Anoikis: an emerging hallmark in health and diseases.

Anoikis is a programmed cell death occurring upon cell detachment from the correct extracellular matrix, thus disrupting integrin ligation. It is a critical mechanism in preventing dysplastic cell growth or attachment to an inappropriate matrix. Anoikis prevents detached epithelial cells from colonizing elsewhere and is thus essential for tissue homeostasis and development. As anchorage-independent growth and epithelial-mesenchymal transition, two features associated with anoikis resistance, are crucial steps during tumour progression and metastatic spreading of cancer cells, anoikis deregulation has now evoked particular attention from the scientific community. The aim of this review is to analyse the molecular mechanisms governing both anoikis and anoikis resistance, focusing on their regulation in physiological processes, as well as in several diseases, including metastatic cancers, cardiovascular diseases and diabetes.

COVID-19 epidemic strongly affected cancer research in Italy: a survey of the Italian Cancer Society (SIC).

BACKGROUND: Italy was among the first countries hit by the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The application of strict lockdown measures disproportionately affected both cancer patient care as well as basic and translational cancer research. MATERIALS AND METHODS: The Italian Cancer Society (SIC) conducted a survey on the effect of lockdown on laboratories involved in cancer research in Italy. The survey was completed by 570 researchers at different stages of their career, working in cancer centers, research institutes and universities from 19 Italian regions. RESULTS: During the lockdown period, the impact of the COVID-19 pandemic emergency on face-to-face research activities was high, with a complete (47.7%) or partial (36.1%) shutdown of the laboratories. In the post-lockdown period, research activities were resumed in most of the respondents' institutions (80.4%), though with some restrictions (77.2%). COVID-19 testing was offered to research personnel only in ~50% of research institutions. Overall, the response to the pandemic was fragmented as in many cases institutions adopted different strategies often aimed at limiting possible infections without a clearly defined contingency plan. Nevertheless, research was able to provide the first answers and possible ways out of the pandemic, also with the contribution of many cancer researchers that sacrificed their research programs to help overcome the pandemic by offering their knowledge and technologies. CONCLUSIONS: Given the current persistence of an emergency situation in many European countries, a more adequate organization of research centers will be urgent and necessary to ensure the continuity of laboratory activities in a safe environment.

Metabolic cell communication within tumour microenvironment: models, methods and perspectives.

Environmental cues are essential in defining tumour malignancy, by promoting tumour initiation, progression and metastatic spreading. Stromal cells may metabolically cooperate or compete with cancer cells, playing a mandatory role in defining cancer metabolic plasticity, potentially dictating the final tumour outcome. Assessing shared nutrients between different tumoural or stromal compartments is essential to understand the impact of environmental nutrients on the metabolic plasticity of tumours. Here, we review analytical and computational approaches for studying the tumour metabolic microenvironment, the destiny of nutrients shared among tumour and stromal populations, as well as the molecular modules of these metabolic relationships.

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival.

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.

Lactate modulates CD4+ T-cell polarization and induces an immunosuppressive environment, which sustains prostate carcinoma progression via TLR8/miR21 axis.

Leukocyte infiltration plays an active role in controlling tumor development. In the early stages of carcinogenesis, T cells counteract tumor growth. However, in advanced stages, cancer cells and infiltrating stromal components interfere with the immune control and instruct immune cells to support, rather than counteract, tumor malignancy, via cell-cell contact or soluble mediators. In particular, metabolites are emerging as active players in driving immunosuppression. Here we demonstrate that in a prostate cancer model lactate released by glycolytic cancer-associated fibroblasts (CAFs) acts on CD4+ T cells, shaping T-cell polarization. In particular, CAFs exposure (i) reduces the percentage of the antitumoral Th1 subset, inducing a lactate-dependent, SIRT1-mediated deacetylation/degradation of T-bet transcription factor; (ii) increases Treg cells, driving naive T cells polarization, through a lactate-based NF-kB activation and FoxP3 expression. In turn, this metabolic-based CAF-immunomodulated environment exerts a pro-invasive effect on prostate cancer cells, by activating a previously unexplored miR21/TLR8 axis that sustains cancer malignancy.

Insulin inhibits platelet-derived growth factor-induced cell proliferation.

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

Role of a Preorganized Scaffold Presenting Four Residues of a GM-3 Lactone Mimetic on Melanoma Progression.

Two tetravalent architectures, the glycocalix 7 and the RAFT 9, presenting four residues of a GM-3 ganglioside lactone mimetic, target the host compartment of melanoma and significantly abrogate the effect induced by cancer-associated fibroblasts (CAFs) contact + hypoxia in the motility and invasiveness of tumor cells. The data reported support the involvement of glycosphingolipids (GSLs) in hypoxia and show an interesting role played by compound 9 in targeting melanoma cells thereby interfering with melanoma progression. The unprecedented findings reported for the glycocluster 9 may contribute to the understanding of the critical and complex interactions between tumor cells and their local environment paving the way for new therapeutic agents.

The role of Cys-17 in the pyridoxal 5'-phosphate inhibition of the bovine liver low M(r) phosphotyrosine protein phosphatase.

Mammalian tissues contain two low M(r) phosphotyrosine protein phosphatase isoforms (type-1 and type-2) that differ in the 40-73 amino-acid sequence. Only one isoform (type-2) is strongly inhibited by pyridoxal 5'-phosphate, whereas the other is poorly inhibited by this compound. The mechanism of pyridoxal 5'-phosphate inhibition of the bovine liver enzyme (a type-2 isoform) has been studied by kinetic methods using a series of pyridoxal 5'-phosphate analogues. These studies indicate that pyridoxal 5'-phosphate interacts with the enzyme in both the phosphate and aldehyde groups. Active site-directed mutagenesis has been used to investigate the sites of pyridoxal 5'-phosphate binding. Our results indicate that Cys-17, essential for enzyme activity, interacts with the phosphate moiety of pyridoxal 5'-phosphate. On the other hand, Cys-12, which is also involved in the catalytic mechanism, does not participate in pyridoxal 5'-phosphate binding.

Acylphosphatase is involved in differentiation of K562 cells.

The level of both isoforms of acylphosphatase was evaluated in the human erythroleukemia K562 cell line during differentiation. K562 cells were treated with PMA, which induces megakaryocytic differentiation, and with aphidicolin or hemin, which stimulate erythrocytic differentiation. While the MT isoform showed an average 10-fold increase independently of the differentiating agent used, only hemin treatment caused a similar increase of the CT isoform, suggesting a different role of the two isoforms in the cell. Treatment with either hemin or aphidicolin of K562 cells overexpressing the two acylphosphatase isoforms suggested the possibility that acylphosphatases play a role in the onset of differentiation.

In vivo inactivation of phosphotyrosine protein phosphatases by nitric oxide.

The effect of NO on phosphotyrosine protein phosphatases (PTPases) has been investigated in vivo. NO production is induced in interferon-gamma and lipopolysaccharide stimulated RAW-264.7 macrophages as indicated by the increase of NO2- in the medium. Our results demonstrate an inhibition of p-nitrophenylphosphatase activity as a consequence of macrophages activation. Under the described experimental conditions, most of the hydrolysis of p-nitrophenylphosphate can be ascribed to the action of cellular PTPases. The presence of NG-mono-methyl-L-arginine, a specific inhibitor of NO synthase decreases the inactivation rate of both membrane-bound and soluble PTPases. This evidence further confirms the ability of NO to inactivate PTPases and suggests a possible role of NO in the regulation of cellular processes involving this class of phosphatases.

The inhibitory effect of the 5' untranslated region of muscle acylphosphatase mRNA on protein expression is relieved during cell differentiation.

Previous experiments suggested that the upstream AUG triplet present in the 5' untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5'UTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5'UTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5'UTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5'UTR of the muscle acylphosphatase mRNA.

Cloning and expression of the cDNA coding for the erythrocyte isoenzyme of human acylphosphatase.

Three independent cDNAs coding for the erythrocyte isoform of human acylphosphatase were isolated and characterized. All the clones were incomplete at the 5' end, but Northern blot analysis using the cDNA as a probe showed the presence of an unusually long mRNA 5'-untranslated region. The transcript was present in a variety of human cell lines of different origins, although at different levels. Southern blot analysis on DNA from different individuals revealed a simple hybridization pattern. Large amounts of pure enzyme with kinetic characteristics very similar to those of the native protein were expressed in E. coli.

Aspartic-129 is an essential residue in the catalytic mechanism of the low M(r) phosphotyrosine protein phosphatase.

The crystal structure of the bovine liver low M(r) phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by 1H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k2) and dephosphorylation (k3) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.

PDGF receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase.

Low M(r) phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW-PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW-PTP substrate/s we have expressed in NIH-3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW-PTP expressing cells has enabled us to demonstrate an association between LMW-PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW-PTP on the phosphorylated platelet derived growth factor receptor.

The 5'-untranslated region of the human muscle acylphosphatase mRNA has an inhibitory effect on protein expression.

The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5'-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5'-untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase.

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.