Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilisation (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA-damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the down-regulation of inhibitor of apoptosis (IAP) proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

Background & AimsPancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.MethodsWe used KRasG12D p53R172H Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.ResultsPancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRasG12D p53R172H Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial−mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.ConclusionsThe actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

SummaryThe individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.

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SummaryThe five‐subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin.

We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly localized to the plasma membrane independent of the MyTH4-FERM domain. Filopodia-inducing Myo10 is implicated in axon guidance and mice lacking the Myo10 cargo protein DCC (deleted in colorectal cancer) have severe commissural defects, whereas MRI (magnetic resonance imaging) of isolated brains revealed intact commissures in Myo10tm2/tm2 mice. However, reminiscent of Waardenburg syndrome, a neural crest disorder, Myo10tm2/tm2 mice exhibited pigmentation defects (white belly spots) and simple syndactyly with high penetrance (>95%), and 24% of mutant embryos developed exencephalus, a neural tube closure defect. Furthermore, Myo10tm2/tm2 mice consistently displayed bilateral persistence of the hyaloid vasculature, revealed by MRI and retinal whole-mount preparations. In principle, impaired tissue clearance could contribute to persistence of hyaloid vasculature and syndactyly. However, Myo10-deficient macrophages exhibited no defects in the phagocytosis of apoptotic or IgG-opsonized cells. RNA sequence analysis showed that Myo10 was the most strongly expressed unconventional myosin in retinal vascular endothelial cells and expression levels increased 4-fold between P6 and P15, when vertical sprouting angiogenesis gives rise to deeper layers. Nevertheless, imaging of isolated adult mutant retinas did not reveal vascularization defects. In summary, Myo10 is important for both prenatal (neural tube closure and digit formation) and postnatal development (hyaloid regression, but not retinal vascularization).

Unconventional myosin‐X (Myo10) is a MyTH4‐FERM and PH domain containing actin‐based motor protein which is widely expressed among vertebrate tissues. MyTH4‐FERM myosins are associated with protrusions enriched in bundled actin filaments. Full‐length (fl) Myo10 is best known for its localization to the tips of filopodia and for its ability to induce and regulate the formation of these dynamic finger‐like structures. At the cellular level, Myo10 has been implicated in diverse functions, including oriented cell migration, axon guidance, melanosome transfer, spindle assembly and phagocytosis. However, the physiological functions at the organismal level of this filopodial cargo carrier are not clear. We studied the phenotypes of Myo10 reporter knockout mice (Myo10tm2), to elucidate Myo10 function in vivo. Using Western blot analysis, we showed that in homozygous knockout Myo10tm2/tm2 mice, the expression of motorized fl Myo10 was deleted, but the brain‐specific headless (hdl) isoform was not abolished. Macroscopic examination of Myo10‐deficient mice revealed ventral coat pigmentation defects (100% penetrance) and a limb malformation, syndactyly, with a high penetrance of >95%. 24% of homozygous KO embryos developed exencephaly, a severe neural tube closure defect that is lethal. Magnetic resonance imaging (MRI) of isolated brains revealed, in contrast to mice lacking the Myo10 cargo protein DCC, intact cerebral commissures. Furthermore, Myo10 KO mice consistently displayed bilateral persistence of the hyaloid vasculature, visualized by MRI and retinal whole mount preparations, whereas retinal vascularization, phagocytosis and chemotactic behavior were not impaired. Confocal spinning disk microscopy confirmed that the hdl isoform of Myo10 does not induce filopodia and we found out that hdl Myo10 strongly localizes to the plasma membrane in dependency of its PH domains, supporting the auto‐inhibition model in which head‐tail interactions of fl Myo10 abolishes inactive monomer binding to PIP3‐rich membranes. In conclusion, Myo10 is important for normal vertebrate development, both prenatal (neural tube closure and digit separation) and postnatal (hyaloid regression).Support or Funding InformationDeutsche Forschungsgemeinschaft (DFG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nursing is the NHS's single largest staff group with over 300,000 full-time equivalent (FTE) registered nurses in the hospital and community sector. Nursing vacancies accounted for over a third of all FTE vacancies in NHS trusts in the quarter to June 2022. A lack of long-term planning and a coordinated workforce strategy has been acknowledged as a major factor for the shortfall. The Health Foundation commissioned Decision Analysis Services to develop a system dynamics model to represent the future supply of nurses across England. The model was designed to take a system-wide view of nurse supply and to consider second order effects The nurse supply model was used to generate projections of future nurse supply in England under three scenarios, with the results published in July 2022. The projections suggested that while the government appears to be on track to meet its 50,000 nurses target by 2023/24, this would still leave the NHS short of around 38,000 FTE nurses relative to projected demand in 2023/24.