T. Maguire

Metalloproteinases: role in breast carcinogenesis, invasion and metastasis.

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. Their primary function is degradation of proteins in the extracellular matrix. Currently, at least 19 members of this family are known to exist. Based on substrate specificity and domain organization, the MMPs can be loosely divided into four main groups: the interstitial collagenases, gelatinases, stromelysins and membrane-type MMPs. Recent data from model systems suggest that MMPs are involved in breast cancer initiation, invasion and metastasis. Consistent with their role in breast cancer progression, high levels of at least two MMPs (MMP-2 and stromelysin-3) have been found to correlate with poor prognosis in patients with breast cancer. Because MMPs are apparently involved in breast cancer initiation and dissemination, inhibition of these proteinases may be of value both in preventing breast cancer and in blocking metastasis of established tumours

Urokinase plasminogen activator: A prognostic marker in multiple types of cancer

Urokinase plasminogen activator (uPA) is a serine protease causally involved in cancer invasion and metastasis. Consistent with its role in cancer spread, uPA has been shown to be a prognostic marker in a variety of malignancies, especially breast cancer. Approximately 20 different groups have shown that high levels of uPA in breast tumor tissue predict poor outcome. As a prognostic marker in breast cancer, uPA provides information that is independent of traditionally used factors such as tumor size, tumor grade, axillary node status and estrogen receptor status. Furthermore, uPA is prognostic in node‐negative patients, and a clinical trial is currently under way to assess whether uPA and its inhibitor, plasminogen activator inhibitor‐1, can differentiate between the majority of node‐negative breast cancer patients who are cured by surgery from the minority who might benefit from adjuvant therapy. uPA is also prognostic in other malignancies, such as gastric, colorectal, esophageal, renal, endometrial, and ovarian cancers. uPA may thus be a prognostic indicator for multiple types of adenocarcinoma. J. Surg. Oncol. 1999:71:130–135.

High levels of tissue inhibitor of metalloproteinase-1 predict poor outcome in patients with breast cancer

Studies from model systems suggest that matrix metalloproteinases (MMPs) are causally involved in tumor progression while tissue inhibitors of MMPs (TIMPs) prevent this progression. Here, we show that concentrations of TIMP‐1 are significantly higher in breast carcinomas than in fibroadenomas. In primary breast cancers, TIMP‐1 concentrations increased with increasing tumor size but showed an inverse relationship with estrogen receptor concentrations. In primary breast cancers also, TIMP‐1 levels were weakly but significantly correlated with those for MMP‐1, proMMP‐2, active MMP‐2, MMP‐3 and proMMP‐9. Contrary to what might be expected from published data on model systems, high concentrations of TIMP‐1 predicted a poor outcome in patients with breast cancer. We conclude that in human breast cancer, endogenous TIMP‐1 does not inhibit tumor progression but may enhance the process. Int. J. Cancer (Pred. Oncol.) 84:44–48, 1999.

Increased gelatinase-A and gelatinase-B activities in malignant vs. benign breast tumors.

Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and enzyme‐inhibitor complexes. Using newly developed quantitative activity assays, the levels of active MMP‐2, total (active and activatable) MMP‐2 and total MMP‐9 were found to be significantly higher in breast carcinomas than in fibroadenomas. In addition, active MMP‐2 and MMP‐9 were detected more frequently in malignant than in benign breast carcinoma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP‐2 and ‐9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis. Int. J. Cancer 86:204–207, 2000.

High levels of TIMP-2 correlate with adverse prognosis in breast cancer.

TIMP‐2 is an endogenous inhibitor of MMPs. Most data from model systems suggest that high levels of this inhibitor prevent metastasis. In human breast cancers, however, we show that high levels of TIMP‐2 correlate with both shortened disease‐free interval and overall survival. In primary breast cancers, TIMP‐2 levels showed no significant relationship with either tumor size or axillary node status but correlated inversely with estrogen receptor levels. TIMP‐2 levels also correlated significantly with those for TIMP‐1. We conclude that high levels of endogenous TIMP‐2, like other protease inhibitors such as PAI‐1 and TIMP‐1, correlate with progression of human breast cancer. Int. J. Cancer (Pred. Oncol.) 89:118–121, 2000.

Overexpression of the Ets-1 transcription factor in human breast cancer

The Ets family of transcription factors regulate expression of multiple genes involved in tumour progression. The aim of this study was to investigate the expression of Ets-1 in a large panel of human breast cancers and relate its levels to the parameters of tumour progression and metastasis. Using RT–PCR, Ets-1 mRNA was detected in 30 out of 42 (71%) fibroadenomas and 131 out of 179 (73%) primary breast carcinomas. Similarly, levels of Ets-1 mRNA were not significantly different in fibroadenomas and primary breast carcinomas. Using Western blotting, four forms of the Ets-1 protein were detected, that is, p33, p42, p51 and p52. Levels of both p51 and p52 but not p33 and p42 were present at significantly higher levels in the carcinomas compared to the fibroadenomas (for p51, P<0.007; for p52, P<0.02; Mann–Whitney U-test). Levels of p52, p51 and p33 correlated significantly with uPA protein levels (P<0.01), while only levels of p52 correlated significantly with HER-2/neu protein levels (P<0.01). Using immunohistochemistry, Ets-1 was found predominantly in tumour cells, but was also detected in some stromal cells surrounding tumour islands. We conclude that, while at the mRNA level, Ets-1 was found at similar levels in fibroadenomas and primary breast carcinomas, higher protein levels were detected in the cancers compared to the benign specimens. Since p52, p51 and p33 correlate with uPA levels, these forms of Ets-1 may play a role in breast cancer metastasis.

Studies on oestrogen receptor-α and -β mRNA in breast cancer

Abstract The oestrogen receptor (ER) is widely used to predict response to tamoxifen in patients with breast cancer. Recently a new form of ER known as ER-β was discovered, the original ER is now designated ER-α. In this investigation, ER-α and ER-β were measured in 107 breast carcinomas and 22 fibroadenomas. Using reverse transcriptase-polymerase chain reaction (RT-PCR), ER-β mRNA, but not ER-α mRNA was expressed more frequently in fibroadenomas than carcinomas. In the carcinomas, ER-β mRNA was present in a greater proportion of samples positive for ER-α mRNA than in those lacking this form of the receptor. ER-α, but not ER-β mRNA, was significantly associated with ER protein-positivity in the cancers. ER-α mRNA was also positively related to progesterone receptors (PR), but ER-β mRNA showed an inverse relationship with PR. We conclude that the presently used enzyme-linked immunosorbent assay (ELISA) for ER appears to be mostly measuring ER-α and is unlikely to be detecting ER-β.

High levels of cathepsin B predict poor outcome in patients with breast cancer.

Cathepsin B (CB) is a thiol-stimulated protease implicated in cancer invasion and metastasis. Other proteases involved in cancer spread such as urokinase-type plasminogen activator (uPA) and cathepsin D have previously been shown to be prognostic markers in breast cancer. CB was assayed by ELISA in 193 patients with primary breast cancer. CB levels were significantly higher in both primary and metastatic breast tumors than in fibroadenomas (p=0.0001). In the primary carcinomas, CB levels showed no significant correlation with either nodal status, tumor size or estrogen receptor (ER) status. Patients with primary breast cancers containing high levels of CB had a significantly shorter disease-free interval (p=0.01, chi-square=6.61) and overall survival (p=0.014, chi-square=6.08) than patients with low levels of the protease. However, in multivariate analysis, using nodal status, tumor size, ER status and urokinase plasminogen activator (uPA), CB was not an independent prognostic marker. In contrast, nodal status, ER status and uPA were prognostic in multivariate analysis. In conclusion, CB, like certain other proteases implicated in cancer metastasis, correlates with poor outcome in patients with breast cancer. These results thus support the evidence from model systems linking CB to cancer spread. Inhibition of CB expression or activity might therefore be exploited for anti-metastatic therapies.

Urokinase plasminogen activator as a predictor of aggressive disease in breast cancer.

Urokinase plasminogen activator (uPA) is a multifunctional protein involved in both extracellular proteolysis and signal transduction. uPA usually mediates its actions while attached to a membrane-bound receptor, termed uPAR. In this study, uPA and its receptor were measured at both protein and mRNA levels in breast cancer. At both levels, concentrations of uPA were significantly correlated with those for uPAR. uPA levels also correlated significantly with cathepsin B and cathepsin D but not with cathepsin L, MMP-8 or MMP-9 levels. Irrespective of the cut-off point used (e.g., median, tertile or quartile values), uPA was a significant prognostic marker for breast cancer.

Assay of E-cadherin by ELISA in human breast cancers.

E-cadherin is a membrane-bound adhesion glycoprotein. Loss of E-cadherin has been correlated with invasion and metastasis in model systems. Using a new ELISA, we found higher levels of E-cadherin in fibroadenomas than in primary breast cancers. Levels in primary cancers showed no significant relationship with either tumour size, nodal status or oestrogen receptor levels. Patients with breast cancers containing low levels of the adhesion protein had a significantly shorter disease-free interval than patients with high levels (P = 0.041). The prognostic value of E-cadherin, for disease-free interval, was also found in node-negative patients as well as in patients presenting with small tumors (< or = 2 cm). In conclusion, loss of E-cadherin expression in human breast cancers is associated with increased metastatic potential as has previously been found in model systems. Loss of E-cadherin is thus likely to contribute to breast cancer progression.