Sensitivity towards HDAC inhibition is associated with RTK / MAPK pathway activation in gastric cancer
2022; Springer Nature; Volume: 14; Issue: 10 Linguagem: Inglês
10.15252/emmm.202215705
ISSN1757-4684
AutoresTherese Seidlitz, Tim Schmäche, Fernando Garcı́a, Joon Ho Lee, Nan Qin, Susan Kochall, Juliane Fohgrub, David Pauck, Alexander Rothe, Bon‐Kyoung Koo, Jürgen Weitz, Marc Remke, Javier Muñoz, Daniel E. Stange,
Tópico(s)Melanoma and MAPK Pathways
ResumoArticle22 August 2022Open Access Transparent process Sensitivity towards HDAC inhibition is associated with RTK/MAPK pathway activation in gastric cancer Therese Seidlitz Therese Seidlitz orcid.org/0000-0001-7367-5525 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Tim Schmäche Tim Schmäche orcid.org/0000-0002-2770-285X Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Fernando Garcίa Fernando Garcίa orcid.org/0000-0003-3888-4326 Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Joon Ho Lee Joon Ho Lee Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Nan Qin Nan Qin Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Susan Kochall Susan Kochall Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Juliane Fohgrub Juliane Fohgrub Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author David Pauck David Pauck Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Formal analysis, Funding acquisition, Visualization, Methodology Search for more papers by this author Alexander Rothe Alexander Rothe orcid.org/0000-0002-1958-6182 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea Contribution: Writing - review & editing Search for more papers by this author Jürgen Weitz Jürgen Weitz Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Writing - review & editing Search for more papers by this author Marc Remke Marc Remke Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Supervision, Writing - review & editing Search for more papers by this author Javier Muñoz Corresponding Author Javier Muñoz [email protected] orcid.org/0000-0003-3288-3496 Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Biocruces Bizkaia Health Research Institute, Barakaldo, Spain Ikerbasque, Basque Foundation for Science, Bilbao, Spain Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Daniel E Stange Corresponding Author Daniel E Stange [email protected] orcid.org/0000-0003-4246-2230 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Therese Seidlitz Therese Seidlitz orcid.org/0000-0001-7367-5525 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Tim Schmäche Tim Schmäche orcid.org/0000-0002-2770-285X Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Fernando Garcίa Fernando Garcίa orcid.org/0000-0003-3888-4326 Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Joon Ho Lee Joon Ho Lee Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Nan Qin Nan Qin Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Susan Kochall Susan Kochall Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Juliane Fohgrub Juliane Fohgrub Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author David Pauck David Pauck Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Formal analysis, Funding acquisition, Visualization, Methodology Search for more papers by this author Alexander Rothe Alexander Rothe orcid.org/0000-0002-1958-6182 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Data curation, Formal analysis, Visualization, Methodology Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea Contribution: Writing - review & editing Search for more papers by this author Jürgen Weitz Jürgen Weitz Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Writing - review & editing Search for more papers by this author Marc Remke Marc Remke Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Contribution: Supervision, Writing - review & editing Search for more papers by this author Javier Muñoz Corresponding Author Javier Muñoz [email protected] orcid.org/0000-0003-3288-3496 Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Biocruces Bizkaia Health Research Institute, Barakaldo, Spain Ikerbasque, Basque Foundation for Science, Bilbao, Spain Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Daniel E Stange Corresponding Author Daniel E Stange [email protected] orcid.org/0000-0003-4246-2230 Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany National Center for Tumor Diseases (NCT), Dresden, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Therese Seidlitz1,†, Tim Schmäche1,2,3,4,†, Fernando Garcίa5,†, Joon Ho Lee1, Nan Qin6, Susan Kochall1, Juliane Fohgrub1, David Pauck6, Alexander Rothe1, Bon-Kyoung Koo7,8, Jürgen Weitz1,2,3,4, Marc Remke6, Javier Muñoz *,5,9,10 and Daniel E Stange *,1,2,3,4 1Department of Visceral, Thoracic and Vascular Surgery, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany 2National Center for Tumor Diseases (NCT), Dresden, Germany 3German Cancer Research Center (DKFZ), Heidelberg, Germany 4Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany 5Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 6Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany 7Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria 8Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea 9Biocruces Bizkaia Health Research Institute, Barakaldo, Spain 10Ikerbasque, Basque Foundation for Science, Bilbao, Spain † These authors contributed equally to this work *Corresponding author. Tel: +34 946007967; E-mail: [email protected] author. Tel: +49 (0)351 458 2742; E-mail: [email protected] EMBO Mol Med (2022)14:e15705https://doi.org/10.15252/emmm.202215705 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Gastric cancer ranks the fifth most common and third leading cause of cancer-related deaths worldwide. Alterations in the RTK/MAPK, WNT, cell adhesion, TP53, TGFβ, NOTCH, and NFκB signaling pathways could be identified as main oncogenic drivers. A combination of altered pathways can be associated with molecular subtypes of gastric cancer. In order to generate model systems to study the impact of different pathway alterations in a defined genetic background, we generated three murine organoid models: a RAS-activated (KrasG12D, Tp53R172H), a WNT-activated (Apcfl/fl, Tp53R172H), and a diffuse (Cdh1fl/fl, Apcfl/fl) model. These organoid models were morphologically and phenotypically diverse, differed in proteome expression signatures and possessed individual drug sensitivities. A differential vulnerability to RTK/MAPK pathway interference based on the different mitogenic drivers and according to the level of dependence on the pathway could be uncovered. Furthermore, an association between RTK/MAPK pathway activity and susceptibility to HDAC inhibition was observed. This finding was further validated in patient-derived organoids from gastric adenocarcinoma, thus identifying a novel treatment approach for RTK/MAPK pathway altered gastric cancer patients. Synopsis The analysis of murine and human gastric tumor organoids uncovered an association between RTK/MAPK pathway activity and susceptibility to HDAC inhibition, thereby identifying a potential new treatment approach for gastric cancer patients. Three murine tumor organoid models with a defined genetic background were generated by altering frequently mutated pathways in gastric cancer. The organoid models were characterized concerning their phenotype, proteome expression pattern, and therapeutic response via a drug screen. RAS activation in murine tumor organoids led to a significantly increased sensitivity to HDAC inhibition. HDAC sensitivity was confirmed in patient derived gastric cancer organoids with MAPK pathway alterations. The paper explained Problem Gastric cancer ranks the fifth most common and third leading cause of cancer-related deaths worldwide. Patient-derived cancer organoids (PDOs) constitute a three-dimensional cell culture system with self-renewal and self-organization capability recapitulating many aspects of the parental tumor. They also retain the complex individual mutational landscape, carrying between a few hundred to several thousand mutations. Data generated in these PDOs can, therefore, often not be generalized, but need to be interpreted bearing in mind the singularity of the analyzed tumor. The Cancer Genome Atlas (TCGA) consortium developed a molecular classification system of gastric cancer by describing four different subtypes with characteristic mutations and associated deregulated pathways. Organoids with defined pathway alterations could overcome the limitations of PDOs. Results We generated three murine organoid models with a defined genetic makeup by activating frequently altered pathways: a RAS-activated (KrasG12D, Tp53R172H), a WNT-activated (Apcfl/fl, Tp53R172H), and a diffuse (Cdh1fl/fl, Apcfl/fl) model. These organoid models were characterized concerning their phenotype, proteome expression, and sensitivity to drug treatment. We observed different organoid morphologies as well as proliferation rates. All three models altered the expression of a significant fraction of their proteome, affecting multiple processes and functions. A divergent response pattern to classical chemotherapy and targeted small molecules was recognized in a drug screen. We analyzed in detail the response of RAS-activated organoids upon interference with the RTK/MAPK pathway at different levels, revealing a sensitivity on the level of B-RAF and MEK1/2, but no differential response on the level of ERK1/2. Furthermore, the RAS-activated organoids showed a significantly increased sensitivity to HDAC inhibition. To evaluate whether this correlation is translatable to human gastric cancer, we analyzed gastric cancer PDOs and could show similar to the murine organoid model a sensitivity of RTK/MAPK-altered PDOs to trametinib and HDAC inhibitors. Impact By using murine and human organoids with RTK/MAPK alterations, an association between MAPK pathway activity and susceptibility to HDAC inhibition was uncovered, delineating a novel treatment approach for RTK/MAPK pathway altered gastric cancer patients. Introduction Gastric cancer ranks as the fifth most common and third leading cause of cancer-related deaths worldwide (Bray et al, 2018; Ferlay et al, 2019). The diagnosis of gastric cancer is often delayed due to the lack of early clinical signs resulting in a high percentage of patients with incurable disease (Hunt et al, 2015). The widely used Lauren classification divided gastric cancer based on the morphological appearance into the intestinal, diffuse, and intermediate types (Lauren, 1965). Next to the histology-based classification, several attempts have been made to use molecular data to classify gastric cancer. The Asian Cancer Research Group (ACRG) classified gastric cancer based on gene expression data into four subtypes: (i) microsatellite instability (MSI), (ii) microsatellite stable and epithelial-to-mesenchymal transition (MSS/EMT), (iii) MSS/TP53 active, and (iv) MSS/TP53 inactive (Cristescu et al, 2015). The Singapore classification system distinguished three subtypes: proliferative, metabolic, and mesenchymal (Lei et al, 2013). The Cancer Genome Atlas (TCGA) consortium developed a molecular classification system based on observed mutational alterations and grouped gastric cancer into four subtypes (The Cancer Genome Atlas Research Network, 2014). One subtype is characterized by an Epstein Barr virus (EBV) infection and named "EBV-positive" subtype. A second subtype shows a high frequency of MSI and is, therefore, named "MSI" subtype. A third subtype is termed "genomically stable" (GS), displaying a diffuse non-coherent cancer morphology due to the loss of proteins involved in cell adhesion, such as CDH1. A fourth molecular subtype, named "chromosomal instability" (CIN) subtype, is characterized by a high number of somatic copy number alterations (SCNA). Of note, none of the classification systems up to today influence clinical decision-making, as no convincing link has been established between individual subtypes and certain treatment schemes. Organoids constitute a three-dimensional (3D) cell culture system directly derived from tissue-resident stem cells (Sato et al, 2009). Cells are embedded in an extracellular matrix (ECM) and exposed to growth factors present in the native microenvironment. Organoid cultures show self-renewal, self-organization, and long-term proliferation capacities while faithfully recapitulating many aspects of the tissue they are derived from. Organoids from healthy tissue remain genomically stable over long periods of time (Huch et al, 2015; Georgakopoulos et al, 2020). Initially developed from intestinal stem cells, protocols have been developed to establish organoids from multiple murine and human organs (Fatehullah et al, 2016; Bartfeld & Clevers, 2017). They represent an excellent model system to be employed in a broad range of research topics from basic to translational science, that is, organ development, infection studies, or disease modeling. In addition, patient-derived cancer organoids (PDOs) have been shown to be predictive of the patient's response to a certain anticancer treatment (Vlachogiannis et al, 2018; Wensink et al, 2021). Each PDO line has an individual pattern of molecular alterations with hundreds of mutations and deregulated signaling pathways. Due to this, they represent unique avatars of a patient, rather than models for a particular cancer (subtype). We thus established organoid models with a defined mutational spectrum altering specific pathways (Seidlitz et al, 2019). Here, these two models were complemented by an additional model and all three were extensively characterized concerning their molecular and functional behavior using proteomics and a drug screen. Specific treatment vulnerabilities were then further validated in PDOs from gastric cancer. Results Generation and phenotypic characterization of murine gastric organoid models with defined oncogenic pathway alterations In order to define the most frequently altered mutations in gastric cancer, we analyzed the TCGA dataset and determined alteration frequencies for the four established molecular subtypes (The Cancer Genome Atlas Research Network, 2014). RTK/MAPK pathway alterations combined with TP53 mutations are characteristic for the CIN subtype, but can also be found in the MSI and GS subtypes. To model this pathway combination, we coupled inducible alleles of KrasG12D and Tp53R172H (RAS-activated model). Cell motility genes such as CDH1 are frequently mutated in the GS subtype, which largely overlaps with the diffuse subtype according to Lauren, and are associated with additional activations of oncogenic pathways, that is, the TGFβ, RTK/MAPK, or WNT (Smyth et al, 2020). We chose to combine a floxed Cdh1 with a floxed Apc allele to model the diffuse subtype. These two models have been previously established (Seidlitz et al, 2019). WNT pathway alterations also frequently occur in other gastric cancer subtypes, with 78% of cases most prominently in the MSI subtype, which also contains TP53 pathway alterations in 77% of cases (The Cancer Genome Atlas Research Network, 2014). A third organoid model was, therefore, established by combining a floxed Apc with an inducible Tp53R172H allele (WNT-activated model). Organoids were generated from the gastric corpus and mutations activated via infection with a Cre/GFP expressing adenovirus (Fig EV1A). They were selected via withdrawal of specific growth factors from the cultivation medium, resulting in the outgrowth of only recombined organoids (Fig EV1B–E). Figure 1. Immunohistological characterization of the organoid models A. Brightfield pictures of normal versus RAS-activated organoids (1 and 2), HE staining (3 and 4), TP53 (5 and 6) and pERK1/2 (7 and 8) (scale bar 15 μm). B. Brightfield pictures of normal versus diffuse organoids (9 and 10), HE staining (11 and 12), E-cadherin (13 and 14), and β-catenin (15 and 16) (scale bar 15 μm). C. Brightfield pictures of normal versus WNT-activated organoids (17 and 18), HE staining (19 and 20), TP53 (21 and 22), and β-catenin (23 and 24) (scale bar 15 μm). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation of organoid models A. Adenoviral infection of organoids with a Cre-GFP expressing recombinase. Fluorescence microscopy 24-h post-infection (scale bar 100 μm). B. Selection of organoids based on altered pathway. The RAS-activated model was selected via EGF removal from the normal cultivation medium. The diffuse and WNT-activated models were enriched by depletion of WNT3A and Rspondin (scale bar 25 μm). C–E. Genotyping PCRs of infected and selected organoid models to document successful recombination. Tp53R172H/+ in the WNT-activated organoids showed loss of heterozygosity of the wild-type allele after activating the R172H mutation. Download figure Download PowerPoint Normal murine gastric corpus organoids had a cystic structure with a thin single-layered epithelium (Fig 1A1 an A3). As described before, the RAS-activated model displayed a multi-layered irregular epithelium (Fig 1A2 and A4; Seidlitz et al, 2019). Contrasting to normal organoids, the Tp53R172H mutation resulted in a nuclear accumulation of TP53 (Fig 1A5 and A6). Due to the EGF in the culture medium, normal organoids showed active epidermal growth factor receptor (EGFR) pathway signaling, demonstrated by phosphorylated nuclear ERK1/2 (Fig 1A7). The KrasG12D mutation in the RAS-activated model resulted in an increase in the ERK1/2 phosphorylation level compared with normal organoids (Fig 1A8). The Cdh1 loss in the diffuse organoid model led to a complete change in organoid morphology toward a grape-like structure (Fig 1B9–B12; Seidlitz et al, 2019). Cdh1, which encodes for the cell–cell junction protein E-cadherin, was absent in the diffuse model organoids (Fig 1B13 and B14), and the activation of the WNT pathway resulted in a nuclear accumulation of β-catenin (Fig 1B15 and B16). The newly established WNT-activated model was characterized phenotypically by an irregular mono-layered structure with a rather small organoid size (Fig 1C17–C20). Due to the Tp53R172H mutation and Apc deletion, a nuclear accumulation of TP53 and β-catenin could be observed (Fig 1C21–C24). Cell cycle and proliferation analyses revealed different proliferation rates between the three organoid models. The RAS-activated and WNT-activated organoids contained with 12.7 and 9.4% a higher number of cells within the S-phase, respectively, compared with their normal counterpart (7.4%) (Fig EV2A and B). EdU incorporation assays confirmed this finding, both models also had a significantly higher proliferation rate compared with normal gastric organoids (RAS-activated 36.1%, two-tailed Student's t-test P = 0.031; WNT-activated 38.1%, P = 0.0017; Fig EV2C). The diffuse organoid model showed the lowest number of proliferation with 6.4% of cells in the S-phase and 21% of EdU positivity (normal organoids 26.4%; Fig EV2A–C). Figure 2. Proteomic characterization of organoid models A–C. Volcano plots of proteomic analyses of the organoid models. Plots indicate up- and downregulated proteins compared with uninduced controls (biological replicates n = 2–3). D. Comparison of significantly altered biological processes (Gene Ontology) identified by gene set enrichment analyses (GSEA) (q-value < 0.1) between the organoid models. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Proliferative capacity of organoid models A. Exemplary cell cycle analysis of normal organoids and organoid models. B. Quantitative representation of cell cycle analysis (two-tailed Student's t-test model vs. normal; *< 0.05; **< 0.01; RAS-activated P4 G2/M phase P = 0.0105, WNT-activated P2 G0/G1 phase P = 0.0061, biological replicate n = 3, data are shown as mean ± SD). C. Proliferation rate of organoids assessed by EdU proliferation assays. Two-tailed Student's t-test organoid models versus normal stomach organoids (*< 0.05; **< 0.01; RAS-activated P = 0.031; WNT-activated P = 0.0017, biological replicates n = 3, data are shown as mean ± SD). Download figure Download PowerPoint Oncogenic pathway activations resulted in individual proteome signatures To understand the underlying molecular biology present in each organoid model, we conducted global proteomic analyses (Fig 2A–C, Dataset EV1). We analyzed the proteomes of the altered organoids with respect to the normal (uninduced) organoids of the same genotype. The RAS-activated model showed an altered expression in 978 proteins (455 down- and 523 upregulated) compared with normal gastric organoids (Fig 2A). In the diffuse organoid model, 635 proteins were differentially expressed (363 down- and 272 upregulated; Fig 2B), and in the WNT-activated organoid model, 341 differentially expressed proteins were found (193 down- and 148 upregulated; Fig 2C). As expected, a significantly downregulation of the CDH1 protein was found in the diffuse organoid model (Log2 diffuse/normal −1.595, Limma significance = DOWN < −0.4, P-value = < 0.05; Dataset EV1). Activating the WNT pathway by Apc deletion resulted in a significant upregulation of the WNT target gene matrix metallopeptidase 7 (MMP7; Log2 WNT-activated/normal 0.624, Limma significance = UP > 0.4, P-value = < 0.05) in the WNT-activated model, which was not seen in the diffuse model (Dataset EV1). To understand the proteomic changes present in each model, we performed gene set enrichment analysis (GSEA) of Gene Ontology (GO) terms and found a large number of biological processes displaying both upregulated and downregulated proteins across the three different models (q-value < 0.1; Fig 2D). In detail, the RAS-activated model carrying the R172H mutation in the tumor suppressor Tp53 showed a downregulation of GO term "cell cycle phase control" (Normalized Enrichment Score (NES) −2.26, q-value < 0.000; Fig 3A; Dataset EV2). Importantly, this was not seen for the WNT-activated model (NES 1.02, q-value = 0.743) harboring the same Tp53R172H mutation. The diffuse model also showed no altered cell cycle control (NES 1.44, q-value = 0.276). Furthermore, in the RAS-activated model a downregulation of the GO term "double strand break repair" (NES −2.01, q-value = 0.007) was seen (Dataset EV2). This was again not observed in the WNT-activated organoids (NES 1.32, q-value = 0.40). For the WNT-activated model, a significant increase in the GO term "nuclear DNA replication" was observed (NES 1.93, q-value = 0.02), while the RAS-activated organoids showed the opposite pattern (NES −1.72, q-value = 0.008; Dataset EV2). An activation of translational processes was detected in the RAS-activated organoids, that is, elongation (NES 2.26, q-value < 0.000) and termination (NES 2.14, q-value = 0.002). This was not recognized for the diffuse (NES −1.39, q-value = 0.316) and WN
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