Rosa Musso

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.

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).

Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells.

BackgroundIn regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.MethodsFibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student’s unpaired t test and considering p < 0.05 as statistically significant.ResultsThe isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of “the stem gene profile”; 84 were differentially expressed and were involved in structural activity.ConclusionsThe fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.

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.

New Insights into the Occurrence of Matrix Metalloproteases -2 and -9 in a Cohort of Breast Cancer Patients and Proteomic Correlations

Matrix metalloproteases (MMPs) are a family of well-known enzymes which operate prevalently in the extracellular domain, where they fulfil the function of remodeling the extracellular matrix (ECM). Within the 26 family members, encoded by 24 genes in humans, MMP-2 and MMP-9 have been regarded as primarily responsible for the basement membrane and peri-cellular ECM rearrangement. In cases of infiltrating carcinomas, which arise from the epithelial tissues of a gland or of an internal organ, a marked alteration of the expression and the activity levels of both MMPs is known to occur. The present investigation represents the continuation and upgrading of our previous studies, now focusing on the occurrence and intensity levels of MMP-2 and -9 and their proteomic correlations in a cohort of 80 breast cancer surgical tissues.