AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

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.

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress.

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.

TRAP1 S-nitrosylation as a model of population-shift mechanism to study the effects of nitric oxide on redox-sensitive oncoproteins.

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.

Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms.

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.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation.

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.

Regulation of redox signaling in HIF-1-dependent tumor angiogenesis.

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.

S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli.

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.