Toshihiko Yokoyama

In Operando X-ray Absorption Fine Structure Studies of Polyoxometalate Molecular Cluster Batteries: Polyoxometalates as Electron Sponges

We carried out in operando Mo K-edge X-ray absorption fine structure measurements on the rechargeable molecular cluster batteries (MCBs) of polyoxometalates (POMs), in which a Keggin-type POM, [PMo(12)O(40)](3-), is utilized as a cathode active material with a lithium metal anode. The POM-MCBs exhibit a large capacity of ca. 270 (A h)/kg in a voltage range between V = 4.0 V and V = 1.5 V. X-ray absorption near-edge structure analyses demonstrate that all 12 Mo(6+) ions in [PMo(12)O(40)](3-) are reduced to Mo(4+) in the discharging process. This means the formation of a super-reduced state of the POM, namely, [PMo(12)O(40)](27-), which stores 24 electrons, and this electron number can explain the large capacity of the POM-MCBs. Furthermore, extended X-ray absorption fine structure analyses reveal the molecular structure of [PMo(12)O(40)](27-), which is slightly reduced in size compared to the original [PMo(12)O(40)](3-) and involves Mo(4+) metal-metal-bonded triangles. Density functional theory calculations suggest that these triangles are formed because of the large number of additional electrons in the super-reduced state.

Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

Malignant pleural mesothelioma (MPM) is an asbestos‐related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC‐MESO‐1, ACC‐MESO‐4, Y‐MESO‐8A and Y‐MESO‐8D) from Japanese patients, with the latter two from the same patient with biphasic‐like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI‐1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC‐MESO‐1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y‐MESO‐8A and Y‐MESO‐8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies. (Cancer Sci 2006; 97)

Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) gene, suppresses anchorage-independent cell growth of lung cancer cells

We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.

YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation

We previously reported the results of bacterial artificial chromosome array comprehensive genomic hybridization of malignant pleural mesotheliomas (MPMs), including two cases with high-level amplification in the 11q22 locus. In this study, we found that the YAP1 gene encoding a transcriptional coactivator was localized in this amplified region and overexpressed in both cases, suggesting it as a candidate oncogene in this region. We analyzed the involvement of YAP1 in MPM proliferation, as well as its functional and physical interaction with Merlin encoded by the neurofibromatosis type 2 (NF2) tumor suppressor gene, which is frequently mutated in MPMs. YAP1-RNA interference suppressed growth of a mesothelioma cell line NCI-H290 with NF2 homozygous deletion, probably through cell-cycle arrest and apoptosis induction, whereas YAP1 transfection promoted the growth of MeT-5A, an immortalized mesothelial cell line. We also found that the introduction of NF2 into NCI-H290 induced phosphorylation at serine 127 of YAP1, which was accompanied by reduction of nuclear localization of YAP1, whereas nuclear localization of a YAP1 S 127A mutant was not affected. Furthermore, results of immunoprecipitation and in vitro pull-down assays indicated a physical interaction between Merlin and YAP1. These results suggest that YAP1 is involved in mesothelial cell growth and that the transcriptional coactivator activity of YAP1 is functionally inhibited by Merlin through the induction of phosphorylation and cytoplasmic retention of YAP1. This is the first report of negative regulatory signaling from Merlin to YAP1 in mammalian cells. Future studies of transcriptional targets of YAP1 in MPMs may shed light on the molecular mechanisms of MPM development and lead to new therapeutic strategies.

Genomic profiling of malignant pleural mesothelioma with array-based comparative genomic hybridization shows frequent non-random chromosomal alteration regions including JUN amplification on 1p32

Genome‐wide array‐based comparative genomic hybridization analysis of malignant pleural mesotheliomas (MPM) was carried out to identify regions that display DNA copy number alterations. Seventeen primary tumors and nine cell lines derived from 22 individuals were studied, some of them originating from the same patients. Regions of genomic aberrations observed in >20% of individuals were 1q, 5p, 7p, 8q24 and 20p with gains, and 1p36.33, 1p36.1, 1p21.3, 3p21.3, 4q22, 4q34‐qter, 6q25, 9p21.3, 10p, 13q33.2, 14q32.13, 18q and 22q with losses. Two regions at 1p32.1 and 11q22 showed a high copy gain. The 1p32.1 region contained a protooncogene, JUN, and we further demonstrated overexpression of JUN with real‐time polymerase chain reaction analysis. As MPM cell lines did not overexpress JUN, our findings suggested that induction of JUN expression was involved in the development of MPM cells in vivo, which also might result in gene amplification in a subset of MPM. Meanwhile, the most frequent alteration was the 9p21.3 deletion, which includes the p16INK4a/p14ARF locus. With polymerase chain reaction analysis, we determined the extent of the homozygous deletion regions of the p16INK4a/p14ARF locus in MPM cell lines, which indicated that the deletion regions varied among cell lines. Our results with array comparative genomic hybridization analysis provide new insights into the genetic background of MPM, and also give some clues to develop a new molecular target therapy for MPM. (Cancer Sci 2007; 98: 438–446)

EGFR point mutation in non‐small cell lung cancer is occasionally accompanied by a second mutation or amplification

Activating mutations of EGFR are found frequently in a subgroup of patients with non‐small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription–polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild‐type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second‐hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light‐smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC. (Cancer Sci 2006; 97: 753–759)

Integrated analysis of genetic and epigenetic alterations reveals CpG island methylator phenotype associated with distinct clinical characters of lung adenocarcinoma

DNA methylation affects the aggressiveness of human malignancies. Cancers with CpG island methylator phenotype (CIMP), a distinct group with extensive DNA methylation, show characteristic features in several types of tumors. In this study, we initially defined the existence of CIMP in 41 lung adenocarcinomas (AdCas) through genome-wide DNA methylation microarray analysis. DNA methylation status of six CIMP markers newly identified by microarray analysis was further estimated in a total of 128 AdCas by bisulfite pyrosequencing analysis, which revealed that 10 (7.8%), 40 (31.3%) and 78 (60.9%) cases were classified as CIMP-high (CIMP-H), CIMP-low and CIMP-negative (CIMP-N), respectively. Notably, CIMP-H AdCas were strongly associated with wild-type epidermal growth factor receptor (EGFR), males and heavy smokers (P = 0.0089, P = 0.0047 and P = 0.0036, respectively). In addition, CIMP-H was significantly associated with worse prognosis; especially among male smokers, CIMP-H was an independent prognostic factor (hazard ratio 1.7617, 95% confidence interval 1.0030-2.9550, P = 0.0489). Compellingly, the existence of CIMP in AdCas was supported by the available public datasets, such as data from the Cancer Genome Atlas. Intriguingly, analysis of AdCa cell lines revealed that CIMP-positive AdCa cell lines were more sensitive to a DNA methylation inhibitor than CIMP-N ones regardless of EGFR mutation status. Our data demonstrate that CIMP in AdCas appears to be a unique subgroup that has distinct clinical traits from other AdCas. CIMP classification using our six-marker panel has implications for personalized medical strategies for lung cancer patients; in particular, DNA methylation inhibitor might be of therapeutic benefit to patients with CIMP-positive tumors.

RhoB is frequently downregulated in non-small-cell lung cancer and resides in the 2p24 homozygous deletion region of a lung cancer cell line.

Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non‐small‐cell lung cancer (NSCLC) cell line, NCI‐H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistry and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly‐ or moderately‐differentiated adenocarcinomas than in well‐differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG for NSCLC.

Clinical usefulness of serum pyridinoline cross-linked carboxyterminal telopeptide of type I collagen for diagnosis of bone metastases in patients with primary lung cancer

Objective:  Recently, various blood and urine markers of bone metabolism have been developed and applied to the diagnosis of bone metastases. However, the cut‐off values for each parameter have not yet been completely defined. In this study, the usefulness of serum pyridinoline cross‐linked carboxyterminal telopeptide of type I collagen (I CTP) was assessed for detecting bone metastases from primary lung cancer and the most efficient cut‐off value for I CTP based on the receiver operating characteristic curve was calculated.

Abstract 61: Integrated analysis of genetic and epigenetic alterations reveals CpG island methylator phenotype associated with clinical outcome of lung adenocarcinoma

Background: Lung cancer is the leading cause of cancer-related death worldwide. While targeted therapy, such as tyrosine kinase inhibitors (TKIs), has been used against the non small cell lung cancers (NSCLCs) patients with epidermal growth factor receptor (EGFR) mutation, not all the patients received benefit from TKIs. Epigenetic alterations have been recognized as one of the important mechanism of NSCLCs. The aim of this study is to clarify the significance of DNA methylation, especially the clinical impacts of CpG island methylator phenotype (CIMP), in NSCLCs. Methods: Using methylated CpG island amplification-microarray (MCAM), a genome-wide DNA methylation analysis, 41 lung adenocarcinomas (AdCas) were analyzed. DNA methylation of newly identified genes from MCAM analysis was further examined in another cohort of 86 AdCas with bisulfate pyrosequencing method. Results: Unsupervised hierarchical cluster analysis revealed that DNA methylation was extensively accumulated in one subgroup (n=4, 9.8%), which may represent CIMP tumors (651 genes vs. 354 genes, P=0.0004). DNA methylation status of newly identified 7 CIMP markers revealed that 8 (9.3%), 12 (14%), and 66 (76.7%) were classified into CIMP-high, CIMP-low, and CIMP-negative tumors, respectively. CIMP-high tumors showed no EGFR mutations and frequent exposure to smoking. In addition, these tumors had worse prognosis than the other tumors (P=0.035), suggesting that CIMP-high tumors have characteristic clinical features, which may represent an alternative mechanism to the EGFR mutation. Treatments of NSCLC cell lines showed that CIMP positive AdCa cell lines are sensitive to DNA demethylating agent regardless of EGFR status. Conclusions: Our data suggest that CIMP in AdCas appears to be a unique subgroup, which has distinguished clinical traits from the other AdCas. Applying DNA demethylating agent to CIMP-high tumors might be a new strategy for the treatment of NSCLCs without EGFR mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2011-61