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International audience; Hepatocellular carcinoma (HCC) usually afflicts individuals in their maturity after a protracted liver disease. Contrasting with this pattern, the age structure of HCC in Andean people displays a bimodal distribution with half of the patients developing HCC in adolescence and early adulthood. To deepen our understanding of the molecular determinants of the disease in this population, we conducted an integrative analysis of gene expression and DNA methylation in HCC developed by 74 Peruvian patients, including 39 adolescents and young adults. While genome-wide hypomethylation is considered as a paradigm in human HCCs, our analysis revealed that Peruvian tumors are associated with a global DNA hypermethylation. Moreover, pathway enrichment analysis of transcriptome data characterized an original combination of signatures. Peruvian HCC forgoes canonical activations of IGF2, Notch, Ras/MAPK, and TGF-β signals to depend instead on Hippo/YAP1, MYC, and Wnt/β-catenin pathways. These signatures delineate a homogeneous subtype of liver tumors at the interface of the proliferative and non-proliferative classes of HCCs. Remarkably, the development of this HCC subtype occurs in patients with one of the four Native American mitochondrial haplogroups A-D. Finally, integrative characterization revealed that Peruvian HCC is apparently controlled by the PRC2 complex that mediates cell reprogramming with massive DNA methylation modulating gene expression and pinpointed retinoid signaling as a potential target for epigenetic therapy.

International audience; An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffinembedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We lasermicrodissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

International audience; Upregulation of the telomerase reverse transcriptase (TERT) gene in human cancers leads to telomerase activation, which contributes to the growth advantage and survival of tumor cells. Molecular mechanisms of TERT upregulation are complex, tumor-specific and can be clinically relevant. To investigate these mechanisms in breast cancer, we sequenced the TERT promoter, evaluated TERT copy number changes and assessed the expression of the MYC oncogene, a known transcriptional TERT regulator, in two breast cancer cohorts comprising a total of 122 patients. No activating TERT promoter mutations were found, suggesting that this mutational mechanism is not likely to be involved in TERT upregulation in breast cancer. The T349C promoter polymorphism found in up to 50% of cases was not correlated with TERT expression, but T349C carriers had significantly shorter disease-free survival. TERT gains (15-25% of cases) were strongly correlated with increased TERT mRNA expression and worse patient prognosis in terms of disease-free and overall survival. Particularly aggressive breast cancers were characterized by an association of TERT gains with MYC overexpression. These results evidence a significant effect of gene copy number gain on the level of TERT expression and provide a new insight into the clinical significance of TERT and MYC upregulation in breast cancer.

// Bastien Jamet 1 , Thomas Carlier 2, 3 , Loic Campion 2, 4 , Emmanuelle Bompas 4 , Sylvie Girault 5 , Fanny Borrely 6 , Ludovic Ferrer 7 , Maxime Rousseau 1 , Yann Venel 6 , Francoise Kraeber-Bodere 1, 2, 3 and Caroline Rousseau 1, 2 1 Nuclear Medicine Unit, ICO Cancer Center Gauducheau, Saint Herblain, France 2 Nantes-Angers Cancer Research Center, INSERM U892, CNRS UMR 6299, University of Nantes, Nantes, France 3 Nuclear Medicine Unit, University Hospital, Nantes, France 4 Oncology Unit, ICO Cancer Center Gauducheau, Saint Herblain, France 5 Nuclear Medicine Unit, ICO Cancer Center Papin, Angers, France 6 Nuclear Medicine Unit, University Hospital Bretonneau, Tours, France 7 Physics Unit, ICO Cancer Center Gauducheau, Saint Herblain, France Correspondence to: Bastien Jamet, email: bastien.jamet@chu-nantes.fr Keywords: FDG-PET/CT, oncology, Ewing sarcoma family of tumors, survival analysis, prognosis Received: May 11, 2017 Accepted: June 27, 2017 Published: August 18, 2017 ABSTRACT Purpose: The aim of this retrospective study was to determine, at baseline, the prognostic value of different FDG-PET/CT quantitative parameters in a homogenous Ewing Sarcoma Family of Tumors (ESFT) adult population, compared with clinically relevant prognostic factors. Methods: Adult patients from 3 oncological centers, all with proved ESFT, were retrospectively included. Quantitative FDG-PET/CT parameters (SUV (maximum, peak and mean), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of the primary lesion of each patient were recorded before treatment, as well as usual clinical prognostic factors (stage of disease, location, tumor size, gender and age). Then, their relation with progression free survival (PFS) and overall survival (OS) was evaluated. Results: 32 patients were included. Median age was 21 years (range, 15 to 61). Nineteen patients (59%) were initially metastatic. On multivariate analysis, high SUV max remained independent predictor of worst OS (p=0.02) and PFS (p=0.019), metastatic disease of worst PFS (p=0.01) and high SUVpeak of worst OS (p=0.01). Optimal prognostic cut-off of SUV peak was found at 12.5 in multivariate analyses for PFS and OS ( p =0.0001). Conclusions: FDG-PET/CT, recommended at ESFT diagnosis for initial staging, can be a useful tool for predicting long-term adult patients outcome through semi-quantitative parameters.

MicroRNAs (miRs) participate in tumor growth and dissemination by regulating expression of various target genes. MiR-223-3p is suspected of being involved in head and neck squamous cell carcinoma (HNSCC) growth although its precise role has not been elucidated. In this study, we showed that miR-223-3p is present in biopsies of HNSCC patients and that its presence is correlated with high neutrophil infiltrate. We found that overexpression of miR-223-3p slightly increased proliferation of the CAL27 squamous carcinoma cell line both in vitro and in vivo. Moreover, miR-223-3p induced CAL27 apoptosis in an orthotopic xenograft mouse model, counteracting the proliferative effect and resulting in no impact on overall tumor growth. We analyzed the effect of miR-223-3p overexpression on signaling pathways and showed that it induced pERK2, pAKT and AKT, consistent with an increase in cell proliferation. In addition, we found that miR-223-3p reduced the STAT3 level correlating with increased cell apoptosis and inhibited vasculature formation. In HNSCC tissues, miR-223-3p expression was inversely correlated to CD31, highlighting the relationship between miR-223 and vessel formation. Finally, we studied the effect of miR-223-3p on response to selected anticancer agents and showed that cells expressing miR-223-3p are more resistant to drugs, notably cetuximab. In conclusion, our study is the first to show the antiangiogenic properties of miR-223-3p in HNSCC patients and to question whether expression levels of miR-223-3p can be evaluated as an indicator of eligibility for non-treatment of HNSCC patients with cetuximab.

International audience; The cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

Targeted therapies have substantially changed the management of non-small cell lung cancer (NSCLC) patients with driver oncogenes. Given the high frequency, EGFR and ALK aberrations were the first to be detected and paved the way for tyrosine kinase inhibitor (TKI) treatments. Other kinases such as ROS1 and more recently RET have emerged as promising targets, and ROS1 and RET TKIs are already available for precision medicine. We screened a large cohort of 713 Caucasian non-squamous NSCLC patients lacking EGFR/KRAS/BRAF/HER2/PI3KCA/ALK aberrations for ROS1 and RET rearrangements using fluorescence in situ hybridization to determine the frequency and clinicopathological characteristics of ROS1- and RET-positive patients. Frequencies of ROS1 and RET rearrangements were 2.1% and 2.52%, respectively. Contrary to common belief, both ROS1 and RET rearrangements were detected in patients with a history of smoking, and the RET-positive patients were not younger than the negative patients. Moreover, RET but not ROS1 rearrangement was associated with the female gender. Nearly half of the ROS1-rearranged patients were successfully treated with ROS1 TKIs. In contrast, only 5/18 RET-positive patients received off-label RET TKIs. Two patients had stable disease, and three experienced disease progression. In addition to the 18 RET-positive cases, 10 showed isolated 5′ signals. The clinical relevance is unknown but if the frequency is confirmed by other groups, the question whether these patients are eligible to TKIs will arise. More potent RET TKIs are under development and may improve the response rate in RET-positive patients. Therefore, we recommend the routine implementation of RET testing in non-squamous NSCLC patients, including those with a history of smoking.

International audience; Glypican-3 (GPC3) is an oncogene, frequently upregulated in liver malignancies such as hepatocellular carcinoma (HCC) and hepatoblastoma and constitutes a potential molecular target for therapy in liver cancer. Using a functional screening system, we identified 10 new microRNAs controlling GPC3 expression in malignant liver cells, five of them e.g. miR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v reduce GPC3 expression. These 5 microRNAs were significantly downregulated in tumoral compared to non-tumoral liver and inhibited tumor cell proliferation. Interestingly, miR-4510 inversely correlated with GPC3 mRNA and protein in HCC samples. This microRNA also induced apoptosis of hepatoma cells and blocked tumor growth in vivo in the chick chorioallantoic membrane model. We further show that the tumor suppressive effect of miR-4510 is mediated through direct targeting of GPC3 mRNA and inactivation of Wnt/β-catenin transcriptional activity and signaling pathway. Moreover, miR-4510 up-regulated the expression of several tumor suppressor genes while reducing the expression of other pro-oncogenes. In summary, we uncovered several new microRNAs targeting the oncogenic functions of GPC3. We provided strong molecular, cellular and in vivo evidences for the tumor suppressive activities of miR-4510 bringing to the fore the potential value of this microRNA in HCC therapy.

International audience; Angiogenesis represents a hallmark of tumor progression in Multiple Myeloma (MM), a still incurable malignancy. Here we analyzed the activity of cytokine-stimulated NK cells against tumor-associated endothelial cells isolated from bone marrow aspirates of MM patients with active disease (MMECs). We show that NK cells activated with optimal doses of IL-15 killed MMECs thanks to the concerted action of multiple activating receptors. In particular, according to the high expression of PVR and Nectin-2 on MMECs, DNAM-1 actively participated in target recognition. Interestingly, in MMECs the surface density of PVR was significantly higher than that detected in endothelium from patients with MM in complete remission or with monoclonal gammopathy of undetermined significance (MGUS). Importantly, IL-27, which unlike IL-15 does not display pro-angiogenic properties, maintained or increased the NK cell functions induced by suboptimal concentrations of IL-15. NK cell properties included killing of MMECs, IFN-γ production as well as a peculiar increase of NKp46 expression on NK cell surface. Finally, IL-27 showed a striking capability of up-regulating the expression of PD-L2 and HLA-I on tumor endothelium, whereas it did not modify that of PD-L1 and HLA-II. Our results suggest that cytokine-activated endogenous or adoptively transferred NK cells might support conventional therapies improving the outcome of MM patients.

International audience; Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

IF 5.168; International audience; HLA-A*0201/DRB1*0101 transgenic mice (A2/DR1 mice) have been developed to study the immunogenicity of tumor antigen-derived T cell epitopes. To extend the use and application of this mouse model in the field of antitumor immunotherapy, we described a tumor cell line generated from a naturally occurring tumor in A2/DR1 mouse named SARC-L1. Histological and genes signature analysis supported the sarcoma origin of this cell line. While SARC-L1 tumor cells lack HLA-DRB1*0101 expression, a very low expression of HLA-A*0201 molecules was found on these cells. Furthermore they also weakly but constitutively expressed the programmed death-ligand 1 (PD-L1). Interestingly both HLA-A*0201 and PD-L1 expressions can be increased on SARC-L1 after IFN-γ exposure in vitro. We also obtained two genetically modified cell lines highly expressing either HLA-A*0201 or both HLA-A*0201/ HLA-DRB1*0101 molecules referred as SARC-A2 and SARC-A2DR1 respectively. All the SARC-L1-derived cell lines induced aggressive subcutaneous tumors in A2DR1 mice in vivo. The analysis of SARC-L1 tumor microenvironment revealed a strong infiltration by T cells expressing inhibitory receptors such as PD-1 and TIM-3. Finally, we found that SARC-L1 is sensitive to several drugs commonly used to treat sarcoma and also susceptible to anti-PD-L1 monoclonal antibody therapy in vivo. Collectively, we described a novel syngeneic tumor model A2/DR1 mice that could be used as preclinical tool for the evaluation of antitumor immunotherapies.

International audience; Recent findings have shed new light on the mechanisms of action through which biguanides exert their anti-aging and cytostatic effects in Caenorhabditis elegans and human cell lines. The drop in energy charge resulting from the metformin mediated inhibition of mitochondrial activity affects the function of the nuclear pore complex, blocks mTOR signaling and enhances the expression of ACAD10. Whether the inhibition of this pathway is truly responsible for the anti-diabetic and cancer effects of the drug in mammals remains to be established.

// Maroua Manai 1, 5, 6, 7 , Jeanne Thomassin-Piana 2, * , Amor Gamoudi 6, * , Pascal Finetti 1, * , Marc Lopez 1 , Radhia Eghozzi 6 , Sinda Ayadi 6 , Olfa Ben Lamine 6 , Mohamed Manai 5 , Khaled Rahal 6 , Emmanuelle Charafe-Jauffret 2, 3 , Jocelyne Jacquemier 2 , Patrice Viens 3, 4 , Daniel Birnbaum 1 , Hamouda Boussen 5, 7 , Max Chaffanet 1 , Francois Bertucci 1, 3, 4 1 Departement d’Oncologie Moleculaire, Centre de Recherche en Cancerologie de Marseille, Aix Marseille Universite, Marseille, France 2 Departement de Bio-Pathologie, Institut Paoli-Calmettes, Marseille, France 3 UFR de Medecine, Aix Marseille Universite, Marseille, France 4 Departement d’Oncologie Medicale, Institut Paoli-Calmettes, Marseille, France 5 Departement de Biologie, Unite de Biochimie et Biologie Moleculaire, Faculte des Sciences de Tunis, Universite de Tunis El Manar, Tunisie 6 Departement d’Oncologie Medicale, Institut Salah Azaiez, Tunis, Tunisie 7 Service d’Oncologie Medicale, Hopital l’Ariana, Tunis, Tunisie * These authors contributed equally to second authors Correspondence to: Francois Bertucci, email: bertuccif@ipc.unicancer.fr Keywords: expression, immunohistochemistry, inflammatory breast cancer, MARCKS, survival Received: July 29, 2016 Accepted: December 14, 2016 Published: December 21, 2016 ABSTRACT Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. Results: Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E−09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. Materials and Methods: We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). Conclusions: MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC.

// Guenaelle Levallet 1 , Fatemeh Dubois 2 , Pierre Fouret 3 , Martine Antoine 4 , Solenn Brosseau 2,5,6 , Emmanuel Bergot 2,5 , Michele Beau-Faller 7 , Valerie Gounant 6,8 , Elisabeth Brambilla 9 , Didier Debieuvre 10 , Olivier Molinier 11 , Francoise Galateau-Salle 1 , Julien Mazieres 12 , Elisabeth Quoix 13 , Jean-Louis Pujol 14 , Denis Moro-Sibilot 15 , Alexandra Langlais 16 , Franck Morin 16 , Virginie Westeel 17 and Gerard Zalcman 5,6,18,19 1 Service d’Anatomie et Cytologie Pathologique, Centre Hospitalier Universitaire de Caen, Normandie Universite, Caen, France 2 Normandie Universite; UMR 1086 INSERM, Caen, France 3 Service d’Anatomie Pathologique, Pitie-Salpetriere, AP-HP, Universite Pierre et Marie Curie (UPMC), Paris, France 4 Service d’Anatomie Pathologique, Hopital Tenon, AP-HP, Universite Pierre et Marie Curie (UPMC), Paris, France 5 Service de Pneumologie et Oncologie Thoracique, Centre Hospitalier Universitaire de Caen, Normandie Universite, Caen, cedex, France 6 Service d’Oncologie Thoracique, Hopital Bichat-Claude Bernard, AP-HP, Universite Paris-Diderot, Paris, France 7 Laboratoire de Biochimie et Biologie moleculaire, Hopital de Hautepierre, Centre Hospitalier Universitaire de Strasbourg, Universite de Strasbourg, BP426 Strasbourg, Cedex, France 8 Service de Pneumologie, Hopital Tenon, AP-HP, Universite Pierre et Marie Curie (UPMC), Paris, France 9 Service d’Anatomie Pathologique, Hopital Albert Michallon, Centre Hospitalier Universitaire de Grenoble, La Tronche, France 10 Service de Pneumologie, GHRMSA-Centre Hospitalier Eric Muller, Mulhouse, France 11 Service de Pneumologie, Centre Hospitalier du Mans, Le Mans, France 12 Service de Pneumologie, Hopital Larrey, Centre Hospitalier Universitaire de Toulouse, Universite de Toulouse III, Toulouse, France 13 Service de Pneumologie, Nouvel Hopital Civil, Hospices Civils de Strasbourg, Universite de Strasbourg, BP426 Strasbourg, Cedex, France 14 Service de Pneumologie, Centre Hospitalier Universitaire Arnaud de Villeneuve, Universite de Montpellier, Montpellier, France 15 Unite d’Oncologie Thoracique–Pneumologie, CHU de Grenoble, INSERM U823, Grenoble, France 16 Intergroupe Francophone de Cancerologie Thoracique (IFCT), Paris, France 17 Service de Pneumologie, Hopital Jean-Minjoz, Centre Hospitalier Universitaire de Besancon, Universite de Franche-Comte, Besancon, France 18 CIC INSERM 1425-CLIP2 Paris-Nord, Hopital Bichat-Claude Bernard, AP-HP, Paris, France 19 On Behalf of the IFCT (Intergroupe Francophone de Cancerologie Thoracique) Correspondence to: Gerard Zalcman, email: // Keywords : non-small cell lung cancer; neo-adjuvant chemotherapy; MSH2; BRCA1; MGMT Received : August 08, 2016 Accepted : December 05, 2016 Published : December 19, 2016 Abstract Introduction: DNA repair is a double-edged sword in lung carcinogenesis. When defective, it promotes genetic instability and accumulated genetic alterations. Conversely these defects could sensitize cancer cells to therapeutic agents inducing DNA breaks. Methods: We used immunohistochemistry (IHC) to assess MSH2, XRCC5, and BRCA1 expression in 443 post-chemotherapy specimens from patients randomized in a Phase 3 trial, comparing two neoadjuvant regimens in 528 Stage I-II non-small cell lung cancer (NSCLC) patients (IFCT-0002). O 6 MGMT promoter gene methylation was analyzed in a subset of 208 patients of the same trial with available snap-frozen specimens. Results: Median follow-up was from 90 months onwards. Only high BRCA1 (n = 221, hazard ratio [HR] = 1.58, 95% confidence interval [CI] [ 1 . 07 - 2 . 34 ], p = 0.02) and low MSH2 expression (n = 356, HR = 1.52, 95% CI [ 1 . 11 - 2 . 08 ], p = 0.008) significantly predicted better overall survival (OS) in univariate and multivariate analysis. A bootstrap re-sampling strategy distinguished three patient groups at high ( n = 55, low BRCA1 and high MSH2, median OS >96 months, HR = 2.5, 95% CI [ 1 . 45 - 4 . 33 ], p = 0.001), intermediate (n = 82, median OS = 73.4 p = 0.0596), and low (high BRCA1 and low MSH2, n = 67, median OS = ND, HR = 0.51, 95% CI [ 0 . 31 - 0 . 83 ], p = 0.006) risk of death. Interpretation: DNA repair protein expression assessment identified three different groups of risk of death in early-stage lung cancer patients, according to their tumor MSH2 and BRCA1 expression levels. These results deserve prospective evaluation of MSH2/BRCA1 theranostic value in lung cancer patients treated with combinations of DNA-damaging chemotherapy and drugs targeting DNA repair, such as Poly(ADP-ribose) polymerase (PARP) inhibitors.

// Anthony Goncalves 1, 2, * , Francois Bertucci 1, 2, 3, * , Arnaud Guille 2, 3 , Severine Garnier 2, 3 , Jose Adelaide 2, 3 , Nadine Carbuccia 2, 3 , Oliver Cabaud 2, 3 , Pascal Finetti 2, 3 , Serge Brunelle 4 , Gilles Piana 4 , Jeanne Tomassin-Piana 5 , Maria Paciencia 5 , Eric Lambaudie 6 , Cornel Popovici 2, 7 , Renaud Sabatier 1, 2, 3 , Carole Tarpin 1 , Magali Provansal 1 , Jean-Marc Extra 1 , Francois Eisinger 2, 7 , Hagay Sobol 2, 7 , Patrice Viens 1, 2 , Marc Lopez 2, 3 , Christophe Ginestier 2, 3 , Emmanuelle Charafe-Jauffret 2, 3, 5 , Max Chaffanet 2, 3, # , Daniel Birnbaum 2, 3, # 1 Department of Medical Oncology, Institut Paoli-Calmettes, Marseille, France 2 Aix Marseille Univ, CNRS U7258, INSERM U1068, Institut Paoli-Calmettes, CRCM, Marseille, France 3 Department of Molecular Oncology, Institut Paoli-Calmettes, Marseille, France 4 Department of Imaging, Institut Paoli-Calmettes, Marseille, France 5 Department of Biopathology, Institut Paoli-Calmettes, Marseille, France 6 Department of Surgical Oncology, Institut Paoli-Calmettes, Marseille, France 7 Department of Oncogenetics, Institut Paoli-Calmettes, Marseille, France * both authors equally contributed # both authors equally supervised Correspondence to: Anthony Goncalves, email: goncalvesa@ipc.unicancer.fr Keywords: precision medicine, advanced breast cancer, NGS, CGH, patient-derived xenograft Received: July 10, 2016 Accepted: September 29, 2016 Published: October 18, 2016 ABSTRACT Background: Routine feasibility and clinical impact of genomics-based tumor profiling in advanced breast cancer (aBC) remains to be determined. We conducted a pilot study to evaluate whether precision medicine could be prospectively implemented for aBC patients in a single center and to examine whether patient-derived tumor xenografts (PDX) could be obtained in this population. Results: Thirty-four aBC patients were included. Actionable targets were found in 28 patients (82%). A targeted therapy could be proposed to 22 patients (64%), either through a clinical trial (n=15) and/or using already registered drugs (n=21). Ten patients (29%) eventually received targeted treatment, 2 of them deriving clinical benefit. Of 22 patients subjected to mouse implantation, 10 had successful xenografting (45%), mostly in triple-negative aBC. Methods: aBC patients accessible to tumor biopsy were prospectively enrolled at the Institut Paoli-Calmettes in the BC-BIO study ( ClinicalTrials.gov , NCT01521676). Genomic profiling was established by whole-genome array comparative genomic hybridization (aCGH) and targeted next-generation sequencing (NGS) of 365 candidate cancer genes. For a subset of patients, a sample of fresh tumor was orthotopically implanted in humanized cleared fat pads of NSG mice for establishing PDX. Conclusions: Precision medicine can be implemented in a single center in the context of clinical practice and may allow genomic-driven treatment in approximately 30% of aBC patients. PDX may be obtained in a significant fraction of cases.

International audience; Pancreatic adenocarcinoma (PDAC) is a dismal disease. The lack of specific symptoms still leads to a delay in diagnosis followed by death within months for most patients. Exon 11 of the bile salt-dependent lipase (BSDL) gene encoding variable number of tandem repeated (VNTR) sequences has been involved in pancreatic pathologies. We hypothesized that BSDL VNTR sequences may be mutated in PDAC. The amplification of BSDL VNTR from RNA extracted from pancreatic SOJ-6 cells allowed us to identify a BSDL amplicon in which a cytosine residue is inserted in a VNTR sequence. This insertion gives rise to a premature stop codon, resulting in a truncated protein and to a modification of the C-terminal amino-acid sequence; that is PRAAHG instead of PAVIRF. We produced antibodies directed against these sequences and examined pancreatic tissues from patients with PDAC and PanIN. Albeit all tissues were positive to anti-PAVIRF antibodies, 72.2% of patient tissues gave positive reaction with anti-PRAAHG antibodies, particularly in dysplastic areas of the tumor. Neoplastic cells with ductal differentiation were not reactive to anti-PRAAHG antibodies. Some 70% of PanIN tissues were also reactive to anti-PRAAHG antibodies, suggesting that the C insertion occurs early during pancreatic carcinogenesis. Data suggest that anti-PRAAHG antibodies were uniquely reactive with a short isoform of BSDL specifically expressed in pre-neoplastic lesions of the pancreas. The detection of truncated BSDL reactive to antibodies against the PRAAHG C-terminal sequence in pancreatic juice or in pancreatic biopsies may be a new tool in the early diagnosis of PDAC.

International audience; Pancreatic cancer is one of the most aggressive human cancers. PD1/PDL1-inhibitors recently showed promising results in different cancers with correlation between PDL1 tumor expression and responses. Expression of programmed cell death receptor ligand 1 (PDL1) has been scarcely studied in pancreatic cancer. In this retrospective study, we analyzed PDL1 mRNA expression in 453 clinical pancreatic cancer samples profiled using DNA microarrays and RNASeq. Compared to normal pancreatic samples, PDL1 expression was upregulated in 19% of cancer samples. Upregulation was not associated with clinicopathological features such as patients' age and sex, pathological type, tumor size, lymph node status, and grade, but was associated with shorter disease-free survival and overall survival in multivariate analyses. Analysis of correlations with biological parameters showed that PDL1 upregulation was associated with some degree of lymphocyte infiltration and signs of anti-tumor T-cell response, but to a lesser extent than what has been reported in breast cancer and GIST. PDL1-up pancreatic cancers displayed profiles of lymphocyte exhaustion, were more enriched in inhibitory molecules and pro-tumor populations (Tregs with upregulation of FOXP3 and IL10, myeloid-derived suppressor cells with upregulation of CD33 and S100A8/A9), and demonstrated a down-modulation of most MHC class I members (HLA-A/B/C, HLA-E/F/G) suggestive of a defect in antigen processing and presentation. In conclusion, our results suggest that PDL1 expression might refine the prediction of metastatic relapse in operated pancreatic cancer, and that PD1/PDL1 inhibitors might reactivate inhibited T-cells to increase the anti-tumor immune response in PDL1-upregulated tumors.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) is the first example of targeted therapy. In fact, the oncogenic fusion-protein (PML-RAR) typical of this leukemia contains the retinoid-nuclear-receptor RARalpha. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARalpha proteins are present in APL blasts, i.e. RARalpha1 and RARalpha2. We developed different cell populations characterized by PML-RAR, RARalpha2 and RARalpha1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARalpha2 results in similar increases in the constitutive expression of several granulocytic differentiation markers. This is accompanied by enhanced expression of the same granulocytic markers upon exposure of the NB4 blasts to ATRA. Silencing of PML-RAR and RARalpha2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARalpha2, RARalpha1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARalpha2 in NB4 cells. RARalpha2 action on myeloid differentiation does not require the presence of PML-RAR, as it is recapitulated also upon knock-down in PML-RAR-negative HL-60 cells. Thus, relative to RARalpha1, PML-RAR and RARalpha2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARalpha2 interact with and inhibit the transcriptional activity of RARalpha1. The interaction surface is located in the carboxy-terminal domain containing the D/E/F regions and it is influenced by phosphorylation of Ser-369 of RARalpha1.

International audience; Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

// Sophie Bustany 1, * , Jerome Bourgeais 2, * , Guergana Tchakarska 1, 4 , Simon Body 1 , Olivier Herault 2, 3 , Fabrice Gouilleux 2 , Brigitte Sola 1 1 Universite de Caen Normandie, EA4652 (MILPAT), MICAH Team, Caen, France 2 Universite Francois Rabelais, CNRS UMR 7292 (GICC), LNox Team, Tours, France 3 Service d'Hematologie Biologique, CHRU Tours, Tours, France 4 Present address: Cytogenetics Laboratory, Research Institute, McGill University Health Centre, Montreal, Canada * Co-first authors Correspondence to: Brigitte Sola, email: brigitte.sola@unicaen.fr Keywords: reactive oxygen species, p44/42 mitogen-activated protein kinase, pomalidomide, carfilzomib, NADPH oxidase Received: February 01, 2016 Accepted: May 28, 2016 Published: June 07, 2016 ABSTRACT The interactions of multiple myeloma (MM) cells with their microenvironment are crucial for pathogenesis. MM cells could interact differentially with their microenvironment depending on the type of cyclin D they express. We established several clones that constitutively express cyclin D1 from the parental RPMI8226 MM cell line and analyzed the impact of cyclin D1 expression on cell behavior. We performed a gene expression profiling study on cyclin D1-expressing vs . control cells and validated the results by semi-quantitative RT-PCR. The expression of cyclin D1 altered the transcription of genes that control adhesion and migration. We confirmed that cyclin D1 increases cell adhesion to stromal cells and fibronectin, stabilizes F-actin fibers, and enhances chemotaxis and inflammatory chemokine secretion. Both control and cyclin D1-expressing cells were more resistant to acute carfilzomib treatment when cultured on stromal cells than in suspension. However, this resistance was specifically reduced in cyclin D1-expressing cells after pomalidomide pre-treatment that modifies tumor cell/microenvironment interactions. Transcriptomic analysis revealed that cyclin D1 expression was also associated with changes in the expression of genes controlling metabolism. We also found that cyclin D1 expression disrupted the redox balance by producing reactive oxygen species. The resulting oxidative stress activated the p44/42 mitogen-activated protein kinase (or ERK1/2) signaling pathway, increased cell adhesion to fibronectin or stromal cells, and controlled drug sensitivity. Our results have uncovered a new function for cyclin D1 in the control of redox metabolism and interactions of cyclin D1-expressing MM cells with their bone marrow microenvironment.

International audience; We characterized the mechanism of action of the neuregulin-non-competitive anti-HER3 therapeutic antibody 9F7-F11 that blocks the PI3K/AKT pathway, leading to cell cycle arrest and apoptosis in vitro and regression of pancreatic and breast cancer in vivo. We found that 9F7-F11 induces rapid HER3 down-regulation. Specifically, 9F7-F11-induced HER3 ubiquitination and degradation in pancreatic, breast and prostate cancer cell lines was driven mainly by the itchy E3 ubiquitin ligase (ITCH/AIP4). Overexpression of the ITCH/AIP4 inhibitor N4BP1 or small-interfering RNA-mediated knockdown of ITCH/AIP4 inhibited HER3 ubiquitination/degradation and PI3K/AKT signaling blockade induced by 9F7-F11. Moreover, 9F7-F11-mediated JNK1/2 phosphorylation led to ITCH/AIP4 activation and recruitment to HER3 for receptor ubiquitination and degradation. ITCH/AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference. Taken together, our results suggest that 9F7-F11-induced HER3 ubiquitination and degradation in cancer cells mainly occurs through JNK1/2-dependent ITCH/AIP4 activation.

International audience; Since its cloning a decade ago, TRPM8 channel has emerged as a promising prognostic marker and a putative therapeutic target in prostate cancer (PCa). However, recent studies have brought to light the complexity of TRPM8 isoforms in PCa. Consequently, the respective role of each TRPM8 isoform needs to be deciphered prior to considering TRPM8 as an attractive therapeutic target. Full-length (6 transmembrane (TM)-domain) TRPM8 channel is overexpressed in early PCa and repressed in advanced prostate tumors whereas the localization of the truncated, 4TM-TRPM8 channel (4 transmembrane (TM)-domain), in the membranes of endoplasmic reticulum (ER) is independent of the pathogenic status of epithelial cells. In the same line, expression of non-channel cytoplasmic small TRPM8 isoforms (namely sM8) is conserved in cancer cells. In this study, we identify sM8s as putative regulator of PCa cell death. Indeed, suppression of sM8 isoforms was found to induce concomitantly ER stress, oxidative stress, p21 expression and apoptosis in human epithelial prostate cancer cells. We furthermore demonstrate that induction of such mechanisms required the activity of 4TM-TRPM8 channels at the ER-mitochondria junction. Our study thus suggests that targeting sM8 could be an appropriate strategy to fight prostate cancer.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib. Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

International audience; Terminal fucosylated motifs of glycoproteins and glycolipid chains are often altered in cancer cells. We investigated the link between fucosylation changes and critical steps in cancer progression: epithelial-to-mesenchymal transition (EMT) and lymph node metastasis. Using mammary cell lines, we demonstrate that during EMT, expression of some fucosylated antigens (e.g.: Lewis Y) is decreased as a result of repression of the fucosyltransferase genes FUT1 and FUT3. Moreover, we identify the fucose-binding bacterial lectin BC2L-C-Nt as a specific probe for the epithelial state. Prolectin (CLEC17A), a human lectin found on lymph node B cells, shares ligand specificities with BC2L-C-Nt. It binds preferentially to epithelial rather than to mesenchymal cells, and microfluidic experiments showed that prolectin behaves as a cell adhesion molecule for epithelial cells. Comparison of paired primary tumors/ lymph node metastases revealed an increase of prolectin staining in metastasis and high FUT1 and FUT3 mRNA expression was associated with poor prognosis. Our data suggest that tumor cells invading the lymph nodes and expressing fucosylated motifs associated with the epithelial state could use prolectin as a colonization factor.

International audience; Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

Introduction treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have improved the natural history of HER2-positive breast cancer. However, except HER2 protein expression and gene amplification, there is no predictive biomarker to guide the HER2-targeted therapies. We developed Parallel reaction monitoring (PRM) a powerful approach, to quantify and evaluate key proteins involved in the HER2 pathway and/or anti-HER2 treatment sensitivity. Results in BCLs, PRM measurements correlated with western blot immunocytochemistry and transcriptomic data. At baseline, higher expression of HER2, EGFR, PTEN and HER3 but lower expression of phospho-HER2 correlated with trastuzumab sensitivity. Under trastuzumab, PRM demonstrated a decrease in HER2 and an increase in phospho-HER2, which correlated with drug sensitivity. The opposite was observed under lapatinib. HER2 quantification was also correlated with immunohistochemistry in PDXs and clinical breast cancer samples. Discussion in conclusion, PRM-based assay, developed to quantify proteins of the HER2 pathway in breast cancer samples revealed a large magnitude of expression, which may have relevance in terms of treatment sensitivity. Materials and Methods we first evaluated PRM in term of sensitivity, linearity and reproducibility. PRM was then applied to breast cancer cell lines (BCLs) including BCLs exposed to anti-HER2 agents, patient-derived xenografts (PDXs) and frozen breast cancer samples.

// Emmanuelle Martinez 1, 2 , Francoise Silvy 1, 2 , Frederic Fina 1, 2, 3 , Marc Bartoli 4 , Martin Krahn 4, 5 , Fabrice Barlesi 1, 2, 6 , Dominique Figarella-Branger 1, 2, 7 , Juan Iovanna 8, 9, 10 , Rene Laugier 11 , Mehdi Ouaissi 1, 2, 12 , Dominique Lombardo 1, 2 , Eric Mas 1, 2 1 Aix-Marseille Universite, CRO2, Centre de Recherche en Oncologie biologique et Oncopharmacologie, F-13005, Marseille, France 2 INSERM, UMR_S 911, F-13005, Marseille, France 3 LBM- Assistance Publique Hopitaux de Marseille, Hopital Nord, Service de Transfert d’Oncologie Biologique, F-13015, Marseille, France 4 Aix-Marseille Universite, INSERM, UMR 910, F-13005, Marseille, France 5 Assistance Publique Hopitaux de Marseille, Hopital de la Timone-Enfants, Departement de Genetique Medicale, F-13005, Marseille, France 6 Assistance Publique Hopitaux de Marseille, Hopital Nord, Service d’Oncologie Multidisciplinaire & Innovation Therapeutique, F-13915, Marseille, France 7 Assistance Publique Hopitaux de Marseille, Hopital de la Timone, Service d’Anatomopathologie, F-13005, Marseille, France 8 Aix-Marseille Universite, CRCM, Centre de Recherche en Cancerologie de Marseille, F-13009, Marseille, France 9 INSERM, UMR_S 1068, F-13009, Marseille, France 10 CNRS, UMR 7258, F-13009, Marseille, France 11 Assistance Publique Hopitaux de Marseille, Hopital de la Timone, Service de Gastroenterologie, F-13005, Marseille, France 12 Assistance Publique Hopitaux de Marseille, Hopital de la Timone, Service de Chirurgie Digestive et Viscerale, F-13005, Marseille, France Correspondence to: Eric Mas, e-mail: eric.mas@univ-amu.fr Dominique Lombardo, e-mail: dominique.lombardo@univ-amu.fr Keywords: SNP, rs488087, pancreatic cancer Abbreviations: BSDL, bile-salt-dependent lipase; PDAC, pancreatic ductal adenocarcinoma; PC, pancreatic cancers Received: June 23, 2015 Accepted: October 05, 2015 Published: October 16, 2015 ABSTRACT Pancreatic cancer (PC) is a devastating disease progressing asymptomatically until death within months after diagnosis. Defining at-risk populations should promote its earlier diagnosis and hence also avoid its development. Considering the known involvement in pancreatic disease of exon 11 of the bile salt-dependent lipase (BSDL) gene that encodes variable number of tandem repeat (VNTR) sequences, we hypothesized upon the existence of a genetic link between predisposition to PC and mutations in VNTR loci. To test this, BSDL VNTR were amplified by touchdown-PCR performed on genomic DNA extracted from cancer tissue or blood samples from a French patient cohort and amplicons were Sanger sequenced. A robust method using probes for droplet digital (dd)-PCR was designed to discriminate the C/C major from C/T or T/T minor genotypes. We report that the c.1719C > T transition (SNP rs488087) present in BSDL VNTR may be a useful marker for defining a population at risk of developing PC (occurrence: 63.90% in the PC versus 27.30% in the control group). The odds ratio of 4.7 for the T allele was larger than those already determined for other SNPs suspected to be predictive of PC. Further studies on tumor pancreatic tissue suggested that a germline T allele may favor Kras G12R/G12D somatic mutations which represent negative prognostic factors associated with reduced survival. We propose that the detection of the T allele in rs488087 SNP should lead to an in-depth follow-up of patients in whom an association with other potential risk factors of pancreatic cancer may be present.

International audience; Renal Cell Carcinomas (RCCs) are heterogeneous tumors with late acquisition of TP53 abnormalities during their evolution. They harbor TP53 abnormalities in their metastases. We aimed to study TP53 gene alterations in tissue samples from primary and metastatic RCCs in 36 patients followed up over a median of 4.2 years, and in xenografted issued from primary RCCs. In 36 primary RCCs systematically xenografted in mice, and in biopsies of metastases performed whenever possible during patient follow-up, we studied p53expressing tumor cells and TP53 gene abnormalities. We identified TP53 gene alterations in primary tumors, metastases and xenografts. Quantification of tumors cells with TP53 gene alterations showed a significant increase in the metastases compared to the primary RCCs, and, strikingly, the xenografts were similar to the metastases and not to the primary RCCs from which they were derived. Using laser-microdissection of p53-expressing tumor cells, we identified TP53mutated tumor cells in the xenografts derived from the primary RCC, and in a lung metastasis later developed in one patient. The mutation enabled us to track back their origin to a minority sub-clone in the primary heterogeneous RCC. Combining in situ and molecular analyses, we demonstrated a clonal expansion in a living patient with metastatic RCC.

International audience; Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were PIK3CA (25%) followed by FBXW7 (15%), FAT1 (15%), and TRIP12 (15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting PIK3CA) and losses of chromosome 11q (affecting ATM). The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.

// Veronique D’Hondt 1, 2 , Magalie Lacroix-Triki 3 , Marta Jarlier 4 , Florence Boissiere-Michot 5 , Carole Puech 1, 2, 6, 7 , Peter Coopman 1, 2, 6, 7 , Dionyssios Katsaros 8 and Gilles Freiss 1, 2, 6, 7 1 IRCM, Institut de Recherche en Cancerologie de Montpellier, Montpellier F-34298, France 2 Institut regional du Cancer de Montpellier, Montpellier F-34298, France 3 Departement de Biologie et Pathologie Medicales, Gustave-Roussy Cancer Campus, 94805 Villejuif cedex, France 4 Unite de Biometrie, Institut regional du Cancer de Montpellier, Montpellier F-34298, France 5 Unite de Recherche Translationnelle, Institut Regional du Cancer de Montpellier, Montpellier F-34298, France 6 INSERM, U 1194, Montpellier F-34298, France 7 Universite de Montpellier, Montpellier F-34090, France 8 Azienda Ospedaliero-Universitaria Cittadella Salute, Presidio S. Anna and Department of Surgical Science, Gynecology, University of Torino, Torino, Italy Correspondence to: Gilles Freiss, email: gilles.freiss@inserm.fr Keywords: high-grade serous ovarian carcinoma; PTPN13; 4q LOH; tyrosine phosphatase; prognosis Received: May 16, 2017 Accepted: August 16, 2017 Published: September 21, 2017 ABSTRACT Background: Chromosome 4q loss of heterozygosity (LOH) is frequently observed in high-grade serous ovarian carcinoma (HGSOC). However, this LOH has not been clearly associated with the inactivation of any tumor suppressor gene(s). As the tumor suppressor gene PTPN13 is located on chromosome 4q21, we investigated its expression in HGSOC. Methods: PTPN13 protein expression was investigated by immunohistochemistry (IHC) in normal ovary epithelium and in 30 HGSOC samples, whereas PTPN13 mRNA expression was quantified by RT-PCR in another independent cohort of 28 HGSOC samples. Patients in both cohorts were followed for more than 8.5 years. Results: PTPN13 protein expression was lower in one third of HGSOC samples compared with normal ovary epithelium. In both cohorts, high PTPN13 expression level (mRNA or protein) in the tumor was associated with favorable outcome and significantly longer survival (HR=0.27; p=0.0087 and HR=0.42; p=0.03, respectively). Conclusion: This study demonstrates, for the first time, that high PTPN13 expression level is a prognostic indicator of favorable outcome in patients with HGSOC. This finding, in conjunction with our previous mechanistic studies, suggests that PTPN13 loss, possibly by 4q LOH, enhances HGSOC aggressiveness and highlight the interest of studying PTPN13 signaling in HGSOC to identify new potential therapeutic targets.

International audience; By gating the traffic of molecules and cells across the vessel wall, endothelial cells play a central role in regulating cardiovascular functions and systemic homeostasis and in modulating pathophysiological processes such as inflammation and immunity. Accordingly, the loss of endothelial cell integrity is associated with pathological disorders that include atherosclerosis and cancer. The p38 mitogen-activated protein kinase (MAPK) cascades are major signaling pathways that regulate several functions of endothelial cells in response to exogenous and endogenous stimuli including growth factors, stress and cytokines. The p38 MAPK family contains four isoforms p38α, p38β, p38γ and p38δ that are encoded by four different genes. They are all widely expressed although to different levels in almost all human tissues. p38α/MAPK14, that is ubiquitously expressed is the prototype member of the family and is referred here as p38. It regulates the production of inflammatory mediators, and controls cell proliferation, differentiation, migration and survival. Its activation in endothelial cells leads to actin remodeling, angiogenesis, DNA damage response and thereby has major impact on cardiovascular homeostasis, and on cancer progression. In this manuscript, we review the biology of p38 in regulating endothelial functions especially in response to oxidative stress and during the metastatic process.

// Guilhem Bousquet 1, 2, 3, 4 , Morad El Bouchtaoui 2 , Tan Sophie 2 , Christophe Leboeuf 1, 2 , Cedric de Bazelaire 1, 2, 5 , Philippe Ratajczak 1, 2 , Sylvie Giacchetti 6 , Anne de Roquancourt 1, 2, 7 , Philippe Bertheau 1, 2, 7 , Laurence Verneuil 1, 2 , Jean-Paul Feugeas 8 , Marc Espie 1, 6 , Anne Janin 1, 2, 7 1 Universite Paris Diderot, Sorbonne Paris Cite, Laboratoire Pathologie, Paris, France 2 INSERM, Paris, France 3 Universite Paris 13, Villetaneuse, France 4 AP, HP, Avicenne, Service Oncologie, Paris, France 5 AP HP Hopital Saint-Louis, Service Radiologie, Paris, France 6 AP HP Hopital Saint-Louis, Centre Maladies Sein, Paris, France 7 AP HP Hopital Saint-Louis, Service Pathologie, Paris, France 8 INSERM, Paris, France Correspondence to: Guilhem Bousquet, email: guilhem.bousquet@aphp.fr Anne Janin, email: anne.janin1165@gmail.com Keywords: breast cancer stem cells, TNBC, chemoresistance, autophagy, hypoxia Received: March 21, 2017 Accepted: March 29, 2017 Published: April 07, 2017 ABSTRACT There is growing evidence for the role of cancer stem-cells in drug resistance, but with few in situ studies on human tumor samples to decipher the mechanisms by which they resist anticancer agents. Triple negative breast cancer (TNBC) is the most severe sub-type of breast cancer, occurring in younger women and associated with poor prognosis even when treated at a localized stage. We investigated here the relationship between complete pathological response after chemotherapy and breast cancer stem-cell characteristics in pre-treatment biopsies of 78 women with triple negative breast carcinoma (TNBC). We found that chemoresistance was associated with large numbers of breast cancer stem-cells, and that these cancer stem-cells were neither proliferative nor apoptotic, but in an autophagic state related to hypoxia. Using relevant pharmacological models of patient-derived TNBC xenografts, we further investigated the role of autophagy in chemoresistance of breast cancer stem-cells. We demonstrated that hypoxia increased drug resistance of autophagic TNBC stem-cells, and showed that molecular or chemical inhibition of autophagic pathway was able to reverse chemoresistance. Our results support breast cancer stem-cell evaluation in pre-treatment biopsies of TNBC patients, and the need for further research on autophagy inhibition to reverse resistance to chemotherapy.

International audience; Due to its abundant chondrogenic matrix and hypoxic tissue, chondrosarcoma is chemo-and radio-resistant. Our group has developed a proteoglycan targeting strategy by using a quaternary ammonium (QA) function as a carrier of DNA alkylating agents to chondrosarcoma environment. Here, we assessed the relevance of this strategy applied to hypoxia-activated prodrugs, by conjugating a QA to 2-nitroimidazole phosphoramidate. This derivative, named as 8-QA, was evaluated respectively to its non-QA equivalent and to a QA-conjugated but non-hypoxia activated. Firstly binding to aggrecan was confirmed from dissociation constant determined by Surface Plasmon Resonance. In vitro, in HEMC-SS chondrosarcoma cells cultured in monolayer and in spheroids, 8-QA showed higher cytotoxic activity in hypoxia versus normoxia, and led to a strong accumulation of cells in S phase and apoptosis. In vivo, a HEMC-SS xenograft model was implanted on SCID mice and characterized for hypoxia by photoacoustic imaging as well as proteoglycan content. When HEMC-SS bearing mice were given 8-QA at 47 μmol/kg according to a q4d x 6 schedule, a significant 62.1% inhibition of tumor growth was observed, without associated hematological side effects. Mechanistic studies of treated tumors highlighted decrease in mitotic index associated to increase in both p21 and p53S15 markers. Interestingly, 8-QA treatment induced an increase of DNA damages as measured by γH2AX predominantly found in pimonidazole-positive hypoxic areas. These preclinical results are the first to demonstrate the interest of addressing hypoxia-activated prodrugs selectively to proteoglycan of chondrogenic tumor tissue, as a promising therapeutic strategy.

International audience; Understanding the molecular and cellular processes underlying melanoma plasticity and heterogeneity is of paramount importance to improve the efficiency of current treatment and to overcome resistance to chemotherapy drugs. The notion of plasticity and heterogeneity implies the existence of melanoma cell populations with different phenotypic and tumorigenic properties. Using melanoma cell lines and melanoma cells freshly isolated from patient biopsies, we investigated the relationship between ABCB5+, CD271+ and low-MITF, expressing populations that were reported to display melanoma initiating cell properties. Here, we showed that ABCB5+ and CD271+ populations poorly overlap. However, we found that the CD271+ population is enriched in low-MITF cells and expresses a higher level of stemness genes, such as OCT4, NANOG and NES. These features could explain the increased tumorigenicity of the CD271+ cells. The rapid conversion of CD271+ to CD271- cells in vitro demonstrates the plasticity ability of melanoma cells. Finally, we observed that the transient slow-growing population contains only CD271+ cells that are highly tumorigenic. However, the fast growing/CD271+ population exhibits a poor tumorigenic ability. Taking together, our data show that CD271 is an imperfect marker for melanoma initiating cells, but may be useful to identify melanoma cells with an increased stemness and tumorigenic potential.

// Mickael Ohanna 1,2 , Caroline Bonet 1,2 , Karine Bille 1,2 , Maryline Allegra 1,2 , Irwin Davidson 3 , Philippe Bahadoran 2,4 , Jean-Philippe Lacour 2,4 , Robert Ballotti 1,2 and Corine Bertolotto 1,2 . 1, INSERM, U1065 (equipe 1), Equipe labelisee Ligue Contre le Cancer, C3M, Nice, France. 2, Universite de Nice Sophia-Antipolis, UFR Medecine, Nice, France. 3, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS, INSERM, Universite de Strasbourg, Illkirch, France. 4, Centre Hospitalier Universitaire, Service de Dermatologie, Nice, France Correspondence: Corine Bertolotto, email: // Keywords : melanoma, MITF, SIRT1, PLX4032, treatment Received : February 7, 2014 Accepted :February 18, 2014 Published : February 19, 2014 Abstract SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF -i nduced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAF V600E -mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAF V600E -mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option.

// Lucie Hebert 1 , Dorine Bellanger 1 , Chloe Guillas 1 , Antoine Campagne 2 , Florent Dingli 3 , Damarys Loew 3 , Alice Fievet 1, 4 , Virginie Jacquemin 1 , Tatiana Popova 1 , Didier Jean 5 , Fatima Mechta-Grigoriou 1 , Raphael Margueron 2 and Marc-Henri Stern 1, 4 1 Department of Genetics and Biology of Cancers, INSERM U830, Institut Curie, PSL Research University, Paris 75248, France 2 Department of Developmental Biology and Genetics, CNRS UMR 3215/INSERM U934, Institut Curie, PSL Research University, Paris 75248, France 3 Mass Spectrometry and Proteomics facility, Institut Curie, PSL Research University, Paris 75248, France 4 Department of Genetics, Institut Curie, Paris 75248, France 5 INSERM UMR-1162, Paris 75010, France Correspondence to: Marc-Henri Stern, email: marc-henri.stern@curie.fr Keywords: BAP1, tumor suppressor, de-ubiquitination, proteomics, protein stability Received: October 29, 2016 Accepted: July 23, 2017 Published: August 03, 2017 ABSTRACT The tumor suppressor BAP1 associates with ASXL1/2 to form the core Polycomb complex PR-DUB, which catalyzes the removal of mono-ubiquitin from several substrates including histone H2A. This complex also mediates the poly-deubiquitination of HCFC1, OGT and PCG1-α, preventing them from proteasomal degradation. Surprisingly, considering its role in a Polycomb complex, no transcriptional signature was consistently found among BAP1 -inactivated tumor types. It was hypothesized that BAP1 tumor suppressor activity could reside, at least in part, in stabilizing proteins through its poly-deubiquitinase activity. Quantitative mass spectrometry and gene expression arrays were used to investigate the consequences of BAP1 expression modulation in the NCI-H226 mesothelioma cell line. Analysis of differentially expressed proteins revealed enrichment in cytoskeleton organization, mitochondrial activity and ROS management, while gene expression analysis revealed enrichment in the epithelial-to-mesenchymal transition pathway. Functional assessments in BAP1 inactivated, BAP1 wild-type and BAP1 catalytically dead-expressing NCI-H226 and QR mesothelioma cell lines confirmed alteration of these pathways and demonstrated that BAP1 deubiquitinase activity was mandatory to maintain these phenotypes. Interestingly, monitoring intracellular ROS levels partly restored the morphology and the mitochondrial activity. Finally, the study suggests new tumorigenic and cellular functions of BAP1 and shows for the first time the interest of studying the proteome as readout of BAP1 inactivation.

// Clotilde Dumars 1,2,3 , Jean-Michel Ngyuen 2,3 , Aurelie Gaultier 3 , Rachel Lanel 1,2 , Nadege Corradini 4 , Francois Gouin 1,2,3 , Dominique Heymann 1,2,3,5 and Marie-Francoise Heymann 1,2,3,5 1 INSERM, UMR 957, Equipe LIGUE Nationale Contre le Cancer, Nantes, France 2 Universite de Nantes, Nantes atlantique universites, Pathophysiology of Bone Resorption and Therapy of Primary Bone Tumors, Nantes, France 3 CHU de Nantes, Nantes University Hospital, France 4 Centre de Lutte Contre le Cancer, Leon Berard, Lyon, France 5 INSERM, European Associated Laboratory “Sarcoma Research Unit”, Department of Oncology and Metabolism, University of Sheffield, Medical School, Sheffield, UK Correspondence to: Marie-Francoise Heymann, email: // Dominique Heymann, email: // Keywords : osteosarcoma, tumour associated macrophage, osteoprotegerin, Pathology Section Received : September 24, 2016 Accepted : October 27, 2016 Published : November 13, 2016 Abstract Osteosarcoma (OS) is the most common bone sarcoma in adolescents, and has poor prognosis. A vicious cycle is established between OS cells and their microenvironment in order to facilitate the tumor growth and cell spreading. The present work aims to better characterize the tumor microenvironment in OS in order to identify new therapeutic targets relating to metastatic process. Tissue microarrays of pre-chemotherapy OS biopsies were used for characterizing the tumor niche by immunohistochemistry. Parameters studies included: immune cells (M1, M2-subtypes of tumor-associated macrophages (TAM); T, B lymphocytes; mast cells), vascularization (endothelial, perivascular cells), OPG, RANKL, and mitotic index. Two groups of patients were defined, 22 localized OS (OS Meta-) and 28 metastatic OS (OS Meta+). The OS Meta- group was characterized by a higher infiltration of INOS + M1-polarizedmacrophages and upregulated OPG immunostaining. OS Meta+ tumors showed a significant increase in CD146 + cells. INOS + M1-macrophages were correlated with OPG staining, and negatively with the presence of metastases. CD163 + M2-macrophages were positively correlated with CD146 + cells. In multivariate analysis, INOS and OPG were predictive factors for metastasis. An older age, non-metastatic tumor, good response to chemotherapy, and higher macrophage infiltration were significantly associated with better overall survival. TAMs are associated with better overall survival and a dysregulation of M1/M2 polarized-macrophages in favor of M1 subtype was observed in non-metastatic OS.

International audience; Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound β-escin as a selective inhibitor of GIC viability. Indeed, β-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, β-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that β-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the β-escin-induced reduction in sphere formation or stemness marker activity, indicating that β-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned β-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM.

// Ikhlak Ahmed 1 , Thasni Karedath 1 , Simeon S. Andrews 1 , Iman K. Al-Azwani 2 , Yasmin Ali Mohamoud 2 , Denis Querleu 3 , Arash Rafii 4 , Joel A. Malek 1, 2 1 Department of Genetic medicine, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 2 Genomics Core, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 3 Department of Gynecologic Oncology, Universite Montepllier 1, Montpellier, France 4 Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar Correspondence to: Joel A. Malek, e-mail: jom2042@qatar-med.cornell.edu Keywords: circular RNA, ovarian cancer, peritoneal metastasis, lymph node metastasis, miRNA Received: December 07, 2015 Accepted: April 02, 2016 Published: April 22, 2016 ABSTRACT Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF-β typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis.

International audience; Triple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy.

International audience; The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

// Charlotte Kervoelen 1, 2 , Emmanuelle Menoret 1 , Patricia Gomez-Bougie 2, 3 , Regis Bataille 2 , Catherine Godon 4 , Severine Marionneau-Lambot 5 , Philippe Moreau 2, 3 , Catherine Pellat-Deceunynck 2, 3 , Martine Amiot 2, 3 1 Myelomax, Nantes, France 2 CRCNA, University of Nantes, INSERM, UMR892, CNRS, UMR 6299, Nantes, France 3 Hematology Clinic, University Hospital, Nantes, France 4 Hematology Laboratory, University Hospital, Nantes, France 5 Plate-forme in vivo , Canceropole Grand Ouest, Nantes, France Correspondence to: Martine Amiot, e-mail: martine.amiot@inserm.fr Keywords: multiple myeloma, glucocorticoid, glucocorticoid receptor, GILZ, Bim Received: March 27, 2015 Accepted: July 06, 2015 Published: July 16, 2015 ABSTRACT Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines ( n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.

The implication of the Notch pathway in cancer has been known since it was found hyperactivated in acute T-cell lymphoblastic leukemias (T-ALL) one decade ago. During this time, the link between the Notch pathway and cancer has been extended to many types of malignancies. A unique feature of the pathway is the fact that it can be oncogenic or tumor suppressive depending on the tumor type. For example, NOTCH receptors present gain-of-function mutations that make the receptor constitutively active in T-ALL, B-cell chronic lymphocytic leukemia, or lung adenocarcinoma. In contrast, NOTCH receptors undergo loss-of-function mutations in myeloid leukemias and in squamous cell carcinomas (SCCs) from esophagus, skin, lung or head and neck [1]. Two recent reports, from the group of Dr. Klinakis [2] and from us [3], have addressed the role of the Notch pathway in urinary bladder cancer demonstrating that it plays a relevant tumor suppressive role. The Klinakis group performed an extensive mutational analysis of up to nine components of the Notch pathway and copy number variations of the NOTCH1 gene in a cohort of human bladder cancers [2]. Remarkably, they found that up to 60% of bladder cancers have loss-of-function alterations in components of the Notch pathway (43% overall mutation incidence, being more than half concentrated in NOTCH1 and NOTCH2) and NOTCH1 gene copy losses. In support of this, we also showed that NOTCH1 and NOTCH2 mutations previously found in bladder cancers are functionally defective [3]. Of further relevance, both studies found a correlation between low activity of the Notch pathway, higher cancer aggressiveness and shorter patient survival [2,3]. To support these findings, the two groups used mouse genetics to demonstrate that inactivation of the canonical Notch pathway at different levels promotes the development of bladder cancer in mice [2,3]. As it was the case in human cancers, Notch-deficient murine bladder cancers were also highly infiltrating [2,3]. The two studies performed mechanistic analyses that shed light on different aspects of the biology of Notch in bladder cancer. We focused on the aggressive behavior of the cancers with loss of Notch function. By manipulating the Notch pathway in cultured cells, we found that inhibition of the Notch pathway results in the upregulation of key mediators and effectors of the epithelial-mesenchymal-transition (EMT), including SNAIL, SLUG, ZEB2 and VIMENTIN, and the concomitant downregulation of the epithelial marker E-CADHERIN [3]. The transcriptional repressor HES1 is one of the critical transcriptional targets of the Notch pathway, and we showed that reduced HES1 activity upon Notch inhibition is responsible for the de-repression of the EMT program in bladder cancer cells [3]. Therefore, we conclude that Notch represses EMT in bladder, where it acts as a tumor suppressive pathway. In this regard, it is interesting to note that a large body of evidence indicates that Notch induces EMT in those tissues where Notch is oncogenic. It appears that Notch acts in opposite directions in different tissues: it represses EMT in tissues where Notch is tumor suppressive; and it activates EMT in tissues where it is oncogenic. We will come back to this issue at the end of this commentary. A sizeable proportion of human bladder cancers have gain-of-function mutations in FGFR3, HRAS, KRAS or PIK3CA genes. These mutations potently activate the canonical MAPK cascade and, accordingly, are associated to high levels of phospho-ERK (pERK1/2). The Notch signaling pathway is not generally considered a potent activator of the MAPK cascade. Unexpectedly, Klinakis' group made the intriguing observation that Notch-deficient bladder cancers (with no other concomitant mutations in FGFR3 or RAS) present high levels of pERK1/2, even higher than those in FGFR3/RAS-mutant bladder cancers [2]. Mechanistic analyses in culture cells led the authors to conclude that the Notch pathway transcriptionally activates several members of the dual-specificity phosphatases (DUSPs), including DUSP1, by direct binding of the activated NOTCH receptors to their promoter regions [2]. The DUSP phosphatases are responsible for the dephosphorylation of pERK1/2 and, therefore, when the Notch pathway is not functional DUSP expression decreases and there is an increase of pERK1/2 in bladder cancer cells [2]. Of note, previous work from others and us in lung adenocarcinoma, where Notch is oncogenic, has shown that the Notch pathway, through HES1, represses the DUSP1 promoter [4,5]. Again, as in the case of EMT, it appears that Notch acts in opposite directions depending on the tissues: it activates DUSPs in tissues where it is tumor suppressive; and it represses DUSPs in tissues where it is oncogenic. Based on the above-described sets of mechanistic studies, it is tempting to speculate that the dual actions of the Notch pathway, oncogenic or tumor suppressive, reflect opposite actions on the same transcriptional targets. This situation is not new to researchers working on Notch in Drosophila, where it is well established that the Notch pathway can act positively and negatively on the same genes. Specifically, Notch-regulated genes often have binding sites for NOTCH/RBPJ (positive) and for the Notch target HES1 (negative), and this is thought to allow transient transcriptional effects [6]. In this context, alterations in the activation/repression equilibrium in different cell types may dramatically change the outcome of Notch activity. For example, this may well be the case of DUSP1: in those settings where the Notch pathway is tumor suppressive (as in bladder), the activation promoted by NOTCH1 is dominant; in contrast, in tissues where Notch is oncogenic (as in lung adenocarcinoma), the repressive action of HES1 is the dominant one. Conceivably, understanding the epigenetic status of critical Notch target genes could eventually explain the function, oncogenic or tumor suppressive, of the Notch pathway in cancer. In summary, a new tumor suppressive role has been found for the Notch pathway in the urinary bladder, and this has yielded important mechanistic insights about bladder cancer and the biology of the Notch pathway.