Biology subjects

Nixon, C.

Publications and source records attributed to Nixon, C..

At least 37 records · Page 2Linked to original sources

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↗

bioRxiv2023

ERBB signalling contributes to immune evasion in KRAS-driven lung adenocarcinoma

Immunotherapy is increasingly viewed as treatment of choice for lung cancer, however, clinical responses to immune checkpoint blockade remain highly unpredictable and are largely transient. A deeper mechanistic understanding of the dynamics of tumour:immune interactions is needed to drive rational development of improved treatment strategies. Progress is hampered by a paucity of autochthonous model systems in which to interrogate the 2-way interactions of immune responses to evolving tumours and vice-versa. Specifically, commonly used genetically engineered mouse models typically lack the genetic diversity needed to drive an adaptive immune response. APOBEC mutagenesis signatures are prominent in lung cancer and APOBEC activity is predicted to drive immune visibility through Cytidine deaminase activity, coupled with inaccurate DNA-repair responses. We therefore generated a CRE-inducible APOBEC3B allele, interbred with multiple oncogenic drivers of lung adenocarcinoma, and used the resulting mice to investigate the response to PD1 blockade at single cell resolution. SIGNIFICANCE Using our novel immune-visible model of KRas-driven autochthonous lung adenocarcinoma, we uncovered a surprising increase in tumour-cell expression of EGFR/ERBB ligands following treatment with -PD1 and present evidence that transient ERBB blockade can restore immune surveillance in KRas mutant LuAd and combine effectively with immune checkpoint blockade.

cancer biology↗

bioRxiv2023

Characterising neutrophil subtypes in cancer using human and murine single-cell RNA sequencing datasets

Neutrophils are a highly heterogenous cellular population. However, a thorough examination of the different transcriptional neutrophil states, between health and malignancy, has not been performed. We utilised single-cell RNA-sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and their developmental lineages in health and malignancy. Datasets of lung, breast and colorectal cancer (CRC) were integrated to establish and validate the reproducibility of neutrophil gene-signatures. Pseudo-time analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand-receptor interactions and signalling pathways between neutrophils and other immune cell populations in primary CRC and metastatic CRC were investigated. We define two main neutrophil subtypes in primary tumours: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumour-specific subtype. This signature is conserved in murine and human cancer, across different tumour types. In CRC metastases, neutrophils are more heterogenous, exhibiting additional transcriptomic subtypes. Pseudo-time analysis implicates an IL1B/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Assessment of global communication signalling identified CD4+ T-cells and macrophages as dominant regulators of the immunosuppressive, metastatic niche, whereas CD8+ T-cells are receivers of signals from other immune cells. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by an IL1/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of the neutrophil transcriptomic programming could optimise immunotherapeutic interventions into early and late interventions, targeting different neutrophil subtypes.

bioinformatics↗

bioRxiv2023

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins

A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs - questioning the differences and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the overall consensus protein is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted region is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order couplings using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

biophysics↗

bioRxiv2023

Paracrine secretion of MUFAs prevents ferroptosis in triple-negative breast cancer and reveals selenocysteine synthesis dependency for lung metastasis

The limited availability of therapeutic options for patients with triple-negative breast cancer (TNBC) contributes to the high rate of metastatic recurrence and poor prognosis. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation and counteracted by the antioxidant activity of the selenoprotein GPX4. Here, we show that TNBC cells secrete an anti-ferroptotic factor in the extracellular environment when cultured at high cell densities but are primed to ferroptosis when forming colonies at low density. We found that secretion of the anti-ferroptotic factors, identified as monounsaturated fatty acid (MUFA) containing lipids, and the vulnerability to ferroptosis of single cells depends on the low expression of stearyl-CoA desaturase (SCD) that is proportional to cell density. Finally, we show that the inhibition of Sec-tRNAsec biosynthesis, an essential step for selenoprotein production, causes ferroptosis and impairs the lung seeding of circulating TNBC cells that are no longer protected by the MUFA-rich environment of the primary tumour.

cancer biology↗

bioRxiv2023

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target

With colorectal cancer (CRC) being the second most common cause of cancer-related deaths worldwide1, there is an urgent need for better diagnostic tools and new, more targeted therapies. Here we used genetically engineered mouse models (GEMMs), and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that unsupervised metabolic profiling can stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging (MSI) to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-mutant CRC, and propose one of its enzymes, i.e. adenosylhomocysteinase (AHCY), as a new therapeutic target. Collectively, we show that the profound genotype-dependent alterations in both lipid and small molecule metabolism in CRC may be exploited for tissue classification with no need for ion identification, and we applied further data analysis to expose a novel metabolic vulnerability of CRC.

cancer biology↗

bioRxiv2023

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we unveil a mechanism of cell-cell communication between CAFs with myofibroblast phenotype and endothelial cells (ECs) based on intercellular protein transfer through extracellular vesicles (EVs). CAFs transfer proteins to ECs, including plasma membrane receptors, which we have identified by using mass spectrometry- based proteomics. Using THY1 as an example of transferred plasma membrane-bound protein, we show that CAF-derived proteins can influence how ECs interact with other cell types. Here, we show that CAFs produce high amounts of matrix-bound EVs that have a key role in protein transfer. Hence, our work paves the way for further studies to understand how CAF-derived matrix-bound EVs influence tumor pathology by regulating functions of neighboring cancer, stromal and immune cells. One sentence summaryCAFs with a myofibroblastic-like phenotype transfer proteins to ECs, including plasma membrane receptors, through matrix-bound EVs

cancer biology↗

bioRxiv2022

CYRI-B mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake

Pancreatic ductal adenocarcinoma carries a dismal prognosis, with high rates of metastasis and few treatment options. Hyperactivation of KRAS in almost all tumours drives RAC1 activation, conferring enhanced migratory and proliferative capacity as well as macropinocytosis. Macropinocytosis is well understood as a nutrient scavenging mechanism, but little is known about its functions in trafficking of signaling receptors. We find that CYRI-B is highly expressed in pancreatic tumours in a mouse model of KRAS and p53- driven pancreatic cancer. Deletion of CYRI-B accelerates tumourigenesis, leading to enhanced ERK and JNK-induced proliferation in precancerous lesions, indicating a role as a buffer of RAC1 hyperactivation in early stages. However, as disease progresses, loss of CYRI-B inhibits metastasis. CYRI-B depleted tumour cells show reduced chemotactic responses to lysophosphatidic acid, a major driver of tumour spread, due to impaired macropinocytic uptake of LPAR1 receptor. Overall, we implicate CYRI-B as a mediator of growth and signaling in pancreatic cancer, providing new insights into pathways controlling metastasis.

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

Spatially Resolved Transcriptomics Deconvolutes Histological Prognostic Subgroups in Patients with Colorectal Cancer and Synchronous Liver Metastases

BackgroundPatients demonstrating strong immune responses to primary colorectal cancer (CRC) have a survival benefit following surgery, while those with predominantly stromal microenvironments do poorly. Biomarkers to identify patients with colorectal cancer liver metastases (CRLM) who have good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary CRC and CRLM, and to interrogate the underlying functional biology that drives disease progression. MethodsPatients undergoing synchronous resection of primary CRC and CRLM underwent detailed histological assessment, panel genomic and bulk transcriptomic assessment, immunohistochemistry (IHC) and GeoMx Spatial Transcriptomics (ST) analysis. Integration with genomic features, pathway enrichment analysis and immune deconvolution were performed. ResultsHigh-immune metastases were associated with improved cancer specific survival (HR, 0.36, P=0.01). Bulk transcriptomic analysis was confounded by stromal content but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for Type II Interferon signalling (NES=-2.05 P.Adj<0.005) and MHC-Class II Antigen Presentation (NES=-2.09 P.Adj<0.005). In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T-cells and neutrophils with enrichment of Notch (NES=2.2 P.Adj=0.022) and TGF-{beta} (NES=2.2 P.Adj=0.02) signalling pathways at the metastatic tumor centre. ConclusionsHistological assessment stratifies outcome in patients undergoing synchronous resection of CRLM. ST analysis reveals significant intra-tumoral and inter-lesional heterogeneity with underlying transcriptomic programmes identified in driving each phenotype. TRANSLATIONAL RELEVANCEThe current study demonstrates that accurate histological assessment of immune cell infiltration and stromal content can define survival in patients following resection of oligometastatic liver disease when presenting synchronously with primary colorectal cancer. A spatial transcriptomic approach has demonstrated heterogeneity between patients, between matched lesions in the same patient and within individual lesions. Patients with high immune infiltrates at the invasive margin demonstrated lymphocytic infiltration and associated upregulated adaptive immune pathways in long term survivors. In specimens with low immune infiltrate at the tumor edge a significant reduction in survival was observed, this was determined by upregulated immunosuppressive pathways and a predominance of innate immune cells surrounding metastases. Spatial transcriptomics can be used to examine drivers of metastatic progression in CRC and identifies patients with reactive and suppressed immune microenvironments. Application across a larger cohort will build the cartography of CRLM, while in future, studies may assess application of this technology to pre and post treatment biopsy samples with the aim of predicting individual therapeutic responses. The current study has highlighted discrepancies between bulk and ST derived data whilst demonstrating accuracy of deconvoluted transcriptome to determine immune profiling. Now that ST strategies are becoming more achievable at scale, this has implications for the interpretation of the bulk transcriptomic signatures both of primary and metastatic CRC.

cancer biology↗

bioRxiv2022

Collagen-VI expression is negatively mechanosensitive in pancreatic cancer cells and supports the metastatic niche

Pancreatic cancer is a deadly disease with high rates of metastasis, though how tumor cells establish metastatic lesions is not fully understood. A key feature of primary pancreatic tumors is extensive fibrosis due to deposition of extracellular matrix. While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment as well as adaptation to a softer microenvironment upon reaching distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing of soft substrates. Through use of engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that Collagen-VI is specifically upregulated on soft substrates, due to a lack of integrin engagement and low YAP1 activity. Collagen-VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 expression, were characterised by stromal cell-derived collagen-VI deposition, suggesting that collagen-VI, either cancer or stroma derived, is an essential component of the metastatic niche. Summary StatementCollagen-VI is expressed by pancreatic tumors and metastases in a mechanosensitive way to promote niche colonisation.

cancer biology↗

bioRxiv2022

CXCR2 inhibition enables NASH-HCC immunotherapy

ObjectiveHepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DesignNeutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. ResultsNeutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoral XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoral immunity, this was confirmed by loss of therapeutic effect upon genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a pro-tumor to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. ConclusionCXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC. Significance of this studyWhat is already known on this subject? O_LIImmune checkpoint inhibition therapy is emerging as a promising new therapy for the treatment of advanced hepatocellular carcinoma (HCC). C_LIO_LIOnly a minority of HCC patients will respond to immune checkpoint inhibition (ICI) therapy and recent data suggest that HCC on the background of NASH may have reduced sensitivity to this treatment strategy. C_LIO_LINeutrophils are a typical myeloid component of the liver in NASH and are found either within the HCC tumour microenvironment or in a peritumoural location. C_LIO_LINeutrophils have considerable phenotypic plasticity and can exist in both tumour promoting and tumour suppressing states. C_LIO_LINeutrophils may have the ability to influence ICI therapy. C_LI What are the new findings? O_LICXCR2+ neutrophils are found in human NASH and within the tumour of both human and mouse models of NASH-HCC. C_LIO_LIThe resistance of NASH-HCC to anti-PD1 therapy is overcome by co-treatment with a CXCR2 small molecule inhibitor, with evidence of reduced tumour burden and extended survival. C_LIO_LIAnti-PD1 and CXCR2 inhibitor combine to selectively reprogramme tumour-associated neutrophils (TANs) from a pro- to an anti-tumour phenotype. C_LIO_LIReprogrammed TANs proliferate locally within Granzyme B+ immune clusters that contain physically associating CD8+ T cells and antigen presenting cells. C_LIO_LIConventional XCR1+ dendritic cells (cDC1s) are found to be elevated in anti-PD1 and CXCR2 inhibitor treated HCCs and together with CD8+ T cells are required for therapeutic benefit. C_LI How might it impact on clinical practice in the foreseeable future? O_LITANs can be selectively manipulated to adopt an anti-tumour phenotype which unlocks their potential for cancer therapy. The ability of CXCR2 antagonism to combine with ICI therapy to bring about enhanced therapeutic benefit in NASH-HCC (and potentially in HCC of other aetiologies) warrents clinical investigation. C_LI

cancer biology↗

bioRxiv2022

Inhibiting Runx1 protects heart function after myocardial infarction

Myocardial infarction is a major cause of death worldwide. Effective treatments are required that limit adverse cardiac remodelling and preserve cardiac contractility following myocardial infarction, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile myocardium bordering a newly created infarct is functionally distinct from the remote surviving myocardium; it is also a major determinant of adverse cardiac remodelling and whole heart contractility. Expression of the transcription factor RUNX1 is increased in the border zone at 1 day after myocardial infarction, suggesting potential for targeted therapeutic intervention. Here we demonstrate that RUNX1 drives reductions in cardiomyocyte contractility, sarcoplasmic reticulum-mediated calcium release, mitochondrial density, and the expression of genes important for oxidative phosphorylation. Antagonising RUNX1 expression via short-hairpin RNA interference preserved cardiac contractile function following myocardial infarction when delivered either via direct adenoviral delivery into the border zone or via an adeno-associated virus vector administered intravenously. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with the essential co-factor CBF{beta}. Both tamoxifen-inducible Runx1-deficient and Cbf{beta}-deficient cardiomyocyte-specific mouse models demonstrated that antagonising RUNX1 function preserves the expression of genes important for oxidative phosphorylation following myocardial infarction. Our results confirm the translational potential of RUNX1 as a novel therapeutic target in myocardial infarction, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

pathology↗

bioRxiv2021

Increased apoptotic priming of glioblastoma enables therapeutic targeting by BH3-mimetics

IDH wild-type glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. Tackling this, we investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti- apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non- malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti- apoptotic BCL-2 family members. Surprisingly, high anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein- targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3- mimetics.

cancer biology↗

bioRxiv2021

THEM6-mediated lipid remodelling sustains stress resistance in cancer

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. In patients, THEM6 expression correlates with progressive disease and is associated with poor survival. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, THEM6 is located at the endoplasmic reticulum (ER) membrane and controls lipid homeostasis by regulating intracellular levels of ether lipids. Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated induction of ATF4. Finally, we show that THEM6 is required for the establishment of the MYC-induced stress response. Thus, similar to PCa, THEM6 loss significantly impairs tumorigenesis in the MYC-dependent subtype of triple negative breast cancer. Altogether, our results highlight THEM6 as a novel component of the treatment-induced stress response and a promising target for the treatment of CRPC and MYC-driven cancer.

cancer biology↗

bioRxiv2021

Genetic manipulation using hepatocyte-targeting adeno-associated viral vectors has minimal off-target effects.

Mice are a widely used pre-clinical model system in large part due to their potential for genetic manipulation. The ability to manipulate gene expression in specific cells under temporal control is a powerful experimental tool. The liver is central to metabolic homeostasis and a site of many diseases, making the targeting of hepatocytes attractive. Adeno-Associated Virus 8 (AAV8) vectors are valuable instruments for the manipulation of hepatocellular gene expression. However, their off-target effects in mice have not been thoroughly explored. Here, we sought to identify the short-term off-target effects of AAV8 administration in mice. To do this, we injected C57BL/6J Wild-Type mice with either recombinant AAV8 vectors expressing Cre recombinase or empty AAV8 vectors and characterised the changes in general health and in liver physiology, histology and transcriptomics compared to uninjected controls over 1 week. We observed an acute and transient reduction in homeostatic liver proliferation together with induction of the DNA damage marker {gamma}H2AX following AAV8 administration. The latter was enhanced upon Cre recombinase expression by the vector. Furthermore, we observed transcriptional changes in genes involved in circadian rhythm and response to infection. Notably, there were no additional transcriptomic changes upon expression of Cre recombinase by the AAV8 vector. Overall, there was no evidence of liver injury, dysfunction or leukocyte infiltration following AAV8 infection. These data support the use of AAV8-based Cre recombinase delivery as a specific tool for hepatocellular gene manipulation with minimal effects on murine physiology but highlight the off target effects of these systems. Summary statementThis paper provides a comprehensive characterisation of the short-term effects of administration of Adeno-Associated Virus 8 on murine physiology, liver histology and liver transcriptome.

cell biology↗

bioRxiv2020

Absent expansion of pericentral hepatocytes and altered physiology in Axin2CreERT2 mice

Abstract/IntroductionUnderstanding how the liver regenerates is a key biological question. Hepatocytes are the principle regenerative population in the liver. Recently, numerous lineage tracing studies (which apply genetic tagging to a restricted population and track its descendants over time) have reported conflicting results using a variety of hepatocyte based reporting systems in mice1,2. The first significant lineage tracing from a distinct subpopulation of hepatocytes in homeostasis reported hyper-proliferation of self-renewing pericentral hepatocytes with their subsequent expansion across the liver lobule3. This study used a CreERT2 construct knocked into the endogenous Axin2 locus; here termed Axin2CreERT2. Subsequent studies, using either a different pericentral marker (Lgr54) or a different AxinCreERT2 transgene5, did not show lineage tracing. Here we aim to reconcile these discrepancies by re-evaluating lineage tracing in the Axin2CreERT2 knock-in model and explore the physiological consequences of this mutant allele. We were unable to find evidence of expansion of an Axin2CreERT2 labelled population and show that this population, whilst zonated, is spread throughout the lobule rather than being zonally restricted. Finally, we report that this allele results in profound perturbation of the Wnt pathway and physiology in the mouse.

cell biology↗

bioRxiv2020

Schlafen family member 5 (SLFN5) regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer

Androgen-deprivation therapy (ADT) is the standard of care for the treatment of non-resectable prostate cancer (PCa). Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we perform a comparative proteomic analysis of three in vivo, androgen receptor (AR)-driven, orthograft models of CRPC. Differential proteomic analysis reveals that distinct molecular mechanisms, including amino acid (AA) and fatty acid (FA) metabolism, are involved in the response to ADT between the different models. Despite this heterogeneity, we identify SLFN5 as an AR-regulated biomarker in CRPC. SLFN5 expression is high in CRPC tumours and correlates with poor patient outcome. In vivo, SLFN5 depletion strongly impairs tumour growth in castrated condition. Mechanistically, SLFN5 interacts with ATF4 and regulates the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreases intracellular levels of essential AA and impairs mTORC1 signalling in a LAT1-dependent manner.

cancer biology↗