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1.
Cell ; 152(1-2): 25-38, 2013 Jan 17.
Article in English | MEDLINE | ID: mdl-23273993

ABSTRACT

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.


Subject(s)
Cell Dedifferentiation , Cell Transformation, Neoplastic , Colonic Neoplasms/pathology , Neoplastic Stem Cells/pathology , Animals , Colon/pathology , Epithelial Cells/pathology , Female , Humans , Male , Mice , NF-kappa B/metabolism , Wnt Signaling Pathway
2.
Nature ; 607(7919): 548-554, 2022 07.
Article in English | MEDLINE | ID: mdl-35831497

ABSTRACT

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.


Subject(s)
Cell Count , Cell Movement , Intestines , Stem Cells , Animals , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intestines/cytology , Mice , Receptors, G-Protein-Coupled , Stem Cells/cytology , Wnt Proteins
3.
Nature ; 594(7863): 430-435, 2021 06.
Article in English | MEDLINE | ID: mdl-34079124

ABSTRACT

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.


Subject(s)
Cell Competition , Cell Transformation, Neoplastic , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Esterases/metabolism , Genes, APC , Mutation , Adenoma/genetics , Adenoma/pathology , Adenomatous Polyposis Coli Protein/genetics , Animals , Cell Competition/genetics , Cell Differentiation , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Culture Media, Conditioned , Disease Progression , Esterases/antagonists & inhibitors , Esterases/genetics , Female , Humans , Ligands , Male , Mice , Mice, Inbred C57BL , Organoids/cytology , Organoids/metabolism , Organoids/pathology , Stem Cells/cytology , Stem Cells/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway
4.
Nat Chem Biol ; 19(3): 292-300, 2023 03.
Article in English | MEDLINE | ID: mdl-36280791

ABSTRACT

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.


Subject(s)
Glutamine , Neoplasms , Humans , Mice , Animals , Glutamine/metabolism , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Ammonia , Glutamic Acid/metabolism , Liver/metabolism , Neoplasms/metabolism , Homeostasis , Mammals
5.
Genes Dev ; 31(2): 172-183, 2017 01 15.
Article in English | MEDLINE | ID: mdl-28143833

ABSTRACT

Senescence is a form of cell cycle arrest induced by stress such as DNA damage and oncogenes. However, while arrested, senescent cells secrete a variety of proteins collectively known as the senescence-associated secretory phenotype (SASP), which can reinforce the arrest and induce senescence in a paracrine manner. However, the SASP has also been shown to favor embryonic development, wound healing, and even tumor growth, suggesting more complex physiological roles than currently understood. Here we uncover timely new functions of the SASP in promoting a proregenerative response through the induction of cell plasticity and stemness. We show that primary mouse keratinocytes transiently exposed to the SASP exhibit increased expression of stem cell markers and regenerative capacity in vivo. However, prolonged exposure to the SASP causes a subsequent cell-intrinsic senescence arrest to counter the continued regenerative stimuli. Finally, by inducing senescence in single cells in vivo in the liver, we demonstrate that this activates tissue-specific expression of stem cell markers. Together, this work uncovers a primary and beneficial role for the SASP in promoting cell plasticity and tissue regeneration and introduces the concept that transient therapeutic delivery of senescent cells could be harnessed to drive tissue regeneration.


Subject(s)
Cell Plasticity/physiology , Cellular Senescence/physiology , Regeneration/physiology , Secretory Pathway/physiology , Animals , Biomarkers/metabolism , Cell Plasticity/genetics , Cells, Cultured , Cellular Senescence/genetics , Epithelial Cells/cytology , Epithelial Cells/physiology , Female , Gene Deletion , Gene Expression Regulation, Developmental/genetics , Keratinocytes/cytology , Keratinocytes/physiology , Liver/cytology , Liver/physiology , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , Phenotype , Regeneration/genetics , Secretory Pathway/genetics , Stem Cells/metabolism
6.
Cell ; 139(7): 1327-41, 2009 Dec 24.
Article in English | MEDLINE | ID: mdl-20064378

ABSTRACT

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.


Subject(s)
Cell Movement , Integrin alpha5beta1/metabolism , Neoplasm Metastasis , Tumor Suppressor Protein p53/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Line, Tumor , ErbB Receptors/metabolism , Humans , Membrane Proteins/metabolism , Mutation , Pseudopodia/metabolism , Tumor Suppressor Protein p53/genetics
7.
Nature ; 563(7733): 719-723, 2018 11.
Article in English | MEDLINE | ID: mdl-30464341

ABSTRACT

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Mannose/metabolism , Mannose/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Administration, Oral , Animals , Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Body Weight/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/drug effects , Drug Synergism , Female , Glucose/metabolism , Glycolysis/drug effects , Humans , Mannose/administration & dosage , Mannose/therapeutic use , Mannose-6-Phosphate Isomerase/deficiency , Mannose-6-Phosphate Isomerase/genetics , Mannose-6-Phosphate Isomerase/metabolism , Mannosephosphates/metabolism , Mice , Mice, Inbred C57BL , Mice, Nude , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasms/classification , Neoplasms/pathology , RNA Interference , bcl-X Protein/metabolism
8.
Nucleic Acids Res ; 50(19): e112, 2022 10 28.
Article in English | MEDLINE | ID: mdl-35979952

ABSTRACT

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.


Subject(s)
Protein Biosynthesis , Ribosomes , Ribosomes/genetics , Ribosomes/metabolism , Polyribosomes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptome
9.
Genes Dev ; 30(1): 52-63, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26679840

ABSTRACT

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.


Subject(s)
Intestines/cytology , Neuropeptides/metabolism , Proteins/metabolism , Reactive Oxygen Species/metabolism , Wnt Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , Adenomatous Polyposis Coli Protein/genetics , Animals , Apoptosis Regulatory Proteins , Cell Proliferation/physiology , Gene Expression Regulation, Neoplastic/genetics , Mice , Phosphoric Monoester Hydrolases
10.
Br J Cancer ; 128(7): 1333-1343, 2023 03.
Article in English | MEDLINE | ID: mdl-36717674

ABSTRACT

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.


Subject(s)
Colorectal Neoplasms , Humans , Animals , Mice , Colorectal Neoplasms/pathology , Disease Models, Animal , Signal Transduction
11.
J Hepatol ; 78(5): 1028-1036, 2023 05.
Article in English | MEDLINE | ID: mdl-36702176

ABSTRACT

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.


Subject(s)
Focal Nodular Hyperplasia , Liver Regeneration , Male , Female , Humans , Liver Regeneration/physiology , Hepatocytes/metabolism , Liver/metabolism , Homeostasis , Cell Proliferation , Axin Protein/genetics
12.
J Cell Sci ; 134(1)2021 01 13.
Article in English | MEDLINE | ID: mdl-33441326

ABSTRACT

Cell division, differentiation and function are largely dependent on accurate proteome composition and regulated gene expression. To control this, protein synthesis is an intricate process governed by upstream signalling pathways. Eukaryotic translation is a multistep process and can be separated into four distinct phases: initiation, elongation, termination and recycling of ribosomal subunits. Translation initiation, the focus of this article, is highly regulated to control the activity and/or function of eukaryotic initiation factors (eIFs) and permit recruitment of mRNAs to the ribosomes. In this Cell Science at a Glance and accompanying poster, we outline the mechanisms by which tumour cells alter the process of translation initiation and discuss how this benefits tumour formation, proliferation and metastasis.


Subject(s)
Neoplasms , Ribosomes , Eukaryotic Initiation Factors/metabolism , Humans , Neoplasms/genetics , Neoplasms/metabolism , Peptide Chain Initiation, Translational , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomes/genetics , Ribosomes/metabolism
14.
Nature ; 544(7650): 372-376, 2017 04 19.
Article in English | MEDLINE | ID: mdl-28425994

ABSTRACT

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.


Subject(s)
Glycine/deficiency , Intestinal Neoplasms/diet therapy , Intestinal Neoplasms/metabolism , Lymphoma/diet therapy , Lymphoma/metabolism , Serine/deficiency , Animals , Antioxidants/metabolism , Biguanides/pharmacology , Cell Line, Tumor , Diet , Disease Models, Animal , Female , Food Deprivation , Glycine/metabolism , Humans , Intestinal Neoplasms/genetics , Intestinal Neoplasms/pathology , Lymphoma/pathology , Male , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Nutritional Status , Oxidative Phosphorylation/drug effects , Pancreatic Neoplasms/diet therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Serine/biosynthesis , Serine/metabolism , Serine/pharmacology , Survival Rate
15.
Nature ; 541(7636): 233-236, 2017 01 12.
Article in English | MEDLINE | ID: mdl-28052056

ABSTRACT

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.


Subject(s)
Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Genome/genetics , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Animals , Anion Transport Proteins/deficiency , Cell Line, Tumor , Cell Movement , Disease Models, Animal , Female , Genomics , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lymphopenia/genetics , Lymphopenia/pathology , Lysophospholipids/metabolism , Male , Mice , Sphingosine/analogs & derivatives , Sphingosine/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Tumor Microenvironment
16.
Gut ; 71(12): 2502-2517, 2022 12.
Article in English | MEDLINE | ID: mdl-35477539

ABSTRACT

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.


Subject(s)
Biomarkers, Tumor , Colonic Neoplasms , Humans , Biomarkers, Tumor/genetics , Stromal Cells/pathology , Neoplasm Recurrence, Local/prevention & control , Neoplasm Recurrence, Local/pathology , Colonic Neoplasms/pathology , Prognosis
17.
Gut ; 2022 Apr 27.
Article in English | MEDLINE | ID: mdl-35477863

ABSTRACT

OBJECTIVE: Hepatocellular 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. DESIGN: Neutrophil 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. RESULTS: Neutrophils 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 intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on 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 protumour 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. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

18.
Gastroenterology ; 160(1): 245-259, 2021 01.
Article in English | MEDLINE | ID: mdl-32941878

ABSTRACT

BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/+ mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/+mice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apc+/+ mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fc-transduced ApcMin/+ mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/+ mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/+ mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/+ mice expressing RSPO1-Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/+ mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.


Subject(s)
Adenoma/pathology , Intestinal Neoplasms/pathology , Thrombospondins/metabolism , Transforming Growth Factor beta/physiology , Wnt Signaling Pathway/physiology , Adenoma/etiology , Animals , Disease Models, Animal , Intestinal Neoplasms/etiology , Mice , Organoids
19.
Anal Chem ; 94(3): 1795-1803, 2022 01 25.
Article in English | MEDLINE | ID: mdl-35005896

ABSTRACT

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Carcinoma, Pancreatic Ductal/diagnostic imaging , Carcinoma, Pancreatic Ductal/drug therapy , Cell Line, Tumor , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Mice , Multimodal Imaging , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Tumor Microenvironment , Gemcitabine
20.
Genes Dev ; 28(24): 2712-25, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25512559

ABSTRACT

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.


Subject(s)
Cell Cycle Proteins/metabolism , Cellular Senescence/physiology , Histone Chaperones/metabolism , Transcription Factors/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/genetics , Cell Cycle Proteins/genetics , Cell Line , Cell Proliferation , Cellular Senescence/genetics , Chromatin/metabolism , Female , Gene Expression Regulation/drug effects , Genetic Markers , Histone Chaperones/genetics , Histones/genetics , Histones/metabolism , Humans , Male , Mice , Papilloma/pathology , Skin Neoplasms/pathology , Tamoxifen/pharmacology , Transcription Factors/genetics
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