Mitogen-activated protein kinases (MAPKs) are essential for proper cell functioning as they regulate many molecular effectors. Careful regulation of MAPKs is therefore required to avoid MAPK pathway dysfunctions and pathologies. The mammalian genome encodes about 200 phosphatases, many of which dephosphorylate the MAPKs and bring them back to an inactive state. In this review, we focus on the normal and pathological functions of dual-specificity phosphatase 9 (DUSP9)/MAP kinase phosphatases-4 (MKP-4). This cytoplasmic phosphatase, which belongs to the threonine/tyrosine dual-specific phosphatase family and was first described in 1997, is known to dephosphorylate ERK1/2, p38, JNK and ASK1, and thereby to control various MAPK pathway cascades. As a consequence, DUSP9 plays a major role in human pathologies and more specifically in cardiac dysfunction, liver metabolic syndromes, diabetes, obesity and cancer including drug response and cell stemness. Here, we recapitulate the mechanism of action of DUSP9 in the cell, its levels of regulation and its roles in the most frequent human diseases, and discuss its potential as a therapeutic target.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumoral tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also discovered that the blood capillary controls the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for new investigations in an emerging field that we call ‘onconanotomy’.

Background: EZH2 is a histone methyltransferase and a key component of polycomb repressive complex 2 (PRC2). It plays a critical role in genome remodeling, gene regulation and acts through PRC2-dependent and independent mechanisms, which comprise methylation of histone and non-histone substrates, and transcriptional activation through different transcriptional complexes. EZH2 is involved in many cancers, but its role in hepatoblastoma is poorly understood. Methods: Potential correlation between EZH2 mRNA expression and clinical parameters was analyzed by computational and histological approaches using seven published transcriptomic datasets and tissue samples from patients with hepatoblastoma. EZH2 molecular function was deciphered using molecular approaches, gain- and loss-of-function genetic tools, proteomics, immunohistochemistry, pharmacological drugs, 2D cell- and spheroid-based assays, and four different animal models. Results: Our data show that EZH2 mRNA expression correlated with poor prognostic markers such as tumor proliferation, and patients’ death and shorter survival. EZH2 protein potentiated hepatoblastoma cell proliferation, migration, survival and cisplatin resistance through its histone methyltransferase activity by repressing DUSP5 , and transcriptionally inducing DUSP9 and HMGCR . In vivo EZH2 sustained tumor cell proliferation, and tumor development and angiogenesis. The EZH2 inhibitor GSK126 synergized with HMG-CoA reductase inhibitor statins to eradicate hepatoblastoma cells in vitro and block tumor development in mice. This combination was also very effective on various hepatoblastoma and non-hepatoblastoma tumor cell lines. Conclusion: Collectively, our data showed that the protein EZH2 promotes hepatoblastoma development, partly through its histone methyltransferase activity, by differentially modulating the expression of DUSP5 , DUSP9 and HMGCR genes and by supporting the MAPK/ERK pathway in hepatoblastoma cells already displaying high Wnt signal activity. EZH2 inhibitors triggered lipid synthesis in hepatoblastoma cells and synergized with cholesterol-lowering statins to block hepatoblastoma development in vitro and in vivo. Therefore, we demonstrate the key role of EZH2 in proliferative hepatoblastoma and the therapeutic benefit of combining EZH2 inhibitor and statin to treat patients with cancer.

L’hépatoblastome (HB) est un cancer rare qui touche principalement les enfants âgés de 6 mois à 3 ans et dont la survie globale à 5 ans est d’environ 80%. Le traitement de référence de ce cancer associe une chimiothérapie à base de Cisplatine et une résection chirurgicale de la tumeur ou une transplantation hépatique. Dans le cas des tumeurs de haut risque (métastatique, agressive, touchant la globalité du foie, sujet âgé), cette chimiothérapie est utilisée à fortes doses avec des conséquences néfastes pour la qualité de vie des enfants après la maladie et elle ne garantit pas l’absence de récidive.En 2018, notre équipe a effectué une étude transcriptomique sur 25 échantillons d’HB et de foies normaux adjacents par RNA-seq (PMID : 29152775). L'analyse a mis en évidence l'existence de 3 groupes indépendants d'HB dénommés C1, C2A et C2B. Au niveau moléculaire, le groupe hautement prolifératif et de mauvais pronostic C2A est caractérisé par une augmentation spécifique de plusieurs composants du complexe répressif polycomb-2 (PRC2), et notamment de la méthyl-transférase EZH2 et de ses cofacteurs SUZ12 et EED.Grâce à l'utilisation complémentaire d'approches cellulaires 2D et 3D et d'un modèle in vivo chez l'embryon de poulet, nous avons confirmé le rôle oncogénique d'EZH2 dans l'HB C2A. Nous avons ensuite démontré le lien entre ce gène et la résistance des cellules tumorales au Cisplatine. La combinaison du cisplatine et d’un inhibiteur spécifique d'EZH2 a un effet additif pour bloquer la prolifération et la survie des cellules HB, montrant qu’EZH2 est une cible thérapeutique dans ce cancer. Par protéomique, nous avons identifié de nouveaux gènes régulés par EZH2 dans les cellules d’HB. Parmi ces gènes, nous nous sommes intéressés aux phosphatases à double spécificité (Dual-specificity Phosphatases) DUSP5 et DUSP9, qui sont connues pour inhiber la voie MAPK/ERK en déphosphorylant les kinases ERK1/2. L'expression de ces deux DUSPs est fortement corrélée à l'expression d'EZH2 dans les échantillons d’HB. Nos données montrent également que la dérégulation d'EZH2 et des DUSPs DUSP5 et DUSP9 dans l’HB est associée à plusieurs paramètres de mauvais pronostic et notamment la baisse de survie globale des patients, la récidive tumorale et la présence de métastases. Le rôle de ces DUSPs dans l'HB C2A est actuellement à l'étude et ces travaux devraient conduire à l'identification de nouvelles cibles thérapeutiques dans le traitement des patients présentant un HB de haut risque.Parallèlement à ce premier projet, nous avons analysé l'organisation spatiale de tissus de xénogreffes dérivées de patients HB (patient-derived xenograft, HB-PDX) par microscopie électronique à balayage de blocs en série (SFB-SEM). A l’aide de l’imagerie tridimensionnelle (3D), d’approches de segmentation manuelle et semi-automatique basées sur l'apprentissage et l’intelligence artificielle, d’approches mathématiques et infographiques, nous avons reconstruit sous format digital un fragment de tissu d'HB comprenant un capillaire sanguin, une structure proche d’un canalicule biliaire, des dizaines de cellules tumorales ainsi que leurs principaux organelles (cytoplasme, noyau, mitochondries). Les résultats obtenus permettent d’avoir une vue détaillée de l’organisation architecturale du tissu d’HB. Nous avons notamment découvert que la taille des cellules tumorales est corrélée à celle de leur noyau, de leur cytoplasme et de leur réseau mitochondrial. Nous avons également découvert que le capillaire sanguin influence l'alignement anatomique et la taille des cellules tumorales au sein du tissu et qu’un groupe de cellules d’HB polarise en direction d’une structure qui ressemble à un canalicule biliaire. Cette étude pilote par imagerie 3D a permis d'identifier des paramètres bioarchitecturaux qui définissent l'organisation interne et spatiale des tumeurs, ouvrant ainsi la voie à des études originales dans un domaine émergent que nous avons appelé « onconanotomie ».