The influence of MMP-14, TIMP-2 and MMP-2 expression on breast cancer prognosis

Introduction Matrix metalloproteinase (MMP)-2 is very active at degrading extracellular matrix. It is under the influence of an activator, membrane type 1 MMP (MMP-14), and the tissue inhibitor of metalloproteases (TIMP)-2. We hypothesized that the individual expression of these three markers or their balance may help to predict breast cancer prognosis. Methods MMP-2, MMP-14 and TIMP-2 expression has been evaluated by 35S mRNA in situ hybridization on paraffin material of 539 breast cancers without distant metastasis at diagnosis and with a median follow-up of 9.2 years. Results MMP-2 and MMP-14 mRNA was detected primarily in reactive stromal cells whereas TIMP-2 mRNA was expressed by both stromal and cancer cells. Of the three molecules, an adjusted Cox model revealed that high MMP-14 mRNA (≥ 10% cells) alone predicted a significantly shorter overall survival (p = 0.031) when adjusted for clinical factors (tumor size and number of involved lymph nodes). Prognostic significance was lost when further adjusted for Her-2/neu and urokinase-type plasminogen activator (p = 0.284). Furthermore, when all three components were analyzed together, the survival was worst for patients with high MMP-2/high MMP-14/low TIMP-2 (5 year survival = 60%) and best with low MMP-2/low MMP-14/high TIMP-2 (5 year survival = 74%), but the difference did not reach statistical significance (p = 0.3285). Conclusion Of the MMP-14/TIMP-2/MMP-2 complex, MMP-14 was the factor most significantly associated with the outcome of breast cancer and was an independent factor of poor overall survival when adjusted for clinical prognostic factors, but not for certain ancillary markers.


Introduction
In breast cancer, matrix metalloproteinase (MMP)-2 is a protease produced essentially by stromal cells. In vitro studies have clearly demonstrated that it degrades molecules that are abundant in the extracellular matrix (ECM) [1]. MMP-2 is also one of the major targets of recently developed synthetic MMP inhibitors [2,3]. However, recent literature demonstrates that the mechanism of action of MMP-2 is complex and that other molecules modulate its activity [4,5].
MMP-2 is secreted in an inactive pro-enzymatic form and, unlike other MMPs, its activity is modulated by tissue inhibitor of metalloproteases (TIMP)-2 [6] and the membrane type 1 MMP (MMP-14) [7]. Using zymography, breast cancers were found to express higher levels of activated MMP-2 than benign lesions [8,9] and in vitro studies showed that activated MMP-2 only is associated with an aggressive potential in breast cancer cell lines [10].
Animal studies showed that carcinoma cell lines transfected with MMP-14 produced higher levels of active MMP-2 and developed more lung metastases compared to parent tumor cells [11]. TIMP-2 inhibits most if not all activated MMPs and was shown to form a complex specifically with pro-MMP-2 [12]. However, the role of TIMP-2 is ambiguous since, in addition to its inhibitory effect, it is a main player in the MMP-2 bp = base-pair; ECM = extracellular matrix; HSP = heat-shock protein; MMP = matrix metalloproteinase; OS = overall survival; TIMP = tissue inhibitor of metalloproteases; uPA = urokinase-type plasminogen activator. activation cascade [13]. However, clinical studies on breast cancer are rare, limited in size and do not address the potential interaction of all three factors (MMP-2, MMP-14 and TIMP-2).
This prompted us to test the hypothesis that the individual expression or the balance between MMP-2, MMP-14 and TIMP-2 expression may help to predict breast cancer prognosis.

Population
The patients included in this study had node-positive or nodenegative disease proven by histological examination of axillary lymph nodes, but with absence of distant metastases at diagnosis. The surgery for breast carcinoma was performed between 1 January 1980 and 31 December 1986. Clinical information was obtained from the patients' charts by experienced research nurses. The tumor size, and number of examined and positive lymph nodes were obtained from the pathology reports. The histological types along with the histological and nuclear grades were reassessed by one of us (BT). The project has been approved by the Laval University Ethical Review board.
In situ hybridization MMP-2, MMP-14 and TIMP-2 expression was analyzed by in situ hybridization. The technique used was a modification of the method of Wolf and colleagues [14] as described before [15]. S 35 labeled antisense RNA probes were used. Plasmids were kindly provided by the late Prof. Paul Basset, IGBMC, Illkirch, France (MMP-2 and MMP-14) and the late Dr Anna Kossakowska, Calgary, Alberta (TIMP-2). cDNA inserts were prepared from breast cancer cDNA libraries, subcloned in pBluescript II (MMP-2 and MMP-14) and pBS KS-(TIMP-2) vectors and were used as templates for in vitro transcription to generate sense and anti-sense probes. The 2,124 base-pair (bp) MMP-2 cDNA was subcloned in the EcoRI site and extended from nucleotide 26 to 2,150; the 1,159 bp MMP-14 cDNA was subcloned in the EcoR1 site and extended from nucleotide 454 to 1,613; and the 210 bp TIMP-2 cDNA was subcloned in the HindIII/BamH1 site. Reduction of the probe length was achieved by partial alkaline hydrolysis. The quality of RNA preservation has been assessed with the use of antisense RNA probes for β-actin and negative controls were obtained with the use of sense probes. In situ hybridization for MMP-11 and urokinase-type plasminogen activator (uPA) were performed on all cases as previously described [15].
Flow cytometry DNA flow cytometric analysis was performed in all cases on formalin fixed-paraffin embedded tissues using the method described by Dressler and colleagues [16]. Ploidy and Sphase fraction were determined on single parameter histograms with the use of ModFit ® (Verity Software House Inc., Topsham, ME, USA). Debris were excluded with the use of the 'Single Cut' algorithm. G0, G1 and G2 M were defined from a Gaussian curve while S-phase fraction was evaluated with the trapezoid model. All cases with a coefficient of variation exceeding 8% were excluded; the coefficient of variation averaged 6.1% (range 2.5% to 8.0%).

Hormone receptors
Estrogen and progesterone receptor measurement was obtained from the patient charts. In the early 1980s, analyses were performed using the hydroxylapatite method [17]. Positivity was defined by hormone levels above 10 fmol/mg proteins.

Interpretation
The light microscopic interpretation of in situ hybridization was done by two of us (BT, DT) without knowledge of the clinical information. The scoring was assessed separately on cancer and stromal cells using a semi-quantitative scale similar to that used for immunohistochemistry. For each cell compartment, the percentage of cancer or stromal cells expressing the marker (0%, <10%, 10% to 50%, >50%) was evaluated on the whole tumor surface of one representative section.

Statistical analysis
Distant-metastasis-free and overall survival (OS) curves were obtained for each protease according to Kaplan and Meier [18]. The difference between the curves was assessed using the log-rank test [19]. Correlation between protease expression and other recognized prognostic variables in breast cancer was determined by the Chi-square test [19]. Distantmetastasis-free and OS curves were generated using 10% as the cutoff point between those expressing low (negative) or high (positive) protease levels by stromal cells, as defined before [15,20]. The Cox proportional hazard model was used to evaluate the relationship between the expression of each protease, or a combination of more than one protease, and recurrence or death, adjusting for other known or suspected prognostic factors [21]. Hazard ratios for the occurrence of distant metastases or death of patients with any of the above (page number not for citation purposes) combinations were obtained by univariate and multivariate analyses.

Results
In our tumor bank, information for MMP-2 was available from 610 breast cancers, from 544 for MMP-14 and from 549 cases for TIMP-2. Results for all three molecules were available from 539 cases and all analyses have been performed on this group of patients. The patients' ages ranged from 29 to 72 years (average, 57.3). Of these patients, 190 (35.3%) were node-negative, 340 (63.1%) had node invasion, and the node status was unknown in 9 (1.6%). At last follow-up, 258 (47.9%) were alive and the follow-up of surviving patients ranged from 5.2 to 14.6 years (average, 9.5 years); 21 (3.9%) were lost to follow-up. MMP-14 expression by stromal cells was significantly associated with a younger age, lymph node involvement, a ductal histology, HER-2/neu overexpression, and cathepsin D, MMP-11 (stromelysin-3) and uPA expression by stromal cells (Table 1). MMP-2 expression by stromal cells was associated with lymph node involvement, ductal histology, HER-2/neu and HSP-27 overexpression as well as cathepsin D, MMP-11 and uPA expression by stromal cells. TIMP-2 expression by stromal cells was associated with a ductal histology, and expression of HSP-27 and uPA. TIMP-2 expression by cancer cells was associated with peritumoral lymphovascular invasion, and expression of HSP-27, cathepsin D and uPA by stromal cells. Figure 2 shows the OS curves for each of the three proteases. MMP-2 and MMP-14 expression by cancer cells was not evaluated because too few cases expressed those markers. No difference in OS was found between high and low MMP-2 and between high and low stromal and cancer TIMP-2 expression. However, high MMP-14 expression by stromal cells predicted a shorter survival (p = 0.05). In fact, the 5 year survival reached 72.2% for patients whose tumors had low MMP-14 levels as opposed to 64.6% for those with high MMP-14 levels. By multivariate analysis, using the Cox model adjusting for tumor size and number of involved lymph nodes, high MMP-14 expression was clearly a significant factor of poor survival ( Table 2). The statistical significance was lost when HER-2/neu and uPA were included in the model. A trend was found for the association of MMP-14 expression and metastasis-free survival (p = 0.08). Indeed, the 5 year metastasis-free survival was 64.0% for patients with low MMP-14 as opposed to 54.3% for those with high MMP-14. Figure 3 shows the overall survival curve considering the balance between MMP-2, MMP-14 and TIMP-2. Of the different combinations, OS was worst for those patients with high stromal MMP-2/high stromal MMP-14/low stromal TIMP-2 (60.3% survival at 5 years) and best with low stromal MMP-2/low stromal MMP-14/high stromal TIMP-2 (74.4% survival at 5 years).  This difference did not, however, reach significance (p = 0.25).

Discussion
This study confirms the complexity of the role of proteases on breast cancer prognosis. However, the prognostic influence of MMP-2 expression is not clear in the literature. Talvensaari are among the few investigators who demonstrated a significant association between high MMP-2 mRNA or protein expression levels and a poor outcome. In our study of close to 600 cases, MMP-2 overexpression predicted a 15% greater risk of recurrence [15] but the association of MMP-2 expression with prognosis was not statistically significant. Other studies have failed to relate MMP-2 expression to a poorer prognosis [2,15,25]. These data are consistent with the complexity of the biology of MMP-2. In our study, they may also be explained in part by the fact that in situ hybridization cannot differentiate between the active and inactive forms.
In our study, when each factor was taken separately, and after adjustment for clinical prognostic factors, only MMP-14 was a significant factor of survival, suggesting that MMP-14 may be  HSP, heat-shock protein; MMP, matrix metalloproteinase; Pos, positive; TIMP, tissue inhibitor of metalloproteases; uPA, urokinase-type plasminogen activator.  In our study, TIMP-2 did not provide independent prognostic information. In the literature on breast cancer, the role of TIMP-2 was controversial. This may explain contradictory results in which patients with higher TIMP-2 expression either experienced low cancer recurrence/progression [44,45] or a poor prognosis [25, [46][47][48][49], emphasizing the overall activating or inhibitory role of TIMP-2. These contradictory findings may be explained by the versatile role of TIMP-2 [12]. Indeed, TIMP-2 increases growth rates of murine, bovine and human cells [50], but also inhibits tumor growth and angiogenesis in melanoma B16F10 cells [51], attenuates migration of MDA-MB23 breast cancer cells through a bone marrow fibroblast monolayer [52] and abolishes the tumor-promoting effect of fibroblasts on MCF7 cells injected with matrigel in nude mice [53].

Association of high protease expression by stromal cells with other prognostic factors
The relationship of survival with the combination of MMP-2, MMP-14 and TIMP-2, although not statistically significant, showed that survival was worst for those patients with high MMP-2/high MMP-14/low TIMP-2 and best with low MMP-2/ low MMP-14/high TIMP-2. This lack of statistical significance may be explained by the sample size, which may be too small to reach significance, or by the versatile role of TIMP-2, which may be either a favorable or an unfavorable factor. Different combinations of proteases and protease inhibitors or activators have been investigated in the literature but clinical studies are limited and involve few patients. Clinical studies suggest that TIMP-2, or the ratio of MMP-2 and TIMP-2 expression, may play a critical role on cancer progression. For example, higher MMP-2/TIMP-2 ratios were associated with recurrences in patients with bladder [54] or uterine cervix [55] cancer. In a study on 14 patients, Onisto and colleagues [56] report that a high MMP-2/TIMP-2 ratio correlated with lymph node metastases in breast cancer while it predicted a better outcome in another study [25].

Conclusion
Our data show that, of the three proteases studied, MMP-14 was most strongly associated with breast cancer prognosis but was not independent of HER-2/neu or uPA, which may limit its usefulness as a prognostic marker. When all three proteases are studied in combination, a tendency was found for tumors with high MMP-2, high MMP-14 and low TIMP-2 expression to predict a poor prognosis but the results did not reach significance.