PROTEOMIC IDENTIFICATION OF NOVEL MARKERS IN BREAST AND COLON CANCER

Background: Discovery of new biomarker represent the greatest promise for the detection and management of cancer. Although progress in cancer biology has been rapid during the past few years, the complete understanding of molecular basis for cancer initiation, progression and efficacious treatments is still lacking. In this context, the application of proteomic strategies is now holding a focal position. The main reason is that proteins are the functional players that drive cancer phenotypes. Among cancers, breast and colon represent the most frequent forms. The evolution of these type of cancer are not easily predictable since there are several types that behave differently among patients. The biological heterogeneity is consistent with observed varied responses to therapies across patients, also. On the other hand, drug delivery is an emergent field focused on targeting drugs to a desirable group of cells, in order to minimize undesirable side-effects and maximize the therapeutic activity. Metallic nanoparticles, in particular silver nanoparticles (Ag-NPs) exhibit low toxicity to mammalian cells (Mahapatra and Karak, 2008) and are good candidate as smart therapeutics. Based on these evidences, the first part of the study was aimed to discover new potential protein biomarkers in breast and colon cancer tissues and sera, using proteomic techniques, useful as diagnostic and prognostic factors in vivo. The second part of the study was focused on the in vitro cytotoxic effects of silver nanoparticles Ag-NPs embedded on Klebsiella Oxytoca DSM29614 (KO) Exopolysaccaride (EPS), produced in aerobic versus anaerobic conditions. Methods: Diagnostic biomarkers in breast and colon cancer: Taken advantage from previous results by the proteomic analysis performed on 13 breast cancer tissues and their matched non-tumoral adjacent tissues (Pucci-Minafra et al., 2007), we first analyze by 2D-DIGE pool of both breast and colon cancer tissues extracts compared to the matched pool of non tumoral adjacent tissues extracts. Differentially expressed proteins, identified by Maldi-TOF/TOF, were functionally clustered. We also investigate the activity levels of MMP-2 and MMP-9 in breast and colon tissues as well as in sera of the same patients. Prognostic biomarkers in breast and colon cancer: In order to identity putative proteomic signatures for colorectal cancer (CRC) metastasis, a comparative profiling of a colon cancer tissue paired with the non tumoral adjacent mucosa and with the liver metastasis from the same patient was performed. A three-step approach (normal versus tumoral versus metastasis) was used to select unique proteins involved in liver metastasis. For breast cancer, a large proteomic investigation performed on a large sample set of breast cancer patients (Cancemi et al., 2010, 2012), pointed the important role of S100 protein members in breast cancer progression. Using on line tools, for instance GOBO and breast cancer Kaplan Meir-plotter we assessed gene expression levels and clinical correlations of S100 proteins in breast patients. Cytotoxic effects of silver nanoparticles biosynthesized from KO (Ag-NPs-EPS) in SK-BR3 breast cancer cell line: We monitored cell proliferation inhibition rate by MTT assay, morphological changes and proteomic modulation. Results: Diagnostic biomarkers in breast and colon cancer: Differentially breast and bolon proteomic profiling revealed several proteins involved in common pathways among the type of cancer. The important role of MMPs in tumorigenesis was confirmed by our observations regarding their major expressions in cancer tissues compared to the normal tissues. Prognostic biomarkers in breast and colon cancer: Among the differentially expressed proteins between normal-tumor and liver metastasis, Cathepsin D expression was further analyzed as prognostic factor in CRC. Moreover, integrating results obtained by bioinformatics analysis performed on breast cancer gene expression dataset confirmed the important role of S100 proteins in breast cancer progression. Cytotoxic effects of silver nanoparticles (AgNPs) biosynthesized from KO in SK-BR3 breast cancer cell line: The most important effects were obtained by aerobically AgNPs-EPS treatment, due to the major release of Ag+1, as verified by voltammetry analysis. Morphological alteration were consistent with apoptotic features. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Conclusions: Conclusively, the present study contribute to the implementation of the panel of new proteomic biomarkers useful for diagnostic and prognostic applications in breast and colon cancer, providing new informations about the effects of the biosynthesized Ag-NPs-EPS on breast cancer cells

PROTEOMIC IDENTIFICATION OF NOVEL MARKERS IN BREAST AND COLON CANCER

Background: Discovery of new biomarker represent the greatest promise for the detection and management of cancer. Although progress in cancer biology has been rapid during the past few years, the complete understanding of molecular basis for cancer initiation, progression and efficacious treatments is still lacking. In this context, the application of proteomic strategies is now holding a focal position. The main reason is that proteins are the functional players that drive cancer phenotypes. Among cancers, breast and colon represent the most frequent forms. The evolution of these type of cancer are not easily predictable since there are several types that behave differently among patients. The biological heterogeneity is consistent with observed varied responses to therapies across patients, also. On the other hand, drug delivery is an emergent field focused on targeting drugs to a desirable group of cells, in order to minimize undesirable side-effects and maximize the therapeutic activity. Metallic nanoparticles, in particular silver nanoparticles (Ag-NPs) exhibit low toxicity to mammalian cells (Mahapatra and Karak, 2008) and are good candidate as smart therapeutics. Based on these evidences, the first part of the study was aimed to discover new potential protein biomarkers in breast and colon cancer tissues and sera, using proteomic techniques, useful as diagnostic and prognostic factors in vivo. The second part of the study was focused on the in vitro cytotoxic effects of silver nanoparticles Ag-NPs embedded on Klebsiella Oxytoca DSM29614 (KO) Exopolysaccaride (EPS), produced in aerobic versus anaerobic conditions. Methods: Diagnostic biomarkers in breast and colon cancer: Taken advantage from previous results by the proteomic analysis performed on 13 breast cancer tissues and their matched non-tumoral adjacent tissues (Pucci-Minafra et al., 2007), we first analyze by 2D-DIGE pool of both breast and colon cancer tissues extracts compared to the matched pool of non tumoral adjacent tissues extracts. Differentially expressed proteins, identified by Maldi-TOF/TOF, were functionally clustered. We also investigate the activity levels of MMP-2 and MMP-9 in breast and colon tissues as well as in sera of the same patients. Prognostic biomarkers in breast and colon cancer: In order to identity putative proteomic signatures for colorectal cancer (CRC) metastasis, a comparative profiling of a colon cancer tissue paired with the non tumoral adjacent mucosa and with the liver metastasis from the same patient was performed. A three-step approach (normal versus tumoral versus metastasis) was used to select unique proteins involved in liver metastasis. For breast cancer, a large proteomic investigation performed on a large sample set of breast cancer patients (Cancemi et al., 2010, 2012), pointed the important role of S100 protein members in breast cancer progression. Using on line tools, for instance GOBO and breast cancer Kaplan Meir-plotter we assessed gene expression levels and clinical correlations of S100 proteins in breast patients. Cytotoxic effects of silver nanoparticles biosynthesized from KO (Ag-NPs-EPS) in SK-BR3 breast cancer cell line: We monitored cell proliferation inhibition rate by MTT assay, morphological changes and proteomic modulation. Results: Diagnostic biomarkers in breast and colon cancer: Differentially breast and bolon proteomic profiling revealed several proteins involved in common pathways among the type of cancer. The important role of MMPs in tumorigenesis was confirmed by our observations regarding their major expressions in cancer tissues compared to the normal tissues. Prognostic biomarkers in breast and colon cancer: Among the differentially expressed proteins between normal-tumor and liver metastasis, Cathepsin D expression was further analyzed as prognostic factor in CRC. Moreover, integrating results obtained by bioinformatics analysis performed on breast cancer gene expression dataset confirmed the important role of S100 proteins in breast cancer progression. Cytotoxic effects of silver nanoparticles (AgNPs) biosynthesized from KO in SK-BR3 breast cancer cell line: The most important effects were obtained by aerobically AgNPs-EPS treatment, due to the major release of Ag+1, as verified by voltammetry analysis. Morphological alteration were consistent with apoptotic features. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Conclusions: Conclusively, the present study contribute to the implementation of the panel of new proteomic biomarkers useful for diagnostic and prognostic applications in breast and colon cancer, providing new informations about the effects of the biosynthesized Ag-NPs-EPS on breast cancer cells

Expression of Alpha-Enolase (ENO1), Myc Promoter-Binding Protein-1 (MBP-1) and Matrix Metalloproteinases (MMP-2 and MMP-9) Reflect the Nature and Aggressiveness of Breast Tumors

Breast cancer is a complex and heterogeneous disease: Several molecular alterations cause cell proliferation and the acquisition of an invasive phenotype. Extracellular matrix (ECM) is considered essential for sustaining tumor growth and matrix metalloproteinases (MMPs) have been identified as drivers of many aspects of the tumor phenotype. Mounting evidence indicates that both α-enolase (ENO1) and Myc promoter-binding protein-1 (MBP-1) also played pivotal roles in tumorigenesis, although as antagonists. ENO1 is involved in cell growth, hypoxia tolerance and autoimmune activities besides its major role in the glycolysis pathway. On the contrary, MBP-1, an alternative product of ENO1, suppresses cell proliferation and the invasive ability of cancer cells. Since an important task in personalized medicine is to discriminate a different subtype of patients with different clinical outcomes including chances of recurrence and metastasis, we investigated the functional relationship between ENO1/MBP-1 expression and MMP-2 and MMP-9 activity levels in both tissues and sera of breast cancer patients. We focused on the clinical relevance of ENO1 and MMPs (MMP-2 and MMP-9) overexpression in breast cancer tissues: The association between the higher ENO1, MMP-2 and MMP-9 expression with a worse prognosis suggest that the elevated ENO1 and MMPs expression are promising biomarkers for breast cancer. A relationship seems to exist between MBP-1 expression and the decrease in the activity levels of MMP-9 in cancer tissues and MMP-2 in sera. Moreover, the sera of breast cancer patients grouped for MBP-1 expression differentially induced, in vitro, cell proliferation and migration. Our findings support the hypothesis of patient's stratification based on ENO1, MBP-1 and MMPs expression. Elucidating the molecular pathways through which MBP-1 influences MMPs expression and breast cancer regression can lead to the discovery of new management strategies

Prognostic and Functional Significant of Heat Shock Proteins (HSPs) in Breast Cancer Unveiled by Multi-Omics Approaches

Heat shock proteins (HSPs) are a well-characterized molecular chaperones protein family, classified into six major families, according to their molecular size. A wide range of tumors have been shown to express atypical levels of one or more HSPs, suggesting that they could be used as biomarkers. However, the collective role and the possible coordination of HSP members, as well as the prognostic significance and the functional implications of their deregulated expression in breast cancer (BC) are poorly investigated. Here, we used a systematic multi-omics approach to assess the HSPs expression, the prognostic value, and the underlying mechanisms of tumorigenesis in BC. By using data mining, we showed that several HSPs were deregulated in BC and significantly correlated with a poor or good prognosis. Functional network analysis of HSPs co-expressed genes and miRNAs highlighted their regulatory effects on several biological pathways involved in cancer progression. In particular, these pathways concerned cell cycle and DNA replication for the HSPs co-expressed genes, and miRNAs up-regulated in poor prognosis and Epithelial to Mesenchymal Transition (ETM), as well as receptors-mediated signaling for the HSPs co-expressed genes upregulated in good prognosis. Furthermore, the proteomic expression of HSPs in a large sample-set of breast cancer tissues revealed much more complexity in their roles in BC and showed that their expression is quite variable among patients and confined into different cellular compartments. In conclusion, integrative analysis of multi-omics data revealed the distinct impact of several HSPs members in BC progression and indicate that collectively they could be useful as biomarkers and therapeutic targets for BC management

New Synthetic Nitro-Pyrrolomycins as Promising Antibacterial and Anticancer Agents

Pyrrolomycins (PMs) are polyhalogenated antibiotics known as powerful biologically active compounds, yet featuring high cytotoxicity. The present study reports the antibacterial and antitumoral properties of new chemically synthesized PMs, where the three positions of the pyrrolic nucleus were replaced by nitro groups, aiming to reduce their cytotoxicity while maintaining or even enhancing the biological activity. Indeed, the presence of the nitro substituent in diverse positions of the pyrrole determined an improvement of the minimal bactericidal concentration (MBC) against Gram-positive (i.e., Staphylococcus aureus) or -negative (i.e., Pseudomonas aeruginosa) pathogen strains as compared to the natural PM-C. Moreover, some new nitro-PMs were as active as or more than PM-C in inhibiting the proliferation of colon (HCT116) and breast (MCF 7) cancer cell lines and were less toxic towards normal epithelial (hTERT RPE-1) cells. Altogether, our findings contribute to increase the knowledge of the mode of action of these promising molecules and provide a basis for their rationale chemical or biological manipulation

A multiomics analysis of S100 protein family in breast cancer

The S100 gene family is the largest subfamily of calcium binding proteins of EFhand type, expressed in tissue and cell-specific manner, acting both as intracellular regulators and extracellular mediators. There is a growing interest in the S100 proteins and their relationships with different cancers because of their involvement in a variety of biological events closely related to tumorigenesis and cancer progression. However, the collective role and the possible coordination of this group of proteins, as well as the functional implications of their expression in breast cancer (BC) is still poorly known. We previously reported a large-scale proteomic investigation performed on BC patients for the screening of multiple forms of S100 proteins. Present study was aimed to assess the functional correlation between protein and gene expression patterns and the prognostic values of the S100 family members in BC. By using data mining, we showed that S100 members were collectively deregulated in BC, and their elevated expression levels were correlated with shorter survival and more aggressive phenotypes of BC (basal like, HER2 enriched, ER-negative and high grading). Moreover a multi-omics functional network analysis highlighted the regulatory effects of S100 members on several cellular pathways associated with cancer and cancer progression, expecially immune response and inflammation. Interestingly, for the first time, a pathway analysis was successfully applied on different omics data (transcriptomics and proteomics) revealing a good convergence between pathways affected by S100 in BC. Our data confirm S100 members as a promising panel of biomarkers for BC prognosis

Integrated Multi-Omics Investigations of Metalloproteinases in Colon Cancer: Focus on MMP2 and MMP9

Colorectal cancer (CRC) develops by genetic and epigenetic alterations. However, the molecular mechanisms underlying metastatic dissemination remain unclear and could benefit from multi-omics investigations of specific protein families. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in ECM remodeling and the processing of bioactive molecules. Increased MMP expression promotes the hallmarks of tumor progression, including angiogenesis, invasion, and metastasis, and is correlated with a shortened survival. Nevertheless, the collective role and the possible coordination of MMP members in CRC are poorly investigated. Here, we performed a multi-omics analysis of MMP expression in CRC using data mining and experimental investigations. Several databases were used to deeply mine different expressions between tumor and normal tissues, the genetic and epigenetic alterations, the prognostic value as well as the interrelationships with tumor immune-infiltrating cells (TIICs). A special focus was placed on to MMP2 and MMP9: their expression was correlated with immune markers and the interaction network of co-expressed genes disclosed their implication in epithelial to mesenchymal transition (EMT) and immune response. Finally, the activity levels of MMP2 and MMP9 in a cohort of colon cancer samples, including tissues and the corresponding sera, was also investigated by zymography. Our findings suggested that MMPs could have a high potency, as they are targeted in colon cancer, and might serve as novel biomarkers, especially for their involvement in the immune response. However, further studies are needed to explore the detailed biological functions and molecular mechanisms of MMPs in CRC, also in consideration of their expression and different regulation in several tissues