Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: involvement in the proliferation of cancer cells through the E2F/RB pathway

Molecular Cancer Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: involvement in the proliferation of cancer cells through the E2F/ RB pathway Shinya Hayami Masanori Yoshimatsu Abhimanyu Veerakumarasivam Motoko Unoki 2 Yukiko Iwai Tatsuhiko Tsunoda 1 Helen I Field 0 John D Kelly 3 David E Neal Hiroki Yamaue Bruce AJ Ponder Yusuke Nakamura Ryuji Hamamoto 0 Department of Genetics, University of Cambridge , Downing Street, Cambridge CB2 3EH , UK 1 Laboratory for Medical Informatics, RIKEN , 1-7-22 Suehirocho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 , Japan 2 Laboratory for Biomarker, RIKEN , 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108- 8639 , Japan 3 Division of Surgery & Interventional Science, UCL Medical School, University College London , 74 Huntley Street, London, WC1E 6AU , UK Background: Although an increasing number of histone demethylases have been identified and biochemically characterized, their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. We investigated the role of KDM5B, a JmjC histone demethylase, in human carcinogenesis. Quantitative RT-PCR and microarray analyses were used to examine the expression profiles of histone demethylases in clinical tissue samples. We also examined the functional effects of KDM5B on the growth of cancer cell lines treated with small interfering RNAs (siRNAs). Downstream genes and signal cascades induced by KDM5B expression were identified from Affymetrix Gene Chip experiments, and validated by real-time PCR and reporter assays. Cell cycle-dependent characteristics of KDM5B were identified by immunofluorescence and FACS. Results: Quantitative RT-PCR analysis confirmed that expression levels of KDM5B are significantly higher in human bladder cancer tissues than in their corresponding non-neoplastic bladder tissues (P < 0.0001). The expression profile analysis of clinical tissues also revealed up-regulation of KDM5B in various kinds of malignancies. Transfection of KDM5B-specific siRNA into various bladder and lung cancer cell lines significantly suppressed the proliferation of cancer cells and increased the number of cells in sub-G1 phase. Microarray expression analysis indicated that E2F1 and E2F2 are downstream genes in the KDM5B pathway. Conclusions: Inhibition of KDM5B may affect apoptosis and reduce growth of cancer cells. Further studies will explore the pan-cancer therapeutic potential of KDM5B inhibition. - Background Histone methylation plays an important dynamic role in regulating chromatin structure. Precise coordination and organization of open and closed chromatins are crucial for normal cellular processes such as DNA replication, repair, recombination and transcription. Until recently, histone methylation was considered to be a static modification, but the identification of histone demethylases has revealed that this modification is dynamically regulated [1,2]. Histone demethylases regulate not only the modification itself but also its extended function, by antagonizing the binding of effector proteins to modified chromatin. This is exemplified by JHDM3A/ JMJD2A, which displaces HP1 from chromatin by demethylating the H3K9 methylation and thereby preventing the spread of H3K9 methylation to the surrounding chromatin by HP1 [3,4]. A highly-conserved family of proteins containing the JmjC domain was recently characterized to possess a histone demethylase activity [5]. Despite a large body of information for the prominent role of histone demethylases in transcriptional regulation, their physiological function, and their involvement in human disease is still not wellunderstood. We previously reported that SMYD3, a histone methyltransferase, stimulates cell proliferation through its methyltransferase activity and plays a crucial role in human carcinogenesis [6-10]. Although dysfunction of histone methylation status was indicated to contribute to human carcinogenesis [11-13], the relationship between abnormal histone demethylation and human carcinogenesis is still largely unclear. In order to find demethylases that contribute to human carcinogenesis, we examined the expression profiles of several proteins containing a JmjC histone demethylase domain in clinical tissues and found that expression levels of KDM5B were significantly up-regulated, compared with their corresponding normal tissues, in many types of cancer. KDM5B, also named JARID1B or PLU-1, is one of the four JARID family members [14,15], and contains domains common to transcriptional regulators such as a JmjN domain, a Bright/Arid domain, a C5H2C zinc finger motif, and several PHD domains in addition to a JmjC domain. All four members of the JARID family possess the H3K4 demethylase activity [16-20]. Each member might participate in different biological processes through recruitment to different chromosomal regions and differing enzymatic activities [5]. Here we demonstrate a novel function of KDM5B in human carcinogenesis and show that it is related to the cell cycle through regulation of E2F expression and cell growth. Results KDM5B expression is up-regulated in clinical cancer tissues We first examined expression levels of five jumonji histone demethylase genes included in JARID family, KDM5A (JARID1A), KDM5B (JARID1B), KDM5C (JARID1C), KDM5D (JARID1D) and JARID2, in a small subset of clinical bladder cancer samples and found a significant difference in expression levels between normal and cancer cells only for the KDM5B gene (data not shown). Therefore, we analyzed 123 bladder cancer samples and 23 normal control samples (British) and confirmed significant elevation of KDM5B expression in tumor cells compared with in normal cells (P < 0.0001, Mann-Whitneys U-test) (Figure 1A and Additional file 1). No significant difference was observed in expression levels among different grades and stages (Table 1 and Additional file 1). This suggests that KDM5B expression was up-regulated in an early stage of bladder carcinogenesis, and remained high in the advanced stages of the disease. Subclassification of tumors according to gender, smoking history, metastasis status, and recurrence status identified no significant difference in the expression levels of KDM5B (Table 1). We then analyzed the expression patterns of KDM5B in a number of clinical samples derived from Japanese bladder cancer subjects examined by cDNA microarray (Figure 1B and 1C), and confirmed its significant overexpression (P < 0.0001, Mann-Whitneys U-test). To evaluate protein expression levels of KDM5B in bladder tissues, we performed immunohistochemical analysis using anti-KDM5B specific antibody (Figure 1D). We observed strong KDM5B staining mainly in the nucleus of malignant cells, but no significant staining in non-neoplastic tissues. To further validate this result, we conducted tissue microarray experiments using 29 bladder tissue sections (Figure 2 and Additional file 2), and observed st (...truncated)