Patrizia Cancemi

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.

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.

Large-scale proteomic identification of S100 proteins in breast cancer tissues

BackgroundAttempts 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.MethodsSamples 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.ResultsThe 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.ConclusionsThis 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.

Proteomic profiling of 13 paired ductal infiltrating breast carcinomas and non-tumoral adjacent counterparts

According to recent statistics, breast cancer remains one of the leading causes of death among women in Western countries. Breast cancer is a complex and heterogeneous disease, presently classified into several subtypes according to their cellular origin. Among breast cancer histotypes, infiltrating ductal carcinoma represents the most common and potentially aggressive form. Despite the current progress achieved in early cancer detection and treatment, including the new generation of molecular therapies, there is still need for identification of multiparametric biomarkers capable of discriminating between cancer subtypes and predicting cancer progression for personalized therapies. One established step in this direction is the proteomic strategy, expected to provide enough information on breast cancer profiling. To this aim, in the present study we analyzed 13 breast cancer tissues and their matched non‐tumoral tissues by 2‐DE. Collectively, we identified 51 protein spots, corresponding to 34 differentially expressed proteins, which may represent promising candidate biomarkers for molecular‐based diagnosis of breast cancer and for pattern discovery. The relevance of these proteins as factors contributing to breast carcinogenesis is discussed.

Proteomic differentiation pattern in the U937 cell line

The U937 cell line, originally established from a histiocytic lymphoma, has been widely used as a powerful in vitro model for haematological studies. These cells retain the immature cell phenotype and can be induced to differentiate by several factors, among which 12-O-tetradecanoyl-13-phorbol acetate (TPA). Fully differentiated cells acquire the adherent phenotype and exhibit various properties typical of macrophages. However, in spite of a great deal of research devoted to the U937 cellular model, the molecular basis of biological processes involved in the monocyte/macrophage differentiation remains unclear. The present study has been undertaken to contribute to this knowledge, in order to identify proteomic-based differentiation pattern for the U937 cells exposed to TPA. Present results have highlighted that the U937 cell differentiation is correlated with a significant proteomic modulation, corresponding to about 30% of the identified proteins, including both over- and down-regulated proteins. Negative modulation regarded proteins involved in the regulation of cell proliferation and in metabolic processes. Proteins appearing incremented in macrophagic phenotype include calcium- and phospholipid-binding proteins and several proteins related to the phagocytic activity. Conclusively, we suggest that this new set of differentially expressed proteins may represent meaningful myelo-monocytic differentiation markers to be applied to the study of several haematological diseases.

Decorin Transfection Induces Proteomic and Phenotypic Modulation in Breast Cancer Cells 8701-BC

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decorin.

Anticancer activity of biogenerated silver nanoparticles: an integrated proteomic investigation

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPSaer) and anaerobic conditions (AgNPs-EPSanaer). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPSaer as the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPSaer mode of action. In particular, AgNPs-EPSaer induced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag+ accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPSaer toxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation.

Differential proteomic and phenotypic behaviour of papillary and anaplastic thyroid cell lines

Thyroid carcinomas account for a minority of all malignant tumours but, after those of the gonads, they represent the most common forms of endocrine cancers. They include several types, among which the papillary thyroid cancer (PTC) and the anaplastic thyroid cancer (ATC) are the best known. The two hystotypes display significant biological and clinical differences: PTC is a well differentiated form of tumour with a high incidence and a good prognosis, while the ATC is less frequent but represents one of the most aggressive endocrine tumours with morphological features of an undifferentiated type. To date, as far as we know, no conclusive studies, useful to design arrays of molecular markers, have been published illustrating the phenotypic and proteomic differences between these two tumours. The aim of this work was to perform a comparative analysis of two thyroid cancer cell lines, derived respectively from papillary (BCPAP) and anaplastic (8505C) thyroid carcinomas. The comparative analysis included cell behaviour assays and proteomic analysis by 2D-PAGE and mass spectrometry. The results have highlighted a new proteomic signature for the anaplastic carcinoma-derived cells, consistent with their high proliferation rate, motility propensity and metabolic shift, in relation to the well-differentiated PTC cells.

Differential occurrence of S100A7 in breast cancer tissues: A proteomic-based investigation

The present study reports for the first time a large‐scale proteomic screening of the occurrence, subcellular localization and relative quantification of the S100A7 protein among a group of 100 patients, clinically grouped for the diagnosis of infiltrating ductal carcinoma (IDC).

In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.

Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival. The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R) by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling. In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated β-cell death. This knowledge may be of potential benefit for patients with T1DM. In particular, SUMO4 could be used as a therapeutical target.