Contact: Campus Technologies Freiburg GmbH Responsible Scientist: Prof. Dr. Roland Schüle
Stefan-Meier-Str. 8 | D-79104 Freiburg Gynecological Hospital
phone: +49 (0)761 203-4987
email: [email protected] AZ: ZEE20060418 Stand: Apr-10
C J
New Therapy in Prostate ancer
MJD2C controls Prostate Tumour Growth
Albert-Ludwigs-University Freiburg
Technology The Jumonji C (JMJC) domain-containing protein JMJD2C is the first histone tridemethylase regulating androgen receptor function. Knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumor cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities.Lysine specific Demethylase 1 (LSD1) co-localises with AR in normal human prostate and in prostate tumor. LSD1 interacts with AR and stimulates AR-dependent transcription. We identify pargyline as an inhibitor of LSD1 that blocks AR-dependent transcription. Furthermore LSD1 knockdown by RNAi abrogates androgen induced transcriptional activation and cell proliferation.
Innovation
• better treatment of prostate tumor
Application
Modulation of JMJD2C activity is tissues where AR has a pivotal physiological role i.e.: • Treatment of prostate tumor • Control of fertility • Treatment of Alzheimer´s disease • Treatment of Parkinson´s disease
Market Potential
Prostate cancer represents the most frequent malignant disease in men worldwide and the second leading cause of death from malignant tumors.
Proof of Co
ncept Please contact us for further information.
Patent Status
• European Patent Application Pending • Filed (PRD): January 26th, 2007 • International Patent Application (PCT)
Co-operative demethylation by JMJD2C and LSD1 promotesandrogen receptor-dependent gene expression
Melanie Wissmann, Na Yin, Judith M. Müller, Holger Greschik, Thomas Günther, Barna D. Fodor, Thomas Jenuwein, Reinhard Buettner, Eric Metzger, and Roland Schüle
Klinikum der Albert-Ludwigs Universität, Universitäts-Frauenklinik, Abteilung Experimentelle Gynäkologie und Geburtshilfe,ZKF Zentrum für Klinische Forschung, Breisacherstrasse 66, 79106 Freiburg, Germany.
Figure 4 JMJD2C knockdown blocks AR-induced transcriptionalactivity and tumor cell proliferation. In LNCaP cells, miRNA-mediatedJMJD2C knockdown reduces expression of the endogenous PSA gene(a, left panel), AR-dependent reporter activity (b), and R1881-inducedcell proliferation (c). Knockdown of JMJD2C was verified by Westernblot analysis (a, right panel) using α-JMJD2C or α-AR antibodies. Barsrepresent mean +SD (n>4).
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Figure 3 JMJD2C controls AR-induced transcriptional activity. CV1 (a, b, d), or LNCaP (c) cells were transfectedwith AR-dependent reporters in the presence or absence of R1881. CV1 cells were co-transfected with AR expressionplasmid (a, b, d). JMJD2C but not the other JMJD2 family members JMJD2A, JMJD2B, or JMJD2D (b) controlsAR-induced transcriptional activity on different natural AR-regulated promoters and cell lines. Limited amounts ofJMJD2C, JMJD2C H190A, or LSD1 were tested for co-operative stimulation of AR-dependent reporter activity (d).Bars represent mean +SD (n>5).
Figure 1 JMJD2C co-localizes and interacts with both, AR and LSD1. a,Immunohistochemical staining of JMJD2C, AR, and LSD1 in human normaland tumor prostate. JMJD2C (B, F, J), AR (C, G, K), and LSD1 (D, H, L)immunoreactivity is detected in the secretory epithelium of normal prostate (B,C, D, arrows) and prostate carcinoma cells (F, G, H, J, K, L arrows). Hematoxilin-eosin (HE) stained sections are shown in A, E, and I. All sections were takenfrom the same radical prostatectomy specimen. Magnification: x250. b, JMJD2Cinteracts with AR (left panel) and LSD1 (right panel) in vivo. Extracts from mouseprostate were immunoprecipitated with either α-JMJD2C, α-LSD1, α-cyclin Aantibodies or rabbit IgG. Western blots were decorated with α-AR, α-JMJD2C,or a-LSD1 antibodies as indicated. c, d, GST pull-down assays were performedwith labeled JMJD2C and the bacterially expressed GST-AR or GST-LSD1fusion proteins. GST and GST-Nix1 proteins were used as control. (NTD; N-terminal domain, DBD; DNA-binding domain, LBD; ligand-binding domain, AO;amine oxidase domain). Coomassie blue staining shows the amounts of GSTfusion proteins used.
Figure 2 JMJD2C interacts with chromatin and demethylates H3K9. LNCaP cells were incubated with or without theAR agonist R1881 (a, b), and transfected with stealth RNAi (b). ChIP or Re-ChIP was performed with the indicatedantibodies. The precipitated chromatin was amplified by PCR using primers flanking the promoter region (ARE I+II) andthe enhancer region (ARE III) of the PSA gene, the promoter region (ARE) of the KLK2 gene, or the promoters of theunrelated GAPDH and U6 genes. Western blot analysis (b, right panel) verified the specific siRNA-mediated knockdownof JMJD2C.c, Core histones or nucleosomes from HeLa cells were incubated with recombinant JMJD2C (aa 12-349)or mutant JMJD2C H190A (aa 12-349). Western blots were decorated with the indicated antibodies against α-mono-,α-di-, or α-trimethyl H3K9.
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Conclusion:
1) JMJD2C and LSD1 co-operatively regulate AR transcriptional activity2) JMJD2C demethylates trimethyl histone H3 at Lysine 9 and thereby generates the substrate for LSD13) JMJD2C controls androgen-dependent proliferation of prostate tumour cells4) JMJD2C is a potential target to block prostate tumour growth
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AbstractPosttranslational modifications of histones such as methylation regulatechromatin structure and gene expression. Methylation of histones had beenconsidered to be permanent until recently when lysine-specific demethylase1 (LSD1), the first histone demethylase, was discovered. LSD1 interacts withthe androgen receptor (AR) and promotes androgen-dependent transcriptionof target genes by ligand-induced demethylation of mono- and dimethylatedhistone H3 at lysine 9 (H3K9). In contrast, trimethylated histone H3K9 is notdemethylated by LSD1 suggesting that androgen-dependent demethylationof trimethyl H3K9 is controlled by an as yet unknown histone demethylase.Here we identify the Jumonji C (JMJC) domain-containing protein JMJD2Cas the first histone tridemethylase regulating AR function. JMJD2C interactswith AR in vitro and in vivo. Furthermore, ligand-dependent assembly of ARand JMJD2C on AR target genes results in demethylation of trimethyl H3K9in vivo and in stimulation of AR-dependent transcription. Conversely,knockdown of JMJD2C in prostate cancer cells inhibits androgen-inducedremoval of trimethyl H3K9, transcriptional activation, and tumor cell proliferation.Importantly, JMJD2C not only co-localizes with AR but also with LSD1 innormal prostate and prostate carcinoma. In addition, AR, JMJD2C, and LSD1assemble on chromatin to remove methyl groups from mono-, di-, andtrimethylated H3K9 and both demethylases co-operatively stimulate AR-dependent gene transcription. Thus, our data suggest that specific generegulation requires the assembly and coordinate action of demethylases withdistinct substrate specificities. Furthermore, regulation of JMJD2C activityalone or in combination with LSD1 might be a promising therapeutic strategyto control AR activity in prostate cancer.
Wissmann et al., Nat. Cell Biol. 9, (2007)