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Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance., Competing Interests: Competing interests statement:P.L. is Chief Scientific Officer for AMLo Biosciences Ltd.

Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite clear evidence that ribosome collision occurs stochastically at a cellular and organismal level, physiologically relevant sources of such aberrations are poorly understood. Here we show that a burst of the cellular signaling molecule nitric oxide (NO) reduces translational activity and causes ribosome collision in human cell lines. This is accompanied by activation of the ribotoxic stress response, resulting in ZAKα-mediated activation of p38 and JNK kinases. In addition, NO production is associated with ZNF598-mediated ubiquitination of the ribosomal protein RPS10 and GCN2-mediated activation of the integrated stress response, which are well-described responses to the collision of ribosomes. In sum, our work implicates a novel role of NO as an inducer of ribosome collision and activation of ribosomal surveillance mechanisms in human cells., (© 2023. The Author(s).)

S-nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S-nitrosothiol (SNO) adduct. S-nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S-nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S-nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S-nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S-nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows ( https://github.com/ELELAB/SNO_investigation_pipelines ) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S-nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S-nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field., (© 2023. The Author(s).)

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Angiogenesis is the process of blood vessel growth. The angiogenic switch consists of new blood vessel formation that, in carcinogenesis, can lead to the transition from a harmless cluster of dormant cells to a large tumorigenic mass with metastatic potential. Hypoxia, that is, the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating hypoxia-inducible factor-1 (HIF-1), a transcription factor triggering de novo angiogenesis. HIF-1 and the angiogenic molecules that are expressed upon its activation are modulated by redox status. Modulations of the redox environment can influence the angiogenesis signaling at different levels, thereby impinging on the angiogenic switch. This review provides a molecular overview of the redox-sensitive steps in angiogenic signaling, the main molecular players involved, and their crosstalk with the unfolded protein response. New classes of inhibitors of these modulators which might act as antiangiogenic drugs in cancer are also discussed., (© 2021 Federation of European Biochemical Societies.)

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

One-dimensional nonlinear scattering by a confined distribution of dislocations interacting with point defects distributed along their glide planes or their cores, and of microcracks with or without adhesion between their faces is predicted theoretically. The nonlinear properties of these forms of damage are modeled by macroscopic effective constitutive relationships, and a perturbation approach is used to solve the scattering problem. Backward and forward scattered fields are investigated and use of their main properties towards the development of selective imaging methods which are tailored to hysteretic material damage is briefly discussed. This model is also used to verify recent experimental results on steel samples containing small cracks or confined regions of macrograins. The mechanism associated to hysteresis of large grains is modeled by the interaction between dislocations and point defects along their glide planes. Theoretical predictions confirm that the exponent of a power law, which links the energy dissipated nonlinearly to the energy of a scaled linear field, is sensitive to the physical nature of damage. In turn, experimental findings are shown to provide convincing support to the effective constitutive relationships proposed in this work. Interactions of dislocations with glide and core PDs are characterized by different values of the exponent., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This work examines the linear acoustic behavior of ultrasound contrast agents made of three types of poly (vinyl alcohol) (PVA) shelled microbubbles manufactured at different pH and temperature conditions. Backscattered power, attenuation coefficient and phase velocity of ultrasonic waves propagating through suspensions of PVA contrast agents were measured at temperature values ranging between 24 degrees C and 37 degrees C in a frequency range from 3 MHz to 13 MHz. Enhancement of the backscattered power higher than 20 dB and displaying a weak dependence on temperature was observed. Attenuation and phase velocity, on the other hand, showed higher sensitivity to temperature variations. A modified version of the Church model, which accounts for the dispersion of the dynamic modulus of the PVA shells, was developed to simultaneously fit the attenuation and phase velocity data at 24 degrees C. The frequency dependence of the storage modulus was found to be that of semiflexible polymeric networks. On the other hand, the frequency dependence of the dynamic loss modulus suggests that additional mechanisms, which may be related to the finite dimensions of the shell and/or to its inhomogeneity, may play a significant role in the dissipation of the acoustic energy. For the microbubbles of interest, this model predicts frequency dependent resonance frequency higher than 100 MHz.

Knowledge of the magnitude of the peak negative pressure, P(thr), at which ultrasound contrast agents fracture is relevant for using these microbubbles both as devices for contrast enhancement purposes, as well as carriers of drugs to be delivered locally. In the second part of this communication, the acoustic properties of three types of microbubbles stabilized by poly (vinyl alcohol) (PVA) shells are further investigated. In particular, the dependence of P(thr) on system parameters such as the number of cycles, frequency and exposure is examined. The effects of temperature, blood and, wherever data are available, of the dimension of the microbubbles on P(thr) are also considered. The large shell thickness notwithstanding, the results of this investigation show that at room temperature, PVA contrast agents fracture at negative peak pressure values within the recommended safety limit. Furthermore, P(thr) decreases with increasing temperature, radius of the microbubbles and number of cycles of the incident wave. Fatigue seems to be a physical mechanism playing a dominant role in the fracture process. The effect of blood on P(thr) varies according to condition under which the microbubbles have been synthesized, although stiffening of the shell is observed in most cases. In conclusion, these results suggest that PVA-shelled microbubbles may offer a potentially viable system to be employed for both imaging and therapeutic purposes.

Beyond a characteristic value of the negative peak pressure, ultrasound fracture the shell of ultrasonic contrast agents (UCAs). Existing criteria for ascertaining this threshold value exploit the dependence of the amplitude of the UCA acoustic response on the incident pressure. However, under the common experimental conditions used in this work, these criteria appear to be unreliable when they are applied to UCAs that are stabilized by a thick polymeric shell. An alternative criterion for determining the onset of shell fracture is introduced here, which uses variations of the shape of the acoustic time-domain response of an UCA suspension. Experimental evidence is presented that links the changes of the cross-correlation coefficient between consecutive time-domain signals to the fracture of the shells, and consequent release of air microbubbles. In principle, this criterion may be used to characterize similar properties of other types of particles that cannot undergo inertial cavitation.

The results presented in this communication show that the conventional configuration adopting a shear vertical wave at 45 degrees of incidence to detect cracks breaking the surface opposite the one on which the inspecting transducer is placed is not optimal when the crack depth is of the order of one wavelength of the inspecting wave or smaller. Angles of incidence proximal to the critical angle of longitudinal wave should be used instead.

A theoretical model describing the nonlinear scattering of acoustic waves by surface-breaking cracks with faces in partial contact is presented. The nonlinear properties of the crack are accounted for by suitable boundary conditions that are derived from micromechanical models of the dynamics of elastic rough surfaces in contact. Both linear and nonlinear responses of the crack are shown to be largest for a shear vertical wave incident on the surface containing the crack at an angle just above the critical angle for longitudinal waves. These findings question the fitness for the purpose of a conventional inspection method, which utilizes shear vertical waves at 45 degrees of incidence to search for surface-breaking cracks in many engineering components. For angles of incidence proximal to the critical angle of longitudinal waves, the efficiency of the second harmonic's generation appears to be the highest. Thanks to the increased sensitivity to surface-breaking cracks, this configuration seems to offer a solution to the localization problem, a task that has eluded nonlinear techniques operating under other circumstances. Finally, this model suggests a simple interpretation of the highly localized nonlinear response of delaminations in composite materials.

A theoretical investigation of the nonlinear interaction between an acoustic plane wave and an interface formed by two rough, nonconforming surfaces in partial contact is presented. The macroscopic elastic properties of such a nonlinear interface are derived from micromechanical models accounting for the elastic interaction that is characteristic of spherical bodies in contact. These results are used to formulate set of boundary conditions for the acoustic field, which are to be enforced at the imperfect interface. The scattering problem is solved for plane wave incidence by using a simple perturbation approach and the harmonic balance method. Sample results are presented for arbitrary wave polarization and angle of incidence. The relative magnitude of the nonlinear signals and their potential use toward the nondestructive evaluation of imperfect interfaces are assessed. In particular, attention is drawn to the enhanced nonlinear response of an interface insonified by a shear vertical wave in the neighborhood of the longitudinal critical angle. The motivation for this investigation is provided by the need to develop nondestructive methods to detect and localize small, partially closed cracks in metals with coarse microstructures.

The scattering-induced attenuation coefficient of a beam of longitudinal waves propagating through an austenitic steel plate is measured as a function of the texture angle. The experimental data were obtained by mapping the incident and the transmitted ultrasonic field, and by evaluating the energy loss experienced by each plane wave component of the beam. Contrary to the behavior of data obtained by means of conventional techniques, that of the data reported in this work agrees qualitatively with the theoretical predictions. The reasons for the disagreement between theory and data obtained by means of conventional techniques are also discussed.

Heuristic in nature, the quasistatic approximation (QSA) describes the interaction of ultrasonic waves with imperfect interfaces by modeling the interfacial imperfection as distributions of springs and masses. The QSA does not provide any relationship between the interfacial stiffness constants and the micromechanics of the defects. The aims of this paper are threefold. First, a derivation from first principles of the QSA boundary conditions on a cracked interface is presented. Relationships linking the interfacial constants to the mechanical and geometrical properties of the distributed cracks are also obtained. Second, the stiffness dependence of a cracked interface between a layer and a substrate on the layer thickness is investigated. It is shown that the interfacial stiffnesses cannot be regarded as intrinsic properties of the interface, but they may also depend on the structural properties of the hosting system. Finally, the effect of the thickness dependence of the interfacial stiffnesses on the phase velocity of the lowest mode supported by the layered structure is investigated.