Epigenetic inactivation of MCJ (DNAJD1) in malignant paediatric brain tumours Janet C. Lindsey 1 , Meryl E. Lusher 1 , Gordon Strathdee 2 , Robert Brown 2 , Richard J. Gilbertson 3 , Simon Bailey 4 , David W. Ellison 1 and Steven C. Clifford 1 * 1 Northern Institute for Cancer Research, The Medical School, University of Newcastle, Newcastle upon Tyne, United Kingdom 2 Centre for Oncology and Applied Pharmacology, Cancer Research UK Beatson Laboratories, Glasgow University, Glasgow, United Kingdom 3 Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA 4 Sir James Spence Institute of Child Health, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, United Kingdom MCJ (DNAJD1) is a recently discovered member of the DNAJ pro- tein family whose expression is controlled epigenetically by meth- ylation of a CpG island located within the 5 0 transcribed region of its gene. Methylation-dependent transcriptional silencing of MCJ has been observed in ovarian cancers and associated with increased resistance to chemotherapeutic agents; however, its role in other cancer types has not been widely investigated. We exam- ined the status of MCJ in intracranial primitive neuroectodermal tumours [PNETs, comprising cerebellar PNETs (medulloblasto- mas) and supratentorial PNETs (stPNETs)] and ependymomas, together representing the most common malignant brain tumours of childhood. Evidence of MCJ hypermethylation was found in all 3 tumour types [medulloblastomas, 3/9 (33%) cell lines, 2/28 (7%) primary tumours; stPNETs, 2/2 (100%) cell lines, 3/10 (30%) pri- mary tumours; and ependymomas, 2/20 (10%) primary tumours] but not in nonneoplastic brain tissues (n 5 11), indicating that MCJ methylation is a tumour-specific event. In methylated cases, the distribution of methylated CpG sites across the CpG island could be broadly divided into 2 patterns: (i) extensive methylation of the majority of CpG sites across the island or (ii) limited meth- ylation of individual CpG sites concentrated towards the 5 0 end of the island. Extensive methylation patterns were associated with the methylation-dependent transcriptional silencing of MCJ in medulloblastoma and stPNET cell lines. Further investigations of the mechanism of MCJ inactivation revealed that its loss could occur either through biallelic epigenetic methylation or by methyl- ation in association with genetic loss of its second allele. These data indicate that epigenetic inactivation of MCJ may play a role in the development of a range of paediatric brain tumour types, and its role in disease pathogenesis and chemotherapeutic resistance should now be investigated further. ' 2005 Wiley-Liss, Inc. Key words: MCJ; DNAJD1; primitive neuroectodermal tumour; medulloblastoma; ependymoma; hypermethylation Medulloblastoma is a PNET which arises in the cerebellum and accounts for approximately 20% of childhood brain tumours. 1 stPNETs are histologically similar to medulloblastomas but arise in the cerebrum or pineal body and appear to exhibit different genetic aberrations. 2,3 Medulloblastomas and stPNETs are highly malignant, and overall 5-year survival rates following multimodal therapy are poor (approx. 60%). Current treatment regimes are also associated with long-term neurologic and endocrinologic side effects, and disease course is difficult to predict based on current clinical and pathologic indices. 1,4 Thus, a better understanding of the molecular events underlying the development of these tumours could provide the basis of an improved outlook for these patients, through identification of molecular markers for disease stratifica- tion and development of molecularly targeted therapeutic regimens. There has been significant interest in the epigenetic inactivation of TSGs in cancer development, of which transcriptional silencing caused by hypermethylation of promoter-associated CpG islands represents the best-defined mechanism. 5 Unlike genetic mecha- nisms of TSG inactivation (e.g., gene mutation or deletion), TSG silencing by DNA hypermethylation is reversible by DNA methyl- transferase inhibitors or demethylating agents and, therefore, may represent a potential therapeutic target. 6 Previous studies have shown that epigenetic TSG inactivation plays a role in medullo- blastoma development. 7–15 To date, 3 genes have demonstrated strong and consistent evidence of epigenetic inactivation by pro- moter methylation in a significant proportion of medulloblasto- mas: RASSF1A, CASP8 and HIC-1. 8–12,14,15 Together these data suggest that further epigenetic events are involved in medulloblas- toma; however, the role of hypermethylation events in stPNET pathogenesis has not been widely investigated. MCJ (also DNAJD1) is a recently discovered gene on chromo- some 13q14.1 which is inactivated in over 50% of ovarian cancer cell lines and primary tumours by epigenetic transcriptional silencing (through hypermethylation of a CpG island which lies within the 5 0 transcribed region of the MCJ gene) and genetic deletion. 16,17 MCJ contains a highly conserved 70–amino acid DNAJ domain (or J domain), first described in the Escherichia coli Hsp DNAJ. J-domain proteins interact with the Hsp70 family of chaperone proteins and act as cochaperones, recruiting Hsp70 to specific substrate proteins. Members of the J-domain superfam- ily participate in many complex biologic processes, such as regu- lation of protein kinases, protein folding, protein translocation and cell cycle control by DNA tumour viruses. 18 Although the specific cellular function(s) of MCJ is not well established, loss of MCJ expression causes in vitro resistance to the chemotherapeutic agents cisplatin, topotecan and paclitaxel. 16,17 Moreover, high lev- els of MCJ methylation in ovarian cancer patients are associated with both response to chemotherapy (cisplatin/carboplatin with or without a taxoid) and overall survival. 19 However, the role of MCJ hypermethylation in the development of other cancer types has not been widely investigated. In this study, we investigated the methylation status of the MCJ CpG island in medulloblastoma and stPNET primary tumours and cell lines and in nonneoplastic cerebellar and noncerebellar brain tissues, to assess whether MCJ hypermethylation is a tumour-spe- cific event. Furthermore, we established relationships between the presence and pattern of MCJ methylation and transcriptional silencing in PNET cell lines. Finally, we extended our analysis of MCJ methylation to ependymomas, the second most common Grant sponsor: North of England Children’s Cancer Research Fund; Grant sponsor: Samantha Dickson Research Trust; Grant sponsor: Char- lie’s Challenge; Grant sponsor: American Lebanese Syrian Associated Charities. *Correspondence to: Northern Institute for Cancer Research, Paul O’Gorman Building, The Medical School, Framlington Place, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK. Fax: 144-191-246-4301. E-mail: s.c.clifford@ncl.ac.uk Received 24 March 2005; Accepted after revision 29 April 2005 DOI 10.1002/ijc.21353 Published online 27 July 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: 5-aza CdR, 5 0 -aza-2 0 -deoxycytidine; CASP8, caspase-8; COBRA, combined bisulphite and restriction analysis; HIC-1, hyperme- thylated in cancer-1; Hsp, heat shock protein; LOH, loss of heterozygosity; MCJ, methylation-controlled J protein; PNET, primitive neuroectodermal tumour; RASSF1A, ras association domain protein-1, isoform A; st, supra- tentorial; TBE, 0.09 M TRIS borate 1 0.002 M EDTA; TSG, tumour- suppressor gene. Int. J. Cancer: 118, 346–352 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer