The epithelial monolayer of the intestine is a selective barrier permitting nutrient and electrolyte absorption yet acting to protect the underlying tissue compartments and cellular components from ...attack and infiltration by antigens, bacteria and bacterial products present in the lumen. Disruption of this barrier has been associated with inflammatory bowel disease (IBD). The adherens junction (AJ), together with tight junctions (TJ) and desmosomes, form an apical junction complex that controls epithelial cell-to-cell adherence and barrier function as well as regulation of the actin cytoskeleton, intracellular signalling pathways and transcriptional regulation. Numerous studies and reviews highlight the responses of TJs to physiological and pathological stimuli. By comparison, the response of AJ proteins, and the subsequent consequences for barrier function, when exposed to the IBD inflammatory milieu, is less well studied. In this review, we will highlight the roles and responses of the AJ proteins in IBD and provide suggestions for future studies. We will also consider recently proposed therapeutic strategies to preserve or restore epithelial barrier functions to prevent and treat IBD.
Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed ...neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.
Akt signalling regulates glycolysis and drives the Warburg effect in cancer, thus decreased glucose utilisation is a pharmacodynamic marker of Akt inhibition. However, cancer cells can utilise ...alternative nutrients to glucose for energy such as lactate, which is often elevated in tumours together with increased acidity. We therefore hypothesised that lactic acidosis may confer resistance to Akt inhibition.
The effect of the pan-Akt inhibitor uprosertib (GSK2141795), on HCT116 and LS174T colon cancer cells was evaluated in the presence and absence of lactic acid in vitro. Expression of downstream Akt signalling proteins was determined using a phosphokinase array and immunoblotting. Metabolism was assessed using
H nuclear magnetic resonance spectroscopy, stable isotope labelling and gas chromatography-mass spectrometry.
Lactic acid-induced resistance to uprosertib was characterised by increased cell survival and reduced apoptosis. Uprosertib treatment reduced Akt signalling and glucose uptake irrespective of lactic acid supplementation. However, incorporation of lactate carbon and enhanced respiration was maintained in the presence of uprosertib and lactic acid. Inhibiting lactate transport or oxidative phosphorylation was sufficient to potentiate apoptosis in the presence of uprosertib.
Lactic acidosis confers resistance to uprosertib, which can be reversed by inhibiting lactate transport or oxidative metabolism.
The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of ...cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin.
Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control saline; n = 3 mice per treatment group) were used. Statistical tests were two-sided.
Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval CI = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity.
Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use.
Ovarian high-grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer with limited therapeutic options and a poor prognosis. In recent years, poly-ADP ribose polymerase (PARP) ...inhibitors have demonstrated significant clinical benefits, especially in patients with BRCA1/2 mutations. However, acquired drug resistance and relapse is a major challenge. Indisulam (E7070) has been identified as a molecular glue that brings together splicing factor RBM39 and DCAF15 E3 ubiquitin ligase resulting in polyubiquitination, degradation, and subsequent RNA splicing defects. In this work, we demonstrate that the loss of RBM39 induces splicing defects in key DNA damage repair genes in ovarian cancer, leading to increased sensitivity to cisplatin and various PARP inhibitors. The addition of indisulam also improved olaparib response in mice bearing PARP inhibitor-resistant tumors. These findings demonstrate that combining RBM39 degraders and PARP inhibitors is a promising therapeutic approach to improve PARP inhibitor response in ovarian HGSC.
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•RBM39 regulates mRNA splicing of DNA damage repair genes such as ATM and BRCA1•RBM39 depletion through indisulam synergizes with cisplatin and PARP inhibitors•Olaparib-induced activation of ATM, Chk2, and Chk1 is suppressed by indisulam•Indisulam improves response to olaparib treatment in a murine HGSC model
Xu et al. show that RBM39 modulates correct splicing of various DNA damage repair factors in ovarian cancer cells. Depletion of RBM39 through molecular glues such as indisulam leads to RNA splicing defects in these genes and improves PARP inhibitor response in vitro and in vivo.
Intestinal fibrosis with stricture formation is a complication of CD (Crohn's disease) that may mandate surgical resection. Accurate biomarkers that reflect the relative contribution of fibrosis to ...an individual stricture are an unmet need in managing patients with CD. The miRNA-29 (miR-29) family has been implicated in cardiac, hepatic and pulmonary fibrosis. In the present study, we investigated the expression of miR-29a, miR-29b and miR-29c in mucosa overlying a stricture in CD patients (SCD) paired with mucosa from non-strictured areas (NSCD). There was significant down-regulation of the miR-29 family in mucosa overlying SCD compared with mucosa overlying NSCD. miR-29b showed the largest fold-decrease and was selected for functional analysis. Overexpression of miR-29b in CD fibroblasts led to a down-regulation of collagen I and III transcripts and collagen III protein, but did not alter MMP (matrix metalloproteinase)-3, MMP-12 and TIMP (tissue inhibitor of metalloproteinase)-1 production. TGF (transforming growth factor)-β1 up-regulated collagen I and III transcripts and collagen III protein as a consequence of the down-regulation of miR-29b, and TGF-β1-induced collagen expression was reversed by exogenous overexpression of miR-29b. Furthermore, serum levels of miR-29 were lower in patients with stricturing disease compared with those without. These findings implicate the miR-29 family in the pathogenesis of intestinal fibrosis in CD and provide impetus for the further evaluation of the miR-29 family as biomarkers.
RNA‐binding motif protein 39 (RBM39) is an RNA‐binding protein involved in transcriptional co‐regulation and alternative RNA splicing. Recent studies have revealed that RBM39 is the unexpected target ...of aryl sulphonamides, which act as molecular glues between RBM39 and the DCAF15‐associated E3 ubiquitin ligase complex leading to selective degradation of the target. Loss of RBM39 leads to aberrant splicing events and differential gene expression, thereby inhibiting cell cycle progression and causing tumour regression in a number of preclinical models. Many clinical studies have shown that aryl sulphonamides were well tolerated, but their clinical performance was limited due to an insufficient understanding of the target, RBM39 biology and a lack of predictive biomarkers. This review summarises the current knowledge of RBM39 function and discusses the therapeutic potential of this spliceosome target in cancer therapy.
LINKED ARTICLES
This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc
In a recent issue of Cell, Mossmann et al.
describe a novel role for an emerging cancer target, RNA-binding motif protein 39, as a metabolic sensor of the conditionally essential amino acid arginine.
Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of ...Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor β1 (TGFβ) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. β-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin-positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.
Abstract
There is a growing appreciation that typical cell culture conditions reflect a hyperoxic condition for the majority of cell types. In an effort to more accurately reflect the in vivo ...condition, a growing number of researchers are conducting in vitro measurements at what are deemed to be more physiologically relevant oxygen concentrations (1-8% O2). The importance of oxygen concentration in in vitro model design is underscored by observations that O2 levels influence a wide range of cellular phenomena, including metabolic poise, differentiation and senescence while the development of localised hypoxic environments are implicated in a variety of diseases including stroke, and cancer. Impaired O2 supply can reduce O2 concentrations to below normal physiological levels, initiating a graded series of complex interconnected adaptive responses mediated by regulators including the hypoxia-inducible factor (HIF). These processes are particularly relevant within solid tumours, where oncogene-driven proliferation causes nutrient and oxygen deprivation, aberrant angiogenesis, and the activation of O2-sensitive survival pathways.
Despite the importance of cellular oxygenation, research using in vitro models makes the assumption that oxygenation can be defined by simply altering the oxygen condition applied to the in vitro model. To test this key assumption, cellular oxygenation measurements are performed using a nanoparticulate intracellular oxygen probe (MitoXpress®-Intra), while tumour hypoxia/oygenation is modelled on a fluorescence plate reader equipped with an atmospheric control unit (CLARIOstar®). Data is presented illustrating that the depth of hypoxia experienced by the cell model is impacted significantly by respiratory activity, and that this additional oxygen deprivation is a dynamic process, effected by respiratory substrate availability and related metabolic poise. Data is also presented demonstrating that, in in vitro models, additional respiration-induced hypoxia directly impacts glycolytic flux and other relevant metabolic pathways. Together these data invalidate the assumption that cellular oxygenation can be inferred from ambient oxygen measurements. If ignored, the significant and dynamic deviations between ambient O2 and oxygenation at the cellular level leads to erroneous collusions regarding the relationship between oxygen concentration, HIF stabilisation and related metabolic adaptations. This in turn can impair the physiological relevance of experimental observations.
Citation Format: Michelle Potter, James Hynes, Anke Nijhuis, Conn Carey, Christos Zois, Adrian Harris, Hector Keun, Karl Morten. The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2439.
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