Salvatore Minafra

Zymographic detection and clinical correlations of MMP-2 and MMP-9 in breast cancer sera

Matrix metalloproteinases, in particular the gelatinases MMP-2 and MMP-9, have received great attention in recent years as putative tumour markers for clinical applications. The main reason for the observed interest is their easy detection in body fluids. Moreover, recent evidence has shown multiple functions of MMPs, rather than simply degrading ECM, which include the mobilisation of growth factors and processing of surface molecules. Several authors have reported increased levels of MMPs in a number of cancers, but clinical correlations in breast cancer are still fragmentary. Thus, the aim of the present research was to investigate the activity levels of circulating gelatinases in the sera of breast cancer patients by means of zymographic analysis, and correlate data with clinicopathological parameters. In all, 80 patients and 22 healthy volunteers were involved in this study. Sera were obtained prior to surgery. The clinical variables were: grading of tumours, tumour size, lymph node involvement, tumour staging, oestrogen and progesterone receptor levels (76 out of 80 cases), and c-erbB-2 levels (46 cases). The densitometric measures of MMP-2 and MMP-9 activity levels indicated that the average values of both gelatinase activities were significantly higher in breast cancers than in control sera (P<0.0001). In addition, our analysis showed for the first time that elevated activity levels of both gelatinases correlated only with c-erbB-2 overexpression (P=0.0273 for MMP-2 and P=0.0075 for MMP-9). An inverse correlation was observed with regard to oestrogen receptor expression (P=0.0075 for MMP-2 and P=0.0273 for MMP-9). Moreover, a borderline inverse correlation was observed between the activity levels of both enzymes and nuclear grade (P=0.0511 for MMP-2 and P=0.0794 for MMP-9). In conclusion, the present data suggest that serum measures of MMPs activity may have diagnostic value for discriminating subgroups of breast cancer patients and support the hypothesis that ERBB2 amplification and/or overexpression enhance signalling pathways that may lead to increased production of gelatinases in c-erbB-2 positive breast cancers with higher metastatic potentialities.

Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and two-dimensional electrophoresis

Gelatinase A (MMP-2) and gelatinase B (MMP-9) play a key role in the proteolytic cascade leading to ECM degradation during invasion and metastasis. The enzyme activity is regulated both at the intra- and extra-cellular level. Extracellular regulation is achieved mainly through the balance between proenzyme activation and inhibition, which appears to be altered in cancer patients. One of the mechanisms of MMP inhibition is the binding of the enzymes to appropriate tissue inhibitors (TIMP). In the recent literature, it has been suggested that MMP-2 and/or MMP-9 are indeed over-produced in many carcinomas, while the identity of the various enzymatic forms (latent, activated and enzyme/inhibitor complexes) remains to be elucidated. In this study we have analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of patients with colon carcinoma, as well as the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissue). Proteins were separated by means of mono-dimensional or bidimensional electrophoresis, and the enzymes detected by gelatin zymography and immunological assays. The results of densitometric analyses demonstrate that proMMP-9, but not proMMP-2, is significantly higher in the oncologic sera vs. the normal sera. In addition, several oligomeric circulating and tissue forms of MMP-9 are preferentially found in the oncologic samples, both in mono- and second-dimension zymograms. The activated forms of MMP-2 and MMP-9 are uniquely present in the primary tumor extracts, thus confirming the involvement of the tissue microenvironment in gelatinase activation and function.

Proteomic patterns of cultured breast cancer cells and epithelial mammary cells.

Abstract: Breast cancer is one of the leading causes of death from cancer among women in western countries. The different types of breast cancer are grouped into invasive and noninvasive forms. Among the invasive types, ductal infiltrating carcinoma (DIC) is the most common and aggressive form. Using an in vitro model consisting of a DIC‐derived cell line (8701‐BC) and a nontumoral mammary epithelial cell line (HB2), we used the proteomics approach to search for homology and differences in protein expression patterns between tumoral and nontumoral phenotypes. Within an analysis window comprising 1,750 discernible spots we have currently catalogued 140 protein spots of potential interest. Fifty‐eight of them were identified by gel matching with reference maps, immunodetection, or N‐terminal microsequencing and classified into four functional groups. Twelve proteins were found differentially expressed in two cell lines: four were uniquely present in the neoplastic cell proteome and eight in epithelial cells. In addition, 53 proteins displayed different relative expression levels between the two cell lines, that is, 44 were more elevated in cancer cells and 9 in HB2 cells. Among proteins with greater relative abundance in cancer cells we identified glycolytic enzymes (or their isoforms), which may indicate that the known metabolic dysregulation in cancer can reflect oncogenic‐related defects of glycolytic gene expression.

Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen☆

Type V collagen is one of the minor components of the extracellular matrix (ECM) whose content is increased in cases of ductal infiltrating carcinomas of the breast. In order to clarify its biological role, we have investigated the effect of this molecule, both as substrate and as soluble factor, on the behaviour of a breast carcinoma cell line (8701-BC) grown in vitro. Cell-collagen adhesion was monitored for 24 h from plating in the absence or presence of serum. The influence of type V collagen on cell growth was followed during 9 days of culture, and the actin-vinculin arrangement was studied by simultaneous fluorescent immuno-staining. The results indicate that type V collagen is not a permissive substrate for neoplastic cell proliferation and dissemination in vitro.

A contribution to breast cancer cell proteomics: Detection of new sequences

Ductal infiltrating carcinoma (DIC) of the breast is the most common and potentially aggressive form of cancer. Knowledge of proteomic profiles, attained both in vivo and in vitro, is fundamental to acquire as much information as possible on the proteins expressed in these pathologic conditions. We used the breast cancer cell line 8701‐BC, established from a primary DIC, with the aim of contributing to the databases on mammary cancer cells, which in turn will be very useful for the identification of differentially expressed proteins in normal and neoplastic cells. Within an analysis window comprising about 1750 discernible spots, we have at present catalogued 84 protein spots. The proteins for which an identity was assigned were identified essentially using gel comparison, N‐terminal (Nt) microseqencing and immune detection. Among the protein spots Nt‐microsequenced, sixteen corresponded to known proteins, four resulted as modified, relative to matching sequences deposited on databases, and seven were unknown. These modified or novel sequences are thus of potential interest to the knowledge of breast cancer proteomics and its applications.

Retrospective Proteomic Screening of 100 Breast Cancer Tissues

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The specimens were selected from patients with invasive ductal carcinoma of the breast, the most frequent and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers potentially useful for clinical applications. The proteomic screening; by 2D-IPG and mass spectrometry; allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients; and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is reasonably supported by concurrent events: the inhibition of apoptosis and stimulation of cellular proliferation, and the increased expression of glycolytic enzymes with multiple functions. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. We suggest that this approach on large-scale 2D-IPG proteomics of breast cancer is currently a valid tool that offers the opportunity to evaluate on the same assay the presence and recurrence of individual proteins, their isoforms and short forms, to be proposed as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.