Tommaso Galeotti

Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion

Signal transduction by reactive oxygen species (ROS; “redox signaling”) has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. A variety of mitogenic signals, including those released by receptor tyrosine kinase (RTKs) ligands and oncogenic H-Ras, involve as a critical downstream event the intracellular generation of ROS. Signaling by integrins is also essential for the growth of most cell types and is constantly integrated with growth factor signaling. We provide here evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during fibroblast adhesion and spreading. Moreover, we propose a synergistic action of integrins and RTKs for redox signaling. Integrin-induced ROS are required to oxidize/inhibit the low molecular weight phosphotyrosine phosphatase, thereby preventing the enzyme from dephosphorylating and inactivating FAK. Accordingly, FAK phosphorylation and other downstream events, including MAPK phosphorylation, Src phosphorylation, focal adhesion formation, and cell spreading, are all significantly attenuated by inhibition of redox signaling. Hence, we have outlined a redox circuitry whereby, upon cell adhesion, oxidative inhibition of a protein tyrosine phosphatase promotes the phosphorylation/activation and the downstream signaling of FAK and, as a final event, cell adhesion and spreading onto fibronectin.

Metastasis: cancer cell's escape from oxidative stress.

According to a “canonical” view, reactive oxygen species (ROS) positively contribute, in different ways, to carcinogenesis and to malignant progression of tumor cells: they drive genomic damage and genetic instability, transduce, as signaling intermediates, mitogenic and survival inputs by growth factor receptors and adhesion molecules, promote cell motility and shape the tumor microenvironment by inducing inflammation/repair and angiogenesis. Chemopreventive and tumor-inhibitory effects of endogenous, diet-derived or supplemented antioxidants largely support this notion. However, emerging lines of evidence indicates that tumor cells also need to defend themselves from oxidative damage in order to survive and successfully spread at distance. This “heresy” has recently received important impulse from studies on the role of antioxidant capacity in cancer stem cells self-renewal and resistance to therapy; additionally, the transforming activity of some oncogenes has been unexpectedly linked to their capacity to maintain elevated intracellular levels of reduced glutathione (GSH), the principal redox buffer. These studies underline the importance of cellular antioxidant capacity in metastasis, as the result of a complex cell program involving enhanced motility and a profound change in energy metabolism. The glycolytic switch (Warburg effect) observed in malignant tissues is triggered by mitochondrial oxidative damage and/or activation of redox-sensitive transcription factors, and results in an increase of cell resistance to oxidants. On the other hand, cytoskeleton rearrangement underlying cell motile and tumor-aggressive behavior use ROS as intermediates and are therefore facilitated by oxidative stress. Along this line of speculation, we suggest that metastasis represents an integrated strategy for cancer cells to avoid oxidative damage and escape excess ROS in the primary tumor site, explaning why redox signaling pathways are often up-regulated in malignancy and metastasis.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS)

Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.

Growth-related lipid peroxidation in tumour microsomal membranes and mitochondria

Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPH-dependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the O2- and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD+-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.

Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lepOb/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lepOb/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.

Mitochondrial superoxide dismutase: A promising target for new anticancer therapies

Compelling experimental and epidemiological evidence involves oxygen radicals in carcinogenesis, acting reactive oxygen species both as endogenous genotoxins during cell initiation and as messenger molecules in mitogenesis and in tumor promotion. Moreover, oxidants stimulate neoangiogenesis, which is a prerequisite for tumor growth. However, while several natural as well as synthetic antioxidant compounds appear to be chemopreventive in mutagenicity assays, antioxidant-based treatments for the prevention or cure of cancer have led to non-conclusive if not disappointing results. This is likely due to the fact that oxygen radicals have also a major role in the natural defences against the propagation of cancer cells, i.e. tumor cell apoptosis and immune surveillance, and mediate the beneficial cytotoxic effect of both the chemo-and radio-therapy. In recent years, the mitochondrial antioxidant enzyme, Manganous Superoxide Dismutase (MnSOD), has received a growing attention as a negative modulator of cellular apoptosis and as a survival factor for cancer cells. In fact, while overexpression of this enzyme in cancer cells decreases proliferation and tumor incidence in transgenic models, it is clear that even small amounts of this enzyme are crucial for cell resistance to inflammatory stimuli and anticancer drugs, and prevent oncogene-induced apoptosis triggered by the tumor suppressor protein p53. A previously unexpected oncogenic potential of MnSOD is also suggested by the elevated levels of this enzyme in several classes of human neoplasms, in a fashion which often correlates with the degree of their malignancy. This review focuses on the debated issue of the pro- and/or anti-tumoral effect of MnSOD, with special emphasis on recent observations suggesting that pharmacological inhibition of MnSOD may represent an effective strategy to selectively kill cancer cells and to circumvent their resistance to the commonly used anticancer treatments.

Superoxide dismutase content and microsomal lipid composition of tumours with different growth rates

The content of cytosolic superoxide dismutase has been determined in Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and in ascites tumour cells (Novikoff hepatoma and Ehrlich-Lettré). The enzyme is decreased in all the tumours examined. The lowest amounts were found in the tumours with the fastest growth rates. Measurements of the lipid composition and fluidity of microsomal membranes isolated from Morris hepatomas show that also these parameters are changed in relation to the growth rate. The lipid to protein ratio and the degree of fatty acid unsaturation decrease gradually from rat liver to hepatoma 44 and 3924A microsomes. The different lipid composition is reflected also by differences in the physical environment of the bilayer, as indicated by data obtained with spin-labeled fatty acids. It is proposed that the changes in the membrane lipid composition and organization are consequent to the decrease in the protective effect of cytosolic superoxide dismutase against the O2- induced lipid peroxidation.

Defective gene expression of MnSOD in cancer cells

Reactive oxygen species (ROS) have been frequently implicated in the initiation and promotion phases of carcinogenesis. Antioxidant enzymes, which can antagonize this process, are lowered in a number of malignancies. The enzyme most commonly decreased is the mitochondrial Mn-containing superoxide dismutase (MnSOD) encoded by a nuclear gene mapped on the band 6q21, a region frequently deleted in several human tumours. The close association of del(6q) with diminution of MnSOD has led to suggest that MnSOD might be a new type of tumour-suppressor gene. This hypothesis is also sustained by the finding that transfection of MnSOD cDNA into human melanoma cell lines suppress the malignant phenotype. There are, however, conflicting observations that tend to ascribe the deficiency of the MnSOD activity more to a defect in the expression of the gene than to its deletion. In many transformed cell lines, including some with marked del(6q), there is no change in the dosage of the MnSOD gene and the enzyme is highly inducible by various pro-oxidant agents. Transition metals (Mn, Fe) have been found to be highly deficient in human and rodent tumours. Owing to the second messenger function of ROS in activating transcription factors (NF-kB, AP-1) and to the ability of Mn to facilitate the dismutation of O2- to H2O2 and of Fe to participate in the Fenton reaction, we propose that in the early stage of carcinogenesis an impairment of the signal transduction machinery, related to the metal deficiency, might limit the binding to DNA of transcription factors and cause the defect in the MnSOD gene expression.

Lipid peroxidation and fluidity of plasma membranes from rat liver and Morris hepatoma 3924A

Plasma membranes isolated from the fast‐growing, maximal‐deviation, Morris hepatoma 3924A exhibit remarkable changes in lipid composition, lipid peroxidation and to some extent in the physical state with respect to rat liver plasmalemmas. A correlation appears to exit between the lower phospholipid: protein ratio, higher cholesterol: phospholipid ratio, lower rate of lipid peroxidation and decrease in fluidity in tumor plasma membranes.