Biology subjects

Norman, J. C.

Publications and source records attributed to Norman, J. C..

12 recordsLinked to original sources

bioRxiv2025

Increased mRNA translation delays lung adenocarcinoma initiation and exposes a therapeutic vulnerability to MEK inhibitors

Although protein synthesis inhibitors are being evaluated as anti-cancer agents, the dynamics of mRNA translation in early tumorigenesis are still poorly understood. We report that deletion of the mRNA-translation repressor, eIF4A2 in early KRAS-driven lung adenocarcinoma leads to a dysregulated protein synthesis landscape characterised by a strongly upregulated secretome, enlarged secretory compartments, increased oxidative metabolism and acquisition of senescence-like characteristics. Paradoxically, this overdriven protein synthesis landscape delays tumorigenesis and leads to appearance of clusters of non-proliferative, p21-positive KRAS-expressing cells in the lung. Administration of rapamycin to reduce mRNA translation suppresses senescence and restores tumorigenesis following eIF4A2 deletion. Importantly, some eIF4A2 knockout cells overcome senescence to form tumours that exhibit enhanced MAP-kinase signalling and, in contrast to eIF4A2+/+ lesions, these may be eradicated by administration of a MEK inhibitor. Thus, dysregulated mRNA translation exposes a potential therapeutic vulnerability in KRAS-driven lung adenocarcinoma by forcing cancer cells to rely on MEK signalling. Statement of significanceThe requirement for anabolism in cancers has led to the search for inhibitors of mRNA translation as anti-cancer agents. However, we report that increased rates of mRNA translation promote a senescence-like phenotype in KRAS-driven lung cancer which delays tumorigenesis and renders the resulting tumours sensitive to MEK inhibition.

cancer biology↗

bioRxiv2025

EPHA2 and scavenger receptor-directed trafficking enhances endosomal leakiness and antisense therapy delivery

The potential for using therapeutic antisense oligonucleotides (ASOs) has been hampered by lack of understanding of how they enter cells and subsequently access their targets. Endocytosis contributes to ASO uptake, but the machinery mediating subsequent ASO trafficking to permit suppression of their target mRNAs has not been described. Here, we show that ASO engagement with a scavenger receptor (CD44) activates the ERK-RSK axis to promote serine phosphorylation of a receptor tyrosine kinase (EPHA2). Serine phosphorylation of EPHA2 permits endocytosis, trafficking, and accumulation of ASOs in nuclear-adjacent endosomes. These endosomes then become leaky, allowing ASOs to escape and effectively suppress target mRNA expression. Inhibition of stress granule-mediated repair of leaky endosomes further enhances ASO effectiveness. These data identify an endocytic route to the nucleus which may be exploited to maximise effectiveness of ASO-mediated therapies.

cell biology↗

bioRxiv2024

Uridine Phosphorylase-1 supports metastasis of mammary cancer by altering immune and extracellular matrix landscapes of the lung

Understanding the mechanisms that facilitate early events in metastatic seeding is key to developing therapeutic approaches to reduce metastasis - the leading cause of cancer-related death. Using whole animal screens in genetically engineered mouse models of cancer we have identified circulating metabolites associated with metastasis. Specifically, we highlight the pyrimidine uracil as a prominent metastasis-associated metabolite. Uracil is generated by neutrophils expressing the enzyme uridine phosphorylase-1 (UPP1), and neutrophil specific Upp1 expression is increased in cancer. Altered UPP1 activity influences expression of adhesion molecules on the surface of neutrophils, leading to decreased neutrophil motility in the pre-metastatic lung. Furthermore, we find that UPP1-expressing neutrophils suppress T-cell proliferation, and the UPP1 product uracil can increase fibronectin deposition in the extracellular microenvironment. Consistently, knockout or inhibition of UPP1 in mice with mammary tumours increases the number of T-cells and reduces fibronectin content in the lung and decreases the proportion of mice that develop lung metastasis. These data indicate that UPP1 influences neutrophil behaviour and extracellular matrix deposition in the lung and suggest that pharmacological targeting of this pathway could be an effective strategy to reduce metastasis.

cancer biology↗

bioRxiv2024

Metabolic adaptations of micrometastases alter EV production to generate invasive microenvironments

Altered cellular metabolism has been associated with acquisition of invasive phenotypes during metastasis. To study this, we combined a genetically engineered mouse model of mammary carcinoma with syngeneic transplantation and primary tumour resection to generate isogenic cells from primary tumours and their corresponding lung micrometastases. Metabolic analyses indicated that micrometastatic cells increase proline production at the expense of glutathione synthesis leading to a reduction in total glutathione levels. Micrometastatic cells also have altered sphingomyelin metabolism leading to increased intracellular levels of specific ceramides. The combination of these two metabolic adaptations alters small extracellular vesicle (sEV) production to drive generation of an invasive microenvironment. Indeed, micrometastatic cells shut-down Rab27-dependent production of sEVs and, instead, switch-on neutral sphingomyelinase-2 (nSM2)-dependent sEV release. sEVs released in a nSM2-dependent manner from micrometastatic cells, in turn, influence the ability of fibroblasts to deposit extracellular matrix which promotes cancer cell invasiveness. These data provide evidence that metabolic rewiring drives invasive processes in metastasis by influencing sEV release. SummaryBreast cancer cells isolated from lung micrometastases have altered metabolism which influences extracellular vesicle production to generate invasive microenvironments.

cancer biology↗

bioRxiv2024

Integrin inactivation slows down neutrophils congesting the pre-metastatic lung in a model of breast cancer

Neutrophils are thought to be critical to the process whereby breast cancers establish an immunosuppressive and tumour cell nurturing pre-metastatic niche before overt metastasis can be detected. However, the spatial localization of neutrophils and their interaction with other cell types in the lung pre-metastatic niche is not well described. We used a spontaneously metastatic mammary cancer model combined with a multiplexed three- and four-dimensional imaging approach to investigate the behaviour of neutrophils in the pre-metastatic niche. Volume fixed tissue three-dimensional imaging showed that approximately 40% of CD8+ T cells are adjacent to neutrophils at this stage. In live tissue, we found neutrophils with impaired intravascular motility congested the capillaries of pre-metastatic lungs potentially obstructing CD8+ T cells. Slowed neutrophil transit was dependent on the conformation of {beta}2-integrin and could be recapitulated by treating non-tumour bearing mice with G-CSF, a potent systemic mediator of granulopoiesis. We found a decrease in L-selectin (CD62L) on neutrophils in the lungs of both mammary tumour bearing and G-CSF treated mice. Finally, we observed differential accumulation of intravenously injected micro-beads in the lung, suggestive of transient circulatory dead spaces which were also dependent on {beta}2-integrin inactivation. Overall, our study proposes that integrin-mediated neutrophil congestion of the alveolar capillaries could contribute to the generation of the pulmonary pre-metastatic niche.

immunology↗

bioRxiv2023

The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

Increased protein synthesis supports growth of established tumours. However, how mRNA translation contributes to early tumorigenesis remains unclear. Here we show that following oncogene activation, hepatocytes enter a non-proliferative/senescent-like phase characterized by 5{beta}1 integrin-dependent deposition of fibronectin-rich extracellular matrix (ECM) niches. These niches then promote exit from oncogene-induced senescence to permit progression to proliferating hepatocellular carcinoma (HCC). Removal of eIF4A2, a negative regulator of mRNA translation, boosts the synthesis of membrane/secretory proteins which drives a compensatory increase in the turnover/degradation of membrane proteins including 5{beta}1 integrin. This increased membrane protein degradation, in turn, compromises generation of ECM-rich tumour initiation niches, senescence-exit and progression to proliferating HCC. Consistently, pharmacological inhibition of mRNA translation following eIF4A2 loss restores ECM deposition and reinstates HCC progression. Thus, although inhibition of protein synthesis may be an effective way to reduce tumour biomass and the growth of established tumours, our results highlight how agents which reduce mRNA translation, if administered during early tumorigenesis, may awaken senescent cells and promote tumour progression.

cancer biology↗

bioRxiv2022

PTEN deficiency exposes a requirement for an ARF GTPase module in integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in ovarian carcinomas. Loss of the tumour suppressor PTEN in high-grade serous ovarian carcinoma (HGSOC) is associated with a patient subgroup with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We utilise long-term time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency does not affect proliferation but rather induces PI(3,4,5)P3-rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor {beta}1-integrin (ITGB1) as key ARF6 interactors regulating the PTEN loss-associated invasion phenotype. ARF6 functions to promote invasion by controlling the recycling of internalised, active {beta}1-integrin complexes to maintain invasive activity into the ECM. The expression of the ARF6-centred complex in HGSOC patients is inversely associated with outcome, allowing identification of patient groups with improved versus poor outcome. ARF6 may represent a new therapeutic vulnerability in PTEN- depleted HGSOC tumours.

cancer biology↗

bioRxiv2022

Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

The glycocalyx component and sialomucin Podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumour types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for Galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high versus low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

cell biology↗

bioRxiv2022

Glioblastoma exosomes influence glial cell hyaluronic acid deposition to promote invasiveness

BackgroundInfiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. MethodsCRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. ResultsGBM cells expressing a p53 mutant (p53273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. ConclusionsThis work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue. KEY POINTSThe p53R273H oncogene encourages GBM cells to release EVs containing podocalyxin. Podocalyxin-containing EVs from GBM increase hyaluronic acid production by astrocytes. Hyaluronic acid production by astrocytes drives GBM migration. IMPORTANCE OF THE STUDYThe infiltrative behaviour of glioblastoma (GBM) leads to widespread dissemination of cancer cells throughout the brain. Thus, even following successful resection of the primary tumour these disseminated cells inevitably contribute to post-surgical relapse. In this study, we have discovered a new mechanism through which GBM can release small extracellular vesicles (EVs) to reprogramme extracellular matrix (ECM) production by astrocytes in a way that supports increased invasive behaviour of the GBM cells. Moreover, we have discovered several key components of the pathway which contribute to this EV-mediated GBM-glial cell communication. Principal amongst these, we show that a particular mutant of the p53 tumour suppressor, p53273H drives the release of EVs which foster the deposition of pro-invasive ECM by astrocytes. This study provides mechanistic insight into why brain tumours expressing p53273H are associated with particularly poor patient survival and highlights the possibility of deploying agents which target astrocyte ECM deposition to reduce the morbidity of p53273H- expressing GBM.

cancer biology↗

bioRxiv2021

Maturation, developmental site, and pathology dictate murine neutrophil function

Neutrophils have been implicated in poor outcomes in cancer and severe inflammation. We found that neutrophils expressing intermediate levels of Ly6G (Ly6GInt) were present in mouse cancer models and more abundant in those with high rates of spontaneous metastasis. Maturation, age, tissue localization and functional capacity all drive neutrophil heterogeneity. Recent studies have proposed various markers to distinguish between these heterogeneous sub-populations; however, these markers are limited to specific models of inflammation and cancer. Here, we identify and define Ly6G expression level as a robust and reliable marker to distinguish neutrophils at different stages of maturation. Ly6GInt neutrophils were bona fide immature neutrophils with reduced immune regulatory and adhesion capacity. Whereas the bone marrow is a more recognised site of granulopoiesis, the spleen also produces neutrophils in homeostasis and cancer. Strikingly, neutrophils matured faster in the spleen than in the bone marrow with unique transcriptional profiles. We propose that developmental origin is critical in neutrophil identity and postulate that neutrophils that develop in the spleen supplement the bone marrow by providing an intermediate more mature reserve before emergency haematopoiesis.

immunology↗

bioRxiv2020

RAL GTPases mediate EGFR/MAPK signalling-driven intestinal stem cell proliferation and tumourigenesis upstream of RAS activation.

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

cancer biology↗

bioRxiv2020

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.

cancer biology↗