Clinical relevance of (circulating) proteases in breast cancer: matrix metalloproteinases and cathepsins

Breast cancer remains the most common deadly form of cancer in Europeanwomen. Thanks to early diagnosis and administration of adjuvant systemic therapy, its mortality rate is dropping in the Western world. The majority of treatment failures and deaths are attributed to metastasis or spread of tumour cells to distant organs. Currently, the established prognostic factors for operable breast cancer are tumour size, histologic grade, steroid hormone receptors, Her2 expression and lymph node involvement. In spite of their strong association with recurrence-free and overallsurvival, these parameters do not allow us to identify all patients at high risk of metastasis. Therefore, a great deal of research is devoted to the identification of new prognostic markers or biomarkers for breastcancer. In particular, circulating biomarkers have been the focus of interest since they are readily accessible and permit serial assessment for monitoring of therapy response and disease progression. Proteases suchas matrix metalloproteinases (MMPs) and cathepsins have been suggested as candidate biomarkers of cancer due to their numerous functions in tumour progression and metastasis. Hence, we evaluated their clinical relevance in breast cancer. In a first study, we explored the mRNA expression profile of a panel of 6 MMPs in luminal A invasive ductal carcinomas and correlated it with clinical and pathological prognostic markers. We observed a strong positive correlation between tumour MMP2 and MMP11 expression and the postmenopausal status which is in line with a MMP sex steroid dependent regulation. At first sight, an increase in MMP expression in luminal A breast tumours of postmenopausal women seems paradoxical. First, luminal A tumours are primarily found in postmenopausal women being associated witha good prognosis. Second, increased MMP expression has been associated with a worse prognosis in various tumour types. However, this apparent paradoxical finding might suggest that luminal A breast tumours of premenopausal women have a more aggressive biological behaviour than those of postmenopausal women. In a second study, we evaluated the prognostic value of plasma MMP2 andMMP9 in breast cancer, discriminating the expression of the active MMP fraction from the total expression. We demonstrated a significant increase in MMP2 activity in plasma from patients with a Her2 positive tumour.Hence, we confirmed the link between the Her2 and MMP pathway in human samples, a relation that was previously solely observed in in vitro systems and in animal models. In a third study, we investigated whether measurement of circulating levels of the collagenases MMP1, MMP8 and MMP13 may be a useful tool for breast cancer diagnosis and prognosis. Plasma MMP1 and MMP8 levels enabled the diagnosis of breast cancer, including inflammatory breast cancer. We found differences in MMP1 and MMP8 levels between patients with different clinical and pathological characteristics. Furthermore, MMP8 levelswere higher in patients with moderate lymph node involvement (pN1, pN2)than in healthy controls, while the opposite was true for patients withan extended lymph node metastasis (pN3) and a strong predisposition to distant metastasis. Based on the hypothesis of a reverse association of blood and tissue protein levels, with lower blood levels in case of concentration in the tumour and/or stroma, our observations support previousstudies that MMP8 might influence the metastatic behaviour of breast cancer cells. Moreover, our results suggest a greater protective effect against lymph node metastasis than against distant metastasis. In a fourth study, we examined the expression of cathepsins B, H, X andtheir endogenous inhibitor cystatin C in serum of early stage and inflammatory breast cancer patients. As chemotherapy is known to have adverseside effects, the debate is ongoing whether patients with favourable prognostic characteristics, i.e. tumours smaller than 5 cm and without lymph node involvement, should receive adjuvant chemotherapy. On the other hand, inflammatory breast cancer is as yet a rare but very aggressive disease with unknown underlying pathology. Since cathepsins have been shown to play an important role in cancer, their serum levels might provide additional prognostic information. We found a correlation between lower serum cathepsin B levels and poor histologic grade, and observed higher cystatin C levels in postmenopausal women. Serum cystatin C levels indicated that early stage breast carcinomas can be classified into 2 groups:tumours smaller than 2cm and those of 2cm or larger; the latter being associated with higher cystatin C levels. The increase in cystatin C levels may result from a compensation mechanism, although an apparently inefficient one, for elevated cathepsin activity in the tumour. Furthermore,we observed lower levels of cathepsins H and X and a trend for lower cathepsin B and cystatin C levels in inflammatory breast cancer patients; suggesting that cathepsins H and X might be involved in the inflammatory-dependent processes of this aggressive breast disease. In a fifth study, we studied the impact of single nucleotide polymorphisms (SNPs) in the MMP8 gene on the expression and paradoxical protectiverole of MMP8 in metastasis. We found that carriers of the minor allele of the rs11225395 promoter SNP had a reduced risk of lymph node metastasis. In vitro functional assays revealed a significantly higher promoter activity in the presence of the minor allele and in support of this, we found binding of nuclear proteins at the polymorphic site for the minor allele but not for the common allele. Moreover, we observed a reduced risk of relapse as well as a better recurrence-free and overall survival among carriers of the minor allele and in particular in those with early stage breast cancer. Hence, our results suggest that MMP8 gene variationmay affect breast cancer prognosis and support the notion that MMP8 hasan anti-metastatic role in cancer.status: publishe