Journal of Pathology J Pathol 2014; 233: 392–401 Published online 16 June 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4376 ORIGINAL PAPER Deregulation of RB1 expression by loss of imprinting in human hepatocellular carcinoma Sumadi Lukman Anwar, 1# Till Krech, 1 Britta Hasemeier, 1 Elisa Schipper, 1 Nora Schweitzer, 2 Arndt Vogel, 2 Hans Kreipe 1 and Ulrich Lehmann 1 * 1 Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany 2 Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany *Correspondence to: U Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. E-mail: Lehmann.Ulrich@MH-Hannover.de # Present address: Department of Surgery, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Abstract The tumour suppressor gene RB1 is frequently silenced in many different types of human cancer, including hepatocellular carcinoma (HCC). However, mutations of the RB1 gene are relatively rare in HCC. A systematic screen for the identiication of imprinted genes deregulated in human HCC revealed that RB1 shows imprint abnormalities in a high proportion of primary patient samples. Altogether, 40% of the HCC specimens (16/40) showed hyper- or hypomethylation at the CpG island in intron 2 of the RB1 gene. Re-analysis of publicly available genome-wide DNA methylation data conirmed these indings in two independent HCC cohorts. Loss of correct DNA methylation patterns at the RB1 locus leads to the aberrant expression of an alternative RB1–E2B transcript, as measured by quantitative real-time PCR. Demethylation at the intron 2 CpG island by DNMT1 knock-down or aza-deoxycytidine (DAC) treatment stimulated expression of the RB1–E2B transcript, accompanied by diminished RB1 main transcript expression. No aberrant DNA methylation was found at the RB1 locus in hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5) and their corresponding adjacent liver tissue specimens. Deregulated RB1 expression due to hyper- or hypomethylation in intron 2 of the RB1 gene is found in tumours without loss of heterozygosity and is associated with a decrease in overall survival (p = 0.032) if caused by hypermethylation of CpG85. This unequivocally demonstrates that loss of imprinting represents an important additional mechanism for RB1 pathway inactivation in human HCC, complementing well-described molecular defects. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: retinoblastoma gene (RB1); loss of imprinting (LOI); hepatocellular carcinoma; DNA methylation; epigenetics Received 19 February 2014; Revised 30 April 2014; Accepted 11 May 2014 No conlicts of interest were declared. Introduction RB1 is the irst identiied tumour suppressor gene that is frequently inactivated in several major cancers (see [1] and references therein). Germline mutations in this gene are associated with a high risk of developing retinoblastoma [2]. The pRb protein plays a key role in cell cycle regulation, particularly during the S–G 2 transition. Unphosphorylated pRb binds the E2F tran- scription factor, thereby down-regulating E2F target genes. Phosphorylation of pRb by cyclin-dependent kinases (CDKs) results in the release of E2F proteins from the complex to initiate transcription of genes involved in cell-cycle progression [3]. In addition, pRb regulates several downstream pathways involved in cell differentiation [4], apoptosis [5], DNA damage response [6], senescence [7,8] and embryogenesis [9]. In HCC, mutations in RB1 are rare events, although the pRb pro- tein is frequently down-regulated in HCC cells [10 – 12]. Several mechanisms have been proposed for the absence of RB1 expression in HCC, including genetic loss, epi- genetic defects and post-transcriptional degradation [13–17]. A recent study using genome-wide DNA methy- lation analysis in a patient with extensive imprinting defects discovered that the human RB1 gene is actually imprinted [18]. Genomic imprinting is a biological phenomenon leading to parent-of-origin-speciic gene expression, which is frequently disturbed in many diseases, including cancer [19,20]. The differentially methylated region (DMR) showing parent-of-origin- speciic DNA methylation at the RB1 gene is located within a CpG island in intron 2 (called ‘CpG85’; see Figure 1A). In humans, this region is speciically methy- lated on the maternal allele, whereas in the mouse (and rat) genome RB1 is not imprinted, due to the absence of the retrotransposon-derived differentially methylated region in exon 2 of the RB1 gene [21]. Differential Copyright © 2014 Pathological Society of Great Britain and Ireland. J Pathol 2014; 233: 392–401 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com