Fuzzy modeling and global optimization to predict novel therapeutic targets in cancer cells.

MOTIVATION: The elucidation of dysfunctional cellular processes that can induce the onset of a disease is a challenging issue from both the 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 emergent dynamical behaviors of highly heterogeneous systems in unperturbed and perturbed conditions, regardless of the availability of quantitative parameters, and (2) determine a minimal set of system components whose perturbation can lead to a desired system response, therefore facilitating the design of a more appropriate experimental strategy. RESULTS: We applied this method to investigate what drives K-ras-induced cancer cells, displaying the typical Warburg effect, to death or survival upon progressive glucose depletion. The optimization analysis allowed to identify new combinations of stimuli that maximize pro-apoptotic processes. Namely, our results provide different evidences of an important protective role for protein kinase A in cancer cells under several cellular stress conditions mimicking tumor behavior. The predictive power of this method could facilitate the assessment of the response of other complex heterogeneous systems to drugs or mutations in fields as medicine and pharmacology, therefore paving the way for the development of novel therapeutic treatments. AVAILABILITY AND IMPLEMENTATION: The source code of FUMOSO is available under the GPL 2.0 license on GitHub at the following URL: https://github.com/aresio/FUMOSO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Central metabolism of functionally heterogeneous mesenchymal stromal cells.

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 potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

Inhibition of the Hexosamine Biosynthetic Pathway by targeting PGM3 causes breast cancer growth arrest and apoptosis.

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 of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N- and O-glycosylation level that cause also a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with the activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice, supporting the advantage of targeting HBP for therapeutic purpose and encouraging further investigation about the use of this small molecule as a promising compound for breast cancer therapy.

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

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 to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.

Reversal of Warburg Effect and Reactivation of Oxidative Phosphorylation by Differential Inhibition of EGFR Signaling Pathways in Non-Small Cell Lung Cancer.

PURPOSE: One of the hallmarks of cancer cells is the excessive conversion of glucose to lactate under normoxic conditions, also known as the 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). EXPERIMENTAL DESIGN: Sensitive (HCC827) and resistant (H1975 and H1993) NSCLC cells were treated with a panel of EGFR or MET inhibitors, and then tested for changes of EGFR signaling, glycolytic cascade, and mitochondrial function. Silencing of key glycolytic enzymes was then performed with targeted siRNAs. Furthermore, tumor-bearing nude mice treated with EGFR inhibitors were evaluated with (18)F-FDG PET/CT and tumors were analyzed for glycolytic and mitochondrial proteins. RESULTS: Effective inhibition of EGFR signaling in NSCLC cells induced a dramatic reduction of hexokinase II (HKII) and phospho-pyruvate kinase M2 (p-PKM2, Tyr105) levels as well as an upregulation of mitochondrial complexes subunits (OXPHOS). Accordingly, a decreased lactate secretion and increased intracellular ATP levels were also observed in response to EGFR inhibitors. Downregulation of HKII and PKM2 by targeted siRNA transfection did not cause upregulation of OXPHOS but enhanced the effects of EGFR TKIs. Conversely, selective inhibition of AKT and ERK1/2 caused OXPHOS upregulation and glycolysis inhibition, respectively. Similar findings were obtained in tumors from animals treated with appropriate EGFR inhibitors. CONCLUSIONS: Our findings indicate that EGFR inhibitors may reactivate oxidative phosphorylation of cancer cells and provide a mechanistic clue for the rational combination of agents targeting EGFR-dependent proliferation and glucose metabolism in cancer therapy.

New Insights into the Connection Between Histone Deacetylases, Cell Metabolism, and Cancer.

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 that these proteins are involved in the regulation of several other cellular processes by their ability to deacetylate hundreds of proteins with different functions in both the cytoplasm and the nucleus. Importantly, recent high-throughput studies have identified as important target proteins several enzymes involved in different metabolic pathways. Conversely, it has been also shown that metabolic intermediates may control HDACs activity. Consequently, the acetylation/deacetylation of metabolic enzymes and the ability of metabolic intermediates to modulate HDACs may represent a cross-talk connecting cell metabolism, transcription, and other HDACs-controlled processes in physiological and pathological conditions. CRITICAL ISSUES: Since metabolic alterations and HDACs deregulation are important cancer hallmarks, disclosing connections among them may improve our understanding on cancer mechanisms and reveal novel therapeutic protocols against this disease. FUTURE DIRECTIONS: High-throughput metabolic studies performed by using more sophisticated technologies applied to the available models of conditional deletion of HDACs in cell lines or in mice will fill the gap in the current understanding and open directions for future research.

Urokinase receptor promotes ovarian cancer cell dissemination through its 84-95 sequence.

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 uPAR84-95 sequence to invasion of ovarian cancer cells. Now, we provide evidence that the ability of uPAR-expressing ovarian cancer cells to cross extra-cellular matrix and mesothelial monolayers is prevented by specific inhibitors of the uPAR84-95 sequence. To specifically investigate uPAR84-95 function, uPAR-negative CHO-K1 cells were stably transfected with cDNAs coding for uPAR D2 and D3 regions exposing (uPARD2D3) or lacking (uPAR∆D2D3) the 84-95 sequence. CHO-K1/D2D3 cells were able to cross matrigel, mesothelial and endothelial monolayers more efficiently than CHO-K1/∆D2D3 cells, which behave as CHO-K1 control cells. When orthotopically implanted in nude mice, tumor nodules generated by CHO-K1/D2D3 cells spreading to peritoneal cavity were more numerous as compared to CHO-K1/∆D2D3 cells. Ovarian tumor size and intra-tumoral microvessel density were significantly reduced in the absence of uPAR84-95. Our results indicate that cell associated uPAR promotes growth and abdominal dissemination of ovarian cancer cells mainly through its uPAR84-95 sequence.

Energy metabolism characterization of a novel cancer stem cell-like line 3AB-OS.

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 on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate (mitochondrial specific substrates) leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSC energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism.

Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide.

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 dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.

Analysis of secretome changes uncovers an autocrine/paracrine component in the modulation of cell proliferation and motility by c-Myc.

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 c-Myc expression on the secretome of a nontransformed human epithelial cell line (hT-RPE). By using a quantitative proteomic approach, we have identified 125 proteins in conditioned media of hT-RPE/MycER cells upon c-Myc induction. Analysis of the 49 proteins significantly down-regulated by c-Myc revealed a marked enrichment of factors associated with growth inhibition and cellular senescence. Accordingly, media conditioned by hT-RPE cells expressing c-Myc show an increased ability to sustain hT-RPE cellular proliferation/viability. We also find a marked down-regulation of several structural and regulatory components of the extracellular matrix (ECM), which correlates with an increased chemotactic potency of the conditioned media toward fibroblasts, a major cellular component of tumor stroma. In accordance with these data, the expression of the majority of the genes encoding proteins down-regulated in hT-RPE was significantly reduced also in colorectal adenomatous polyps, early tumors in which c-Myc is invariably overexpressed. These findings help to elucidate the significance of c-Myc overexpression at early stages of tumor development and uncover a remarkable autocrine/paracrine component in the ability of c-Myc to stimulate proliferation, sustain tumor maintenance, and modulate cell migration.

Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

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 turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Modulation of cellular migration and survival by c-Myc through the downregulation of urokinase (uPA) and uPA receptor.

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 invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

Structure-based design of an urokinase-type plasminogen activator receptor-derived peptide inhibiting cell migration and lung metastasis.

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 inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH(2) (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe-dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an alphav integrin-dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/alphav association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein-expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy.

Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation.

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 Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.