Ken Masuda

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 (P < 0.0001), 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 (P = 0.0239). 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)

Identification of Nectin-4 Oncoprotein as a Diagnostic and Therapeutic Target for Lung Cancer

Gene expression profile analysis of lung cancers revealed the transactivation of an immunoglobulin-like molecule Nectin-4 in the majority of non-small cell lung cancers (NSCLC). Immunohistochemical staining of 422 NSCLCs showed that a high level of Nectin-4 expression was associated with poor prognosis for NSCLC patients (P < 0.0001), and multivariate analysis confirmed its independent prognostic value (P < 0.0001). We established an ELISA to measure serum Nectin-4 and found that serum Nectin-4 levels were significantly higher in NSCLC patients than in healthy volunteers. The proportion of the serum Nectin-4-positive cases was 88 of 164 (53.7%) NSCLCs, whereas only 3 of 131 (2.3%) healthy volunteers were falsely diagnosed as positive, which was superior to carcinoembryonic antigen (CEA) and cytokeratin 19-fragment (CYFRA21-1) in sensitivity and specificity. A combined ELISA for both Nectin-4 and CEA increased sensitivity and classified 65.0% of lung adenocarcinomas as positive with false-positive rate of 4.6%. The use of both Nectin-4 and CYFRA21-1 classified 68.3% of lung squamous cell carcinomas as positive with false-positive rate of 6.1%. Treatment of lung cancer cells with small interfering RNAs against Nectin-4 suppressed its expression and cell growth. In addition, exogenous expression of Nectin-4 increased the lamellipodia formation and the invasive ability of mammalian cells through activation of small GTPase Rac1. Nectin-4 might play a significant role in lung carcinogenesis, and it should be a new candidate serum and tissue biomarker, as well as a therapeutic target.

Wnt Inhibitor Dickkopf-1 as a Target for Passive Cancer Immunotherapy

Dickkopf-1 (DKK1) is an inhibitor of Wnt/beta-catenin signaling that is overexpressed in most lung and esophageal cancers. Here, we show its utility as a serum biomarker for a wide range of human cancers, and we offer evidence favoring the potential application of anti-DKK1 antibodies for cancer treatment. Using an original ELISA system, high levels of DKK1 protein were found in serologic samples from 906 patients with cancers of the pancreas, stomach, liver, bile duct, breast, and cervix, which also showed elevated expression levels of DKK1. Additionally, anti-DKK1 antibody inhibited the invasive activity and the growth of cancer cells in vitro and suppressed the growth of engrafted tumors in vivo. Tumor tissues treated with anti-DKK1 displayed significant fibrotic changes and a decrease in viable cancer cells without apparent toxicity in mice. Our findings suggest DKK1 as a serum biomarker for screening against a variety of cancers, and anti-DKK1 antibodies as potential theranostic tools for diagnosis and treatment of cancer.

Vaccination with multiple peptides derived from novel cancer-testis antigens can induce specific T-cell responses and clinical responses in advanced esophageal cancer.

We previously identified three novel HLA‐A24‐restricted epitope peptides, which were derived from three cancer‐testis antigens, TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and insulin‐like growth factor (IGF)‐II mRNA binding protein 3 (IMP‐3), as targets for cancer vaccination against esophageal squamous cell carcinoma (ESCC). To examine the safety, immunogenicity, and antitumor effect of vaccine treatment using a combination of these three peptides, 10 HLA‐A2402‐positive advanced ESCC patients who failed to standard therapy were enrolled in a phase I clinical trial. Each of the three peptides (1 mg each) was intradermally administered with 1 mL of incomplete Freunds adjuvant to the neck in three separate regions weekly for 5 weeks. The cancer vaccination therapy was well tolerated without any treatment‐associated adverse events of grade 3 or 4. The TTK‐, LY6K‐, and/or IMP‐3‐specific T‐cell immune responses were observed by enzyme‐linked immunospot assay in peripheral blood lymphocytes obtained from nine of the 10 ESCC patients after their vaccination. The median survival time after the vaccination was 6.6 months. The vaccination could induce good clinical responses in 50% of the 10 patients. One patient experienced a complete response in hepatic metastasis lasting 7 months, one showed objective responses in all lung metastasis lesions, and three patients revealed a stable disease condition for at least 2.5 months. The cancer vaccine therapy using these three peptides demonstrated satisfactory safety and good immunogenicity as well as promising disease control rate, and therefore warrants further clinical studies. (Cancer Sci 2009)

Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.

Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer

BackgroundThe research emphasis in anti-cancer drug discovery has always been to search for a drug with the greatest antitumor potential but fewest side effects. This can only be achieved if the drug used is against a specific target located in the tumor cells. In this study, we evaluated Minichromosome Maintenance Protein 7 (MCM7) as a novel therapeutic target in cancer.ResultsImmunohistochemical analysis showed that MCM7 was positively stained in 196 of 331 non-small cell lung cancer (NSCLC), 21 of 29 bladder tumor and 25 of 70 liver tumor cases whereas no significant staining was observed in various normal tissues. We also found an elevated expression of MCM7 to be associated with poor prognosis for patients with NSCLC (P = 0.0055). qRT-PCR revealed a higher expression of MCM7 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpressed MCM7 by cDNA microarray analysis. Suppression of MCM7 using specific siRNAs inhibited incorporation of BrdU in lung and bladder cancer cells overexpressing MCM7, and suppressed the growth of those cells more efficiently than that of normal cell strains expressing lower levels of MCM7.ConclusionsSince MCM7 expression was generally low in a number of normal tissues we examined, MCM7 has the characteristics of an ideal candidate for molecular targeted cancer therapy in various tumors and also as a good prognostic biomarker for NSCLC patients.

The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene.

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogensis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real‐time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di‐methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down‐regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G1 arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.

Chondrolectin is a novel diagnostic biomarker and a therapeutic target for lung cancer.

Purpose: This study aims to identify molecules that might be useful as diagnostic/prognostic biomarkers and as targets for the development of new molecular therapies for lung cancer. Experimental Design: We screened for genes that were highly transactivated in a large proportion of 120 lung cancers by means of a cDNA microarray representing 27,648 genes and found chondrolectin (CHODL) as a candidate. Tumor tissue microarray was applied to examine the expression of CHODL protein and its clinicopathologic significance in archival non–small cell lung cancer (NSCLC) tissues from 295 patients. A role of CHODL in cancer cell growth and/or survival was examined by siRNA experiments. Cellular invasive effect of CHODL on mammalian cells was examined by Matrigel assays. Results: Immunohistochemical staining revealed that strong positivity of CHODL protein was associated with shorter survival of patients with NSCLC (P = 0.0006), and multivariate analysis confirmed it to be an independent prognostic factor. Treatment of lung cancer cells with siRNAs against CHODL suppressed growth of the cancer cells. Furthermore, induction of exogenous expression of CHODL conferred growth and invasive activity of mammalian cells. Conclusions: CHODL is likely to be a prognostic biomarker in the clinic and targeting CHODL might be a strategy for the development of anticancer drugs. Clin Cancer Res; 17(24); 7712–22. ©2011 AACR.