Giuseppina Votta

• Università degli Studi di Milano-Bicocca

Motivation: The elucidation of dysfunctional cellular processes that can induce the onset of a disease is a challenging issue from both experimental and computational perspectives. Here we introduce a novel computational method based on the coupling between fuzzy logic modeling and a global optimization algorithm, whose aims are to (1) predict the...

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentia...

An understanding of the mechanism of apoptosis has received a substantial amount of interest over the past decades. It is evident from many works that mitochondria play a pivotal role in keeping cell homeostasis in both physiological and pathological conditions. The ability of mitochondria to reprogram cell metabolism is a key function to cell life...

Introduction Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumour progression. Recent studies reported an association between the tumorigenic potential, metastasis and chemoresistance of several type of breast cancer cells...

A library of GlcNAc-6 or 1P analogues was designed and each compound was computationally evaluated through docking studies for its binding affinity to AGM1/PGM3. The compounds with the highest binding affinity ranked through a docking score, were synthesized and screened for their ability to inhibit the production of UDP-GlcNAc. A glycofused oxazol...

Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumor progression. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Here we report the preclinical evaluation...

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading...

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading...

The genes regulated by FSK in Transformed cells show a low degree of connection. In the figure the network of predicted associations for all DEGs-encoded proteins in TF/T comparison is shown. The STRING analysis of the protein-protein interactions was performed to DEGs with fold change ≥2 in the comparison. (PDF)

The treatment with FSK induces a relevant change in the expression of genes related to the glutamine metabolism. Transcriptional data from microarray analysis regarding glutamine metabolism-related genes in Transformed cells at 72h of culture in LG, daily treated with DMSO or FSK. Data express the ratio in TF/T comparison. (PDF)

FSK-treated MDA-MB-231 cells show a different expression of glutamine metabolism-related genes as compared to untreated cells. The expression of genes related to the glutamine metabolism was performed in MDA-MB-231 cells daily treated with DMSO or FSK after 48h and 72h of cultivation in LG. mRNA expression of glutamine metabolism-related genes was...

PKA and autophagy activation in suspended MDA-MB-231 cells. A MDA-MB-231 cells treated with FSK were collected and stained with fluorescent substrate of the active caspase-3 and then newly attached to the coverslip using cytospin. Then they were fixed and labeled with the antibody for PKA substrates. The images were visualized by using fluorescent...

Genes and proteins identified in our study and used for the Gene set enrichment analysis. Transcriptional and proteomic data have been mapped in the 202 KEGG pathways (metabolic and signaling). (XLS)

Graphical representation of the experimental workflow -/+FSK. A, D For the analyses upon treatment with FSK Normal and Transformed cells (A) as well as MDA-MB-231 (D) were cultured in LG medium (1mM as initial concentration) 16h after seeding. Then, starting from 24h the cells were daily treated with DMSO (vehicle) or 10μM FSK. When required, at 72...

The genes regulated by FSK in Normal cells show a high degree of connection. In the figure the network of predicted associations for all DEGs-encoded proteins in NF/N comparison is shown. The STRING analysis of the protein-protein interactions was performed to DEGs with fold change ≥2 in the comparison. (PDF)

Network of predicted associations for all the differentially expressed proteins identified by 2-DIGE. The STRING analysis of the protein-protein interactions was performed using proteins with spot variation ≥10% in NF/N (A) and TF/T (B) comparisons. (PDF)

The inhibition of PKA prevents the resistance to glucose deprivation and anoikis. A Graphical representation of the experimental workflow for analyses in anoikis-resistant floating cells. MDA-MB-231 cells were cultured in LG. At 72h, when a significant part of the cell population was detached from the plate, the suspended cells were collected and t...

PKA pathway is endogenously activated in Transformed cells cultured in glucose deprivation, mediating their resistance to anoikis. A At indicated time points of culture in LG, floating cells were stained with trypan blue and counted to verify whether floating cells were resistant to anoikis. Percentage of floating and dead cells was reported over t...

Glucose depletion induces low anoikis-resistance in MIA PaCa-2 when cultivated on standard support. A MIA PaCa-2 cells were cultured in LG (1mM glucose as initial concentration). The amount of glucose in the medium was measured using an enzymatic kit. B-C MIA PaCa-2 cells cultured in HG and LG were counted at different time points with Trypan blue,...

Highly differentially expressed genes in the two comparisons NF/N and TF/T. The two spreadshits represent the genes with logFC > = 1 in the two comparisons NF/N and TF/T and shown in the heatmap of Fig 1B (XLS)

List of primers used for qPCR amplification of target genes. Table A: Normal and Transformed cells. Table B: MDA- MB-231 cells (PDF)

Analysis of transcriptomic and proteomic data using PIANO method. The heatmap shows the result obtained by applying the PIANO tool to gene (A) and protein (B) datasets separately. In particular, the top 10-ranked pathways associated to each comparison, NF/N and TF/T, are shown. The different color of the heatmap represents the rank position of the...

The FSK treatment attenuates UPR in both Normal and Transformed cells. The analysis shown here was performed in cells cultured for 72h in LG and daily treated with DMSO or 10μM FSK. A-B mRNA expression of UPR-related genes was analyzed by qPCR for Transformed (A) and Normal (B) cells. mRNA expression levels in FSK-treated cells are reported as chan...

The induction of the PKA pathway mediates the autophagy activation in Transformed cells in glucose deprivation. A PKA activation was evaluated by Western blot analysis of p-(Ser/Thr) PKA substrates and pCREB S133 in Transformed cells daily treated with FSK and/or 2μM H89. B The cellular morphology of the cells -/+ FSK and -/+H89 was observed at 96h...

FSK-protective role is not mediated by Epac. A Schematic representation of FSK-mediated activation of PKA and Epac proteins upon intracellular cAMP enhancement. To analyze the effect of Epac inhibition, the cells were daily treated with FSK and/or 1μM Epac inhibitor (ESI-09). B Cells -/+FSK and -/+ESI-09 were counted with trypan blue at 72h of cult...

PKA activation protects MIA PaCa-2 pancreatic cancer cells by glucose deprivation-induced cell death. A-H For the analyses of -/+FSK MIA PaCa-2 cells were cultured in LG and daily treated with DMSO or FSK. A Microscopy images of cell morphology. B PKA activity after FSK treatment was evaluated by Western blot analysis of p-(Ser/Thr) PKA substrates...

Proteins identified by mass spectrometry in the two comparisons NF/N and TF/T. The two spreadsheets show the mass spectrometry data, the UniProtKB accession number and percentage of the volume spot variation. (XLS)

Total gene expression analysis. The table show the 17669 transcripts used for PIANO analysis and represented as hetamap in Fig 1C. N: Normal cells; N+F: Normal cells + FSK; T: Transformed cells; T+F: Transformed cells + FSK. (XLS)

Supplementary methods. (PDF)

The inhibition of PKA counteracts the protective effects of FSK in MDA-MB-231. MDA-MB-231 cells were analyzed upon daily treatment with FSK and 2μM H89. A PKA activation by Western blot analysis of p-(Ser/Thr) PKA substrates and pCREB S133. B Microscopy images of the cells were collected at 72h of culture. C Western blot analysis of Grp78 and CHOP...

The table reports the rankings obtained by each KEGG pathway (rows) in the two comparisons (N+F vs N and T+F vs T) in the transcript, in the proteomic, and in the integrated/aggregated dataset for the three directionality classes (down- and up-regulation, and non-directional regulation). Pathways in the first 10 positions for the transcript and int...

Pathways used for Venn diagram. (XLS)

NMR identified metabolite in N, N+F, T and T+F cells. Metabolite identified by NMR analysis for three independent biological replicates and mean and standard deviation thereof. (XLS)

One of the hallmarks of cancer cells is the excessive conversion of glucose to lactate under normoxic conditions, also known as Warburg effect. Here we tested whether the targeted inhibition of EGFR may revert this effect and reactivate mitochondrial oxidative phosphorylation in non-small cell lung cancer (NSCLC). Sensitive (HCC827) and resistant (...

The clinical relevance of the urokinase receptor (uPAR) as a prognostic marker in ovarian cancer is well documented. We had shown that the uPAR sequence corresponding to 84-95 residues, linking D1 and D2 domains (uPAR84-95), drives cell migration and angiogenesis in a protease-independent manner. This study was aimed at defining the contribution of...

Significance: Histone deacetylases (HDACs) activity and cell metabolism are considered important targets for cancer therapy, as both are deregulated and associated with the onset and maintenance of tumors. Recent advances: Besides the classical function of HDACs as HDAC enzymes controlling the transcription, it is becoming increasingly evident t...

Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence...

Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally diss...

Proteins secreted by cancer cells are a major component of tumor microenvironment. However, little is known on the impact of single oncogenic lesions on the expression of secreted proteins at early stages of tumor development. Because c-Myc overexpression is among the most frequent alterations in cancer, here we investigated the effect of sustained...

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity...

It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiven...

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser(88)-Arg-Ser-Arg-Tyr(92) is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibit...

We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J C...

It is now beyond reasonable doubt that plasminogen activation, catalyzed by urokinase-type plasminogen activator (uPA), plays an important role in the growth and dissemination of malignant tumours. The plasmin generated facilitates spread of tumour cells by catalyzing degradation of basement membranes and the extracellular matrix (ECM). In addition...