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Cancer cells rely on invasive growth to survive in a hostile microenvironment; this growth is characterised by interconnected processes such as epithelial-to-mesenchymal transition and migration. A master regulator of these events is the MET oncogene, which is overexpressed in the majority of cancers; however, since mutations in the MET oncogene are seen only rarely in cancers and are relatively infrequent, the mechanisms that cause this widespread MET overexpression remain obscure. Here, we show that the 5' untranslated region (5'UTR) of MET mRNA harbours two functional stress-responsive elements, conferring translational regulation by the integrated stress response (ISR), regulated by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) at serine 52. ISR activation by serum starvation, leucine deprivation, hypoxia, irradiation, thapsigargin or gemcitabine is followed by MET protein overexpression. We mechanistically link MET translation to the ISR by (i) mutation of the two uORFs within the MET 5'UTR, (ii) CRISPR/Cas9-mediated mutation of eIF2α (S52A), or (iii) the application of ISR pathway inhibitors. All of these interventions reduce stress-induced MET overexpression. Finally, we show that blocking stress-induced MET translation blunts MET-dependent invasive growth. These findings indicate that upregulation of the MET oncogene is a functional requirement linking integrated stress response to cancer progression., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Targeting nuclear mechanics is emerging as a promising therapeutic strategy for sensitizing cancer cells to immunotherapy. Inhibition of the mechano-sensory kinase ATR leads to mechanical vulnerability of cancer cells, causing nuclear envelope softness and collapse and activation of the cGAS-STING-mediated innate immune response. Finding novel compounds that interfere with the non-canonical role of ATR in controlling nuclear mechanics presents an intriguing therapeutic opportunity. We carried out a multiparametric high-content screen to identify small molecules that affect nuclear envelope shape and to uncover novel players that could either ameliorate or further compromise the nuclear mechanical abnormalities of ATR-defective cells. The screen was performed in HeLa cells genetically depleted for ATR. Candidate hits were also tested in combination with the chemical inhibition of ATR by AZD6738, and their efficacy was further validated in the triple-negative breast cancer cell lines BT549 and HCC1937. We show that those compounds enhancing the abnormal nuclear shape of ATR-defective cells also synergize with AZD6738 to boost the expression of interferon-stimulated genes, highlighting the power of multiparametric screens to identify novel combined therapeutic interventions targeting nuclear mechanics for cancer immunotherapy., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors., Competing Interests: Competing interests statement:M.P. is of the board of Directors and stakeholder of CheckmAb s.r.l. and is a recipient of grants under a research agreement with Bristol-Myers Squibb and Macomics.

Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1 ATR DNA damage response (DDR). We study DDR and NER in aging cells and find that old cells struggle to repair DNA and activate Mec1 ATR . We employ pharmacological and genetic approaches to rescue DDR and NER during aging. Conditions activating Snf1 AMPK rescue DDR functionality, but not NER, while inhibition of the TORC1-Sch9 S6K axis restores NER and enhances DDR by tuning PP2A activity, specifically in aging cells. Age-related repair deficiency depends on Snf1 AMPK -mediated phosphorylation of Sch9 S6K on Ser160 and Ser163. PP2A activity in old cells is detrimental for DDR and influences NER by modulating Snf1 AMPK and Sch9 S6K . Hence, the DDR and repair pathways in aging cells are influenced by the metabolic tuning of opposing AMPK and TORC1 networks and by PP2A activity. Specific Sch9 S6K phospho-isoforms control DDR and NER efficiency, specifically during aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3 hPif1 and Sen1 hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Combined inhibition of oxidative phosphorylation (OXPHOS) and glycolysis has been shown to activate a PP2A-dependent signaling pathway, leading to tumor cell death. Here, we analyze highly selective mitochondrial complex I or III inhibitors in vitro and in vivo to elucidate the molecular mechanisms leading to cell death following OXPHOS inhibition. We show that IACS-010759 treatment (complex I inhibitor) induces a reactive oxygen species (ROS)-dependent dissociation of CIP2A from PP2A, leading to its destabilization and degradation through chaperone-mediated autophagy. Mitochondrial complex III inhibition has analogous effects. We establish that activation of the PP2A holoenzyme containing B56δ regulatory subunit selectively mediates tumor cell death, while the arrest in proliferation that is observed upon IACS-010759 treatment does not depend on the PP2A-B56δ complex. These studies provide a molecular characterization of the events subsequent to the alteration of critical bioenergetic pathways and help to refine clinical studies aimed to exploit metabolic vulnerabilities of tumor cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

Chromatin metabolism is frequently altered in cancer cells and facilitates cancer development. While cancer cells produce large amounts of histones, the protein component of chromatin packaging, during replication, the potential impact of histone density on cancer biology has not been studied systematically. Here, we show that altered histone density affects global histone acetylation, histone deactylase inhibitor sensitivity and altered mitochondrial proteome composition. We present estimates of nuclear histone densities in 373 cancer cell lines, based on Cancer Cell Line Encyclopedia data, and we show that a known histone regulator, HMGB1, is linked to histone density aberrations in many cancer cell lines. We further identify an E3 ubiquitin ligase interactor, DCAF6, and a mitochondrial respiratory chain assembly factor, CHCHD4, as histone modulators. As systematic characterization of histone density aberrations in cancer cell lines, this study provides approaches and resources to investigate the impact of histone density on cancer biology., (© 2022. The Author(s).)

Ageing is intimately connected to the induction of cell senescence 1,2 , but why this is so remains poorly understood. A key challenge is the identification of pathways that normally suppress senescence, are lost during ageing and are functionally relevant to oppose ageing 3 . Here we connected the structural and functional decline of ageing tissues to attenuated function of the master effectors of cellular mechanosignalling YAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation of YAP/TAZ in these cells leads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation of YAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent that inhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signalling relies on the unexpected role of YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which is involved in building the peri-nuclear actin cap. The findings demonstrate that declining YAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STING signalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limiting senescence-associated inflammation and improving healthy ageing., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterized by impaired left ventricular (LV) diastolic function, with normal LV ejection fraction. Aortic valve stenosis can cause an HFpEF-like syndrome by inducing sustained pressure overload (PO) and cardiac remodeling, as cardiomyocyte (CM) hypertrophy and fibrotic matrix deposition. Recently, in vivo studies linked PO maladaptive myocardial changes and DNA damage response (DDR) activation: DDR-persistent activation contributes to mouse CM hypertrophy and inflammation, promoting tissue remodeling, and HF. Despite the wide acknowledgment of the pivotal role of the stromal compartment in the fibrotic response to PO, the possible effects of DDR-persistent activation in cardiac stromal cell (C-MSC) are still unknown. Finally, this novel mechanism was not verified in human samples. This study aims to unravel the effects of PO-induced DDR on human C-MSC phenotypes. Human LV septum samples collected from severe aortic stenosis with HFpEF-like syndrome patients undergoing aortic valve surgery and healthy controls (HCs) were used both for histological tissue analyses and C-MSC isolation. PO-induced mechanical stimuli were simulated in vitro by cyclic unidirectional stretch. Interestingly, HFpEF tissue samples revealed DNA damage both in CM and C-MSC. DDR-activation markers γH2AX, pCHK1, and pCHK2 were expressed at higher levels in HFpEF total tissue than in HC. Primary C-MSC isolated from HFpEF and HC subjects and expanded in vitro confirmed the increased γH2AX and phosphorylated checkpoint protein expression, suggesting a persistent DDR response, in parallel with a higher expression of pro-fibrotic and pro-inflammatory factors respect to HC cells, hinting to a DDR-driven remodeling of HFpEF C-MSC. Pressure overload was simulated in vitro , and persistent activation of the CHK1 axis was induced in response to in vitro mechanical stretching, which also increased C-MSC secreted pro-inflammatory and pro-fibrotic molecules. Finally, fibrosis markers were reverted by the treatment with a CHK1/ATR pathway inhibitor, confirming a cause-effect relationship. In conclusion we demonstrated that, in severe aortic stenosis with HFpEF-like syndrome patients, PO induces DDR-persistent activation not only in CM but also in C-MSC. In C-MSC, DDR activation leads to inflammation and fibrosis, which can be prevented by specific DDR targeting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stadiotti, Santoro, Scopece, Pirola, Guarino, Polvani, Maione, Ascione, Li, Delia, Foiani, Pompilio and Sommariva.)

Mechanosignaling, initiated by extracellular forces and propagated through the intracellular cytoskeletal network, triggers signaling cascades employed in processes as embryogenesis, tissue maintenance and disease development. While signal transduction by transcription factors occurs downstream of cellular mechanosensing, little is known about the cell intrinsic mechanisms that can regulate mechanosignaling. Here we show that transcription factor PREP1 (PKNOX1) regulates the stiffness of the nucleus, the expression of LINC complex proteins and mechanotransduction of YAP-TAZ. PREP1 depletion upsets the nuclear membrane protein stoichiometry and renders nuclei soft. Intriguingly, these cells display fortified actomyosin network with bigger focal adhesion complexes resulting in greater traction forces at the substratum. Despite the high traction, YAP-TAZ translocation is impaired indicating disrupted mechanotransduction. Our data demonstrate mechanosignaling upstream of YAP-TAZ and suggest the existence of a transcriptional mechanism actively regulating nuclear membrane homeostasis and signal transduction through the active engagement/disengagement of the cell from the extracellular matrix., (© 2022. The Author(s).)

Several cytotoxic agents used in cancer therapy cause DNA damage and replication stress. Understanding the metabolic determinants of the cell response to replication stress-inducing agents could have relevant implications for cancer treatment. In a recent study, we showed that cell survival during replication stress is influenced by the availability of amino acids, as well as by TORC1 and Gcn2-mediated amino acid sensing pathways. Amino acid starvation, or TORC1 inhibition, sensitizes cells to replication stress conditions, whereas Gcn2 ablation promotes cell survival by stimulating protein synthesis. The Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG phosphatidylinositol-3-phosphate (PtdIns3P) complex at the endosomes sets the balance between survival and death signals during replication stress and amino acid starvation. The Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG axis promotes the degradation of amino acid transporters, thus sensitizing cells to amino acid starvation, while Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG inactivation promotes cell survival by enabling synthesis of stress response proteins mediating survival under replication stress conditions. Our study unravels an autophagy-independent mechanism through which Vps34-Vps30/Atg6/BECN1 promotes lethal events during replication stress.

Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)