ERα-36 regulates progesterone receptor activity in breast cancer.

BACKGROUND: Alterations in estrogen and progesterone signaling, via their respective receptors, estrogen receptor alpha (ERα) and progesterone receptor (PR), respectively, are largely involved in the development of breast cancer (BC). The recent identification of ERα-36, a splice variant of ERα, has uncovered a new facet of this pathology. Although ERα-36 expression is associated with poor prognosis, metastasis development, and resistance to treatment, its predictive value has so far not been associated with a BC subtype and its mechanisms of action remain understudied. METHODS: To study ERα-36 expression in BC specimens, we performed immunochemical experiments. Next, the role of ERα-36 in progesterone signaling was investigated by generating KO clones using the CRISPR/CAS9 technology. PR signaling was also assessed by proximity ligation assay, Western blotting, RT-QPCR, and ChIP experiments. Finally, proliferation assays were performed with the IncuCyte technology and migration experiments using scratch assays. RESULTS: Here, we demonstrate that ERα-36 expression at the plasma membrane is correlated with a reduced disease-free survival in a cohort of 160 BC patients, particularly in PR-positive tumors, suggesting a crosstalk between ERα-36 and PR. Indeed, we show that ERα-36 interacts constitutively with PR in the nucleus of tumor cells. Moreover, it regulates PR expression and phosphorylation on key residues, impacting the biological effects of progesterone. CONCLUSIONS: ERα-36 is thus a regulator of PR signaling, interfering with its transcriptional activity and progesterone-induced anti-proliferative effects as well as migratory capacity. Hence, ERα-36 may constitute a new prognostic marker as well as a potential target in PR-positive BC

A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer’s Disease

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer’s disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor β (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline</p

A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine

Étude de la régulation de la méthylation du récepteur aux œstrogènes de type alpha dans le cancer du sein

Estrogen receptor a {ERa}, belonging to the superfamily of hormone nuclear receptors, regulates many physiological processes, notably the growth and survival of breast tumor cells, acting as a ligand-dependent transcription factor. Besides to the well described transcriptional effects, estrogen also mediate extranuclear events called non genomic signaling via its receptor. /n fact, team shows that ERa is methylated and that this event is a prerequisite for the recrutement of Src and P/3K and the activation of Akt which orchestrate cell proliferation and survival. During my PhD, / demonstrated that the non genomic signaling complex mERa/Src/P/3K exists in vivo and is operative. /n addition, the complex is found to be an independent prognostic factor for disease free survival. This is an emergent concept that estrogen non genomic pathway is operative in vivo and can constitute a new therapeutic targets. The search for new partners of the complex has allowed us to identify the tumor suppressor LKB1 and arginine demethylase JMJD6. Expression of LKB1 in immunohistochemistry revealed dual properties based on its subcellular localization. When LKB1 is complexed with mERa/Src/P/3K it may acquire oncogenic properties. /n addition, JMJD6 interacts with methylated ERa when the receptor is associated with Src and P/3K, and allows the demethylation of ERa and the dissociation of the complex mERa/Src/P/3K. This work showed that estrogenic non genomic players can constitute new therapeutic targets in Breast tumorsLe cancer du sein représente une cause de mortalité élevée chez la femme. Le cancer du sein est un cancer hormono-dépendant. De ce fait, il est extrêmement important de définir le rôle joué par les différents acteurs protéiques de la signalisation hormonale, notamment la signalisation œstrogénique. Parallèlement aux effets nucléaires de ERa où l'hormone lie le récepteur nucléaire et régule la transcription génique, il existe une voie dite non génomique. L'équipe a montré que les œstrogènes induisent la méthylation de ERa, qui est un prérequis au recrutement de la Pl3K et de la tyrosine kinase Src, conduisant à l'activation de molécules de signalisation telles que les MAPK et Akt, induisant prolifération et survie cellulaire. Durant ma thèse, j'ai pu démontrer que le complexe mERa/Src/Pl3K existe in vivo et constitue un nouveau biomarqueur indépendant de mauvais pronostique. La recherche de nouveaux partenaires du complexe mERa/Src/Pl3K nous a permis d'identifier le suppresseur de tumeur LKB1 et l'arginine déméthylase JMJD6. De façon surprenante, l'étude de l'expression de LKB1 par immunohistochimie dans une cohorte de tumeurs mammaires a montré une dualité fonctionnelle selon sa localisation subcellulaire. De plus, nous avons démontré que JMJD6 s'associe à ERa méthylé lorsque le récepteur est complexé à Src et Pl3K, et permet ainsi la déméthylation de ERa et la dissociation du complexe mERa/Src/Pl3K. Ce travail a ainsi pu mettre en évidence que les différents acteurs de cette signalisation peuvent constituer des éléments clés au diagnostique mais également lors de la décision thérapeutique, puisque qu'il existe des drogues peuvant cibler cette voie de signalisatio

Regulation of estrogen receptor alpha methylation in breast cancer

Le cancer du sein représente une cause de mortalité élevée chez la femme. Le cancer du sein est un cancer hormono-dépendant. De ce fait, il est extrêmement important de définir le rôle joué par les différents acteurs protéiques de la signalisation hormonale, notamment la signalisation œstrogénique. Parallèlement aux effets nucléaires de ERa où l'hormone lie le récepteur nucléaire et régule la transcription génique, il existe une voie dite non génomique. L'équipe a montré que les œstrogènes induisent la méthylation de ERa, qui est un prérequis au recrutement de la Pl3K et de la tyrosine kinase Src, conduisant à l'activation de molécules de signalisation telles que les MAPK et Akt, induisant prolifération et survie cellulaire. Durant ma thèse, j'ai pu démontrer que le complexe mERa/Src/Pl3K existe in vivo et constitue un nouveau biomarqueur indépendant de mauvais pronostique. La recherche de nouveaux partenaires du complexe mERa/Src/Pl3K nous a permis d'identifier le suppresseur de tumeur LKB1 et l'arginine déméthylase JMJD6. De façon surprenante, l'étude de l'expression de LKB1 par immunohistochimie dans une cohorte de tumeurs mammaires a montré une dualité fonctionnelle selon sa localisation subcellulaire. De plus, nous avons démontré que JMJD6 s'associe à ERa méthylé lorsque le récepteur est complexé à Src et Pl3K, et permet ainsi la déméthylation de ERa et la dissociation du complexe mERa/Src/Pl3K. Ce travail a ainsi pu mettre en évidence que les différents acteurs de cette signalisation peuvent constituer des éléments clés au diagnostique mais également lors de la décision thérapeutique, puisque qu'il existe des drogues peuvant cibler cette voie de signalisationEstrogen receptor a {ERa}, belonging to the superfamily of hormone nuclear receptors, regulates many physiological processes, notably the growth and survival of breast tumor cells, acting as a ligand-dependent transcription factor. Besides to the well described transcriptional effects, estrogen also mediate extranuclear events called non genomic signaling via its receptor. /n fact, team shows that ERa is methylated and that this event is a prerequisite for the recrutement of Src and P/3K and the activation of Akt which orchestrate cell proliferation and survival. During my PhD, / demonstrated that the non genomic signaling complex mERa/Src/P/3K exists in vivo and is operative. /n addition, the complex is found to be an independent prognostic factor for disease free survival. This is an emergent concept that estrogen non genomic pathway is operative in vivo and can constitute a new therapeutic targets. The search for new partners of the complex has allowed us to identify the tumor suppressor LKB1 and arginine demethylase JMJD6. Expression of LKB1 in immunohistochemistry revealed dual properties based on its subcellular localization. When LKB1 is complexed with mERa/Src/P/3K it may acquire oncogenic properties. /n addition, JMJD6 interacts with methylated ERa when the receptor is associated with Src and P/3K, and allows the demethylation of ERa and the dissociation of the complex mERa/Src/P/3K. This work showed that estrogenic non genomic players can constitute new therapeutic targets in Breast tumor

PRMT5 prognostic value in cancer

International audienceProtein arginine methyltransferases (PRMTs) catalyze the methylation of arginine residues on both histones and non-histone proteins. PRMT5, a member of the PRMT family, is overexpressed in a wide variety of cancers and its activity is associated with cell transformation. Moreover, its expression is associated with a decrease in patient survival in several cancers, a rationale for developing highly potent inhibitors of its enzymatic activity. However, most studies do not take into account the subcellular localization of PRMT5, which can modify its properties. Indeed, our team recently showed that PRMT5 nuclear expression is associated with prolonged survival. These results corroborated findings in prostate cancer, in which the nuclear fraction of PRMT5 was responsible for inhibiting cell growth, while the cytoplasmic fraction promoted cell growth. In conclusion, this criterion should be evaluated prior to administering PRMT5 inhibitors, which may have adverse effects

Structure, Activity, and Function of PRMT1

International audiencePRMT1, the major protein arginine methyltransferase in mammals, catalyzes monomethylation and asymmetric dimethylation of arginine side chains in proteins. Initially described as a regulator of chromatin dynamics through the methylation of histone H4 at arginine 3 (H4R3), numerous non-histone substrates have since been identified. The variety of these substrates underlines the essential role played by PRMT1 in a large number of biological processes such as transcriptional regulation, signal transduction or DNA repair. This review will provide an overview of the structural, biochemical and cellular features of PRMT1. After a description of the genomic organization and protein structure of PRMT1, special consideration was given to the regulation of PRMT1 enzymatic activity. Finally, we discuss the involvement of PRMT1 in embryonic development, DNA damage repair, as well as its participation in the initiation and progression of several types of cancers

Structure, Activity, and Function of the Protein Lysine Methyltransferase G9a

International audienceG9a is a lysine methyltransferase catalyzing the majority of histone H3 mono- and dimethylation at Lys-9 (H3K9), responsible for transcriptional repression events in euchromatin. G9a has been shown to methylate various lysine residues of non-histone proteins and acts as a coactivator for several transcription factors. This review will provide an overview of the structural features of G9a and its paralog called G9a-like protein (GLP), explore the biochemical features of G9a, and describe its post-translational modifications and the specific inhibitors available to target its catalytic activity. Aside from its role on histone substrates, the review will highlight some non-histone targets of G9a, in order gain insight into their role in specific cellular mechanisms. Indeed, G9a was largely described to be involved in embryonic development, hypoxia, and DNA repair. Finally, the involvement of G9a in cancer biology will be presented

Targeting AKT in ER-Positive HER2-Negative Metastatic Breast Cancer: From Molecular Promises to Real Life Pitfalls?

International audienceThe AKT protein kinase plays a central role in several interconnected molecular pathways involved in growth, apoptosis, angiogenesis, and cell metabolism. It thereby represents a therapeutic target, especially in hormone receptor-positive (HR) breast cancers, where the PI3K/AKT signaling pathway is largely hyperactivated. Moreover, resistance to therapeutic classes, including endocrine therapy, is associated with the constitutive activation of the PI3K/AKT pathway. Improved knowledge on the molecular mechanisms underlying resistance to endocrine therapy has led to the diversification of the therapeutic arsenal, notably with the development of PI3K and mTOR inhibitors, which are currently approved for the treatment of advanced HR-positive breast cancer patients. AKT itself constitutes a novel pharmacological target for which AKT inhibitors have been developed and tested in clinical trials. However, despite its pivotal role in cell survival and anti-apoptotic mechanisms, as well as in endocrine therapy resistance, few drugs have been developed and are available for clinical practice. The scope of the present review is to focus on the pivotal role of AKT in metastatic breast cancer through the analysis of its molecular features and to discuss clinical implications and remaining challenges in the treatment of HR-positive metastatic breast cancer