High Tissue Inhibitor Of Metalloproteinases-1 Levels Research Articles

Tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) and survival following adjuvant chemotherapy in pre-menopausal lymph node-positive breast cancer patients (N=525).

Abstract Abstract #6054 Predictive markers are needed to guide planning of adjuvant therapy for breast cancer patients. We have recently shown that high tumor tissue levels of TIMP-1 are associated with decreased response to chemotherapy in metastatic breast cancer (Schrohl et al, Clin Cancer Res, 2006) suggesting that TIMP-1 may be a predictive marker in breast cancer patients.
 Purpose: This study investigates the association of tumor tissue TIMP-1 levels with response to adjuvant chemotherapy with CMF (cyclophosphamide/methotrexate/5-fluorouracil) or an anthracycline-based regimen.
 Patients and Methods: 525 pre-menopausal lymph node-positive patients were included; 324 patients received adjuvant CMF, 99 received an adjuvant anthracycline-containing regimen and 102 had no adjuvant chemotherapy. Total TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. Using the untreated patient group as a reference group we analyzed the benefit of adjuvant CMF and anthracyclines in TIMP-1 high and low patients, respectively. The median TIMP-1 concentration was used to dichotomize patients into high and low TIMP-1 groups. End points were disease-free and overall survival (DFS, OS).
 Results: The median TIMP-1 level in the total patient group was 12,54 ng/mg of total protein (range, 0 – 112,9 ng/mg). TIMP-1 levels in subgroups according to adjuvant therapy were not significantly different (P=0,20). In a multivariate model including basic clinico-pathological parameters, TIMP-1 low and high patients benefited differentially from adjuvant CMF and anthracyclines when compared to untreated patients. In particular, patients with high tumor levels of TIMP-1 had little benefit from adjuvant anthracyclines. Hazard ratios (HR) and 95% confidence intervals (CI) are given in the table for the analysis of DFS. A similar pattern was seen in the analyses of OS. In the CMF-treated group, both TIMP-1 low and high patients had significantly better survival than untreated patients (P<0,01). Among anthracycline-treated patients those with TIMP-1 low tumors appeared to benefit more from the adjuvant therapy than TIMP-1 high patients although these results did not reach significance in the present analyses.
 
 Conclusion: This study suggests that high tumor tissue TIMP-1 levels are associated with decreased benefit from adjuvant chemotherapy. Especially in the group treated with anthracycline-based therapy there is a strong tendency for TIMP-1 high tumors to be less sensitive to the treatment. This group, however, is small and should be enlarged to confirm our results. In the group treated with CMF all patients benefit from the therapy with TIMP-1 high patients having slightly less benefit than TIMP-1 low patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6054.

Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n=647) with primary breast cancer.

Abstract Abstract #6042 Background: Randomized trials have in general demonstrated that anthracycline-based chemotherapy prolongs disease-free and overall survival as compared to CMF-based regimens. In the Danish Breast Cancer Cooperative Group (DBCG) 89D randomised trial a 21% improvement in overall survival was observed from substitution of methotrexate in the CMF combination with epirubicin. This suggests that the additional effect of anthracyclines is confined to a subset of the patients. We have previously shown that in vitro grown cancer cells devoid of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) are more sensitive towards chemotherapy than cells expressing TIMP-1 (Davidsen et al., Br J Cancer, 2005). In addition, we have recently published (Schrohl et al., Clin Cancer Res., 2006), that patients with metastatic breast cancer and high levels of TIMP-1 in their primary tumor tissues, have a significantly reduced likelihood of obtaining an objective response to chemotherapy.
 Purpose: The aim of the present study was to evaluate if Tissue Inhibitor of Metalloproteinses-1 (TIMP-1) tumor cell immunoreactivity could be used to identify a subset of patients who benefits from adjuvant chemotherapy.
 Patients and Methods: Formalin fixed paraffin embedded tissue micro arrays from 647 patients who were enrolled in the Danish Breast Cancer Cooperative Group randomized trial 89D comparing adjuvant CMF versus adjuvant CEF were analysed for tumor cell TIMP-1 immunoreactivity. The primary end-point was invasive disease free survival (IDFS). Immunohistochemistry was performed using the anti-TIMP-1 monoclonal antibody VT7 as described previously (Sørensen et al., J. Hist. Cytochem., 2006) and the slides were scored as + or – for positive immunoreactivity.
 Results: Tumor cell TIMP-1 immunoreactivity was found in 75% of the tumor samples. In the CEF treated patients, individuals with TIMP-1 negative tumors had a significant longer IDFS than patients with TIMP-1 positive tumors (p=0.047). The multivariate Cox regression analysis of IDFS showed that CEF was superior to CMF among patients with TIMP-1 negative tumors (HR: 0.51; 95%CI: 0.31 to 0.84, p=0.008), while no significant difference could be demonstrated among patients with TIMP-1 positive tumors (HR: 0.86; 95%CI: 0.66 to 1.11, p=0.24). However, only a non-significanct trend could be demonstrated between TIMP-1 status and CEF versus CMF (p=0.07 for interaction).
 Conclusion: Lack of TIMP-1 tumor cell immunoreactivity seems to predict a favourable effect of epirubicin containing adjuvant therapy in primary breast cancer. However, an independent study is awaited to validate the potential predictive value of TIMP-1 immunoreactivity. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6042.

TIMP-1 and responsiveness of estrogen receptor negative breast cancer to preoperative epirubicin and cyclophosphamide with or without gefitinib.

Abstract Abstract #6072 Background: Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is a naturally occuring proteinase inhibitor, which inhibits most matrix metalloproteinases. It has previously been reported that high levels of tumor TIMP-1 is associated with lack of clinical benefit from anthracycline based chemotherapy in metastatic breast cancer and preliminary data suggest that this is also true for adjuvant treatment. Plasma TIMP-1 has also been shown to predict response to endocrine therapy in patients with metastatic breast cancer. No data regarding TIMP-1 response to neo-adjuvant treatment exists.
 Material and Methods: NICE (1839IL/0712) is a randomized phase II trial, assigning 144 patients per protocol with primary operable estrogen receptor negative breast cancer and tumor ≥ 2 cm to either four cycles of EC (epirubicin 90 mg/m2 and cyclophosphamide 600 mg/m2) plus placebo or EC plus gefitinib 250 mg daily. The primary endpoint was pCR (no invasive residual) at surgery and secondary endpoints were complete (CR) and overall objective (OR) tumor response rates (CR+PR) assessed by palpation, mammography and ultrasonography (the last prevailed in case of disagreements). Plasma TIMP-1 levels were measured (blinded and in duplicates) before and after preoperative EC using a validated enzyme-linked immunosorbent assay.
 Results: 'pCR was attained by 8/71 patients in the EC + gefitinib group and 8/73 patients in the EC + placebo group, and OR by 50 patients in each treatment group. The addition of gefitinib to pre-operative EC was not associated with any significant benefit, thereby justifying pooling of the 2 groups of patients. TIMP-1 ranged from 55.1 to 519 ng/ml, median 126.8 ng/ml at baseline. Patients who obtained pCR had numeric higher plasma TIMP-1 values than non-responders (155.2ng/ml against 139.9 ng/ml, p=0.35). Dichotomizing patients by the plasma TIMP-1 median showed, that patients with elevated TIMP-1 had a numeric increased likelihood of obtaining pCR and OR: Patients with plasma TIMP-1 above the median had a numeric higher pCR rate (6.6% against 4.4%; odds ratio 1.60, p=0.39), and OR (51.0% against 49.0%; odds ratio 1.20, p=0.61). A small decline in TIMP-1 was observed after four cycles of EC (median difference 4.5, 25% and 75% quartile of -28.6 and 27.9, p=0.85) and the change was nearly the same in responders as in non-responders.
 Conclusion: This study could not demonstrate a predictive role of pre-treatment plasma TIMP-1 in neo-adjuvant chemotherapy for primary breast cancer. A change in plasma levels of TIMP-1 during 4 cycles of EC was not associated with response to the treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6072.

TIMP-1 expression in human colorectal cancer is associated with TGF-B1, LOXL2, INHBA1, TNF-AIP6 and TIMP-2 transcript profiles

The balance of activity between the endogenous enzyme inhibitors known as tissue inhibitors of metalloproteinases and their targets, the matrix metalloproteinases, in the extracellular matrix is thought to play an important role in tumour cell invasion. Supporting this notion, we have shown that colorectal cancer patients have increased plasma levels of the tissue inhibitor of metalloproteinases-1 (TIMP-1), and that high plasma TIMP-1 levels are associated with short colorectal cancer patient survival. However, although TIMP-1 has been extensively studied in cancer, very little is known about how it is regulated. To further elucidate potential mechanisms of regulation of this protein, we did a number of experiments to look at associations between the transcript profile of TIMP-1 with known matrix metalloproteinases ( MMPs) as well as with expression profiles of other genes differentially regulated in human colorectal cancer (CRC) and the other TIMPs 2–4, which have also been associated with the progression of colorectal cancer. Genome-wide expression profiling of 172 CRC and normal mucosa samples was used to identify transcript changes for the genes under investigation. We found that TIMP-1 was up-regulated in CRC samples compared with normal tissue, while TIMP-2 was down-regulated. Eight MMPs were up-regulated in CRC compared with normal tissue. Correlating up-regulated genes with the TIMP-1 transcript, we identified 13 that were also up-regulated in cancerous tissue. Among these were genes associated with the synthesis of extracellullar matrix, genes involved in the TGF-beta signalling pathway, and genes that are likely transcribed by the tumour cells. These insights add to the complex picture emerging about the regulation of TIMPs in colorectal cancer.

High serum TIMP-1 correlates with poor prognosis in breast carcinoma – A validation study

A number of studies have demonstrated that high tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) are associated with a poor prognosis in breast cancer, suggesting that TIMP-1 could be a valid prognostic marker in this disease. Recently, our laboratories have presented results showing that TIMP-1 also carries prognostic information when measured in serum. This is an important finding, since serum is a much more preferable material compared with tumor tissue extracts. The aim of the present study was to validate the previous results concerning the prognostic value of TIMP-1 in serum obtained preoperatively from 68 patients with primary breast cancer. This was done by measuring the same serum samples as in the previous study but in a different laboratory using a different ELISA assay. We confirmed that patients with the highest serum levels of TIMP-1 (> 197.7 ng/ml) had significantly shorter disease-specific survival compared with patients with low serum TIMP-1 levels. In the group of node-negative patients, 53% of the patients with high levels of TIMP-1 survived after 10 years of follow-up compared to 92% of the patients with low levels. This study thus confirms the reproducibility across laboratories of the results concerning the prognostic value of TIMP-1 in serum. We also investigated whether measurements of the specific fraction of uncomplexed TIMP-1 improved the prognostic value of TIMP-1 in serum, as has been shown to be the case for tumor tissue extracts. However, including information of the level of uncomplexed TIMP-1 did not seem to provide additional prognostic information to that already provided by total TIMP-1.

Primary Tumor Levels of Tissue Inhibitor of Metalloproteinases-1 Are Predictive of Resistance to Chemotherapy in Patients with Metastatic Breast Cancer

Only about 50% of metastatic breast cancer patients benefit from cytotoxic chemotherapy. Today, no validated markers exist for prediction of chemotherapy sensitivity/resistance in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis, and the purpose of the present study was to test the hypothesis that tumors expressing high levels of TIMP-1 are protected against apoptosis-inducing agents and thus less sensitive to apoptosis-inducing chemotherapeutic drugs. We investigated the association between primary tumor expression levels of TIMP-1 protein and objective response to first-line chemotherapy in 173 patients with metastatic breast cancer. When analyzed as a continuous log-transformed variable, increasing TIMP-1 levels were significantly associated with lack of response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy (P = 0.01; odds ratio, 2.0; 95% confidence interval, 1.1-3.3). In a multivariate model, including lymph node status, steroid hormone receptor status, menopausal status, dominant metastases site, type of chemotherapy, and disease-free interval, TIMP-1 was significantly associated with resistance to treatment (P = 0.03; odds ratio, 1.7; 95% confidence interval, 1.1-3.3). In the present exploratory study, we showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to chemotherapy. By using TIMP-1, we identified a group of patients with metastatic breast cancer, which hardly respond to the most frequently used chemotherapy regimes (i.e., cyclophosphamide/methotrexate/5-fluorouracil and anthracyclines).

Tissue inhibitor of metalloproteinases-1 in the postoperative monitoring of colorectal cancer

The aim of this study was to investigate whether the pre- and postoperative plasma levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) were associated with outcome in colorectal cancer (CRC). Pre- and postoperative plasma TIMP-1 from 280 curatively resected CRC patients and carcinoembryonic antigen (CEA) in corresponding serum samples were measured and correlated with patient outcome (death, local recurrence (LR) and distant metastases (DM)). The results showed that the course of plasma TIMP-1 from pre- to postoperative levels correlated with patient outcome ( P = 0.005). However, postoperative plasma TIMP-1 alone was strongly associated with patient outcome, high TIMP-1 predicting short survival ( P = 0.002). Combining postoperative TIMP-1 and CEA demonstrated that high TIMP-1 and CEA levels predicted poor outcome ( P < 0.0001); multivariate analysis identifying both parameters as strong prognostic factors for survival, LR and DM ( P < 0.0001). In conclusion, postoperative plasma TIMP-1 predicts patient outcome both alone and in combination with CEA. Postoperative TIMP-1 may be a marker of residual disease after primary surgery for CRC.

Localization of tissue inhibitor of metalloproteinases 1 (TIMP-1) in human colorectal adenoma and adenocarcinoma.

Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied. In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP-1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP-1 showed good correlation between TIMP-1 mRNA and immunoreactivity. Combining TIMP-1 in situ hybridization with immunohistochemical staining for alpha-smooth muscle actin or CD68 showed TIMP-1 mRNA in myofibroblasts but not in macrophages. TIMP-1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial-stromal interface. In conclusion, TIMP-1 expression is a rare event in benign human colon tissue but is highly expressed by myofibroblasts in association with invading colon cancer cells.

Association between preoperative plasma levels of tissue inhibitor of metalloproteinases 1 and rectal cancer patient survival: a validation study

The level of the tissue inhibitor of metalloproteinases 1 (TIMP-1) has previously been demonstrated to predict the survival of early stage colorectal cancer patients. The present study was undertaken to further validate plasma TIMP-1 as a prognostic marker in rectal cancer. Preoperative plasma from 352 rectal cancer patients were analysed using an immunoassay for TIMP-1. The TIMP-1 immunoassay demonstrated robustness and good reproducibility with low interassay coefficients of variation (CV). The rectal cancer patients had a mean plasma TIMP-1 level of 184 μg/l (standard deviation (SD): 70 μg/l). There were no significant differences in TIMP-1 levels between patients with Dukes’ stage A, B or C disease, whereas Dukes’ stage D patients had significantly increased TIMP-1 levels (P<0.0001); however, high levels of TIMP-1 were not restricted to those with advanced disease. Univariate analysis demonstrated an increasing risk of mortality with increasing TIMP-1 levels Hazard Ratio (HR)=2.9; 95% Confidence Interval (CI): 1.7–5.0; P<0.0001). Including additional covariates, multivariate analysis identified plasma TIMP-1 as an independent prognostic marker (HR=2.2; 95% CI: 1.2–4.1 (P=0.01). This study showed a highly significant and independent association between preoperative plasma TIMP-1 levels and survival in rectal cancer patients, thus confirming our previous findings. Furthermore, the TIMP-1 immunoassay proved to be stable and reproducible in this confirmatory study.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits tumor growth and angiogenesis in the TIMP-1 transgenic mouse model.

The tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) has been recognized as a multifunctional protein. The role of TIMPs in cancer remains the subject of conflicting reports with an antitumor activity or a tumor growth stimulation activity by several mechanisms. The aim of our study is to investigate the effect of ectopic TIMP-1 overexpression on the primary transplanted tumor growth. We employed transgenic mice overexpressing the human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/enhancer (TIMP-Tg-mice) and producing high serum levels of TIMP-1. We used the transplantable Ehrlich tumor cells in the current study. The allograft study revealed that the tumor growth in the TIMP-Tg-mice was more significantly inhibited than control (Cont) mice by associated suppression of neovascularization in the tumor. The in vitro studies showed that the recombinant TIMP-1 (rTIMP-1) did not affect the proliferation of the endothelial cells (ECs) and tumor cells, suggesting that the tumor suppressive effect of TIMP-1 was not due to cytotoxicity. TIMP-1 significantly inhibited EC tubular formation in vitro. Furthermore, TIMP-1 treatment did not affect the levels of matrix metalloproteinase (MMP)-2 and MMP-9 mRNA in the Ehrlich tumor cells in vitro, although these expressions in the tumor were markedly suppressed in the TIMP-Tg-mice, compared to the Cont-mice at the end of the experiment. These results suggested that the ectopically overexpressed TIMP-1 inhibited the tumor growth by angiogenesis suppression.

Tumor Tissue Concentrations of the Proteinase Inhibitors Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and Plasminogen Activator Inhibitor Type 1 (PAI-1) Are Complementary in Determining Prognosis in Primary Breast Cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29-75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3-11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II-IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.

Plasma tissue inhibitor of metalloproteinase-1 levels are elevated in essential hypertension and related to left ventricular hypertrophy

Background: Essential hypertensive patients have an increased heart and arterial collagen concentration. Increased collagen synthesis can be assessed using procollagen III N peptide (PIIINP) and reduced collagen degradation measured using tissue inhibitor of metalloproteinase-1 (TIMP-1). Methods: Plasma TIMP-1 and PIIINP levels were measured in 31 patients with essential hypertension and in 17 normotensive control subjects. The hypertensive patients were either treatment naive ( n = 18) or had been without treatment for 1 month ( n = 13). Both groups of patients were screened to exclude other fibrotic diseases. Results: In the hypertensive patients, TIMP-1 levels were significantly ( P < .0002) elevated (median 380 ng/mL, range 160 to 1560 ng/mL) compared with those of the normotensive control subjects (median 178 ng/mL, range 99 to 330 ng/mL). In hypertensive subjects who had never received antihypertensive therapy there were significant correlations between TIMP-1 and left ventricular posterior wall thickness in diastole (LVPWd) (r = 0.58) ( P < .02) and left ventricular mass index (r = 0.58) ( P < .02). There was no difference in PIIINP levels (mean ± 2 SD) between the hypertensive (0.56 U/mL ± 0.3) and normotensive groups (0.52 U/mL ± 0.2). Conclusions: The increased tissue collagen III levels found in the heart and vessels of hypertensive patients is due to a reduction in collagen degradation because of high TIMP-1 levels, rather than an increase in synthesis of collagen type III. The tissue source of this TIMP-1 is unclear.

Longitudinal evaluation of GCF MMP-3 and TIMP-1 levels as prognostic factors for progression of periodontitis.

To determine whether matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in gingival crevicular fluid (GCF) could serve as prognostic factors for the progression of periodontitis, we monitored GCF MMP-3 and TIMP-1 and periodontal status of selected sites in 40 medically healthy subjects over a 6-month period. Clinical measurements including gingival index (GI), plaque index, bleeding on probing, suppuration, probing depth (PD), attachment loss (AL), and GCF samples were taken from 2 healthy sites (including sites with gingival recession, GI=0 PD < or =3 mm; AL < or =2 mm) and 2 periodontitis sites (GI > or =1; PD > or =5 mm; AL > or =3 mm) of each patient at baseline, 3-month and 6-month visits by means of sterile paper strips. GCF levels of MMP-3 and TIMP-1 were determined by sandwich ELISA assays. The mean amounts of MMP-3 and TIMP-1 in diseased sites were significantly higher than in healthy sites (p<0.0001). Significantly higher GCF levels of MMP-3 and TIMP-1 were found at progressing sites than in nonprogressing periodontitis sites (0.001<p<0.01). A progressing site was defined as a site which had > or =2 mm loss of attachment during 6- month study period. GCF levels of MMP-3 were highly correlated with clinical measurements taken at baseline, 3-month and 6-month visits (p<0.001). TIMP-1 levels were only moderately correlated with probing depth and attachment level (p<0.01). Step-wise multiple regression analysis was performed to construct models for the prediction of probing depth and attachment loss increases. The most parsimonious regression models which had the best R2 values included the following variables and accounted for the indicated % of variability. The regression model for the prediction of probing depth increase included MMP-3, smoking pack-years, TIMP-1 and accounted for 53% of the variability. The best model for the prediction of attachment loss increase included MMP-3, smoking pack-years, age, TIMP-1 and explained 59% of the variability. These data indicate that sites with high GCF levels of MMP-3 and TIMP-1 are at significantly greater risk for progression of periodontitis.