Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis

EMBO Molecular Medicine, Journal Year: 2018, Volume and Issue: 10(11)

Published: Aug. 24, 2018

Research Article24 August 2018Open Access Source DataTransparent process Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis Alexander Greenhough Corresponding Author [email protected] orcid.org/0000-0002-8306-811X UK Colorectal Tumour Biology Group, School of Cellular & Molecular Medicine, Faculty Life Sciences, University Bristol, Epigenetics Laboratory, Search for more papers by this author Clare Bagley Kate J Heesom Proteomics Facility, David B Gurevich Biochemistry, Gay Beatson Institute, Glasgow, Mark Bond Clinical Tracey Collard Chris Paraskeva Paul Martin orcid.org/0000-0002-2665-5086 Physiology, Pharmacology and Neuroscience, Cardiff University, Cardiff, Owen Sansom orcid.org/0000-0001-9540-3010 Institute Karim Malik orcid.org/0000-0002-8965-200X Ann C Williams orcid.org/0000-0002-6009-7137 Information *,1,2,‡, Bagley1,‡, Heesom3, Gurevich4, Gay5, Bond6, Collard1, Paraskeva1, Martin4,7,8, Sansom5,9, *,2 *,1 1Cancer 2Cancer 3Proteomics 4School 5Cancer 6School 7School 8School 9Institute ‡These authors contributed equally work *Corresponding (Lead contact). Tel: +44 (0) 117 331 2043; E-mail: author. 2078; 2070; EMBO Mol Med (2018)10:e8699https://doi.org/10.15252/emmm.201708699 PDFDownload PDF article text main figures. Peer ReviewDownload summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract Hypoxia is hallmark solid tumours key physiological feature distinguishing cancer from normal tissue. However, major challenge remains in identifying tractable molecular targets that hypoxic cells depend on survival. Here, we used SILAC-based proteomics identify orphan G protein-coupled receptor GPRC5A as novel hypoxia-induced protein functions protect apoptosis during oxygen deprivation. Using genetic approaches vitro vivo, reveal HIFs direct activators transcription. Furthermore, find upregulated colonic epithelium patients with mesenteric ischaemia, colorectal cancers high correlates gene signatures poor clinical outcomes. Mechanistically, show enables survival activating Hippo pathway effector YAP its anti-apoptotic target BCL2L1. Importantly, induced depletion can be rescued constitutively active YAP. Our study identifies critical mediator adaptive response potential therapy. Synopsis This hypoxia-activated signalling facilitates tumour low conditions. directly induce expression GPRC5A, which signals via apoptosis. Orphan vivo. are regulators expression. Depletion promotes cells. activates RhoA suppress BCL-XL induction. potentially druggable exploit tumour-associated Introduction (reduced tissue O2 levels) features prominently pathophysiologies associated perturbed blood supply an important (Harris, 2002). Due cancer-specific nature regulatory role growth, has been proposed one best validated cancer-selective not yet exploited oncology (Wilson Hay, 2011). Intratumoral occurs pace growth outstrips availability exacerbated developing vasculature, often poorly formed aberrant flow (Ruoslahti, 2002; McIntyre Harris, 2015). these regions switch transcriptional mediated primarily hypoxia-inducible factors (HIFs) help them survive continue grow (Bottaro Liotta, 2003; Pouyssegur et al, 2006). considered difficult therapeutic targets, attractive prospect would mediators In study, new GPCR microenvironmental stress hypoxia. We (G Protein-coupled Receptor Class C, Group 5, Member A)—an understood regulation function—is bona fide both upregulation protects findings uncover previously unappreciated regulator highlights vulnerability opportunity Results induces Regions frequently found (Yoshimura 2004; Dekervel 2014), but previous omics studies hypoxia-mediated cellular have largely focused breast (Mole 2009; Djidja 2014; Semenza, 2017). To proteins cells, performed SW620 grown normoxia (21% O2) or (1% O2). As shown Fig 1A (and Appendix Table S1), experiments confirmed well-established HIF-regulated (e.g. carbonic anhydrase 9; CA9, ~ 2.6-fold) well reported hypoxia-regulated, (~ 1.6-fold). line our data, Western blotting (Fig 1B), apparent series bands [likely due dimerisation post-translational modifications (Zhou Rigoutsos, 2014)] verified identity using siRNA 1C, note non-specific 60 kDa band henceforth marked asterisk). other types (Tao 2007), immunofluorescence revealed localised plasma membrane 1D). generality panel malignant pre-malignant human lines 1E). Although detectable levels were present normoxia, was robustly all tested These data protein. Figure 1. A. known (red) (green) One-sample t-test performed. B. SILAC lysates. C. Validation blot siRNA. *Non-specific ˜60 depleted D. Confocal microscopy showing (scale bars: 75 μm). E. lines. F. Basal decreased HIF-1/2α depletion. G. HIF-1β H. mimetic DMOG HIF-1/2α, CA9 Dual reduced induction DMOG. I. qRT–PCR demonstrating mRNA (n = 3). normalised HPRT (error bars ± SD). J. K. ChIP-PCR analyses HIF-1α binding promoter region containing putative optimal HRE SD, n Data information: Asterisks (*) indicate band. Level adjustments made images Adobe Photoshop post-acquisition clarity (equal changes applied entire image). Representative examples 3 independent shown. available online figure. 1 [emmm201708699-sup-0003-SDataFig1.pdf] Download figure PowerPoint address whether dependent HIF-α isoforms (HIF-1α HIF-2α). Low basal strongly 1F). (Raval 2005), preferentially diminished individual HIF-2α (Figs 1F, EV1A B) abolished together indicates shared HIF-1/2. also 1G), HIF-1/2-dependent 1H). Quantitative transcript analysis following 1I J, EV1C D). assess represents HIFs, ChIP-qPCR ascertain HIF at primers spanning (Wenger 2005) element (HRE; 5′-B(A/G)CGTGVBBB-3′ [B bases except A; V T]). representing target, bound (−103/+47 relative TSS; 1K) (5′-CACGTGGCTT-3′, −58/−49), RNAPII increased 1K). controls, neither nor recruited downstream non-regulatory locus (+30,762/30,911) positive 1K), described previously. Taken together, suggest target. Click here expand EV1. requires A–D. Knockdown siRNAs (A, (C, D) LS174T Related 1F For (C representative shown; presented mean SD. hypoxia/HIF-induced vivo Having identified vitro, it investigate true context. Firstly, examined CA9) samples characterised acute deprivation (Kaidi 2007). antibodies IHC formalin-fixed paraffin-embedded 2A B). strong staining epithelium, 2C EV2). serial sections, mirrored expression, confirming support Secondly, further establish association took advantage mouse model where genes inducibly deleted specifically intestinal epithelial (Sansom Jackstadt Sansom, 2016). It noted conditional Apc deletion intestine leads Hif1a activation 9 (Newton 2010). Apc-deleted (Villin-CreERT2 Apcfl/fl) Apc/Hif1a-deleted Apcfl/fl;Hif1afl/fl) Gprc5a 70% 2D). expected, preferred Car9 Egln3 2D), Dll4 (a Hif2a target) affected Interestingly, zebrafish model, related homologue (gprc5ba) constitutive 2E; Santhakumar 2012) (Tg[fli1:eGFP;vhl−/−]) upon exposure Tg[fli1:eGFP] 2E F). Finally, bioinformatic transcriptomics dataset (GSE24551) 320 primary correlated 2G H). transcripts closely outcomes 2I). while between patient outcomes, may reflection GPRC5A's activity/hypoxia aggressive tumours, rather than necessarily indicating functional (Kaelin, regulate indicator prognosis patients. 2. A, Expression IHC. Reduced expressions confirm antibody specificity 200 sections ischaemia (strangulated colon). co-expressed 50 RT–PCR Gene housekeeping Tbp. Raw three mice) SEM). Tg[fli1:eGFP; vhl−/−] embryos (5 days post-fertilisation) demonstrate excessive angiogenesis 100 gprc5ba vhl mutant fli1:eGFP exposed 5% (vs. normoxia) 24 h (RT–PCR). G, Bioinformatic GSE24551. set HIF/hypoxia signatures. GSEA datasets Semenza_HIF1_Targets (M12299) Broad_Hallmark_Hypoxia (M5891). Analysis R2 (http://r2.amc.nl). Kaplan–Meier curve Event-free significantly expressing mRNA. 2 [emmm201708699-sup-0004-SDataFig2.pdf] EV2. expressed colon)GPRC5A immunohistochemistry transverse colon (left panels) longitudinal colon. (mesenteric ischaemia), 2A–C. The elevation implicates such evasion Exposure resulted only minor increase apoptosis, determined cleaved caspase-3 PARP 3A). Strikingly, however, displayed substantial increases apoptotic markers phenotypes each caused similar hypoxia-specific caspase-3/PARP cleavage 3B) produced corresponding reductions 3C). rule out off-target effects, generated stably carrying doxycycline-inducible siRNA-resistant (via synonymous mutations) (SW620:GPRC5Asi1R; Figs 3D EV3A–C). GPRC5Asi1R resistant siRNA-mediated knockdown 3D) 3D). accurately quantify resulting depletion, violet ratiometric asymmetry probe cytometry (which optimised apoptosis-inducing drug ABT-737 caspase inhibitor QVD; EV3D). modestly compared non-targeting control (7.82% 1.34 vs. 2.17% 0.23, 3.6-fold increase), markedly (23.37% 2.06 1.84% 0.21; 12.7-fold increase; 3E). pro-apoptotic effect QVD, 3E) 3F) analyses. 3. activation/PARP Three sequences targeting reduces growth/survival. Crystal assays growth/survival GPRC5A-depleted cDNA rescues Upper: doxycycline-induced Lower: generation siRNA1-resistant mutations. probe/dead assay experiments). Caspase QVD prevented SEM. One-way ANOVA Tukey's multiple comparisons test carried [emmm201708699-sup-0005-SDataFig3.pdf] EV3. Generation SW620:GPRC5Asi1R optimisation use Lentivirally transduced puromycin-selected (2.5 μg/ml) 48 flow-sorted (BD Influx) based medium/high TurboGFP (note pCW57-GFP-2A-GPRC5Asi1R uses P2A self-cleaving peptide produce separate cDNA). Pre- post-flow-sorted profiles shown, near 100% 3D, 4H, I, L. Confirmation doxycycline μg/ml)-treated Phase contrast green (TurboGFP) obtained IncuCyte ZOOM live imaging system 300 confirms overexpression doxycycline-treated Cells treated 72 prior harvest. Asterisk Optimisation cytometry. pan-BCL2 family (10 μM). Live, dead gated FlowJo (v10). A example 3E. required gain mechanistic insight into how might promote hypoxia, additional KEGG ontology signalling, particularly later stage (Appendix S2). Since formation (Rosenbluh 2012; Zanconato 2016), total EV4A). determine there connection stability presence absence siRNAs. Ser397 phosphorylation stabilises 4A). Remarkably, phosphorylated persisted suggesting stabilisation this, nuclear localisation activity 8× GTIIC-luciferase (TEAD) reporter. Nuclear abrogated 4B). TEAD 4C) established AREG, CYR61, CTGF BCL2L1 EV4B–E). EV4. lin

Language: Английский

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Degree and site of chromosomal instability define its oncogenic potential

Nature Communications, Journal Year: 2020, Volume and Issue: 11(1)

Published: March 20, 2020

Abstract Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies thus still unclear. Using conditional mouse model for diverse degrees CIN, we find that particular range is sufficient drive very early onset spontaneous adenoma intestine. In mice predisposed intestinal cancer ( Apc Min/+ ), moderate causes remarkable increase burden entire tract especially distal colon, which resembles disease. Strikingly, higher level promotes colon even more than does, but has no effect small Our results show can be potently oncogenic, certain levels contrasting effects distinct tissues.

Language: Английский

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Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors

Nature Communications, Journal Year: 2019, Volume and Issue: 10(1)

Published: May 24, 2019

Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to lack models vivo. Here we show that expression EMT master inducer Snail primary adult Drosophila intestinal tumors leads dissemination tumor cells and formation macrometastases. drives an cells, which, although retaining some epithelial markers, subsequently break through basal lamina midgut, undergo a collective migration seed polyclonal metastases. While metastases re-epithelialize over time, found early are remarkably mesenchymal, discarding requirement for mesenchymal-to-epithelial transition stages metastatic growth. Our results demonstrate flies, identify key role partial-EMTs driving it. This model opens door investigate basic mechanisms underlying metastasis, powerful vivo system suited rapid genetic drug screens.

Language: Английский

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AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer

The Journal of Experimental Medicine, Journal Year: 2020, Volume and Issue: 217(10)

Published: Aug. 4, 2020

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack animal models that faithfully recapitulate different subtypes impedes adequate preclinical testing stratified therapeutic concepts. Here, we demonstrate constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with carcinogen azoxymethane. Gene-expression profiling indicates Trp53ΔIECAktE17K tumors resemble human mesenchymal (CMS4), is characterized by poorest survival rate among four CMSs. are Notch3 up-regulation, treatment NOTCH3-inhibiting antibody reduces metastasis. In patients, NOTCH3 expression correlates positively grading presence lymph node as well distant metastases specifically up-regulated CMS4 tumors. Therefore, suggest putative target for advanced patients.

Language: Английский

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European Groundshot—addressing Europe's cancer research challenges: a Lancet Oncology Commission

The Lancet Oncology, Journal Year: 2022, Volume and Issue: 24(1), P. e11 - e56

Published: Nov. 15, 2022

Language: Английский

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Advances in colon cancer research: in vitro and animal models

Current Opinion in Genetics & Development, Journal Year: 2021, Volume and Issue: 66, P. 50 - 56

Published: Jan. 9, 2021

Modelling human colon cancer has long been the ambition of researchers and oncologists with aim to better replicate disease progression treatment response. Advances in our understanding genetics, stem cell biology, tumour microenvironment immunology have prepared groundwork for recent major advances. In last two years field seen of: using patient derived organoids (alone co-culture) as predictors response; molecular stratification tumours that predict outcome mouse models metastatic disease; transplant can be used de-risk clinical trials. We will discuss these advances this review.

Language: Английский

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An innate IL-25–ILC2–MDSC axis creates a cancer-permissive microenvironment for Apc mutation–driven intestinal tumorigenesis

Science Immunology, Journal Year: 2022, Volume and Issue: 7(72)

Published: June 3, 2022

Interleukin-25 (IL-25) and group 2 innate lymphoid cells (ILC2s) defend the host against intestinal helminth infection are associated with inappropriate allergic reactions. IL-33–activated ILC2s were previously found to augment protective tissue-specific pancreatic cancer immunity. Here, we showed that IL-25–activated created an cancer-permissive microenvironment. Colorectal (CRC) patients higher tumor IL25 expression had reduced survival increased IL-25R–expressing tumor-resident myeloid-derived suppressor (MDSCs) impaired antitumor responses. Ablation of IL-25 signaling tumors, virtually doubling life expectancy in Apc mutation–driven model spontaneous tumorigenesis. Mechanistically, promoted intratumoral ILC2s, which sustained tumor-infiltrating MDSCs suppress Therapeutic antibody-mediated blockade decreased MDSCs, adenoma/adenocarcinoma while increasing adaptive T cell interferon-γ (IFN-γ)–mediated Thus, roles epithelium-derived cytokines IL-33 as well cannot be generalized. The protumoral nature IL-25–ILC2 axis CRC highlights this pathway a potential therapeutic target CRC.

Language: Английский

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Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies

Frontiers in Genetics, Journal Year: 2022, Volume and Issue: 13

Published: Aug. 8, 2022

Cancer is a complex disease whereby multiple genetic aberrations, epigenetic modifications, metabolic reprogramming, and the microenvironment contribute to development of tumor. In traditional anticancer drug discovery pipeline, candidates are usually screened in vitro using two-dimensional or three-dimensional cell culture. However, these methods fail accurately mimic human state. This has led poor success rate drugs preclinical stages since many abandoned due inefficacy toxicity when transitioned whole-organism models. The common fruit fly, Drosophila melanogaster , emerged as beneficial system for modeling cancers. Decades fundamental research have shown evolutionary conservation key genes signaling pathways between flies humans. Moreover, lower redundancy comparison mammals. These factors, addition advancement toolkits manipulating gene expression, allow generation genotypes phenotypes. Numerous studies successfully created models colorectal, lung, thyroid, brain were utilized high-throughput screening FDA-approved which identification several compounds capable reducing proliferation rescuing More noteworthy, also unlocked potential personalized therapies. ‘avatars’ presenting same mutations patient used screen therapeutic agents targeting find most appropriate combination drugs. outcomes translated significant responses patients with adenoid cystic carcinoma metastatic colorectal Despite not being widely utilized, concept vivo making contributions current pipeline. this review, we discuss application platform discovery; special focus on cancer that been generated, libraries status addition, elaborate biological technical limitations system.

Language: Английский

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FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

Advanced Science, Journal Year: 2023, Volume and Issue: 10(35)

Published: Oct. 24, 2023

The forkhead box transcription factor A2 (FOXA2) is a and plays key role in embryonic development, metabolism homeostasis tumor cell proliferation; however, its regulatory potential CRC not fully understood. Here, it found that FOXA2 expression markedly up-regulated samples of patients as compared with the normal tissues, which closely associated worse survival CRC. Notably, positive correlation between nuclear erythroid 2-related 2 (Nrf2)/glutathione peroxidase 4 (GPX4) gene observed patients. Mechanistically, depletion weakens activation Nrf2 pathway decreases GPX4 level cells, thereby leading to ferroptosis, further supported by bioinformatic analysis. More intriguingly, E3 ubiquitin ligase tripartite motif containing 36 (TRIM36) identified suppressor FOXA2, TRIM36 can directly interact induce K48-linked polyubiquitination, resulting protein degradation vitro. Taken together, all studies demonstrate mediated promotes progression inhibiting Nrf2/GPX4 ferroptosis signaling pathway, thus providing new therapeutic target for treatment.

Language: Английский

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

Nature Metabolism, Journal Year: 2023, Volume and Issue: 5(8), P. 1303 - 1318

Published: Aug. 14, 2023

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutant KRAS. Here we used genetically engineered mouse models, multimodal mass spectrometry-based metabolomics to study the impact common genetic drivers CRC on metabolic intestine. We show that untargeted profiling can be applied stratify intestinal tissues according underlying alterations, use spectrometry imaging identify tumour, stromal normal adjacent tissues. By identifying ions drive variation between transformed tissues, found dysregulation methionine cycle a hallmark APC-deficient CRC. Loss Apc intestine was sufficient expression one its enzymes, adenosylhomocysteinase (AHCY), which also transcriptionally upregulated human Targeting AHCY function impaired growth organoids vitro, prevented characteristic hyperproliferative/crypt progenitor phenotype driven acute deletion vivo, even context Kras. Finally, pharmacological inhibition reduced burden

Language: Английский

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