An FGFR 3/ MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers
2018; Springer Nature; Volume: 10; Issue: 4 Linguagem: Inglês
10.15252/emmm.201708163
ISSN1757-4684
AutoresMélanie Mahé, Florent Dufour, Hélène Neyret‐Kahn, Aura Moreno‐Vega, C Béraud, Mingjun Shi, Imène Hamaidi, Virginia Sánchez‐Quiles, Clémentine Krucker, Marion Dorland‐Galliot, Elodie Chapeaublanc, Rémy Nicolle, Hervé Lang, Célio Pouponnot, Thierry Massfelder, François Radvanyi, Isabelle Bernard‐Pierrot,
Tópico(s)Bladder and Urothelial Cancer Treatments
ResumoResearch Article20 February 2018Open Access Source DataTransparent process An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers Mélanie Mahe Mélanie Mahe Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Florent Dufour Florent Dufour Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Hélène Neyret-Kahn Hélène Neyret-Kahn Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Aura Moreno-Vega Aura Moreno-Vega Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Claire Beraud Claire Beraud UROLEAD SAS, School of Medicine, Strasbourg, France Search for more papers by this author Mingjun Shi Mingjun Shi Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Imene Hamaidi Imene Hamaidi Department of Urology, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Virginia Sanchez-Quiles Virginia Sanchez-Quiles Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Clementine Krucker Clementine Krucker Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Marion Dorland-Galliot Marion Dorland-Galliot Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Elodie Chapeaublanc Elodie Chapeaublanc Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Remy Nicolle Remy Nicolle Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Hervé Lang Hervé Lang Department of Urology, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Celio Pouponnot Celio Pouponnot Institut Curie, Orsay, France CNRS UMR3347, Centre Universitaire, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France Search for more papers by this author Thierry Massfelder Thierry Massfelder INSERM UMR_S1113, Section of Cell Signalization and Communication in Kidney and Prostate Cancer, School of Medicine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), INSERM and University of Strasbourg, Strasbourg, France Search for more papers by this author François Radvanyi François Radvanyi Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Isabelle Bernard-Pierrot Corresponding Author Isabelle Bernard-Pierrot [email protected] orcid.org/0000-0002-8967-5092 Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Mélanie Mahe Mélanie Mahe Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Florent Dufour Florent Dufour Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Hélène Neyret-Kahn Hélène Neyret-Kahn Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Aura Moreno-Vega Aura Moreno-Vega Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Claire Beraud Claire Beraud UROLEAD SAS, School of Medicine, Strasbourg, France Search for more papers by this author Mingjun Shi Mingjun Shi Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Imene Hamaidi Imene Hamaidi Department of Urology, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Virginia Sanchez-Quiles Virginia Sanchez-Quiles Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Clementine Krucker Clementine Krucker Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Marion Dorland-Galliot Marion Dorland-Galliot Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Elodie Chapeaublanc Elodie Chapeaublanc Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Remy Nicolle Remy Nicolle Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Hervé Lang Hervé Lang Department of Urology, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Celio Pouponnot Celio Pouponnot Institut Curie, Orsay, France CNRS UMR3347, Centre Universitaire, Orsay, France INSERM U1021, Centre Universitaire, Orsay, France Search for more papers by this author Thierry Massfelder Thierry Massfelder INSERM UMR_S1113, Section of Cell Signalization and Communication in Kidney and Prostate Cancer, School of Medicine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), INSERM and University of Strasbourg, Strasbourg, France Search for more papers by this author François Radvanyi François Radvanyi Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Isabelle Bernard-Pierrot Corresponding Author Isabelle Bernard-Pierrot [email protected] orcid.org/0000-0002-8967-5092 Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Author Information Mélanie Mahe1,2,‡, Florent Dufour1,2,‡, Hélène Neyret-Kahn1,2, Aura Moreno-Vega1,2, Claire Beraud3, Mingjun Shi1,2, Imene Hamaidi4, Virginia Sanchez-Quiles1,2, Clementine Krucker1,2, Marion Dorland-Galliot1,2, Elodie Chapeaublanc1,2, Remy Nicolle1,2, Hervé Lang4, Celio Pouponnot5,6,7, Thierry Massfelder8, François Radvanyi1,2 and Isabelle Bernard-Pierrot *,1,2 1Institut Curie, CNRS, UMR144, Equipe Labellisée Ligue contre le Cancer, PSL Research University, Paris, France 2CNRS, UMR144, Sorbonne Universités, UPMC Université Paris 06, Paris, France 3UROLEAD SAS, School of Medicine, Strasbourg, France 4Department of Urology, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 5Institut Curie, Orsay, France 6CNRS UMR3347, Centre Universitaire, Orsay, France 7INSERM U1021, Centre Universitaire, Orsay, France 8INSERM UMR_S1113, Section of Cell Signalization and Communication in Kidney and Prostate Cancer, School of Medicine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), INSERM and University of Strasbourg, Strasbourg, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 42 34 63 40; Fax: +33 1 42 34 63 49; E-mail: [email protected] EMBO Mol Med (2018)10:e8163https://doi.org/10.15252/emmm.201708163 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 FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3. Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo. A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti-FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation. Synopsis In bladder carcinoma, alterations of FGFR3 receptor are often observed and lead to constitutive activation and oncogene addiction, which can be targeted with a pan-FGFR inhibitor. Our identification and characterization of a FGFR3/MYC positive feedback loop opens new avenues for targeted therapies. MYC is a key master regulator of proliferation activated by aberrantly activated FGFR3. FGFR3-dependent MYC accumulation is dependent on p38, which regulates MYC mRNA levels, and AKT, which stabilizes MYC protein. FGFR3 is directly targeted by MYC. Disrupting the FGFR3/MYC loop using FGFR3, p38, AKT, or BET bromodomain inhibitors decreases cell viability and tumor growth in FGFR3-dependent cell lines. Evidence for the relevance of the FGFR3/MYC feedback loop to human tumors is provided by the decrease in both FGFR3 and MYC levels induced by a pan-FGFR inhibitor in a PDX model bearing an FGFR3 mutation, and by the positive correlation between MYC and FGFR3 levels in human tumors with FGFR3 mutations. Introduction Bladder cancer is the ninth most common cancer worldwide, with approximately 430,000 new cases diagnosed in 2012 and 165,000 deaths annually (Antoni et al, 2017). Non-muscle-invasive carcinomas (NMIBCs) account for 70% of cases at first diagnosis. These tumors often have a favorable prognosis following transurethral resection with or without intravesical chemotherapy or immunotherapy with Bacillus Calmette-Guérin (BCG). NMIBC often recurs (50–60% of cases) and sometimes progresses to a muscle-invasive tumor (5–40% progression, depending on clinical and pathological features). This high recurrence rate and the need for monitoring contribute to the economic burden of bladder cancer treatment. Muscle-invasive bladder carcinoma (MIBC) is a major clinical issue, because, even with radical cystectomy as the standard treatment, overall survival at 5 years is only about 50%, and the combination of this treatment with neoadjuvant and/or adjuvant chemotherapy increases overall survival only moderately. No major improvement in survival has been achieved over the last 20 years (Witjes et al, 2013). A clinical response to immune checkpoint inhibitors has recently been reported, but only a subset of patients respond to such treatment, and it remains unclear how to identify these patients (Powles et al, 2014; Bajorin et al, 2015; Bellmunt et al, 2017a,b; Davarpanah et al, 2017). Some targeted therapies have also yielded promising efficacy results. This is the case, for example, for mTOR inhibitors for patients with TSC1 mutations, anti-HER2 treatments for HER2-amplified MIBC, and anti-FGFR therapies for MIBC with activating FGFR mutations or translocations (Abbosh et al, 2015; Rouanne et al, 2016). The definition of therapeutic strategies to improve treatment outcomes remains of the utmost importance. FGFR3 (fibroblast growth factor receptor) belongs to a family of structurally related tyrosine kinase receptors (FGFR1-4). These receptors regulate various physiological processes, including proliferation, differentiation, migration, and apoptosis. There has been considerable interest in the FGFR family (FGFR1-4), as these receptors are frequently involved, through various mechanisms, in genetic disorders and cancer, leading to their identification as possible targets for treatment (Haugsten et al, 2010). FGFR3 is frequently altered through activating mutations and translocations generating FGFR3-gene fusions (Billerey et al, 2001; Tcga, 2014). Mutations are, by far, the most frequent alterations of FGFR3, occurring in almost 50% of bladder tumors (70% of NMIBCs and 15–20% of MIBCs). The two most frequent mutations are the S249C and Y375C mutations, which affect the extracellular domain of the receptor. FGFR3 translocations leading to the production of FGFR3-TACC3 and FGFR3-BAIAP2L1 fusion proteins were recently identified in 3% of MIBCs (Tcga, 2014). These alterations are thought to be "oncogenic drivers", because the expression of an altered FGFR3 induces cell transformation (Bernard-Pierrot et al, 2006; Williams et al, 2013; Wu et al, 2013; Nakanishi et al, 2015). Furthermore, several preclinical studies in cell lines and xenograft models of bladder cancer have shown that FGFR3 alterations confer sensitivity to FGFR inhibitors, which have anti-proliferative and pro-apoptotic effects (Bernard-Pierrot et al, 2006; Wu et al, 2013; Nakanishi et al, 2015). Together, these findings highlight the critical role of FGFR3 in bladder tumor carcinogenesis, raising the possibility of developing anti-FGFR3 therapies for both NMIBC and MIBC (Chae et al, 2017). Promising results were recently reported for four out of the five patients with FGFR3-mutated bladder cancers enrolled in a phase I clinical trial of the pan-FGFR kinase inhibitor BGJ398 (Nogova et al, 2017). However, based on observations for other targeted therapies (EGFR, BRAF, KIT) for various cancers, including colon and lung cancers, melanoma, and gastrointestinal tumors, FGFR3-targeted therapies will probably turn out to be limited by multiple mechanisms of intrinsic and acquired resistance, such as ERBB2/3 or EGFR activation (Flaherty et al, 2012; Herrera-Abreu et al, 2013; Niederst & Engelman, 2013; Wang et al, 2015). The signaling pathway activated by mutated FGFR3 and FGFR3-fusion proteins is not well characterized, particularly for bladder cancer. Improvements in our understanding of the molecular mechanisms underlying the oncogenic activity of activated FGFR3 in bladder tumors may facilitate the identification of new drug targets that could be acted on together with FGFR3, to increase the efficacy of anti-FGFR3 therapies and/or to prevent potential drug resistance. Such strategies, based on the simultaneous inhibition of two or more targets in a single pathway, have already been explored for many specific pairs of agents, in both clinical and preclinical studies (Flaherty et al, 2012; Li et al, 2014; Ran et al, 2015). In this study, we aimed to characterize the aberrantly activated FGFR3 signaling pathways involved in bladder cancer cell growth/transformation. We studied genes regulated by constitutively activated FGFR3 in two bladder tumor-derived cell lines, MGH-U3 and RT112, harboring an FGFR3 mutation (Y375C) and a fusion gene (FGFR3-TACC3), respectively. We identified MYC as a key transcription factor that is overexpressed and activated in response to FGFR3 activity, and critical for FGFR3-induced cell proliferation. We showed here that FGFR3 is a direct target gene of MYC, which binds to active enhancers located upstream from FGFR3, establishing an FGFR3/MYC positive feedback loop. This loop may be relevant in human tumors, because MYC and FGFR3 expression levels were found to be positively correlated in tumors bearing FGFR3 mutations in two independent transcriptomic datasets (n = 63 and n = 271), and because FGFR3 inhibition in a patient-derived tumor xenograft (PDX) model harboring an FGFR3-S249C mutation decreased the levels of both MYC and FGFR3. We found that MYC mRNA levels and protein stability were dependent on p38 and AKT activation, respectively, downstream from FGFR3 activation. Finally, we showed, in xenograft models, that FGFR3 activation conferred sensitivity to FGFR3 and p38 inhibitors and to a BET bromodomain inhibitor (JQ1) preventing MYC transcription. These findings therefore suggest new treatment options for bladder cancers in which FGFR3 is aberrantly activated. Results MYC is a key master regulator of proliferation in the aberrantly activated FGFR3 pathway We investigated the molecular mechanisms underlying the oncogenic activity of aberrantly activated FGFR3 in bladder carcinomas, by studying the MGH-U3 and RT112 cell lines. These cell lines were derived from human bladder tumors, and they endogenously express a mutated activated form of FGFR3 (FGFR3-Y375C, the second most frequent mutation in bladder tumors) and the FGFR3-TACC3 fusion protein (the most frequent FGFR3 fusion protein in bladder tumors), respectively. The growth and transformation of these cell lines are dependent on FGFR3 activity (Bernard-Pierrot et al, 2006; Williams et al, 2013; Wu et al, 2013). We conducted a gene expression analysis with Affymetrix DNA arrays, in these cell lines, with and without FGFR3 siRNA treatment. We identified 741 and 3,124 genes displaying significant differential expression after FGFR3 depletion in MGH-U3 and RT112 cells, respectively (adjusted P-values < 0.05, |log2(FC)| > 0.5; Dataset EV1). An analysis of these two lists of FGFR3-regulated genes using the upstream regulator function of Ingenuity Pathway Analysis (IPA) software identified upstream regulators activated and inhibited by FGFR3 (Fig 1A, left panel). The top 10 transcriptional regulators with activity modulated by FGFR3 were common to the two cell lines and are listed in the right panel in Fig 1A. The transcription factor predicted to be the most strongly inhibited here after FGFR3 depletion, in both cell lines, was the proto-oncogene MYC, for which mRNA levels were downregulated. This downregulation of MYC mRNA levels after FGFR3 knockdown with siRNA was further confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (30–70% decrease, depending on the cell line used; Fig 1B). Consistent with these results suggesting that MYC mRNA levels are modulated by constitutively activated FGFR3, an analysis of previously described transcriptomic data for our CIT-series ("Carte d'Identité des Tumeurs"; tumor identity card) of bladder tumors revealed a significant upregulation of MYC mRNA levels in tumors harboring an FGFR3 mutation (n = 63) relative to normal urothelium samples (n = 4), whereas no such overexpression was observed for tumors expressing wild-type FGFR3 (n = 122; Fig 1C). Moreover, MYC expression was positively correlated with FGFR3 expression in bladder tumors harboring a mutated FGFR3 (Fig 1D, upper panel), whereas no such correlation was observed in tumors bearing wild-type FGFR3 (n = 122; Fig 1D, lower panel). Similar results were also observed for another publicly available transcriptomic dataset for 416 bladder tumors (271 with FGFR3 mutations) and eight normal samples (Hedegaard et al, 2016; Appendix Fig S1A and B), suggesting that mutated FGFR3 may also regulate MYC expression in human bladder carcinomas. Support for this hypothesis was provided by the significant decrease in MYC mRNA levels induced by 4 days of anti-FGFR treatment in tumors from a PDX model (F659) bearing an FGFR3-S249C mutation (Fig 1E). As in cell lines, FGFR3-S249C expression conferred FGFR3 dependence on the PDX model, in which anti-FGFR treatment with BGJ398 decreased tumor growth by 60% after 29 days of administration (Appendix Fig S2). Figure 1. MYC is a key upstream regulator activated by FGFR3 that is required for FGFR3-induced bladder cancer cell growth Venn diagram showing the number of upstream regulators (transcription factors) significantly predicted by Ingenuity Pathway Analysis to be involved in the regulation of gene expression observed after FGFR3 knockdown in RT112 and MGH-U3 cells (left panel). List of the top 10 upstream regulators modulated by FGFR3 expression in both cell lines. The Log2FC of the transcription factor itself is also indicated. NA indicates that the FC was beyond the threshold defining genes as differentially expressed after FGFR3 depletion (see Materials and Methods). Relative MYC mRNA levels in MGH-U3 and RT112 cells transfected for 72 h with siRNAs targeting FGFR3 or a control siRNA (Ctr). The results presented are the means of two independent experiments carried out in triplicate; the standard errors are indicated. The significance of differences was assessed in unpaired Student's t-tests, *P < 0.05; **0.001 < P < 0.005. MYC mRNA levels in normal human urothelium (n = 4) and in the CIT cohort of human bladder tumors bearing FGFR3 mutations (n = 63) or wild-type FGFR3 (n = 122). The significance of differences was assessed in Mann–Whitney tests, and means and standard errors are represented. MYC and FGFR3 mRNA levels in human bladder tumors harboring either mutated FGFR3 (upper panel) or wild-type FGFR3 (lower panel). Spearman's coefficient and P-values are indicated for the correlations between MYC and FGFR3 mRNA levels in each group. MYC mRNA levels in a PDX model bearing a FGFR3-S249C mutation and treated daily, for 4 days, with 30 mg/kg BGJ398, a pan-FGFR inhibitor, or with vehicle (n = 4 mice per group). Means and standard errors are represented. The significance of differences was assessed in Mann–Whitney tests. Western blot (72 h after transfection) comparing FGFR3 and MYC levels in MGH-U3 and RT112 cells transfected with a control siRNA (Ctr) or with siRNAs targeting FGFR3. Western blot comparing MYC levels in MGH-U3 and RT112 cells, treated for 2 h with DMSO or the pan-FGFR inhibitor, PD173074 (500 nM). Western blot comparing MYC levels in MGH-U3 and RT112 cells treated for 3 h with FGFR inhibitor (0.5 μM PD173074) or proteasome inhibitor (10 μM MG132), alone or in combination. Cell viability assay comparing the impact of MYC and/or FGFR3 downregulation on RT112 (left panel, CellTiter-Glo) and MGH-U3 (right panel, MTT assay) cell viability (72 h post-transfection). The results presented are the means of three independent experiments carried out in triplicate, error bars represent standard deviations. Tukey's multiple comparisons tests were performed to evaluate the significance of differences. The results of the statistical analysis are summarized in Dataset EV2. Source data are available online for this figure. Source Data for Figure 1 [emmm201708163-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint MYC is a key regulator of proliferation and its deregulation can promote oncogenesis in various types of cancer (Dang, 2012). We therefore investigated the role of MYC as a master regulator of proliferation in bladder cell lines expressing aberrantly activated FGFR3. Western blot analysis further showed that FGFR3 depletion resulted in the almost total loss of MYC from both MGH-U3 and RT112 cells (Fig 1F). The discrepancy between the decreases in MYC mRNA (Fig 1B) and protein levels (Fig 1F) suggested that the aberrant activation of FGFR3 regulated MYC not only at mRNA level, but also through stabilization of the protein. This hypothesis was also supported by the time course of MYC expression on Western blots after the inhibition of FGFR3 with PD173074. Indeed, MYC levels decreased rapidly, after 30 min of treatment, in MGH-U3 cells (Appendix Fig S3A), and expression was totally lost after 2 h of treatment, in both MGH-U3 and RT112 cells (Fig 1G and Appendix Fig S3A). MYC protein stability is, thus, tightly controlled by the proteasome. We therefore investigated the possible role of FGFR3 in this process, by treating MGH-U3 and RT112 cells with a pan-FGFR inhibitor (PD173074), either alone or in combination with a proteasome inhibitor (MG132; Fig 1H). Western blot analysis showed that the downregulation of MYC induced by the inhibition of FGFR3 was abolished by MG132, in both cell lines. Overall, our results indicate that the inhibition of aberrantly activated FGFR3 decreases MYC mRNA levels and favors proteolysis of the MYC protein by the proteasome, thereby decreasing its transcriptional activity. We then investigated the possible contribution of MYC to the oncogenic activity of aberrantly activated FGFR3. We compared the effects on viability of depleting FGFR3 and MYC alone or together, with siRNA, in RT112 and MGH-U3 cells (Fig 1I). FGFR3 and MYC siRNAs efficiently knocked down the levels of the targeted proteins (Appendix Fig S3B). The depletion of either MYC or FGFR3 resulted in significantly lower cell viability than for cells treated with the control siRNA (Fig 1I, right and left panels and Dataset EV2 for the P-values). No significant additive effect relative to FGFR3 depletion alone was observed in RT112 and MGH-U3 cells with a simultaneous knockdown of FGFR3 and MYC expression, suggesting that MYC is a key downstream effector of the aberrantly activated FGFR3 pathway mediating cell proliferation. FGFR3 and MYC are involved in a positive feedback loop in which FGFR3 is a direct transcriptional target of MYC in bladder cancer cell lines with constitutively activated FGFR3 Surprisingly, we observed that the treatment of MGH-U3 and RT112 cells with a MYC siRNA strongly decreased FGFR3 levels (Fig 2A). RT–qPCR showed that this loss of FGFR3 expression was due to a decrease in FGFR3 mRNA levels after MYC knockdown (Fig 2B). We investigated whether FGFR3 was a direct transcriptional target of MYC, by analyzing MYC occupancy of the FGFR3 locus by chromatin immunoprecipitation and quantitative PCR (ChIP–qPCR). Using the publicly available ENCODE data for three different cancer cell lines, we designed primers binding to two potential enhancers, the promoter and an intragenic region of FGFR3 (Appendix Fig S4A). According to ENCODE data, the enrichment of MYC and activation marks (H3K27ac) in the E1 and E2 enhancers is correlated with the level of FGFR3 transcription (Appendix Fig S4A). We checked, by ChIP–qPCR, that the selected FGFR3 promoter and enhancers did harbor the expected histone activation marks (H3K27ac and H3K4me3) in RT112 cells (Appendix Fig S4B). Finally, we showed that the two FGFR3 enhancer regions tested were enriched in MYC, consistent with the direct regulation of FGFR3 expression by MYC, at the transcriptional level (Fig 2C). This regulation of FGFR3 by MYC seemed to be quite specific to bladder cancer, because MYC binding to the FGFR3 enhancers or promoter was rarely observed in a publicly available dataset encompassing 118 MYC chromatin immunoprecipitation and sequencing (ChIP-Seq) in different tissues (Appendix Fig S5A). Binding was observed in two known FGFR3-dependent cell lines, MCF7 and HepG2 (Qiu et al, 2005; Tomlinson et al, 2012), in some blood-derived cell lines and in one lung cancer-derived cell line. MYC activation did not seem to be sufficient to induce FGFR3 regulation. Indeed, MYC ChIP-Seq data acquired for two inducible models of MYC overexpression/activation (LNCaP and U2OS cells; Walz et al, 2014; Barfeld et al, 2017) showed no MYC enrichment on the FGFR3 enhancers or promoter after MYC activation (Appendix Fig S5B and C). Our data therefore identify MYC as a master regulator of proliferation activated downstream from FGFR3 (Fig 1
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