Alternative lengthening of telomeres (ALT) cells viability is dependent on C-rich telomeric RNAs.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.

Clonal cooperation through soluble metabolite exchange facilitates metastatic outgrowth by modulating Allee effect.

Cancers feature substantial intratumoral heterogeneity of genetic and phenotypically distinct lineages. Although interactions between coexisting lineages are emerging as a potential contributor to tumor evolution, the extent and nature of these interactions remain largely unknown. We postulated that tumors develop ecological interactions that sustain diversity and facilitate metastasis. Using a combination of fluorescent barcoding, mathematical modeling, metabolic analysis, and in vivo models, we show that the Allee effect, i.e., growth dependency on population size, is a feature of tumor lineages and that cooperative ecological interactions between lineages alleviate the Allee barriers to growth in a model of triple-negative breast cancer. Soluble metabolite exchange formed the basis for these cooperative interactions and catalyzed the establishment of a polyclonal community that displayed enhanced metastatic dissemination and outgrowth in xenograft models. Our results highlight interclonal metabolite exchange as a key modulator of tumor ecology and a contributing factor to overcoming Allee effect-associated growth barriers to metastasis.

A planar polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia.

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.

A planar-polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia.

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.

Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

PillarX: A Microfluidic Device to Profile Circulating Tumor Cell Clusters Based on Geometry, Deformability, and Epithelial State.

Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.

Low Tumor-to-Stroma Ratio Reflects Protective Role of Stroma against Prostate Cancer Progression.

Tumor-to-stroma ratio (TSR) is a prognostic factor that expresses the relative amounts of tumor and intratumoral stroma. In this study, its clinical and molecular relevance was evaluated in prostate cancer (PCa). The feasibility of automated quantification was tested in digital scans of tissue microarrays containing 128 primary tumors from 72 PCa patients stained immunohistochemically for epithelial cell adhesion molecule (EpCAM), followed by validation in a cohort of 310 primary tumors from 209 PCa patients. In order to investigate the gene expression differences between tumors with low and high TSR, we applied multigene expression analysis (nCounter® PanCancer Progression Panel, NanoString) of 42 tissue samples. TSR scores were categorized into low (<1 TSR) and high (≥1 TSR). In the pilot cohort, 31 patients (43.1%) were categorized as low and 41 (56.9%) as high TSR score, whereas 48 (23.0%) patients from the validation cohort were classified as low TSR and 161 (77.0%) as high. In both cohorts, high TSR appeared to indicate the shorter time to biochemical recurrence in PCa patients (Log-rank test, p = 0.04 and p = 0.01 for the pilot and validation cohort, respectively). Additionally, in the multivariate analysis of the validation cohort, TSR predicted BR independent of other factors, i.e., pT, pN, and age (p = 0.04, HR 2.75, 95%CI 1.07-7.03). Our data revealed that tumors categorized into low and high TSR score show differential expression of various genes; the genes upregulated in tumors with low TSR score were mostly associated with extracellular matrix and cell adhesion regulation. Taken together, this study shows that high stroma content can play a protective role in PCa. Automatic EpCAM-based quantification of TSR might improve prognostication in personalized medicine for PCa.

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles.

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

Numerical Evaluation of Human Body Near Field Exposure to a Vehicular Antenna for Military Applications.

BACKGROUND: The use of electromagnetic (EM) technologies for military applications is gaining increasing interest to satisfy different operational needs, such as improving battlefield communications or jamming counterpart's signals. This is achieved by the use of high-power EM waves in several frequency bands (e.g., HF, VHF, and UHF). When considering military vehicles, several antennas are present in close proximity to the crew personnel, which are thus potentially exposed to high EM fields. METHODS: A typical exposure scenario was reproduced numerically to evaluate the EM exposure of the human body in the presence of an HF vehicular antenna (2-30 MHz). The antenna was modeled as a monopole connected to a 3D polygonal structure representing the vehicle. Both the EM field levels in the absence and in the presence of the human body and also the specific absorption rate (SAR) values were calculated. The presence of the operator, partially standing outside the vehicle, was simulated with the virtual human body model Duke (Virtual Population, V.3). Several exposure scenarios were considered. The presence of a protective helmet was modeled as well. RESULTS: In the area usually occupied by the personnel, E-field intensity radiated by the antenna can reach values above the limits settled by international safety guidelines. Nevertheless, local SAR values induced inside the human body reached a maximum value of 14 mW/kg, leading to whole-body averaged and 10-g averaged SAR values well below the corresponding limits. CONCLUSION: A complex and realistic near-field exposure scenario of the crew of a military vehicle was simulated. The obtained E-field values radiated in the free space by a HF vehicular antenna may reach values above the safety guidelines reference levels. Such values are not necessarily meaningful for the exposed subject. Indeed, SAR and E-field values induced inside the body remain well below safety limits.

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

RATIONALE: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. OBJECTIVE: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. METHODS AND RESULTS: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of ß-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. CONCLUSIONS: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle.

Mitochondria change distribution across cells following a variety of pathophysiological stimuli. The mechanisms presiding over this redistribution are yet undefined. In a murine model overexpressing Drp1 specifically in skeletal muscle, we find marked mitochondria repositioning in muscle fibres and we demonstrate that Drp1 is involved in this process. Drp1 binds KLC1 and enhances microtubule-dependent transport of mitochondria. Drp1-KLC1 coupling triggers the displacement of KIF5B from kinesin-1 complex increasing its binding to microtubule tracks and mitochondrial transport. High levels of Drp1 exacerbate this mechanism leading to the repositioning of mitochondria closer to nuclei. The reduction of Drp1 levels decreases kinesin-1 activation and induces the partial recovery of mitochondrial distribution. Drp1 overexpression is also associated with higher cyclin-dependent kinase-1 (Cdk-1) activation that promotes the persistent phosphorylation of desmin at Ser-31 and its disassembling. Fission inhibition has a positive effect on desmin Ser-31 phosphorylation, regardless of Cdk-1 activation, suggesting that induction of both fission and Cdk-1 are required for desmin collapse. This altered desmin architecture impairs mechanotransduction and compromises mitochondrial network stability priming mitochondria transport through microtubule-dependent trafficking with a mechanism that involves the Drp1-dependent regulation of kinesin-1 complex.

Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma.

Inhibition of Cyclin-Dependent Kinase 5: A Strategy to Improve Sorafenib Response in Hepatocellular Carcinoma Therapy.

Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.

Reticulon 3-dependent ER-PM contact sites control EGFR nonclathrin endocytosis.

The integration of endocytic routes is critical to regulate receptor signaling. A nonclathrin endocytic (NCE) pathway of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations and targets receptors to degradation, attenuating signaling. Here we performed an unbiased molecular characterization of EGFR-NCE. We identified NCE-specific regulators, including the endoplasmic reticulum (ER)-resident protein reticulon 3 (RTN3) and a specific cargo, CD147. RTN3 was critical for EGFR/CD147-NCE, promoting the creation of plasma membrane (PM)-ER contact sites that were required for the formation and/or maturation of NCE invaginations. Ca2+ release at these sites, triggered by inositol 1,4,5-trisphosphate (IP3)-dependent activation of ER Ca2+ channels, was needed for the completion of EGFR internalization. Thus, we identified a mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca2+ signaling.

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.