1

Number and percentage of cases stained for active and total matrix metalloproteinase-9 (MMP-9) in papillary thyroid carcinoma (PTC) and nontumor tissues (NT).

Figure 2

Representative micrographs of immunostaining for total and active matrix metalloproteinase-9 (MMP-9) in thyroid tissue samples. (a) Moderate diffuse immunoreaction for total MMP-9 in one case of encapsulated papillary thyroid carcinoma (PTC). (b) Negative immunostaining for active MMP-9 in the same case. (c) Strong diffuse MMP-9 positivity in a case of PTC with extrathyroid invasion. (d) Strong staining for active MMP-9 in a case of PTC with extrathyroid invasion.

Figure 3

An example of the gelatin zymograms used for matrix metalloproteinase-9 (MMP-9) quantitation by densitometry. Gelatin degradation was monitored by sodium dodecyl sulfate (SDS)-8% polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. Gelatinolytic bands of 92, 83, 68, and 62 kDa corresponded to proMMP-9, active MMP-9, proMMP-2, and active MMP-2, respectively: NT – non-tumoral thyroid tissue, Ei – extrathyroid invasion. Lane 1, tissue extract of papillary thyroid carcinoma (PTC) sample without extrathyroid invasion (Ei-). Lane 2, paired non-tumor tissue from the same patient. Lanes 3 and 4, tumor and paired non-tumor tissue extract from another PTC sample with extrathyroid invasion (Ei+). Lane 5, a duplicate gel of sample (Ei+) incubated in the presence of 13.7 nM ARP-100, a general inhibitor of MMP-2 activity. Lane 6, the same sample incubated with 10 mM EDTA.

Figure 4

Activation ratio of promatrix metalloproteinase-9 (MMP-9) in papillary thyroid carcinoma (PTC) measured by densitometric analysis of zymographic gels. The activation ratios of MMP-9 (proMMP-9) were calculated by dividing the density of the band for active MMP-9 by the sum of the density of the bands for both latent and active forms. The boxes represent the interquartile range and vertical lines show the range of observations. The midlines of boxes give the median values of activation ratios. A, B, C, D are groups of PTC patients with the following degrees of neoplastic infiltration: A – totally encapsulated carcinomas; B – non-encapsulated tumors without thyroid capsule invasion; C – tumors with thyroid capsule invasion; D – tumors with extrathyroid invasion. LNM+ are carcinomas with lymph node metastasis, LNM – tumors without metastases; Ei + and Ei- are tumors with and without extrathyroid invasion, respectively.

Figure 5

In situ zymography of gelatinase activity in papillary thyroid carcinomas with different degrees of neoplastic infiltration. (a) encapsulated carcinoma; (b) non-encapsulated tumor without thyroid capsule invasion; (c) tumor with extrathyroid extension. Horizontal layers (1-3) represent the consecutive sections of the same tumor. Left column, image of the hematoxylin/eosin stained papillary thyroid carcinoma (PTC) tissue sections. Middle column, distribution of gelatinolytic activity in serial tissue sections of the same areas of cases as the left column, subjected to fluorescent in situ zymography. Intense fluorescence was observed predominantly in the follicular epithelium, but also in some cells of tumoral stroma. Right column, matrix metalloproteinase-2 inhibitor-treated subsequent sections of the same cases as left. Decreased gelatinolytic activity is seen in thyrocytes and the stromal compartment.