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.

Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

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.

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.

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

ATR is essential for preservation of cell mechanics and nuclear integrity during interstitial migration.

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development.

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.

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.

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.

From injury to full repair: nerve regeneration and functional recovery in the common octopus, Octopus vulgaris.

Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.

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.

Redundant and nonredundant organismal functions of EPS15 and EPS15L1.

EPS15 and its homologous EPS15L1 are endocytic accessory proteins. Studies in mammalian cell lines suggested that EPS15 and EPS15L1 regulate endocytosis in a redundant manner. However, at the organismal level, it is not known to which extent the functions of the two proteins overlap. Here, by exploiting various constitutive and conditional null mice, we report redundant and nonredundant functions of the two proteins. EPS15L1 displays a unique nonredundant role in the nervous system, whereas both proteins are fundamental during embryo development as shown by the embryonic lethality of -Eps15/Eps15L1-double KO mice. At the cellular level, the major process redundantly regulated by EPS15 and EPS15L1 is the endocytosis of the transferrin receptor, a pathway that sustains the development of red blood cells and controls iron homeostasis. Consequently, hematopoietic-specific conditional Eps15/Eps15L1-double KO mice display traits of microcytic hypochromic anemia, due to a cell-autonomous defect in iron internalization.

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.

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.

ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress.

ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and ß3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.