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Through genetically engineered mouse models of melanoma, we identified Autophagy/beclin 1 regulator 1 (Ambra1) as novel tumor-suppressor in melanoma. In these settings, loss of Ambra1 associated with the hyperactivation of focal adhesion kinase 1 (Fak1) signaling, the inhibition of which resulted in reduced tumor growth and invasiveness. We therefore propose FAK1 inhibition for current melanoma therapy in AMBRA1-low tumors. AKT, serine/threonine kinase 1; AMBRA1, autophagy/beclin 1 regulator 1; BRAF, v-raf murine sarcoma viral oncogene homolog; BRAFi, BRAF inhibitor; CCLE, Cancer Cell Line Encyclopedia;g ESTDAB, European Searchable Tumor Line Database; FAK1, focal adhesion kinase 1; FAKi, FAK1 inhibitor; LMC, Leeds Melanoma Cohort; MEK, MAPK/ERK kinase; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homolog; TCGA-SKCM, The Cancer Genome Atlas - Skin Cutaneous Melanoma; YAP, yes-associated protein 1. [ABSTRACT FROM AUTHOR]

The establishment of neuronal polarity and axonal outgrowth are key processes affecting neuronal migration and synapse formation, their impairment likely leading to cognitive deficits. Here we have found that the apoptotic protease activating factor 1 (Apaf1), apart from its canonical role in apoptosis, plays an additional function in cortical neurons, where its deficiency specifically impairs axonal growth. Given the central role played by centrosomes and microtubules in the polarized extension of the axon, our data suggest that Apaf1-deletion affects axonal outgrowth through an impairment of centrosome organization. In line with this, centrosomal protein expression, as well as their centrosomal localization proved to be altered upon Apaf1-deletion. Strikingly, we also found that Apaf1-loss affects trans-Golgi components and leads to a robust activation of AMP-dependent protein kinase (AMPK), this confirming the stressful conditions induced by Apaf1-deficiency. Since AMPK hyper-phosphorylation is known to impair a proper axon elongation, our finding contributes to explain the effect of Apaf1-deficiency on axogenesis. We also discovered that the signaling pathways mediating axonal growth and involving glycogen synthase kinase-3β, liver kinase B1, and collapsing-response mediator protein-2 are altered in Apaf1-KO neurons. Overall, our results reveal a novel non-apoptotic role for Apaf1 in axonal outgrowth, suggesting that the neuronal phenotype due to Apaf1-deletion could not only be fully ascribed to apoptosis inhibition, but might also be the result of defects in axogenesis. The discovery of new molecules involved in axonal elongation has a clinical relevance since it might help to explain neurological abnormalities occurring during early brain development. [ABSTRACT FROM AUTHOR]

The activating molecule in Beclin-1-regulated autophagy (Ambra1), also known as autophagy/Beclin-1 regulator 1, is a highly intrinsically disordered and vertebrate-conserved adapter protein that is part of the autophagy signaling network. It acts in an early step of mammalian target of rapamycin complex 1 (mTORC1)-dependent autophagy by favouring formation of the autophagosome core complex. However, recent studies have revealed that Ambra1 can also coordinate a cell response upon starvation or other stresses that involve translocation of the autophagosome core complex to the endoplasmic reticulum (ER), regulative ubiquitylation and stabilization of the kinase ULK1, selective mitochondria removal and cell cycle downregulation. Moreover, Ambra1 itself appears to be targeted by a number of regulatory processes, such as cullin-dependent degradation, caspase cleavage and several modifications, ranging from phosphorylation to ubiquitylation. Altogether, this complex network of regulation highlights the importance of Ambra1 in crucial physiological events, including metabolism, cell death and cell division. In addition, Ambra1 is an important regulator of embryonic development, and its mutation or inactivation has been shown to correlate with several pathologies of the nervous system and to be involved in carcinogenesis. In this Cell Science at a Glance article and the accompanying poster, we discuss recent advances in the Ambra1 field, particularly the role of this proautophagic protein in cellular pathophysiology. [ABSTRACT FROM AUTHOR]

Significance: When lesions are unrepaired or there are defects in the DNA repair system, DNA damage is often correlated to apoptosis. However, different kinds of lesions and different degrees of lesion severity can trigger numerous signaling responses. Recent Advances: DNA repair proteins involved in specific DNA repair pathways can modulate the function or activity of some apoptotic factors, further emphasizing the crosstalk between DNA damage and cell death. Critical Issues: Here, we discuss the signaling networks that link DNA damage to apoptosis, and we focus on post-translational modifications, leading to crucial changes in protein behavior, following various kinds of DNA damage. Moreover, we analyze the existence of apoptosis-related functions of typical repair proteins, leading to diverse, often-overlapping, DNA damage responses. Future Directions: The better understanding of the regulation and the functionality of key DNA repair proteins, also involved in apoptosis regulation, has the potential of modulating the cell outcomes on DNA damage, particularly in the context of cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Late-stage melanoma is one of the leading causes of death from skin cancers, as it frequently acquires resistance to standard therapies. Melanoma aggressiveness relies both on high intratumoral heterogeneity and on the capability of melanoma cells to switch among different differentiation phenotypes. Recently, melanoma plasticity has indeed been pinpointed as a main cause of resistance to standard therapies. Melanoma takes advantage of extrinsic reprogramming, a common feature exploited also by melanocytes, to promote tumor progression. Unfortunately, extrinsic factors and molecular mechanisms driving phenotype switching upon treatment are yet to be thoroughly characterized. The aim of this review is to support this field of research by providing brand-new insights into melanoma plasticity. Starting from the origin of phenotype switching, we will report up-to-date molecular players and extrinsic factors determining different transcriptional programs. Finally, the latest therapeutic strategies to tackle this mechanism of resistance will be discussed. Melanoma is considered one of the deadliest skin cancers, partly because of acquired resistance to standard therapies. The most recognized driver of resistance relies on acquired melanoma cell plasticity, or the ability to dynamically switch among differentiation phenotypes. This confers the tumor noticeable advantages. During the last year, two new features have been included in the hallmarks of cancer, namely "Unlocking phenotypic plasticity" and "Non-mutational epigenetic reprogramming". Such are inextricably intertwined as, most of the time, plasticity is not discernable at the genetic level, as it rather consists of epigenetic reprogramming heavily influenced by external factors. By analyzing current literature, this review provides reasoning about the origin of plasticity and clarifies whether such features already exist among tumors or are acquired by selection. Moreover, markers of plasticity, molecular effectors, and related tumor advantages in melanoma will be explored. Ultimately, as this new branch of tumor biology opened a wide landscape of therapeutic possibilities, in the final paragraph of this review, we will focus on newly characterized drugs targeting melanoma plasticity. [ABSTRACT FROM AUTHOR]

Oxidative DNA damage is produced by reactive oxygen species (ROS) which are generated by exogenous and endogenous sources and continuously challenge the cell. One of the most severe DNA lesions is the double-strand break (DSB), which is mainly repaired by nonhomologous end joining (NHEJ) pathway in mammals. NHEJ directly joins the broken ends, without using the homologous template. Ku70/86 heterodimer, also known as Ku, is the first component of NHEJ as it directly binds DNA and recruits other NHEJ factors to promote the repair of the broken ends. Neurons are particularly metabolically active, displaying high rates of transcription and translation, which are associated with high metabolic and mitochondrial activity as well as oxygen consumption. In such a way, excessive oxygen radicals can be generated and constantly attack DNA, thereby producing several lesions. This condition, together with defective DNA repair systems, can lead to a high accumulation of DNA damage resulting in neurodegenerative processes and defects in neurodevelopment. In light of recent findings, in this paper, we will discuss the possible implication of Ku in neurodevelopment and in mediating the DNA repair dysfunction observed in certain neurodegenerations. [ABSTRACT FROM AUTHOR]

Abstract: Programmed cell death is an orchestrated form of cell death in which cells are actively involved in their own demise. During neural development in mammals, many progenitor cells, immature cells or differentiated cells undergo the most clearly characterized type of cell death, apoptosis. Several pathways of apoptosis have been linked to neural development, but according to the numerous and striking phenotypes observed when apoptotic genes are inactivated, the mitochondrial death-route is the most important pathway in this context. Here, we discuss the relative importance of pro-growth/pro-death factors in the control of neural tissue development. We also discuss the impact of studying programmed cell death in development in order to better understand the basis of several human diseases and embryonic defects of the nervous system. [Copyright &y& Elsevier]

Mouse tumour models are extensively used as a pre-clinical research tool in the field of oncology, playing an important role in anticancer drugs discovery. Accordingly, in cancer genomics research, the demand for next-generation sequencing (NGS) is increasing, and consequently, the need for data analysis pipelines is likewise growing. Most NGS data analysis solutions to date do not support mouse data or require highly specific configuration for their use. Here, we present a genome analysis pipeline for mouse tumour NGS data including the whole-genome sequence (WGS) data analysis flow for somatic variant discovery, and the RNA-seq data flow for differential expression, functional analysis and neoantigen prediction. The pipeline is based on standards and best practices and integrates mouse genome references and annotations. In a recent study, the pipeline was applied to demonstrate the efficacy of low dose 6-thioguanine (6TG) treatment on low-mutation melanoma in a pre-clinical mouse model. Here, we further this study and describe in detail the pipeline and the results obtained in terms of tumour mutational burden (TMB) and number of predicted neoantigens, and correlate these with 6TG effects on tumour volume. Our pipeline was expanded to include a neoantigen analysis, resulting in neopeptide prediction and MHC class I antigen presentation evaluation. We observed that the number of predicted neoepitopes were more accurate indicators of tumour immune control than TMB. In conclusion, this study demonstrates the usability of the proposed pipeline, and suggests it could be an essential robust genome analysis platform for future mouse genomic analysis. [ABSTRACT FROM AUTHOR]

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis. [ABSTRACT FROM AUTHOR]

Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284). [ABSTRACT FROM AUTHOR]

Abstract: This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson''s disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H2O2, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson''s disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity. [Copyright &y& Elsevier]

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redoxbased perspective on aging with direct therapeutic implications. [ABSTRACT FROM AUTHOR]

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene. [ABSTRACT FROM AUTHOR]

Aims: Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. Results: We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. Innovation: Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. Conclusion: These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer. Antioxid. Redox Signal. 21, 570-587. [ABSTRACT FROM AUTHOR]

Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimer's disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimer's disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3-dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimer's disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages. [ABSTRACT FROM AUTHOR]

The downregulation of the denitrosylating enzyme S- nitrosoglutathione reductase (GSNOR, EC:1.1.1.284), is a feature of hepatocellular carcinoma (HCC). This condition causes mitochondrial rearrangements that sensitize these tumors to mitochondrial toxins, in particular to the mitochondrial complex II inhibitor alpha-tocopheryl succinate (αTOS). It has also been reported the GSNOR depletion impairs the selective degradation of mitochondria through mitophagy; however, if this contributes to GSNOR-deficient HCC cell sensitivity to αTOS and can be applied to anticancer therapies, is still not known. Here, we provide evidence that GSNOR-deficient HCC cells show defective mitophagy which contributes to αTOS toxicity. Mitophagy inhibition by Parkin (EC: 2.3.2.31) depletion enhances αTOS anticancer effects, thus suggesting that this drug could be effective in treating mitophagy-defective tumors. [ABSTRACT FROM AUTHOR]

A correction to the article "AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation" that was published in the 2015 issue is presented.