TGFβ signaling directs serrated adenomas to the mesenchymal colorectal cancer subtype
2016; Springer Nature; Volume: 8; Issue: 7 Linguagem: Inglês
10.15252/emmm.201606184
ISSN1757-4684
AutoresEvelyn Fessler, Jarno Drost, Sander R. van Hooff, Janneke F. Linnekamp, Xin Wang, Marnix Jansen, Felipe de Sousa e Melo, Pramudita R. Prasetyanti, Joep IJspeert, Marek Franitza, Peter Nürnberg, Carel J.M. van Noesel, Evelien Dekker, Louis Vermeulen, Hans Clevers, Jan Paul Medema,
Tópico(s)Colorectal Cancer Treatments and Studies
ResumoResearch Article24 May 2016Open Access Source DataTransparent process TGFβ signaling directs serrated adenomas to the mesenchymal colorectal cancer subtype Evelyn Fessler Evelyn Fessler Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Jarno Drost Jarno Drost Cancer Genomics Center, Amsterdam, The Netherlands Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sander R van Hooff Sander R van Hooff Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Janneke F Linnekamp Janneke F Linnekamp Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Xin Wang Xin Wang Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong Search for more papers by this author Marnix Jansen Marnix Jansen Department of Pathology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Felipe De Sousa E Melo Felipe De Sousa E Melo Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Pramudita R Prasetyanti Pramudita R Prasetyanti Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Joep EG IJspeert Joep EG IJspeert Department of Gastroenterology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Marek Franitza Marek Franitza Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Peter Nürnberg Peter Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Carel JM van Noesel Carel JM van Noesel Department of Pathology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Evelien Dekker Evelien Dekker Department of Gastroenterology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Louis Vermeulen Louis Vermeulen Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Hans Clevers Hans Clevers Cancer Genomics Center, Amsterdam, The Netherlands Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Jan Paul Medema Corresponding Author Jan Paul Medema orcid.org/0000-0003-3045-2924 Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Evelyn Fessler Evelyn Fessler Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Jarno Drost Jarno Drost Cancer Genomics Center, Amsterdam, The Netherlands Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sander R van Hooff Sander R van Hooff Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Janneke F Linnekamp Janneke F Linnekamp Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Xin Wang Xin Wang Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong Search for more papers by this author Marnix Jansen Marnix Jansen Department of Pathology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Felipe De Sousa E Melo Felipe De Sousa E Melo Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Pramudita R Prasetyanti Pramudita R Prasetyanti Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Joep EG IJspeert Joep EG IJspeert Department of Gastroenterology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Marek Franitza Marek Franitza Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Peter Nürnberg Peter Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Carel JM van Noesel Carel JM van Noesel Department of Pathology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Evelien Dekker Evelien Dekker Department of Gastroenterology, AMC, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Louis Vermeulen Louis Vermeulen Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Hans Clevers Hans Clevers Cancer Genomics Center, Amsterdam, The Netherlands Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Jan Paul Medema Corresponding Author Jan Paul Medema orcid.org/0000-0003-3045-2924 Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands Search for more papers by this author Author Information Evelyn Fessler1,2, Jarno Drost2,3, Sander R Hooff1,2, Janneke F Linnekamp1,2, Xin Wang4, Marnix Jansen5,9, Felipe De Sousa E Melo1,2,10, Pramudita R Prasetyanti1,2, Joep EG IJspeert6, Marek Franitza7,8, Peter Nürnberg7,8, Carel JM Noesel5, Evelien Dekker6, Louis Vermeulen1, Hans Clevers2,3 and Jan Paul Medema 1,2 1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands 2Cancer Genomics Center, Amsterdam, The Netherlands 3Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, The Netherlands 4Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong 5Department of Pathology, AMC, University of Amsterdam, Amsterdam, The Netherlands 6Department of Gastroenterology, AMC, University of Amsterdam, Amsterdam, The Netherlands 7Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany 8Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 9Present address: Centre for Tumour Biology, Barts Cancer Institute, University of London, London, UK 10Present address: Department of Molecular Oncology, Genentech Inc., San Francisco, CA, USA *Corresponding author. Tel: +31 20 56 67777; E-mail: [email protected] EMBO Mol Med (2016)8:745-760https://doi.org/10.15252/emmm.201606184 PDFDownload PDF of article text and main figures. 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 The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS. Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFβ plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFβ is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFβ treatment prevails in a genetically engineered organoid culture carrying a BRAFV600E mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFβ signaling is already active in SSA precursor lesions and that TGFβ is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC. Synopsis Sessile serrated adenomas (SSAs)—precursor lesions of the serrated neoplasia pathway to colorectal cancer (CRC)—are predicted to give rise to either the good- or the poor-prognosis CRC consensus molecular subtype (CMS1 and CMS4, respectively). Organoid cultures from patient-derived pre-neoplastic lesions and genetically engineered organoid cultures present valuable model systems for early stage disease. An organoid culture genetically engineered to carry the BRAFV600E mutation served as a model system for the earliest stage of the serrated neoplasia pathway. A TGFβ signature separated patient-derived tubular adenomas—precursor lesions of the classical path to CRC—from SSAs. High activity of the TGFβ signaling pathway was detected in SSAs that would likely have—judged by gene expression-based prediction—developed to CMS4-like CRCs. Introduction Transforming growth factor-β (TGFβ) signaling controls a plethora of physiological programs, influencing cellular behavior and tissue homeostasis. The response to TGFβ is highly context specific and results in distinct or even opposite effects for different cell types (Massague, 2012). The multifunctional nature of this cytokine can also be observed in tumorigenesis, where it plays either a tumor-suppressing or tumor-promoting role (Massague, 2008). Induction of apoptosis, for example, by upregulation of the pro-apoptotic molecule BIM and the death-associated protein kinase (DAPK) (Jang et al, 2002; Ramesh et al, 2008; Heldin et al, 2009), as well as the arrest of proliferation by the induction of cyclin-dependent kinase inhibitors such as p21CIP1, are examples of tumor-suppressive mechanisms (Hannon & Beach, 1994; Datto et al, 1995; Massague, 2008). On the other hand, tumors can benefit from activated TGFβ signaling due to its ability to enhance cancer cell invasion and metastasis. The TGFβ signaling molecule is a well-known inducer of the epithelial–mesenchymal transition (EMT) (Moustakas & Heldin, 2007), a process during which epithelial-organized cells lose their cell–cell junctions and gain a mesenchymal, migratory, and invasive phenotype, allowing them to leave the primary site and spread to distant organs (Thiery, 2002). Recent insight from murine models, however, proposes that EMT may be related to therapy resistance rather than enhanced metastatic spread (Fischer et al, 2015; Zheng et al, 2015). Regardless of the exact mechanism, the mesenchymal phenotype has been linked to dismal prognosis in patients for many cancer types, including colorectal cancer (CRC) (Guinney et al, 2015). CRC samples can be classified into four distinct consensus molecular subtypes (CMSs), of which CMS4 displays a mesenchymal gene expression profile and dismal clinical outcome (Guinney et al, 2015). Even though components of the TGFβ signaling pathway are often inactivated during CRC progression (Markowitz et al, 1995; Fleming et al, 2013), it has been shown that active TGFβ signaling—judged by phosphorylation of the downstream components SMAD2/3 (phospho-SMAD2/3)—can be found in colorectal carcinomas (Brunen et al, 2013). Tumors associated with poor prognosis display significantly higher levels of phospho-SMAD2/3 also in the tumor epithelium and activation of the TGFβ pathway has been linked to resistance to conventional and targeted chemotherapeutics (Huang et al, 2012; Brunen et al, 2013). For a long time, the development of CRC was thought to follow a molecularly well-defined route with the inactivation of the adenomatous polyposis coli (APC) gene as the initiating event followed by the activation of the KRAS oncogene and the inactivation of TP53 as the tumor progresses to a metastatic carcinoma (Fearon & Vogelstein, 1990). The tubular adenoma (TA) was viewed as the main epithelial precursor lesion to spawn colorectal malignancies (Muto et al, 1975). However, over the last two decades, it has become increasingly clear that the heterogeneity observed in colorectal malignancies is also reflected already at the premalignant stage (IJspeert et al, 2015). The classical path of CRC development and progression has been complemented with the serrated neoplasia pathway (Snover, 2011). Several types of serrated polyps have been described (IJspeert et al, 2015), and the sessile serrated adenoma (SSA) could be linked to malignant progression to CRC (Oono et al, 2009; Lash et al, 2010; IJspeert et al, 2015). Histologically and molecularly SSAs present as distinct entities, characterized by a serrated morphology and the activation of the BRAF oncogene (Leggett & Whitehall, 2010; Snover, 2011). Furthermore, serrated lesions often display DNA hypermethylation of CpG islands in promoter regions, leading to silencing of tumor suppressor genes (also known as the CpG island methylator phenotype or CIMP) (Park et al, 2003; Kambara et al, 2004). Specific precursor lesions have been suggested to develop into different types of CRC based on gene expression profiling and SSAs were suggested to harbor the potential to develop into the mesenchymal, poor-prognosis CRC subtype, whereas TAs more closely relate to the chromosomally instable type of CRC (De Sousa E Melo et al, 2013). However, it is unclear what is responsible for promoting subtype-specific transformation and for installing unique features associated with distinct groups of CRC. Gene expression-based characterization of a small set of TA and SSA samples has allowed a glimpse of the wiring of these polyps. Whereas components of the WNT pathway are highly expressed in TAs, SSAs present with high levels of EMT- and TGFβ pathway-associated genes (De Sousa E Melo et al, 2013). Therefore, we set out to determine the response of genetically distinct CRC precursor lesions to TGFβ stimulation in organoid cultures, model systems that closely recapitulate human disease (van de Wetering et al, 2015). We made use of human organoid cultures from normal colon tissue and TA polyps, and the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system to genetically engineer an organoid culture to carry the BRAFV600E mutation, thus modeling different paths of CRC development. In TA organoids, which most frequently progress to the classical CRC subtype, apoptosis was the dominating response upon TGFβ treatment, while induction of a mesenchymal phenotype prevailed in the BRAFV600E-mutated organoid culture, presenting a model system for the serrated path to CRC. SSAs following the serrated neoplasia pathway have been suggested to harbor the potential to progress either to good- or to poor-prognosis CRCs based on molecular markers (Jass, 2007; Phipps et al, 2015). Indeed, using gene expression data, we show that SSAs can be segregated into both CMS1- and CMS4-like lesions, which is associated with activation of the TGFβ pathway. Significantly higher levels of TGFβ pathway activity were detectable in SSAs predicted to progress to CMS4-like CRCs compared to those poised to give rise to CMS1 CRC. Hence, our data point to an important role of TGFβ in the serrated path of CRC development and propose that this cytokine represents a critical cue in directing SSAs to the mesenchymal, poor-prognosis CRC subtype. Results TGFβ induces apoptosis in human tubular adenoma organoid cultures To study the effect of TGFβ at an early stage of tumor development, we obtained TAs from familial adenomatous polyposis (FAP) patients, which would predictably follow the classical path of CRC development (Fearon & Vogelstein, 1990; De Sousa E Melo et al, 2013). Organoid cultures (TA1–TA5) were established from these premalignant lesions and they were propagated in medium without the WNT-ligands WNT3A and R-Spondin-1 to select for transformed cells in which the WNT pathway is constitutively active. Normal non-transformed cells without activated WNT signaling do not survive this selection process (Drost et al, 2015). Of note, besides WNT pathway activation, one of the TA organoid cultures—TA1—also carries a KRASG12V mutation (Appendix Fig S1A). In the classical path of CRC development, activating mutations in the KRAS oncogene are thought to follow aberrant WNT pathway activation (Fearon & Vogelstein, 1990); hence, the origin of the TA1 organoid culture is likely to be a more advanced adenoma or early carcinoma. The TGFβ pathway was not perturbed in the TA organoid cultures, as all five cultures used in this study showed SMAD4 expression and induction of phopho-SMAD2 upon TGFβ stimulation (Appendix Fig S1B and C). In the organoid cultures TA2-TA5, the formation of well-organized structures was disrupted by the addition of TGFβ to the culture medium, leading to disintegration of the organoids (Fig 1A). In the control condition, cleaved Caspase-3-positive, and thus apoptotic, cells could only be found inside the organoids (Fig 1B). These represent old cells that have been replaced by a new generation, undergo apoptosis, and are shed into the lumen. In contrast, the amount of cleaved Caspase-3-positive cells was strongly increased upon TGFβ treatment, highlighting the loss of organization in these structures (Fig 1B). In contrast, the KRAS-mutant TA1 organoid culture did not undergo apoptosis judged by the lack of cleaved Caspase-3, but rather showed growth arrest both morphologically and based on KI-67 expression (Figs 1A and B, and EV1A). We performed gene expression arrays on control and TGFβ-treated samples of three TA organoid cultures and two organoid cultures from normal colon tissue. As expected, genes involved in apoptosis were highly enriched in the TGFβ-treated compared to the control samples judged by gene set enrichment analysis (GSEA) (Mootha et al, 2003; Subramanian et al, 2005) (Fig 1C). Similar results have previously been reported for mouse intestinal organoids carrying an inactivating Apc mutation, in which BCL2-like protein 11 (BCL2L11, BIM) was identified to mediate TGFβ-induced apoptosis (Wiener et al, 2014). Also in the human TA organoid cultures, BIM was induced upon TGFβ treatment (Fig 1D), whereas BID and Puma (BBC3) were not induced or downregulated (Fig EV1A). All TA cultures displayed upregulation of BIM, except the KRAS-mutated TA1 organoid culture (Fig 1D), confirming the previously published results that addition of a KRAS mutation to an APC-mutated background increased resistance to TGFβ-mediated apoptosis by inhibiting the induction of BIM (Wiener et al, 2014). Also human wild-type organoid cultures isolated from normal colon mucosa did not show induction of BIM and cleaved Caspase-3 upon TGFβ treatment (Fig EV1B and C), but slowed proliferation illustrated by reduction of KI-67 expression (Fig EV1C). These results indicate that human cells with activated WNT signaling respond to TGFβ via induction of apoptosis, which is alleviated in the presence of an oncogenic KRAS mutation. Thus, patient-derived organoid cultures from premalignant lesions recapitulate similar phenotypes upon TGFβ stimulation as organoids from genetically engineered mouse models (Wiener et al, 2014). Figure 1. TGFβ induces an apoptotic response in human tubular adenoma (TA) organoids The usually well-organized human TA organoids disintegrate upon TGFβ stimulation (scale bars: 200 μm). Following TGFβ treatment, TA organoid cultures display increased levels of cleaved Caspase-3 (scale bars: 200 μm). Gene expression profiles of TGFβ-treated organoids show enrichment in apoptosis-related genes when compared to the control condition. The pro-apoptotic molecule BIM (BCL2L11) is upregulated in the TGFβ-treated condition in all TA cultures, besides in TA1, which carries a KRASG12V mutation (one representative of ≥ 3 independent experiments is shown, error bars represent SD). Source data are available online for this figure. Source Data for Figure 1 [emmm201606184-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TA and normal colon organoids display growth arrest upon TGFβ treatment, but normal organoids do not show features of apoptosis induction The pro-apoptotic molecules BID and Puma (BBC3) are not induced in TGFβ-treated TA organoids, but KI-67 is downregulated (one representative of ≥ 3 independent experiments is shown, error bars represent SD). Cleaved Caspase-3 was not induced upon TGFβ stimulation in the normal colon organoid culture N3 (scale bars: 200 μm). No induction of pro-apoptotic molecules was detected in TGFβ-treated normal colon cultures (N1-N3), but KI-67 expression was reduced [one (representative) experiment is shown (n = 1 for N1 and N2, and n = 3 for N3), error bars represent SD]. Download figure Download PowerPoint Human colon organoid cultures respond to TGFβ by induction of EMT features TGFβ is a well-known inducer of the EMT program (Moustakas & Heldin, 2007). Indeed, we observed morphological changes in the normal colon organoid cultures that resembled the induction of a mesenchymal phenotype in these usually well-organized epithelial structures (Fig 2A). Also in all five TA organoid cultures morphological changes indicative of EMT induction could be observed in the surviving cells (Fig 2B and Appendix Fig S2A). Applying EMT signatures to the gene expression data obtained from TGFβ-treated and control organoids revealed that genes present in these signatures were enriched in TGFβ-treated compared to control samples (Fig 2C and Appendix Fig S2B) (Taube et al, 2010; Gröger et al, 2012). In accordance, the EMT-inducing transcription factor zinc finger E-box binding homeobox 1 (ZEB1) was upregulated upon TGFβ treatment in both normal colon and TA organoid cultures (Fig 2D and E). Also the mesenchymal marker fibronectin 1 (FN1) was strongly induced upon TGFβ treatment (Fig 2F and Appendix Fig S2C). Taken together, non-transformed as well as transformed colon organoid cultures possess the ability to respond to TGFβ via the induction of the EMT program. Figure 2. TGFβ stimulation of human colon organoids induces EMT features A, B. Morphological changes in (A) normal colon (N1-3) and (B) surviving TA organoids upon TGFβ treatment resemble the induction of a mesenchymal phenotype in these usually well-organized epithelial structures (scale bars: 50 μm). C. Genes present in an EMT signature (Gröger et al, 2012) are enriched in TGFβ-treated compared to control samples. D, E. The EMT-inducing transcription factor ZEB1 is strongly induced upon TGFβ treatment both in (D) normal [one (representative) experiment is shown (n = 1 for N1 and N2, and n = 3 for N3), error bars represent SD] as well as (E) TA (one representative of ≥ 3 independent experiments is shown, error bars represent SD) organoids. F. The mesenchymal marker fibronectin 1 (FN1) is induced following TGFβ stimulation (scale bars: 200 μm). Source data are available online for this figure. Source Data for Figure 2 [emmm201606184-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint TGFβ-treated human organoids adapt a mesenchymal CRC subtype gene expression profile Recently, CRC has been classified into multiple subtypes by several groups (Cancer Genome Atlas Network, 2012; Perez-Villamil et al, 2012; Schlicker et al, 2012; Budinska et al, 2013; De Sousa E Melo et al, 2013; Marisa et al, 2013; Sadanandam et al, 2013; Roepman et al, 2014), and in a large international effort, the CRC subtyping consortium (CRCSC) unified these classifications resulting in the four consensus molecular subtypes (CMSs) of CRC (Guinney et al, 2015). In line with the individual classifications, a mesenchymal subtype (CMS4) was identified with significantly worse recurrence-free survival (RFS) (Guinney et al, 2015). This subtype was characterized by high expression of EMT-associated genes (De Sousa E Melo et al, 2013; Guinney et al, 2015). Additionally, based on gene expression profiling SSAs were predicted to be potential precursor lesions of this mesenchymal, poor-prognosis colon cancer subgroup (De Sousa E Melo et al, 2013). Since the EMT phenotype—a hallmark of the mesenchymal colon cancer subtype—can be induced by TGFβ, and the TGFβ signaling pathway is predicted to be active based on gene expression in CMS4 CRCs (Guinney et al, 2015) and SSAs (De Sousa E Melo et al, 2013), we wondered whether TGFβ might also dictate subtype-specific gene expression. We chose FRMD6 (FERM domain containing 6), a marker that is highly expressed in tumors of the mesenchymal subtype, and caudal-type homeobox 2 (CDX2), which is highly expressed in tumors of the classical group (De Sousa E Melo et al, 2013) and assessed their expression changes upon TGFβ treatment. Normal colon and TA organoid cultures responded to TGFβ treatment with upregulation of FRMD6 (Fig 3A and B). CDX2 levels were strongly reduced upon TGFβ stimulation both on RNA and protein level (Fig 3A–C and Appendix Fig S2D). A direct comparison of the gene expression changes induced by TGFβ in organoids with either genes upregulated in CMS4 cancers (Fig 3D) or the 500 most highly expressed genes in SSAs compared with TAs (Fig 3E) confirmed this switch to a more CMS4/SSA-like profile (Fig 3D and E). Therefore, TGFβ treatment of colon organoids not only induced EMT, but also induced the expression of CMS4/mesenchymal marker genes and downregulated expression of genes associated with the classical, epithelial type of CRC. Figure 3. TA organoids adapt their gene expression profiles to those of CMS4/SSA samples upon TGFβ treatment A, B. FRMD6 expression is strongly induced and CDX2 expression is downregulated both in (A) normal [one (representative) experiment is shown (n = 1 for N1 and N2, and n = 3 for N3), error bars represent SD] and (B) TA (one representative of ≥ 3 independent experiments is shown, error bars represent SD) organoid cultures following TGFβ stimulation. C. The reduction in CDX2 levels in the TGFβ-treated condition is also observed on the protein level (scale bars: 200 μm). D, E. Gene expression profiles of TGFβ-treated samples show enrichment of genes highly expressed in (D) the mesenchymal CMS4 of CRC (log2FC > 2) and (E) SSA precursor lesions when compared to control samples. Source data are available online for this figure. Source Data for Figure 3 [emmm201606184-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint SSA and TA polyps can be separate
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