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Background Attempts to reduce morbidity and mortality in breast cancer is based on efforts to identify novel biomarkers to support prognosis and therapeutic choices. The present study has focussed on S100 proteins as a potentially promising group of markers in cancer development and progression. One reason of interest in this family of proteins is because the majority of the S100 genes are clustered on a region of human chromosome 1q21 that is prone to genomic rearrangements. Moreover, there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast, and this is frequently associated with tumour progression. Methods Samples of breast cancer tissues were obtained during surgical intervention, according to the bioethical recommendations, and cryo-preserved until used. Tissue extracts were submitted to proteomic preparations for 2D-IPG. Protein identification was performed by N-terminal sequencing and/or peptide mass finger printing. Results The majority of the detected S100 proteins were absent, or present at very low levels, in the non-tumoral tissues adjacent to the primary tumor. This finding strengthens the role of S100 proteins as putative biomarkers. The proteomic screening of 100 cryo-preserved breast cancer tissues showed that some proteins were ubiquitously expressed in almost all patients while others appeared more sporadic. Most, if not all, of the detected S100 members appeared reciprocally correlated. Finally, from the perspective of biomarkers establishment, a promising finding was the observation that patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. Conclusions This article reports for the first time the comparative proteomic screening of several S100 protein members among a large group of breast cancer patients. The results obtained strongly support the hypothesis that a significant deregulation of multiple S100 protein members is associated with breast cancer progression, and suggest that these proteins might act as potential prognostic factors for patient stratification. We propose that this may offer a significant contribution to the knowledge and clinical applications of the S100 protein family to breast cancer.

Cross talk between tumor cells and connective tissue plays a key role in tumor progression. The communication is due to the release of signalling molecules from both tumor cells and surrounding stromal cells. Several secreted proteins lack the N-terminal signal peptides and, therefore, they are secreted by alternative unconventional processes such as secretion mechanism mediated by vesicle shedding in the extracellular matrix. Actually, a certain number of proteins, playing roles in some aspects of tumor progression, have been found in shed vesicles. For example, EMMPRIN, carried out in vesicles shed by tumor cells, stimulates matrix metalloproteinase (MMP) production in stromal fibroblasts and endothelial cells as well as in tumor cells themselves [1]. Moreover, MMPs vehicled by tumor shed vesicles in association with Integrin beta1 induce the adhesion to and the degradation of extracellular matrix, thus helping cell migration and tumor invasion [2]. Notably vesicles carry many proangiogenic growth factors, such as VEGF [3] or FGF-2 [4] and enzymes as well as SphK1 [5], which act synergistically on endothelial cells to promote neoangiogenesis. Therefore, in cancer state extracellular shed vesicles play a role in tumor progression, through several mechanisms that are elicited in the connective tissue. Two main kind of vesicles, named membrane ones and exosomes, were described; membrane vesicles are thought to be produced by a mechanism similar to virus budding, and their diameter ranges between 100nm-1 μm, while exosomes are smaller vesicles (10-100nm diameter) deriving from the endosomal pathway. Both kinds of vesicles are involved in transporting signal molecules to the extracellular milieu, but distinct roles are not yet been clarified. As reported by Muralidharan-Chari [6] the two vesicle populations can be separated by differential centrifugation: membrane vesicles are sedimented at relatively low speed centrifugation while exosomes require a 100.000g ultracentrifugation. Since the extensive characterization of those vesicles can enlighten their role in cancer progression, especially for what concerns their interaction with the surrounding connective tissue, this work was focused on the proteomic characterization of vesicles shed in vitro by MDA MB 231, a continuous cell line derived from an invasive breast carcinoma which release vesicles also in serum-free medium [7]. 24 hours conditioned serum-free medium was collected and, after two low speed centrifugations to remove cell debris, it was centrifuged at 15000g for 30 minutes to sediment membrane vesicles. The resulting supernatant was ultracentrifuged at 100.000g for 90 minutes to sediment exosomes. Both pellets were analyzed through a proteomic approach, in parallel with the total cell lysate. This was the first study in which 15000g pellet undergoes proteomic analysis, separately from 100000g pellet. MDA-MB 231 proteome was characterized by comparison with the reference map of 8701 BC cells, another cell line derived from ductal invasive breast cancer which had been extensively characterized [8]. The 2D gels obtained from the 15.000g and the 100.000g sediments were compared with the one of MDA MB 231 total cell lysate. Both proteomic profiles differ from the one of cell lysate and some differences are also observed between each other. In particular, the pellet at 15.000g, which in some other studies was considered as containing cell debris, differs from the proteomic profile of cell lysate in some protein component. The analysis by Image Master software showed also the enrichment in vesicles and exosomes of proteins which were not detectable in the cell lysate. Data from in silico analysis are now subjected to LC-MS analyses in order to confirm presumed identification of proteins observed also in the cell lysate and to identify unknown proteins observed in shed vesicles and exosomes. Those molecules could have important roles regarding the vesicle functions and their characterization could help in elucidating the pathways involved in connective tissue-shed vesicle interactions during cancer progression.

S100 proteins are low molecular weight proteins ranging in size from 9 to 13 kDa. They form homo- and heterodimers and even oligomers and are expressed in tissue and cell-specific manner [1]. It is well documented, infact, that S100 proteins have a broad range of intracellular and extracellular functions. Intracellular functions include regulation of protein phosphorylation, enzyme activity, calcium homeostasis, regulation of cytoskeletal components and regulation of transcriptional factors, so they are involved in several biological processes including cell cycle regulation, cell growth, cell differentiation, and motility [2]. Extracellularly they act in a cytokine like manner through the receptor for advanced glycation end products (RAGE). In consideration of the critical roles played by S100 proteins in such large spectrum of biological activities, is not surprising that improper expression of S100 members is correlated with many pathologies and especially with cancer. A number of S100 proteins have been shown to interact with oncogenes and various proteins involved in cancer progression, including p53, cytoskeletal proteins, MMPs, and others [3,4]. In this study we aimed to perform a large-scale proteomic investigation on breast cancer patients for the screening of multiple forms of S100 proteins. The 100 surgical tissues of ductal infiltrating breast cancer utilized were collected between 2003 and 2007 in the Breast Unit of the La Maddalena Hospital. Tissue extracts were submitted to proteomic preparations for2D-IPG and protein identification was performed by peptide mass finger printing. The S100 protein members identified in different proteomic maps were: S100A2 (protein S-100L), S100A4 (metastasin), S100A6 (Calcyclin, Prolactin receptor-associated protein), two isoforms, S100A7 (psoriasin), two isoforms, S100A8 (Calgranulin-A), S100A11 (Calgizzarin), three isoforms and S100A13 (S100 calcium-binding protein A13) (Fig. 1). Our results have shown that the majority of S100 proteins were present at very low levels, if not absent, in the nontumoral tissues adjacent to the primary tumor. Moreover, we showed that some S100 protein members were ubiquitously expressed in almost all patients, while others appeared more sporadic among the same group of patients. The Association analysis of S100 members with tumor variables (age, tumor size, nodal status, immuno-cytochemical, presence of HER-2, oestrogen receptors, progesterone receptor, and Ki67), showed no significant correlations of except for S100A6 (isoform b) and S100A13 correlating with Ki67. More interestingly, patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. The most robust correlation with metastasis regarded primarily the protein S100A4, and secondly the protein S100A7. We believe that this information may substantially contribute to the progress of protein profiling of breast cancer and help to organize subclasses for clinical applications.

Tumor progression and metastasis represent the leading causes of cancer related death. One of the major features that may contribute to neoplastic cell dissemination is the progressive and local degradation of the extracellular matrix (ECM) surrounding the primary tumour. Degradation of the ECM requires the coordinated action of a number of enzymes produced locally by neoplastic cells and/or stromal cells. Five categories of proteinases have been implicated in the invasive process: serine, cysteine, aspartic, threonine proteinases and matrix metalloproteinases (MMPs), also known as matrixins, which play a key role as terminal effectors of the proteolytic cascade. At present 23 members of the matrixin family have been found in humans and classified into subsets of enzymes, according to their molecular domains and preference for the substrates. Since the classic work of Liotta and collaborators [1], much attention has been focused on MMP-2 and MMP-9 because of their ability to degrade type IV collagen, a major constituent of basement membranes. MMP-9 and MMP-2 are secreted by cells as zymogens; both enzymes are activated, by the cleavage of their amino-terminal pro-domains, through distinct pathways operating at the cell-matrix boundary [2]. Tissue detection of MMPs has suggested that augmented secretion and proteolytic activation of MMPs is often associated with tumor metastasis. Several authors have also detected incremented levels of circulating forms MMP-2 and MMP-9 in oncologic patients [3-6]. However, their clinical applications remain controversial. To confirm and extend our previous observations, in this study we analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of 56 selected breast carcinoma patients, in comparison with the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissues) of same patients. The activity levels of MMP-2 and MMP-9 were detected by gelatin zymography and quantified by ImageQuant TL. Two bands of activity, corresponding to the proforms of MMP-9 and MMP-2, were detected in the serum samples and tissue extracts of all oncologic patients, but with highly variable levels of intensity. In general the intensity of lytic bands was higher in serum than in the tissue extracts. In addition, most serum samples, and some of the tissue extracts, displayed additional proteolytic bands at higher Mw, corresponding likely to complexed forms of MMP-9 [5]. As previously reported for a pilot number of cases, lytic bands corresponding the activated forms of the two gelatinases were uniquely present in the tumor extracts, and absent in the paired normal tissues, thus confirming the hypothesis that activation of the enzymes is a key step during the invasive growth of the tumor. In order to assess the possible association between MMPs activities and other proteins potentially involved in tumor progression, we correlated the activity levels of both MMPs with the proteomic-based expression levels of the S100 protein members. One of the reasons of interest for this family of proteins is because there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast cancer, and this is frequently associated with tumour progression. Interestingly we have observed a correlation between the gelatinase activity levels and expression levels of some S100 proteins. In conclusion, our results, which show higher variability of MMP-9 levels suggest its usefulness as a marker evaluable both in the follow-up and in the prognosis of breast cancer patients.

The turn-over of extracellular matrix is a physiological process, that in normal conditions and in wound healing respond to spatial and temporal regulatory mechanisms, involving several cell-matrix interaction pathways. Profound changes occur both at cellular and extracellular level, during the progression of various forms of invasive carcinomas. Collagen alterations and cellular effects. The ultrastructural and biochemical analyses of the collagenous stroma of invasive ductal breast carcinoma have demonstrated the occurrence of extensive fragmentation of pre-existing collagen fibrils and new deposition of thinner fibrils formed mostly by 1(I)3 homotrimer collagen of type I [1-3], which appears preferentially produced by cancer cells [4]. More recently this collagen has been discovered to be resistant to collagenases, due to less efficient unwinding of homotrimers by MMP-1 [5]. When neoplastic cells are exposed to this collagen in an in vitro system, respond by increasing proliferation and migration rates, and display a proteomic [6,7] and genomic differential profiling versus the control cell cultures. Concurrently, we observed an increase of type V collagen, that when used as a substrate for cell culture, induced opposite effects to that exerted by the homotrimer collagen. A similar restraining effect was obtained when cells were induced to express decorin, a small proteoglycan lysine-rich [8]. Experiments conducted in vivo have revealed that the homotrimer collagen, applied on wound beds of treated guinea pigs, promoted keratynocyte migration into the granulation tissue, therefore confirming its propensity to facilitate local cell invasion and metastasis [9]. Fibrobalst influences. Fibroblasts are the major stromal cells with multiple roles, especially toward both the extracellular matrix and the neighbouring cell population, including neoplastic cells. We recently studied the effects exerted by fibroblasts on cancer cell (8701-BC) proteomics, using a trans-well coculture system. Our results clearly indicated that fibroblasts induce considerable proteomic modulations on 8701-BC, mainly in the cytoskeleton proteins and glycolytic enzymes. Additionally, fibroblast-conditioned medium increased neoplastic cell proliferation and invasion with a concurrent upregulation of the c-Myc oncogene. Collectively these results suggest that fibroblast stimulation may enhance the malignant potential of breast cancer cells in vitro [10]. In conclusion, the present information based on multidisciplinary approaches strongly suggest that the local microenvironment around the primary tumor, emanates influences of opposing signals, which may contribute in directing the neoplastic cells towards

Background: Current clinical parameters for breast cancer diagnosis and therapy are: tumour size, axillary lymph node status, histological grading and presence or absence of metastases. Prognostic/predictive properties, such as oestrogen and progesterone receptor status, and human epidermal growth factor receptor (HER-2/neu) status are currently used for therapeutic decision. Conversely, it is now emerging that the number of genetic mutations and epigenetic deregulations in cancer is far more higher than previously thought. Therefore, proteomic screening for differential protein expression in subsets of tumor samples is an essential tool for generating data bases and biomarker discovery. The aim of present study was to extend to a significant number of proteins and patients our previous data, contributing to the knowledge of biological pathways involved in breast cancer and to the molecular-based classification of breast cancer patients. Methods: Aliquots of breast cancer tissues and their adjacent non-tumoral tissues were obtained during surgical intervention from patients who did not receive any neoadiuvant therapy. Diagnosis of ductal infiltrating breast cancer (DIC) was confirmed by histopathology. Sample preparation for proteomics was performed as described (Pucci-Minafra et al. Proteomics Clinical Applications, 1, 118-129, 2007; Pucci-Minafra et al. Journal of Proteome Research 7, 1412-18, 2008). Quantitative determination of protein spots was normalized for actin. Results: We have collected for the present study 100 proteomic maps from G2/G3-DIC patients, and 13 from non-tumoral mammary tissues. All detected proteins were identified, or confirmed, by mass spectrometry. Collectively we have annotated 209 protein spots, corresponding to 122 genes. Genes were analyzed by the instruments of DAVID Bioinformatics Resources (Dennis et al. 2003; Huang et al. 2009). The Gene Accession Conversion tool recognized 112 unambiguous Gene IDs, over the 122 ones present in our list. The gene list was correlated to 136 functional terms, but only 36 correlations were highly significant (Benjamini values from 1x10-8 to 5.6 x10-3). Nine terms over the 36 corresponded to the function of apoptotic processes and in particular 24 genes were related at the highest significant value (1x10-8) with the regulation of programmed cell death; 50% of the genes belonging to this category, codify for proteins with anti-apoptotic functions. The cluster of anti-apoptotic proteins, corresponding to 12 genes and 22 protein isoforms was compared among the 100 proteomics maps and the 13 reference non-tumoral tissue maps. The comparative proteomic profiling showed: 1) a highly significant overexpression of several members of the anti-apoptotic protein cluster in the cancer tissues vs non tumoral counterparts; 2) a relative variability of the expression levels of the normalized proteins within patients. Among proteins of this category reaching the high levels of expression, we observed the nucleophosmin (NPM), a crucial regulator of p53; the translationally-controlled tumor protein (TCTP1), a protein involved in calcium binding and microtubule stabilization; cofilin (COF1), an actin-binding protein responsible also for the signal translocation from cytoplasm to nucleus; annexin A1 (ANXA1) a calcium/phospholipid-binding protein which promotes membrane fusion and ruffling, and the glutathione s-transferase (GSTP1), which plays important roles in detoxification but having also a role in susceptibility to cancer. Conclusions: The application of the powerful Bioinformatics Resources for gene/protein classification provided by DAVID knowledgebase, while confirming our previous protein classification, introduced new terms for further remodulation of protein clusters on the basis of the multiple functions for individual proteins. In particular we found a high number of proteins with specific biochemical functions, converging towards common pathways. Overall, a predominant pathway was the programmed cell-death, which resulted to be the most robust among patients, both as number of proteins involved and as level of significativity.We believe hat the present collection of human breast cancer proteomics represents a valid contribution for clinical applications to breast cancer.

HER-2 is a cell membrane protein that belongs to the ErbB family of receptor tyrosine kinases (HER-1, HER-2, HER-3, HER-4). The over-expression of HER-2, which results in the 25-30% of breast cancer patients, is considered a predictive and prognostic marker for breast cancer malignancy and invasiveness and makes HER-2 an excellent therapeutic target. In the last years new therapeutic strategies have been improved in order to better deal tumor diseases an to minimize collateral effects due to classic chemotherapy in patients. In this way, a new approach was the somministration of humanized antibodies directed against tumor-associated molecular targets. Among these ones Herceptin, an anti-neoplastic humanized monoclonal antibody (Herceptin®, Roche, CH), has been shown to be active against breast cancer cells over-expressing HER-2 receptor. Unfortunately the initial response to Herceptin-based regimens is followed by drug-resistance occurring within 1 year in the majority of patients. Thus it becomes critical to understand the molecular mechanisms underlying primary or acquired Herceptin resistance in order to improve the survival of breast cancer patients whose tumors overexpress HER2. On these bases, we focused our attention to investigate the effects caused by Herceptin treatment on SK-BR-3 breast cancer cells, which overexpress HER-2. To obtain drug-resistant cells, SK-BR-3 were treated with 10 µgr/ml of drug, until cells growth rate became synchronous compared to untreated parental cells. Parental and resistant cells were then properly collected and subjected to cell-lysis to obtain protein extracts, while respective conditioned media were harvested to perform analyses of cell-released gelatinolic activities, such as MMP-2 and MMP-9. These investigations are justified by previous observation of our group, showing a certain degree of correlation between gelatinase expression and HER-2 overexpression in vivo [1]. Protein extracts were subjected to IPG-2D electrophoresis, firstly to evaluate proteomic profiles of both resistant and parental cells, secondly to detect differentially expressed proteins in two above mentioned experimental conditions and thirdly to identify new Herceptin-resistance related proteins by means of MALDI-TOF mass spectrometry. The results so far obtained, have shown that prolonged Herceptin treatment induces dramatic changes in malignant resistant cells, such as rearrangement of cytoskeletal structures, evident effects on expression of proteins involved in cytoskeleyton remodelling, metabolic processes, stress responses and cell cycle and proliferation control. We suggest that present data may contribute significantly to the knowledge of the mechanisms underlying Herceptin drug resistance on breast cancer cells. [1]La Rocca, G., Pucci-Minafra, I., Marrazzo, A., Taormina, P. & Minafra, S. 2004. British Journal of Cancer, 90: 1414-1421.

The S-100 family of calcium-binding proteins includes about 20 members of low molecular weight characterized by two consecutive EF hands domains. They make interactions with cellular target proteins in a calcium-dependent manner; therefore they are thought to regulate a variety of physiological functions, such as cell proliferation, signal transduction, cell adhesion, motility as well as cancer metastasis.

Fibroblasts are the major mesenchymal cell types committed to the matrix formation and renewal. Moreover they are the main source of paracrine factors that influence the growth of epithelial cells of neighbouring tissues. For these properties they may be involved in tumourigenesis, either by remodelling the tumor-associated extracellular matrix (ECM), and by the production of paracrine factors that influence the growth of carcinoma cells. Studies on fibroblasts associated to carcinomas have documented their phenotypic modifications, including abnormal migratory behaviour in vitro and growth factors altered expression(Schor & Schor, 2001). In addition, fibroblasts often recruit inflammatory cells involved in the stimulation of angiogenesis, probably by the proteolytic release of sequestered angiogenic activators (Tlsty & Hein, 2001). Reciprocally, cancer cells may regulate the biosynthetic activities of fibroblasts, thus altering the ECM of the tumor, which in turn exerts some influence on neoplastic cell behaviour. The aim of the present work was to extend our previous observations on the effects of microenvironment factors on neoplastic cell behaviour (Pucci-Minafra et al.2008,in press), utilizing the well characterized breast cancer-derived cells, 8701-BC (Minafra et al. 1989). The neoplastic cells were exposed to influences of fibroblasts either in a co-culture system or through incubation with the theirs conditioned media. In this report we show that the fibroblasts affect neoplastic cell behaviour by: increasing cell proliferation rate, rising the migration and invasion properties of cells in boyden chamber assays, and inducing a transition of cytoskeletal filament expression. Further, these data were confirmed by proteomic approach that allow to evaluate multiple response of cells subjected to external influences.

The exponentially increasing volume of information extracted from genomic and proteomic applications on cancer, while providing new insights into molecular composition of cancer cells and tissues, imposes new challenges on data rationalization as a tool for clinically relevant biomarker discovery. Breast cancer represents the most frequent and potentially aggressive type of cancer and, moreover, it is one of the most enigmatic and poorly predictable in its evolution, likely because it includes several different forms that behave differently among patients. Current clinical parameters for breast cancer diagnosis and cure are: tumour size, axillary lymph node status, histological grading and presence or absence of metastases. Prognostic/predictive properties, such as oestrogen and progesterone receptor status, and human epidermal growth factor receptor (HER-2/neu) status are currently used for therapeutic decision. Conversely, it is now emerging that the number of genetic mutations and epigenetic deregulations in cancer is far more higher than previously thought. Therefore, proteomic screening for differential protein expression in subsets of tumor samples is an essential tool to generate data bases, to contribute to the knowledge of biological pathways in a given cancer tissue, to allow a molecular classification of cancer for patient stratification and biomarker discovery. To minimize the limitation induced by heterogeneity of breast cancer tissues, we have recently introduced, as objective criterion to compare proteomes of different tissues, the normalization of the expression levels of individual proteins, for actin content in each tissue extract (Pucci-Minafra I. et al., 2008). With the aim to identify candidate markers for stratification of patients and clinical correlations, in the present study we evaluated the surgical tissues proteomic profiles of an increased number of patients diagnosed for ductal infiltrating breast cancer. Differentially expressed proteins were identified by 2D-IPG coupled with Peptide Mass Fingerprint (PMF) and N-terminal microsequencing.

Herceptin, an anti-neoplastic humanized monoclonal antibody (Herceptin®, Roche, CH), has been shown to be active against breast cancer cells over-expressing HER-2 receptor. HER-2 is a cell membrane protein that belongs to the epidermal growth factor receptors family and that results over-expressed in the 25-30% of breast cancer patients. The over-expression of HER-2 is considered a predictive and prognostic marker for breast cancer malignancy and invasiveness. On these bases, we aimed to analyze the effects caused by Herceptin treatment on 8701-BC breast cancer cells (Minafra et al., 1989). Firstly we evaluated the effects of Herceptin on the growth rate of 8701-BC cells. To this purpose, parallel cell cultures were daily treated with 0.5, 1, 1.5 and 3 µgr/ml of drug respectively, for a period of 7 days. Cell number was determined by a MTS assay. The resulting growth curves have shown a dose-dependent pharmacologic response, where cells treated with 3µgr/ml of Herceptin underwent 50% inhibition after 7 days. Therefore this concentration was selected to evaluate phenotypic effects exerted by the drug on the surviving cells. In a branch of experiments, the expression level of a group of pivotal protein classes was assayed, both at proteomic and immunological level. Concurrently, MMP-2 and MMP-9 activity in conditioned media of treated and untreated cells was assayed by zymographic assays. This investigation is justified by previous observation of our group, showing a certain degree of correlation between gelatinase expression and HER-2 overexpression in vivo (La Rocca et al., 2004). The results so far obtained, have shown that Herceptin treatment induces a strong inhibition of gelatinolytic activity and modulates the expression levels of some proteins involved in cancer progression, such as cytoskeletal proteins, cell cycle regulators, membrane receptors and associated proteins and some metabolic enzymes. We suggest that present data may contribute greatly to the knowledge of the multiple effects exerted by Herceptin on breast cancer cells.