The emergence of effective cancer chemotherapy is one of the major medical advances of recent years. Adjuvant chemotherapy for lung, breast or colon cancer can augment the survival benefit afforded by surgical management. Even in patients with advanced solid tumors or recurrences after surgery, chemotherapy can offer lengthened survival of worthwhile quality. However the therapeutic approach for patients with recurrent tumor is limited, and its effects are usually partial and often disappointingly brief. In addition, most antitumor agents cause unexpected detrimental side effects. Therefore, it is critical to discover novel therapeutic targets to extend the capability of cancer chemotherapy and improve patient care.

DNA-histone complexes comprise the fundamental repeating unit of chromatin, and the multiplicity of histone modifications results in chromatin-dependent functions. This idea was previously proposed as the "Histone Code Hypothesis". Histones, especially residues of the amino termini of histones H3 and H4 and the amino and carboxyl termini of histones H2A, H2B and H1, are susceptible to a variety of post-translational modifications such as phosphorylation, acetylation, methylation, ubiquitination, sumoylation, and glycosylation. Among histone modifications, methyl-lysine residues in nucleosomal histones are considered to mediate interactions with the macromolecular complexes that regulate chromatin-template processes such as transcription. Despite a large body of information for the prominent role of histone lysine methylation in transcriptional regulation, the involvement of their alterations in human diseases still remains unclear.

The aim of my research project is to clarify the importance of methyltransferase and demethylase in human carcinogenesis. As demonstrated in Figure 1, several histone-modifying enzymes have been shown to be deregulated in cancer cells. Among them, we had reported that SMYD3, a histone lysine methyltransferase, stimulated proliferation of cells and played an important role in human carcinogenesis through its methyltransferase activity.

In order to investigate possible roles of histone lysine methyltransferases (HKMTases) in human carcinogenesis, I examined the expression profiles of human HKMTases in clinical tissues and found that expression levels of WHSC1L1 (Wolf-Hirschhorn syndrome candidate—like 1) were significantly up-regulated in various types of cancers compared with their corresponding normal tissues. WHSC1L1, also known as NSD3 and WHISTLE, shows high sequence similarity to NSD2/WHSC1 (Wolf-Hirschhorn syndrome candidate-1) and NSD1, particularly in their C-terminal portion, which includes the SET domain responsible for the HMTase activity (Figure 2).

Although WHSC1L1 is known to function as a transcriptional regulator that mediates histone methylation, the biological function of the protein has not been well elucidated. Here, I demonstrated a possible involvement of WHSC1L1 in human cancers.

Firstly, expression levels of WHSC1L1 transcript were significantly elevated in a various human cancers including bladder carcinoma and CML (Figure 3). Immunohistochemical analysis of bladder, lung and liver cancers confirmed overexpression of WHSC1L1 (Figure 4).

Moreover, WHSC1L1-specific small interfering RNAs significantly knocked down its expression and resulted in the suppression of proliferation of bladder and lung cancer cell lines (Figure 5). WHSC1L1 knockdown induced cell cycle arrest at the G2/M phase followed by multinucleation of cancer cells.

To further analyze the physiological function of WHSC1L1-signal pathway, I performed microarray expression analysis using Affymetrix's GeneChip(R) system. Messenger RNA from SW780 or A549 cancer cells were subjected to the analysis using HG-U133 Plus 2.0 Array. The expression profiles of these cells were compared with those of cells treated with control siRNAs (siEGFP and siFFLuc). As a result, WHSC1L1 was shown to affect the expression of a number of genes including CCNG1 and NEK7 those are known to play crucial roles in the cell cycle progression at mitosis. Since WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good therapeutic target of various types of cancer.

Figure 1. Histone-modifying enzymes deregulated in human cancer.

Figure 2. Characteristics of WHSC1L1(Wolf-Hirschhorn Syndrome Candidate 1-like 1 ).

Figure 3. Elevated WHSC1L1 expression in human cancers. (A) Expression levels of WHSC1L1 were analyzed by quantitative real-time PCR, and the result is shown by box-whisker plot (median 50% boxed). Relative mRNA expression shows the value normalized by GAPDH and SDH expressions. Mann-Whitney U test was used for statistical analysis. (B) Expression analysis of WHSC1L1 in chronic myelogenous leukemia. Signal intensity of each sample was analyzed by cDNA microarray, and the result is shown by box-whisker plot (median 50% boxed). Mann-Whitney U test was used for the statistical analysis.

Figure 4. Tissue microarray images of clinical bladder tissues (A), lung tissues (B) and liver tissues (C) stained by standard immunohistochemistry for protein expression of WHSC1L1. Clinical information for each section is represented above histological pictures. All tissue samples were purchased from BioChain. Original magnification, x100 and x200.

Figure 5. Involvement of WHSC1L1 in the growth of bladder and lung cancer cells. (A) Expression of WHSC1L1 in A549 cells treated with two independent specific siRNAs targeting WHSC1L1 (siWHSC1L1#1, #2) was analyzed by quantitative real-time PCR. siRNAs targeting EGFP (siEGFP) and siNegative control (siNC) were used as controls. mRNA expression levels were normalized by GAPDH and SDH expressions, and values are relative to siEGFP (siEGFP = 1). Results are the mean ± SD of three independent experiments. P values were calculated using Student's t-test (***, P < 0.001). (B) Effects of WHSC1L1 siRNA knockdown on the viability of two bladder cancer cell line (SW780, RT4) and three lung cancer cell lines (A549, LC319 and SBC5). Relative cell number shows the value normalized to siEGFP-treated cells (siEGFP = 1). Results are the mean ± SD in three independent experiments. P values were calculated using Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

ヒストンメチル化酵素・脱メチル化酵素により、ヒストンのリジン残基のメチル化レベルが調節され、その結果遺伝子の発現調節を介して発生や分化などに重要な役割を担っていることはよく知られている。近年多くのがん組織でヒストンメチル化酵素・脱メチル化酵素の発現が亢進し、発癌過程に重要な役割を担っていることが明らかになっている。ヒストンメチル化酵素の一つであるWHSC1L1は、これまでヒストンH3のlysine36をメチル化することが報告されているが、WHSC1L1よるヒストンタンパク質修飾の生理的意義、特にヒトがん化における役割に関しては殆ど解明されていない。Kangらはガン組織における発現解析の結果、ヒストンメチル化酵素WHSC1L1のが多種のヒトがんで発現亢進することを明らかとし、さらにその生理的な意義について機能解析を行った。

がん特異的に過剰発現を示すヒストンメチル化酵素およびヒストン脱メチル化酵素を探索するために、膀胱がん臨床検体120例からRNAを単離後、定量的RT - PCRで発現解析を行った結果、正常細胞の発現量よりがん細胞での発現が顕著に高いヒストンメチル化酵素WHSC1L1を同定した。またcDNAマイクロアレイ解析及び免疫組織学的解析により、WHSC1L1は肺がん、肝臓がん、慢性骨髄性白血病などでも高発現していることが明らかとなった。WHSC1L1の発現を特異的に阻害するsiRNAを膀胱がん細胞及び肺がん細胞株に導入したところ、有意にがん細胞の増殖が抑制された。これは、WHSC1L1ががん細胞の増殖において重要な役割を担っていることを示唆する結果である。さらにWHSC1L1が過剰発現したがん細胞でH3のメチル化レベルを検討した所、H3K36 di - methylation が顕著に亢進している事が分かった。この結果はがん細胞でH3K36のメチル化の亢進を同定した初めての報告である。

さらに、WHSC1L1が細胞周期に及ぼす影響を調べるためにフローサイトメトリー解析を行った結果、siRNAにてWHSC1L1をノックダウンしたた癌細胞株ではG2/M期の増加が見られた。さらにWHSC1L1により制御される遺伝子の同定を目的として、SW780細胞とA549細胞を用いてWHSC1L1をノックダウン後の遺伝子発現解析をAffymetrix(R)社のGeneChip(R)を用いて行った。その結果、WHSC1L1によって制御される42遺伝子が同定された。42遺伝子中にG2/M期移行に重要なCCNG1およびNEK7が含まれており、WHSC1L1がこれらの標的遺伝子発現を調節することによりG2/M期移行を制御する可能性が考えられた。

以上本論文において、ヒストンメチル化酵素WHSC1L1が種々の癌組織で発現が亢進している事、またWHSC1L1はCCNG1やNEK7などの下流遺伝子の発現を制御することでG2/M期における細胞周期進行を促進し、細胞の悪性化に重要な役割を果たす可能性があることが解明された。この結果は、ヒストンメチル化酵素WHSC1L1の特異的阻害剤開発などにより、新規がんの分子標的治療薬の創生につながり、臨床応用の側面でも大変重要な知見であると考えられる。

本論文は、Hyun-Soo Cho、豊川剛二、小暮正晴、山根由香、岩井裕希子、速水晋也、角田達彦、Helen I.Field、松田浩一、David E.Neal、Bruce A.J.Ponder、前原喜彦、中村祐輔、浜本隆二との共同研究であるが、論文提出者が主体となって分析及び検証を行ったもので、論文提出者の寄与が充分であると判断する。したがって、博士(生命科学)の学位を授与できると認める。