Cellular geometry and epithelial-mesenchymal plasticity intersect with PIEZO1 in breast cancer cells.

Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells.

Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Immune activation of the p75 neurotrophin receptor: implications in neuroinflammation.

Despite structural similarity with other tumor necrosis factor receptor superfamily (TNFRSF) members, the p75 neurotrophin receptor (p75NTR, TNFR16) mediates pleiotropic biological functions not shared with other TNFRs. The high level of p75NTR expression in the nervous system instead of immune cells, its utilization of co-receptors, and its interaction with soluble dimeric, rather than soluble or cell-tethered trimeric ligands are all characteristics which distinguish it from most other TNFRs. Here, we compare these attributes to other members of the TNFR superfamily. In addition, we describe the recent evolutionary adaptation in B7-1 (CD80), an immunoglobulin (Ig) superfamily member, which allows engagement to neuronally-expressed p75NTR. B7-1-mediated binding to p75NTR occurs in humans and other primates, but not lower mammals due to specific sequence changes that evolved recently in primate B7-1. This discovery highlights an additional mechanism by which p75NTR can respond to inflammatory cues and trigger synaptic elimination in the brain through engagement of B7-1, which was considered to be immune-restricted. These observations suggest p75NTR does share commonality with other immune co-modulatory TNFR family members, by responding to immunoregulatory cues. The evolution of primate B7-1 to bind and elicit p75NTR-mediated effects on neuronal morphology and function are discussed in relationship to immune-driven modulation of synaptic actions during injury or inflammation.

Maternal oxidative stress during pregnancy associated with emotional and behavioural problems in early childhood: implications for foetal programming.

Childhood mental disorders, including emotional and behavioural problems (EBP) are increasingly prevalent. Higher maternal oxidative stress (OS) during pregnancy (matOSpreg) is linked to offspring mental disorders. Environmental factors contribute to matOSpreg. However, the role of matOSpreg in childhood EBP is unclear. We investigated the associations between (i) matOSpreg and offspring EBP; (ii) social and prenatal environmental factors and matOSpreg; and (iii) social and prenatal factors and childhood EBP and evaluated whether matOSpreg mediated these associations. Maternal urinary OS biomarkers, 8-hydroxyguanosine (8-OHGua; an oxidative RNA damage marker) and 8-hydroxy-2'-deoxyguanosine (8-OHdG; an oxidative DNA damage marker), at 36 weeks of pregnancy were quantified by liquid chromatography-mass spectrometry in a population-derived birth cohort, Barwon Infant Study (n = 1074 mother-infant pairs). Social and prenatal environmental factors were collected by mother-reported questionnaires. Offspring total EBP was measured by Child Behavior Checklist Total Problems T-scores at age two (n = 675) and Strengths and Difficulties Questionnaire Total Difficulties score at age four (n = 791). Prospective associations were examined by multivariable regression analyses adjusted for covariates. Mediation effects were evaluated using counterfactual-based mediation analysis. Higher maternal urinary 8-OHGua at 36 weeks (mat8-OHGua36w) was associated with greater offspring total EBP at age four (ß = 0.38, 95% CI (0.07, 0.69), P = 0.02) and age two (ß = 0.62, 95% CI (-0.06, 1.30), P = 0.07). Weaker evidence of association was detected for 8-OHdG. Five early-life factors were associated with both mat8-OHGua36w and childhood EBP (P-range < 0.001-0.05), including lower maternal education, socioeconomic disadvantage and prenatal tobacco smoking. These risk factor-childhood EBP associations were partly mediated by higher mat8-OHGua36w (P-range = 0.01-0.05). Higher matOSpreg, particularly oxidant RNA damage, is associated with later offspring EBP. Effects of some social and prenatal lifestyle factors on childhood EBP were partly mediated by matOSpreg. Future studies are warranted to further elucidate the role of early-life oxidant damage in childhood EBP.

The intersection between cysteine proteases, Ca2+ signalling and cancer cell apoptosis.

Apoptosis is a highly complex and regulated cell death pathway that safeguards the physiological balance between life and death. Over the past decade, the role of Ca2+ signalling in apoptosis and the mechanisms involved have become clearer. The initiation and execution of apoptosis is coordinated by three distinct groups of cysteines proteases: the caspase, calpain and cathepsin families. Beyond its physiological importance, the ability to evade apoptosis is a prominent hallmark of cancer cells. In this review, we will explore the involvement of Ca2+ in the regulation of caspase, calpain and cathepsin activity, and how the actions of these cysteine proteases alter intracellular Ca2+ handling during apoptosis. We will also explore how apoptosis resistance can be achieved in cancer cells through deregulation of cysteine proteases and remodelling of the Ca2+ signalling toolkit.

Intersection between calcium signalling and epithelial-mesenchymal plasticity in the context of cancer.

Epithelial-mesenchymal transition (EMT) is a form of cellular phenotypic plasticity and is considered a crucial step in the progression of many cancers. The calcium ion (Ca2+) acts as a ubiquitous second messenger and is implicated in many cellular processes, including cell death, migration, invasion and more recently EMT. Throughout this review, the complex interplay between Ca2+ signalling and EMT will be explored. An overview of the Ca2+ pathways that are remodelled as a consequence of EMT is provided and the role of Ca2+ signalling in regulating EMT and its significance is considered. Ca2+ signalling pathways may represent a therapeutic opportunity to regulate EMT. However, as will be described in this review, the complexity of these signalling pathways represents significant challenges that must be considered if Ca2+ signalling is to be manipulated with the aim of therapeutic intervention in cancer.

AAV5-miHTT-mediated huntingtin lowering improves brain health in a Huntington's disease mouse model.

Huntingtin (HTT)-lowering therapies show great promise in treating Huntington's disease. We have developed a microRNA targeting human HTT that is delivered in an adeno-associated serotype 5 viral vector (AAV5-miHTT), and here use animal behaviour, MRI, non-invasive proton magnetic resonance spectroscopy and striatal RNA sequencing as outcome measures in preclinical mouse studies of AAV5-miHTT. The effects of AAV5-miHTT treatment were evaluated in homozygous Q175FDN mice, a mouse model of Huntington's disease with severe neuropathological and behavioural phenotypes. Homozygous mice were used instead of the more commonly used heterozygous strain, which exhibit milder phenotypes. Three-month-old homozygous Q175FDN mice, which had developed acute phenotypes by the time of treatment, were injected bilaterally into the striatum with either formulation buffer (phosphate-buffered saline + 5% sucrose), low dose (5.2 × 109 genome copies/mouse) or high dose (1.3 × 1011 genome copies/mouse) AAV5-miHTT. Wild-type mice injected with formulation buffer served as controls. Behavioural assessments of cognition, T1-weighted structural MRI and striatal proton magnetic resonance spectroscopy were performed 3 months after injection, and shortly afterwards the animals were sacrificed to collect brain tissue for protein and RNA analysis. Motor coordination was assessed at 1-month intervals beginning at 2 months of age until sacrifice. Dose-dependent changes in AAV5 vector DNA level, miHTT expression and mutant HTT were observed in striatum and cortex of AAV5-miHTT-treated Huntington's disease model mice. This pattern of microRNA expression and mutant HTT lowering rescued weight loss in homozygous Q175FDN mice but did not affect motor or cognitive phenotypes. MRI volumetric analysis detected atrophy in four brain regions in homozygous Q175FDN mice, and treatment with high dose AAV5-miHTT rescued this effect in the hippocampus. Like previous magnetic resonance spectroscopy studies in Huntington's disease patients, decreased total N-acetyl aspartate and increased myo-inositol levels were found in the striatum of homozygous Q175FDN mice. These neurochemical findings were partially reversed with AAV5-miHTT treatment. Striatal transcriptional analysis using RNA sequencing revealed mutant HTT-induced changes that were partially reversed by HTT lowering with AAV5-miHTT. Striatal proton magnetic resonance spectroscopy analysis suggests a restoration of neuronal function, and striatal RNA sequencing analysis shows a reversal of transcriptional dysregulation following AAV5-miHTT in a homozygous Huntington's disease mouse model with severe pathology. The results of this study support the use of magnetic resonance spectroscopy in HTT-lowering clinical trials and strengthen the therapeutic potential of AAV5-miHTT in reversing severe striatal dysfunction in Huntington's disease.

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses.

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.

Salt-inducible kinase 2 regulates fibrosis during bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC50 to its reported tyrosine kinase targets. Nintedanib also inhibited the related kinases SIK1 and SIK3, although with 12-fold and 72-fold higher IC50s, respectively. To investigate if the inhibition of SIK2 may contribute to the effectiveness of nintedanib in treating lung fibrosis, mice with kinase-inactive knockin mutations were tested using a model of bleomycin-induced lung fibrosis. We found that loss of SIK2 activity protects against bleomycin-induced fibrosis, as judged by collagen deposition and histological scoring. Loss of both SIK1 and SIK2 activity had a similar effect to loss of SIK2 activity. Total SIK3 knockout mice have a developmental phenotype making them unsuitable for analysis in this model; however, we determined that conditional knockout of SIK3 in the immune system did not affect bleomycin-induced lung fibrosis. Together, these results suggest that SIK2 is a potential drug target for the treatment of lung fibrosis.

Superficial and Functional Lymphatic Anatomy of the Upper Extremity.

BACKGROUND: Knowledge of detailed lymphatic anatomy in humans is limited, as the small size of lymphatic channels makes it difficult to image. Most current knowledge of the superficial lymphatic system has been obtained from cadaveric dissections. METHODS: Indocyanine green lymphography was performed preoperatively to map the functional arm lymphatics in breast cancer patients without clinical or objective evidence of lymphedema. A retrospective review was performed to extract demographic, indocyanine green imaging, and surgical data. RESULTS: Three main functional forearm channels with variable connections to two upper arm pathways were identified. The median forearm channel predominantly courses in the volar forearm (99 percent). The ulnar forearm channel courses in the volar forearm in the majority of patients (66 percent). The radial forearm channel courses in the dorsal forearm in the majority of patients (92 percent). Median (100 percent), radial (91 percent), and ulnar (96 percent) channels almost universally connect to the medial upper arm channel. In contrast, connections to the lateral upper arm channel occur less frequently from the radial (40 percent) and ulnar (31 percent) channels. CONCLUSIONS: This study details the anatomy of three forearm lymphatic channels and their connections to the upper arm in living adults without lymphatic disease. Knowledge of these pathways and variations is relevant to any individual performing procedures on the upper extremities, as injury to the superficial lymphatic system can predispose patients to the development of lymphedema.

ORAI1-Regulated Gene Expression in Breast Cancer Cells: Roles for STIM1 Binding, Calcium Influx and Transcription Factor Translocation.

A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.

Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components.

Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.

Increased matrix stiffness suppresses ATP-induced sustained Ca2+ influx in MDA-MB-231 breast cancer cells.

Both matrix stiffening and remodeling of calcium signaling occur in breast cancers, with downstream consequences linked to the progression of the disease. However, the potential intersection between calcium signaling and matrix stiffness has not been fully assessed in models of cancer. Here, we describe the assessment of calcium signaling in breast cancer cells at high and low matrix stiffness using novel gel culture models (gelatin methacryloyl and polydimethylsiloxane) and MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m. Remodeling of ATP-stimulated cytosolic calcium responses in cells on different matrices was assessed using a high throughput fluorescence imaging plate reader. Our data reveal that matrices of higher stiffness attenuate ATP-induced sustained calcium influx in MDA-MB-231 breast cancer cells. This matrix-mediated attenuation of sustained calcium influx was dependent on the store-operated calcium channel component ORAI1. These studies suggest that calcium signaling in breast cancer cells can be altered as a consequence of matrix stiffness; modulation of such pathways may represent a new mechanism to target calcium signaling to regulate tumor progression in breast cancer.

Early life environmental factors associated with autism spectrum disorder symptoms in children at age 2 years: A birth cohort study.

LAY ABSTRACT: Mounting evidence indicates the contribution of early life environmental factors in autism spectrum disorder. We aim to report the prospective associations between early life environmental factors and autism spectrum disorder symptoms in children at the age of 2 years in a population-derived birth cohort, the Barwon Infant Study. Autism spectrum disorder symptoms at the age of 2 years strongly predicted autism spectrum disorder diagnosis by the age of 4 years (area under curve = 0.93; 95% CI (0.82, 1.00)). After adjusting for child's sex and age at the time of behavioural assessment, markers of socioeconomic disadvantage, such as lower household income and lone parental status; maternal health factors, including younger maternal age, maternal pre-pregnancy body mass index, higher gestational weight gain and prenatal maternal stress; maternal lifestyle factors, such as prenatal alcohol and environmental air pollutant exposures, including particulate matter < 2.5 µm at birth, child secondhand tobacco smoke at 12 months, dampness/mould and home heating with oil, kerosene or diesel heaters at 2 years postnatal. Lower socioeconomic indexes for area, later birth order, higher maternal prenatal depression and maternal smoking frequency had a dose-response relationship with autism spectrum disorder symptoms. Future studies on environmental factors and autism spectrum disorder should consider the reasons for the socioeconomic disparity and the combined impact of multiple environmental factors through common mechanistic pathways.

The foundations of immune checkpoint blockade and the ipilimumab approval decennial.

Cancer immunity, and the potential for cancer immunotherapy, have been topics of scientific discussion and experimentation for over a hundred years. Several successful cancer immunotherapies - such as IL-2 and interferon-α (IFNα) - have appeared over the past 30 years. However, it is only in the past decade that immunotherapy has made a broad impact on patient survival in multiple high-incidence cancer indications. The emergence of immunotherapy as a new pillar of cancer treatment (adding to surgery, radiation, chemotherapy and targeted therapies) is due to the success of immune checkpoint blockade (ICB) drugs, the first of which - ipilimumab - was approved in 2011. ICB drugs block receptors and ligands involved in pathways that attenuate T cell activation - such as cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD1) and its ligand, PDL1 - and prevent, or reverse, acquired peripheral tolerance to tumour antigens. In this Review we mark the tenth anniversary of the approval of ipilimumab and discuss the foundational scientific history of ICB, together with the history of the discovery, development and elucidation of the mechanism of action of the first generation of drugs targeting the CTLA4 and PD1 pathways.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

An Emerging Role for Calcium Signaling in Cancer-Associated Fibroblasts.

Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell-cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability.