An autocrine ActivinB mechanism drives TGF β/Activin signaling in Group 3 medulloblastoma
2019; Springer Nature; Volume: 11; Issue: 8 Linguagem: Inglês
10.15252/emmm.201809830
ISSN1757-4684
AutoresMorgane Morabito, Magalie Larcher, Florence M.G. Cavalli, Chloé Foray, Antoine Forget, Liliana Mirabal-Ortega, Mamy Andrianteranagna, Sabine Druillennec, Alexandra Garancher, Julien Masliah‐Planchon, Sophie Leboucher, Abel Debalkew, Alessandro Raso, Olivier Delattre, Stéphanie Puget, François Doz, Michael D. Taylor, Olivier Ayrault, Franck Bourdeaut, Alain Eychène, Célio Pouponnot,
Tópico(s)Bone Tumor Diagnosis and Treatments
ResumoArticle22 July 2019Open Access Source DataTransparent process An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma Morgane Morabito Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Magalie Larcher Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Florence MG Cavalli The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Chloé Foray Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Antoine Forget Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Liliana Mirabal-Ortega Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Mamy Andrianteranagna PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France Translational Research in Pediatric Oncology, Institut Curie SiRIC, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France INSERM, U900, Paris, France MINES ParisTech, CBIO-Centre for Computational Biology, Paris, France Search for more papers by this author Sabine Druillennec Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Alexandra Garancher Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Julien Masliah-Planchon PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Sophie Leboucher Institut Curie, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France Search for more papers by this author Abel Debalkew The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Alessandro Raso Department of Patology, ASL 3 Genovese, SC Laboratorio d'Analisi, Genova, Italy Search for more papers by this author Olivier Delattre PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Stéphanie Puget Université Paris Descartes, Sorbonne Paris Cité, Paris, France Département Neurochirurgie Pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author François Doz Institut Curie, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Michael D Taylor The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Division of Neurosurgery, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Olivier Ayrault Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Franck Bourdeaut PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France Translational Research in Pediatric Oncology, Institut Curie SiRIC, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Alain Eychène orcid.org/0000-0002-6818-7225 Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Celio Pouponnot Corresponding Author [email protected] orcid.org/0000-0002-9795-0496 Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Morgane Morabito Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Magalie Larcher Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Florence MG Cavalli The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Chloé Foray Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Antoine Forget Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Liliana Mirabal-Ortega Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Mamy Andrianteranagna PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France Translational Research in Pediatric Oncology, Institut Curie SiRIC, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France INSERM, U900, Paris, France MINES ParisTech, CBIO-Centre for Computational Biology, Paris, France Search for more papers by this author Sabine Druillennec Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Alexandra Garancher Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Julien Masliah-Planchon PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Sophie Leboucher Institut Curie, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France Search for more papers by this author Abel Debalkew The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Alessandro Raso Department of Patology, ASL 3 Genovese, SC Laboratorio d'Analisi, Genova, Italy Search for more papers by this author Olivier Delattre PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Stéphanie Puget Université Paris Descartes, Sorbonne Paris Cité, Paris, France Département Neurochirurgie Pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author François Doz Institut Curie, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Michael D Taylor The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Division of Neurosurgery, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Olivier Ayrault Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Franck Bourdeaut PSL Research University, Paris, France Institut Curie, Paris, France INSERM U830, Paris, France Translational Research in Pediatric Oncology, Institut Curie SiRIC, Paris, France SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France Search for more papers by this author Alain Eychène orcid.org/0000-0002-6818-7225 Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Celio Pouponnot Corresponding Author [email protected] orcid.org/0000-0002-9795-0496 Institut Curie, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France CNRS UMR 3347, Centre Universitaire, Orsay, France University Paris Sud – Paris-Saclay, Orsay, France PSL Research University, Paris, France Search for more papers by this author Author Information Morgane Morabito1,2,3,4,5, Magalie Larcher1,2,3,4,5, Florence MG Cavalli6,7, Chloé Foray1,2,3,4,5, Antoine Forget1,2,3,4,5, Liliana Mirabal-Ortega1,2,3,4,5, Mamy Andrianteranagna5,8,9,10,11,12,13, Sabine Druillennec1,2,3,4,5, Alexandra Garancher1,2,3,4,5, Julien Masliah-Planchon5,8,9,11, Sophie Leboucher1,4, Abel Debalkew6,7, Alessandro Raso14, Olivier Delattre5,8,9,11, Stéphanie Puget15,16, François Doz8,11,15, Michael D Taylor6,7,17,18, Olivier Ayrault1,2,3,4,5, Franck Bourdeaut5,8,9,10,11, Alain Eychène1,2,3,4,5 and Celio Pouponnot *,1,2,3,4,5 1Institut Curie, Orsay, France 2INSERM U1021, Centre Universitaire, Orsay, France 3CNRS UMR 3347, Centre Universitaire, Orsay, France 4University Paris Sud – Paris-Saclay, Orsay, France 5PSL Research University, Paris, France 6The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada 7Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada 8Institut Curie, Paris, France 9INSERM U830, Paris, France 10Translational Research in Pediatric Oncology, Institut Curie SiRIC, Paris, France 11SIREDO Center (Care, innovation, Research in pediatric, adolescent and young adult oncology), Institut Curie, Paris, France 12INSERM, U900, Paris, France 13MINES ParisTech, CBIO-Centre for Computational Biology, Paris, France 14Department of Patology, ASL 3 Genovese, SC Laboratorio d'Analisi, Genova, Italy 15Université Paris Descartes, Sorbonne Paris Cité, Paris, France 16Département Neurochirurgie Pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, Paris, France 17Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada 18Division of Neurosurgery, The Hospital for Sick Children, Toronto, ON, Canada *Corresponding author. Tel: +33 1 69 86 30 79; Fax: +33 1 69 86 30 51; E-mail: [email protected] EMBO Mol Med (2019)11:e9830https://doi.org/10.15252/emmm.201809830 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 Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFβ/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. Our data demonstrate that the TGFβ/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted. Synopsis TGFβ/Activin signaling is activated in a subset of Group 3 medulloblastoma, through an ActivinB autocrine loop. ActivinB sustains tumor cell growth by inducing the expression of the Smad target gene PMEPA1. TGFβ/Activin pathway can be therapeutically targeted with Galunisertib in this G3 subgroup. A subset of G3 medulloblastoma tumors displays high TGFβ/Activin pathway activation. An ActivinB autocrine loop is responsible for TGFβ/Activin pathway activation in G3 medulloblastoma. G3 medulloblastoma with strong TGFβ/Activin pathway activation are characterized by a high level of PMEPA1 expression. Galunisertib treatment is of potential therapeutic interest for G3 medulloblastoma showing strong TGFβ/Activin pathway activation. Introduction Medulloblastoma (MB), a cerebellar tumor, is one of the most common malignant brain tumors in children (Holgado et al, 2017; Wang et al, 2018). Current therapy associates surgery, chemotherapy, and radiotherapy. This aggressive regimen allowed an increase in the overall survival rate up to 70–80% but induces dramatic long-term side effects (Martin et al, 2014). In addition, the overall survival rate of high-risk patients is far below (Holgado et al, 2017; Wang et al, 2018). It is therefore crucial to identify new treatments that decrease side effects and improve efficacy. Genomic and transcriptomic approaches allowed the stratification of MB patients into 4 different molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), Group 3, and Group 4 (Northcott et al, 2012a; Taylor et al, 2012). These groups display differences in terms of cell of origin, transcriptional, epigenetic, and mutational signatures. They also differ in their clinical characteristics such as histology, overall survival rate, and presence of metastases. Recently, intragroup heterogeneity has been further uncovered, allowing their division into subtypes with some specific clinical parameters as well as genomic alterations (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017). Although the existence of subdivisions within the different groups is clear, the outlines of the different subtypes have not completely reached a consensus so far. The WNT group represents 10% of all MBs and is driven by constitutive activation of the WNT/β-catenin pathway with patients showing the best prognosis. The SHH group accounts for 20–25% of MB and is characterized by mutations involving different mediators of the SHH pathway. It is considered of intermediate prognosis. However, recent sub-classifications identified SHH subtypes with poorer outcomes (Cavalli et al, 2017a; Schwalbe et al, 2017). On the other side, Group 3 and Group 4 are far less characterized due to their genetic and clinical heterogeneity. They display some degrees of overlap with a few samples (~10%) being difficult to specifically assign to either Group. They share some clinical characteristics, such as a high propensity to metastasis and genetic alterations such as OTX2 amplifications or KBTBD4 mutations (Northcott et al, 2017). In contrast to SHH and WNT groups, no deregulation of a given signaling pathway has been yet reported. Group 4 represents 35–40% of all MB patients and shows, in few cases, MYCN and CDK6 amplifications and KDM6A mutations. Recently, it has been shown that genomic alterations involving enhancer hijacking induce PRDM6 overexpression in 15–20% of Group 4 (Northcott et al, 2017). Group 3 represents 20–25% of MB patients and is associated with bad prognosis. This group is highly metastatic and characterized by MYC overexpression, which can be explained in 15–20% of cases by its amplification. However, MYC overexpression is not sufficient to induce Group 3 MB and requires additional cooperating oncogenic events (Kawauchi et al, 2012; Pei et al, 2012). Some of them have been identified, such as GFI1 and GFI1B that are highly expressed in a subset of Group 3 through enhancer hijacking (Northcott et al, 2014). These transcription factors have been demonstrated to drive Group 3 MB tumorigenesis in animal models when associated with MYC overexpression (Northcott et al, 2014). At the transcriptomic level, Group 3 is characterized by the expression of a photoreceptor program defined by genes whose expression is highly restricted to the retina (Kool et al, 2008; Cho et al, 2011). We recently uncovered that this program defines a subtype within Group 3 tumors, which exhibits a functional dependency to this ectopic program through its two main drivers, the retina-specific transcription factors NRL and CRX (Garancher et al, 2018). Thus, Group 3 can be subdivided into 2–3 different subtypes according to the different studies (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017). Cavalli et al (2017a) have identified 3 subtypes, one is composed of tumors with high MYC expression including those with amplification of this gene, named G3γ. This subtype has the worse prognosis. The second subtype, G3β, is overrepresented by tumors with GFI1 alterations, and the last one G3α, by tumors expressing photoreceptor genes in which few amplifications of mediators of the TGFβ/Activin pathway can be found (Cavalli et al, 2017a). Since Group 3 displays the worse prognosis, targeted therapies are actively searched. Different actionable targets have been proposed mainly based on genomic data, including the TGFβ signaling, which has been suggested to be deregulated in few Group 3 MB, although no functional data have been reported so far. A study on structural genomic variations across over 1,000 MB has first described few amplifications of different mediators of the TGFβ/Activin pathway in Group 3 MB (Northcott et al, 2012b). They include ACVR2A and ACVR2B, two type II receptors for Activin, as well as TGFBR1, a type I receptor for TGFβ, highlighting a potential deregulation of Smad2/3 signaling (see below). Additionally, since OTX2 has been demonstrated to be a target gene of this signaling pathway (Jia et al, 2009), it has been proposed that OTX2 amplifications could represent a mechanism by which the pathway is also deregulated downstream (Northcott et al, 2012b). The putative significance of this signaling pathway in Group 3 was reinforced by two subsequent studies, one involving sequencing in a large cohort of MB (Northcott et al, 2017) and the other showing that several components of this signaling pathway could also be deregulated at their expression level, through Group 3-specific enhancers (Lin et al, 2016). Although these studies might indicate a potential deregulation of the Smad2/3 signaling pathway, this could account for only a modest proportion of Group 3 tumors. The TGFβ superfamily is a large family of cytokines divided into two distinct groups of ligands: the TGFβs/Activins and the BMPs. TGFβ/Activin ligands signal through Smad2/3. These ligands bring together two types of serine/threonine kinase receptors, the type I and the type II, which are specific for a set of ligands. The TGFβs (TGFβ1, TGFβ2, and TGFβ3) signal through the TGFBR1 type I and TGFBR2 type II receptors. Activin, encoded by 4 different genes, INHBA, INHBB, INHBC, and INHBE, can activate different couples of receptors including the ACVR2A and ACVR2B type II and ACVR1A (ALK4) and ACVR1C (ALK7) type I receptors. INHA, encoding inhibin-α, is an inhibitor of the Activin ligands. Activin and TGFβ ligands lead to the phosphorylation and activation of the same intracellular mediators, Smad2 and Smad3, which then associate with the co-Smad, Smad4. The hetero-complex translocates to the nucleus, where it activates the transcription of target genes with the help of DNA binding partners (Levy & Hill, 2006; Ross & Hill, 2008). TGFβ/Activin signaling displays pleiotropic functions depending on the cellular and environmental context. Its implication in cancer has been well documented, mainly through TGFβ ligands, although BMPs and Activins ligands can be also involved (Seoane & Gomis, 2017). The role of the TGFβ signaling pathway in cancer is complex, acting either as a tumor suppressor pathway in some instances or as a tumor promoter in others (Massagué, 2008; Seoane & Gomis, 2017). Its oncogenic role is mainly associated with an autocrine (or paracrine) stimulation, due to the strong expression of TGFβ ligands. The TGFβ pathway has been shown to promote cell proliferation in specific context such as in Glioblastoma (Bruna et al, 2007) and cancer stem cell maintenance (Peñuelas et al, 2009; Anido et al, 2010; Lonardo et al, 2011). Studies on the role of Activin ligands in cancer are much more scarce (Wakefield & Hill, 2013). By activating the same mediators Smad2/3, a parallel can be drawn between TGFβ and Activin. Indeed, Activins act both as tumor suppressors and tumor promoters (Chen et al, 2002; Antsiferova & Werner, 2012; Marino et al, 2013; Wakefield & Hill, 2013). Their pro-tumorigenic role has been validated in animal models in which deletion of the activin inhibitor, INHA, led to gonadal tumors in mice as well as cachexia-like syndrome (Matzuk et al, 1994; Vassalli et al, 1994). ActivinB has also been shown to play a role in cancer stem cell maintenance (Lonardo et al, 2011) and in cell dedifferentiation in an insulinoma mouse model and deletion of INHBB encoding ActivinB increases survival (Ripoche et al, 2015). Several observations pinpoint to a potential role of the Smad2/3 signaling pathway in Group 3 MB but no published data have confirmed the deregulation of this signaling pathway, nor its functional involvement in Group 3 biology. In this study, we investigated these aspects to bring the proof of principle that this signaling pathway represents an interesting therapeutic target in MB and to identify patients that could be eligible to such therapy. Results TGFβ/ActivinB signaling pathway is active in Group 3 MB Since different genomic alterations in the TGFβ/Activin pathway have been previously described in Group 3 MB (Northcott et al, 2012b, 2017; Lin et al, 2016), we first investigated whether the pathway is activated in patient samples. We performed WB analysis on 38 medulloblastomas: 7 WNT, 12 SHH, 10 Group 3, and 9 Group 4 tumors. Activation of the pathway, monitored by the level of Smad2 phosphorylation (P-Smad2), was observed in some patient samples from all MB groups (Fig 1A). An inter-tumor heterogeneity was observed in each group, with some samples with high P-Smad2. However, an overall higher level of Smad2 phosphorylation was observed in Group 3 when normalized to β-actin (Fig EV1A, left panel). This was not evidenced when normalized to total Smad2 (Fig EV1A, right panel) since an important variation of Smad2 level was observed (Fig 1A). This is in line with the modification of Smad2 stability by auto-regulatory mechanisms (Yan et al, 2018). Thus, the overall level of P-Smad2/β-actin, which formally reflects the level of nuclear and active Smad2, led us to conclude that TGFβ/Activin pathway is activated in some Group 3 patients. Figure 1. TGFβ/ActivinB pathway is activated in Group 3 MB patients and cell lines A. Immunoblot analysis of phosphorylated Smad2 (P-Smad2) and PMEPA1 (high and low exposures displayed) in MB patient sample lysates from different groups: WNT (blue), SHH (red), Group 3 (yellow), or Group 4 (green). β-Actin was used as a loading control. Relative quantification of P-Smad2 signal to β-actin (P-S2/β-Actin) and total Smad2 (P-S2/Tot-S2) are indicated below the blots. B. Boxplots summarizing the expression of INHBB, TGFB1, and TGFB3 ligands of the TGFβ/Activin pathway in the different groups of MB (blue WNT, red SHH, yellow Group 3, and green Group 4) and in fetal and adult cerebellum (gray) in the dataset of Cavalli et al (Data ref: Cavalli et al, 2017b). C. Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in non-Group 3 (blue) and Group 3 (yellow) MB cell lines on the left panel. The level of total Smad2 (Smad2) was assessed, and β-actin was used as a loading control. On the right panel, relative level of P-Smad2 (P-S2) was quantified to total β-actin. P-Smad2 to total Smad normalization is also provided on Appendix Fig S5. D. RT–qPCR was performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBB (left) and TGFB3 (right). Data information: Wilcoxon rank-sum tests were performed to determine P-values for panel (B). Boxplot center lines show data median; box limits indicate the 25th and 75th percentiles; lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles, respectively. Outliers are represented by individual points (B). The remaining P-values were determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent the mean ± SD. Number of replicates is n ≥ 3. The exact P-values and number of replicates are indicated in Appendix Table S5. Detailed statistics are presented in Appendix Table S1 for panel (B) and Appendix Table S2 for panel (D). Source data are available online for this figure. Source Data for Figure 1 [emmm201809830-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TGFβ/ActivinB pathway is activated in Group 3 MB patients and cell lines A. Boxplots represent the quantification of P-Smad2 normalized to β-actin levels on the left and total Smad2 on the right. B. Boxplots summarizing the expression of major actors of the TGFβ/Activin pathway in the different groups of MB (blue WNT, red SHH, yellow Group 3, and green Group 4) in the dataset from Cavalli et al (Data ref: Cavalli et al, 2017b). C. Boxplots summarizing the protein level of major actors of the TGFβ/Activin pathway detected by mass spectrometry in the different groups of MB (blue WNT, red SHH, yellow Group 3, and green Group 4) in the dataset from Archer et al (Data ref: Archer et al, 2018b). D. RT–qPCR was performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBA, TGFB1, TGFB2, TGFBR1, TGFBR2, ACVR1B, ACVR1C, ACVR2A, and ACVR2B. The expression level relative to that of found in HDMB03 is presented. Data information: Boxplot center lines show data median; box
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