Ryuji Hamamoto

SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells

Colorectal and hepatocellular carcinomas are some of the leading causes of cancer deaths worldwide, but the mechanisms that underly these malignancies are not fully understood. Here we report the identification of SMYD3, a gene that is over-expressed in the majority of colorectal carcinomas and hepatocellular carcinomas. Introduction of SMYD3 into NIH3T3 cells enhanced cell growth, whereas genetic knockdown with small-interfering RNAs (siRNAs) in cancer cells resulted in significant growth suppression. SMYD3 formed a complex with RNA polymerase II through an interaction with the RNA helicase HELZ and transactivated a set of genes that included oncogenes, homeobox genes and genes associated with cell-cycle regulation. SMYD3 bound to a motif, 5′-CCCTCC-3′, present in the promoter region of downstream genes such as Nkx2.8. The SET domain of SMYD3 showed histone H3-lysine 4 (H3-K4)-specific methyltransferase activity, which was enhanced in the presence of the heat-shock protein HSP90A. Our findings suggest that SMYD3 has histone methyltransferase activity and plays an important role in transcriptional regulation as a member of an RNA polymerase complex. Furthermore, activation of SMYD3 may be a key factor in human carcinogenesis.

Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers

A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine‐specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001). cDNA microarray analysis also revealed its transactivation in lung and colorectal carcinomas. LSD1‐specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of various bladder and lung cancer cell lines. Concordantly, introduction of exogenous LSD1 expression promoted cell cycle progression of human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin‐modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.

Enhanced SMYD3 expression is essential for the growth of breast cancer cells

We previously reported that upregulation of SMYD3, a histone H3 lysine‐4‐specific methyltransferase, plays a key role in the proliferation of colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC). In the present study, we reveal that SMYD3 expression is also elevated in the great majority of breast cancer tissues. Similarly to CRC and HCC, silencing of SMYD3 by small interfering RNA to this gene resulted in the inhibited growth of breast cancer cells, suggesting that increased SMYD3 expression is also essential for the proliferation of breast cancer cells. Moreover, we show here that SMYD3 could promote breast carcinogenesis by directly regulating expression of the proto‐oncogene WNT10B. These data imply that augmented SMYD3 expression plays a crucial role in breast carcinogenesis, and that inhibition of SMYD3 should be a novel therapeutic strategy for treatment of breast cancer. (Cancer Sci 2006; 97: 113u2003–u2003118)

Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers

Protein arginine methylation is a novel post‐translational modification regulating a diversity of cellular processes, including histone functions, but the roles of protein arginine methyltransferases (PRMTs) in human cancer are not well investigated. To address this issue, we first examined expression levels of genes belonging to the PRMT family and found significantly higher expression of PRMT1 and PRMT6, both of which are Type I PRMTs, in cancer cells of various tissues than in non‐neoplastic cells. Abrogation of the expression of these genes with specific siRNAs significantly suppressed growth of bladder and lung cancer cells. Expression profile analysis using the cells transfected with the siRNAs indicated that PRMT1 and PRMT6 interplay in multiple pathways, supporting regulatory roles in the cell cycle, RNA processing and also DNA replication that are fundamentally important for cancer cell proliferation. Furthermore, we demonstrated that serum asymmetric dimethylarginine (ADMA) levels of a number of cancer cases are significantly higher than those of nontumor control cases. In summary, our results suggest that dysregulation of PRMT1 and PRMT6 can be involved in human carcinogenesis and that these Type I arginine methyltransferases are good therapeutic targets for various types of cancer.

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

BackgroundAlthough 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.ResultsQuantitative 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.ConclusionsInhibition of KDM5B may affect apoptosis and reduce growth of cancer cells. Further studies will explore the pan-cancer therapeutic potential of KDM5B inhibition.

Demethylation of RB regulator MYPT1 by histone demethylase LSD1 promotes cell cycle progression in cancer cells.

Histone demethylase LSD1 (also known as KDM1 and AOF2) is active in various cancer cells, but its biological significance in human carcinogenesis is unexplored. In this study, we explored hypothesized interactions between LSD1 and MYPT1, a known regulator of RB1 phosphorylation. We found that MYPT1 was methylated in vitro and in vivo by histone lysine methyltransferase SETD7 and demethylated by LSD1, identifying Lys 442 of MYPT1 as a target for methylation/demethylation by these enzymes. LSD1 silencing increased MYPT1 protein levels, decreasing the steady state level of phosphorylated RB1 (Ser 807/811) and reducing E2F activity. MYPT1 methylation status influenced the affinity of MYPT1 for the ubiquitin-proteasome pathway of protein turnover. MYPT1 was unstable in murine cells deficient in SETD7, supporting the concept that MYPT1 protein stability is physiologically regulated by methylation status. LSD1 overexpression could activate RB1 phosphorylation by inducing a destabilization of MYPT1 protein. Taken together, our results comprise a novel cell cycle regulatory mechanism mediated by methylation/demethylation dynamics, and they reveal the significance of LSD1 overexpression in human carcinogenesis.

Critical roles of non-histone protein lysine methylation in human tumorigenesis

Several protein lysine methyltransferases and demethylases have been identified to have critical roles in histone modification. A large body of evidence has indicated that their dysregulation is involved in the development and progression of various diseases, including cancer, and these enzymes are now considered to be potential therapeutic targets. Although most studies have focused on histone methylation, many reports have revealed that these enzymes also regulate the methylation dynamics of non-histone proteins such as p53, RB1 and STAT3 (signal transducer and activator of transcription 3), which have important roles in human tumorigenesis. In this Review, we summarize the molecular functions of protein lysine methylation and its involvement in human cancer, with a particular focus on lysine methylation of non-histone proteins.

Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker

The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side‐effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real‐time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non‐neoplastic tissues (Pu2003<u20030.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non‐small‐cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (Pu2003=u20030.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD‐18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor‐specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow‐up in some lung cancer patients after surgical treatment. (Cancer Sci 2011; 102: 1298–1305)

The Lysine 831 of Vascular Endothelial Growth Factor Receptor 1 Is a Novel Target of Methylation by SMYD3

We previously identified SMYD3 as a histone methyltransferase and showed that its expression was elevated in colorectal, hepatocellular, and breast carcinomas. In the investigation of methyltransferase activity of SMYD3, we have found that vascular endothelial growth factor receptor 1 (VEGFR1) was also methylated by SMYD3. We further identified the methylated residue at VEGFR1 lysine 831, which is located in the kinase domain and is conserved among VEGFR1 orthologues. We also found that the lysine is followed by serine, which is conserved among some of the methylation targets of histone methyltransferases. Furthermore, methylation of VEGFR1 enhanced its kinase activity in cells. These data should be helpful for the profound understanding of the biological role of SMYD3 and regulatory mechanisms of VEGFR1. Additionally our finding may facilitate the development of strategies that may inhibit the progression of cancer cells.

A variable number of tandem repeats polymorphism in an E2F-1 binding element in the 5' flanking region of SMYD3 is a risk factor for human cancers.

Histone modification is a crucial step in transcriptional regulation, and deregulation of the modification process is important in human carcinogenesis. We previously reported that upregulation of SMYD3, a histone methyltransferase, promoted cell growth in human colorectal and hepatocellular carcinomas. Here we report significant associations between homozygosity with respect to an allele with three tandem repeats of a CCGCC unit in the regulatory region of SMYD3 and increased risk of colorectal cancer (P = 9.1 × 10−6, odds ratio = 2.58), hepatocellular carcinoma (P = 2.3 × 10−8, odds ratio = 3.50) and breast cancer (P = 7.0 × 10−10, odds ratio = 4.48). This tandem-repeat sequence is a binding site for the transcriptional factor E2F-1. In a reporter assay, plasmids containing three repeats of the binding motif (corresponding to the high-risk allele) had higher activity than plasmids containing two repeats (the low-risk allele). These data suggest that the common variable number of tandem repeats polymorphism in SMYD3 is a susceptibility factor for some types of human cancer.