Modeling mesothelioma utilizing human mesothelial cells reveals involvement of phospholipase-C beta 4 in YAP-active mesothelioma cell proliferation.

Mesotheliomas are frequently characterized by disruption of Hippo pathway due to deletion and/or mutation in genes, such as neurofibromin 2 ( NF2 ). Hippo disruption attenuates yes-associated protein (YAP) phosphorylation allowing YAP to translocate to the nucleus and regulate gene expression. The role of disrupted Hippo pathway in maintenance of established mesotheliomas has been extensively investigated using cell lines; however, its involvement in development of human mesothelioma has not been explored much. Here, we employed immortalized human mesothelial cells to disrupt Hippo pathway. YAP phosphorylation was reduced on NF2 knockdown and the cells exhibited altered growth in vitro , developing tumors when transplanted into nude mice. Similar results were obtained from enforced expression of wild-type or constitutively active (S127A) YAP, indicating the crucial role of activated YAP in the transformation of mesothelial cells. Gene expression analysis comparing control- and YAP-transduced immortalized human mesothelial cells revealed phospholipase-C beta 4 ( PLCB4 ) to be among the genes highly upregulated by YAP. PLCB4 was upregulated by YAP in immortalized human mesothelial cells and downregulated on YAP knockdown in Hippo-disrupted mesothelioma cell lines. PLCB4 knockdown attenuated the growth of YAP-transduced immortalized mesothelial cells and YAP-active, but not YAP-nonactive, mesothelioma cell lines. Our model system thus provides a versatile tool to investigate the mechanisms underlying mesothelioma development. We suggest that PLCB4 may be an attractive drug target for the treatment of mesothelioma.

Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.

Malignant mesothelioma (MM) shows inactivation of the BRCA1-associated protein 1 (BAP1) gene. In this study, we found BAP1 mutations in 5 (26%) of the 19 cell lines that we established from Japanese MM patients, and examined functional differences between the WT and mutant BAP1. First, we studied the subcellular localization of BAP1, demonstrating that the WT primarily resides in the nucleus and that the mutant BAP1 is found in the cytoplasm of the cells. Transduction of the WT BAP1 vector into MM cells with homozygous deletion at the BAP1 3' side resulted in both inhibition of cell proliferation and anchorage-independent cell growth, whereas BAP1 mutants of a missense or C-terminal truncated form showed impaired growth inhibitory effects. Next, we studied how BAP1 is involved in MM cell survival after irradiation (IR), which causes DNA damage. After IR, we found that both WT and mutant BAP1 were similarly phosphorylated and phospho-BAP1 localized mainly in the nucleus. Interestingly, BRCA1 proteins were decreased in the MM cells with BAP1 deletion, and transduction of the mutants as well as WT BAP1 increased BRCA1 proteins, suggesting that BAP1 may promote DNA repair partly through stabilizing BRCA1. Furthermore, using the MM cells with BAP1 deletion, we found that WT BAP1, and even a missense mutant, conferred a higher survival rate after IR compared to the control vector. Our results suggested that, whereas WT BAP1 suppresses MM cell proliferation and restores cell survival after IR damage, some mutant BAP1 may also moderately retain these functions.

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

BACKGROUND & AIMS: Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. METHODS: Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. RESULTS: No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). CONCLUSIONS: In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes.

Contribution of microRNA-1275 to Claudin11 protein suppression via a polycomb-mediated silencing mechanism in human glioma stem-like cells.

Glioblastomas show heterogeneous histological features, and tumor cells show distinct phenotypic states that confer different functional attributes and an aggressive character. However, the molecular mechanisms underlying the heterogeneity in this disease are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute to the establishment of tumor heterogeneity. Using a GSC model, we investigated differentially expressed microRNAs (miRNAs) and associated epigenetic mechanisms that regulate the differentiation of GSCs. miRNA profiling using microarray technology showed that 13 and 34 miRNAs were commonly up-regulated and down-regulated in two independent GSC lines during differentiation, respectively. Among this set of miRNAs, quantitative PCR analysis showed that miRNA-1275 (miR-1275) was consistently down-regulated during GSC differentiation, along with the up-regulation of its target, CLDN11, an important protein during oligodendroglial lineage differentiation. Inhibition of miR-1275 with a specific antisense oligonucleotide (anti-miR-1275) in GSCs increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) in the primary microRNA-1275 promoter was closely associated with miR-1275 expression. Treatment with 3-deazaneplanocin A, an inhibitor of H3K27 methyltransferase, attenuated CLDN11 induction by serum stimulation in parallel with sustained miR-1275 expression. Our results have illuminated the epigenetic regulatory pathways of miR-1275 that are closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity seen in the formation of glioblastomas. Given that inhibition of miR-1275 induces expression of oligodendroglial lineage proteins and suppresses tumor cell proliferation, this may be a potential therapeutic target for glioblastomas.

Membranous expression of activated leukocyte cell adhesion molecule contributes to poor prognosis and malignant phenotypes of non-small-cell lung cancer.

BACKGROUND: Activated leukocyte cell adhesion molecule (ALCAM) has been shown to correlate with the prognosis of patients with various types of human malignancies. However, the relationship between ALCAM expression and progression of non-small-cell lung cancer (NSCLC) has not been investigated. This study was designed to clarify the prognostic impact of ALCAM expression of NSCLC cells. MATERIALS AND METHODS: The study population consisted of 147 NSCLC patients who underwent complete resection. We performed immunohistochemical staining for ALCAM expression and correlated this to the clinicopathologic parameters and patient survival. The ALCAM expression in NSCLC cell lines was analyzed using quantitative reverse transcription-polymerase chain reaction and Western blot analyses. ALCAM knockdown in NSCLC cell lines was performed with lentivirus-mediated short hairpin RNA transduction. RESULTS: Positive membranous and cytoplasmic ALCAM expressions were detected in 66 (44.9%) and 57 (38.8%) patients, respectively. A significant association of high membranous ALCAM expression with shortened overall survival (OS) was found (P = 0.009). However, patients with cytoplasmic staining of ALCAM showed no significantly shortened OS (P = 0.723). Multivariate analyses showed that membranous expression was adverse prognostic factors for OS (hazard ratio, 2.11; P = 0.046). ALCAM knockdown with short hairpin RNA suppressed cell migration and invasion of NSCLC cell lines in vitro. CONCLUSIONS: Strong membranous ALCAM expression is associated with a poor prognosis in patients with resected NSCLC, and overexpression of ALCAM causes malignant phenotypes of NSCLC.

Aberrant DNA methylation associated with aggressiveness of gastrointestinal stromal tumour.

BACKGROUND AND AIMS: The majority of gastrointestinal stromal tumors (GISTs) have KIT mutations; however, epigenetic abnormalities that could conceivably potentiate the aggressiveness of GISTs are largely unidentified. Our aim was to establish epigenetic profiles associated with the malignant transformation of GISTs. METHODS: Methylation of four tumor suppressor genes, RASSF1A, p16, CDH1, and MGMT was analyzed in GISTs. Additionally, genome-wide DNA methylation profiles were compared between small, malignant-prone, and malignant GISTs using methylated GpG island amplification microarrays (MCAM) in a training set (n=40). Relationships between the methylation status of genes identified by MCAM and clinical features of the disease were tested in a validation set (n=75). RESULTS: Methylation of RASSF1A progressively increased from small to malignant GISTs. p16 was specifically methylated in malignant-prone and malignant GISTs. MCAM analysis showed that more genes were methylated in advanced than in small GISTs (average of 473 genes vs 360 genes, respectively, P=0.012). Interestingly, the methylation profile of malignant GISTs was prominently affected by their location. Two genes, REC8 and PAX3, which were newly-identified via MCAM analysis, were differentially methylated in small and malignant GISTs in the training and validation sets. Patients with methylation of at least REC8, PAX3, or p16 had a significantly poorer prognosis (P=0.034). CONCLUSION: Our results suggest that GIST is not, in epigenetic terms, a uniform disease and that DNA methylation in a set of genes is associated with aggressive clinical behavior and unfavorable prognosis. The genes identified may potentially serve as biomarkers for predicting aggressive GISTs with poor survivability.

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.

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21(WAF1/CIP1) and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.

Distinct profiles of epigenetic evolution between colorectal cancers with and without metastasis.

Liver metastasis is a fatal step in the progression of colorectal cancer (CRC); however, the epigenetic evolution of this process is largely unknown. To decipher the epigenetic alterations during the development of liver metastasis, the DNA methylation status of 12 genes, including 5 classical CpG island methylator phenotype (CIMP) markers, was analyzed in 62 liver metastases and in 78 primary CRCs (53 stage I-III; 25 stage IV). Genome-wide methylation analysis was also performed in stage I-III CRCs and in paired primary and liver metastatic cancers. Methylation frequencies of MGMT and TIMP3 increased progressively from stage I-III CRCs to liver metastasis (P = 0.043 and P = 0.028, respectively). The CIMP-positive cases showed significantly earlier recurrence of disease than did CIMP-negative cases with liver metastasis (P = 0.030), whereas no such difference was found in stage I-III CRCs. Genome-wide analysis revealed that more genes were methylated in stage I-III CRCs than in paired stage IV samples (P = 0.008). Hierarchical cluster analysis showed that stage I-III CRCs and stage IV CRCs were clustered into two distinct subgroups, whereas most paired primary and metastatic cancers showed similar methylation profiles. This analysis revealed distinct methylation profiles between stage I-III CRCs and stage IV CRCs, which may reflect differences in epigenetic evolution during progression of the disease. In addition, most methylation status in stage IV CRCs seems to be established before metastasis.

let-7 and miR-17-92: small-sized major players in lung cancer development.

MicroRNA (miRNA)-encoding small non-coding RNA have been recognized as important regulators of a number of biological processes that inhibit the expression of hundreds of genes. Accumulating evidence also indicates the involvement of miRNA alterations in various types of human cancer, including lung cancer, which has long been the leading cause of cancer death in economically well-developed countries, including Japan. We previously found that downregulation of members of the tumor-suppressive let-7 miRNA family and overexpression of the oncogenic miR-17-92 miRNA cluster frequently occur in lung cancers, and molecular insight into how these miRNA alterations may contribute to tumor development has been rapidly accumulating. The present review summarizes recent advances in elucidation of the molecular functions of these miRNA in relation to their roles in the pathogenesis of lung cancer. Given the crucial roles of miRNA alterations, additional studies are expected to provide a better understanding of the underlying molecular mechanisms of disease development, as well as a foundation for novel strategies for cancer diagnosis and treatment of this devastating disease.

Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers.

Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP-negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal-appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP-negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal-appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray-based genome-wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP-negative distal CRCs and CIMP-positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP-negative distal CRCs were also methylated in the normal-appearing mucosae, indicating that hypermethylation in CIMP-negative distal CRCs is more closely associated with age-related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP-positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.

hDREF regulates cell proliferation and expression of ribosomal protein genes.

Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G(1) to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although expression and activation of receptor tyrosine kinases (RTKs), including MET, have been reported in most MPM, specific RTK inhibitors showed less than the expected response in MPM cells. To determine whether the lack of response of MET inhibitors was due to cooperation with other RTKs, we determined activation status of MET and other RTKs, including epidermal growth factor receptor (EGFR) family of 20 MPM cell lines, and tested whether dual RTK inhibition is an effective therapeutic strategy. We detected MET upregulation and phosphorylation (thus indicating activation) in 14 (70%) and 13 (65%) cell lines, but treatment with MET-specific inhibitors showed weak or modest effect of suppression in most of the cell lines. Phospho-RTK array analysis revealed that MET was simultaneously activated with other RTKs, including EGFR, ErbB2, ErbB3 and platelet-derived growth factor receptor-beta. Combination of MET and EGFR inhibitors triggered stronger inhibition on cell proliferation and invasion of MPM cells than that of each in vitro. These results indicated that coactivation of RTKs was essential in mesothelioma cell proliferation and/or survival, thus suggesting that simultaneous inhibition of RTKs may be a more effective strategy for the development of molecular target therapy for MPM.

Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at 21q11-21 in human lung cancer.

The frequent presence of loss of heterozygosity (LOH) at 21q21 in lung cancer suggests the existence of putative tumor suppressor genes in this genomic region. Furthermore, the identification of a homozygous deletion in this region has lent further support for its potential involvement in pathogenesis. In the present study, extensive screening of a large panel of lung cancer cell lines resulted in the identification of a homozygous deletion at 21q21.1 in the large cell lung carcinoma cell line Calu-6. Subsequent detailed characterization allowed us to narrow down the extent of the shortest region of overlap of homozygous deletions at 21q21.1 to 3.4 Mbp. Together with existing information showing a relationship with the shortest region of overlap and LOH in lung cancer, the overlapping 1.8-Mbp region was suggested to be a prime candidate for a genomic region that may harbor putative tumor suppressor genes. We found frequent downregulation of two coding genes, SAMSN1 and USP25, as well as of three miRNA genes, miR-99a, let-7c, and miR-125b-2, which reside in the commonly deleted region in human lung cancer. In addition, initial attempts were made to investigate their potential alterations and functional involvements in the development of lung cancer.

Lineage-specific dependency of lung adenocarcinomas on the lung development regulator TTF-1.

Emerging evidence, although currently very sparse, suggests the presence of "lineage-specific dependency" in the survival mechanisms of certain cancers. TTF-1 has a decisive role as a master regulatory transcription factor in lung development and in the maintenance of the functions of terminal respiratory unit (TRU) cells. We show that a subset of lung adenocarcinoma cell lines expressing TTF-1, which presumably represent those derived from the TRU lineage, exhibit marked dependence on the persistent expression of TTF-1. The inhibition of TTF-1 by RNA interference (RNAi) significantly and specifically induced growth inhibition and apoptosis in these adenocarcinoma cell lines. Furthermore, a fraction of TTF-1-expressing tumors and cell lines displayed an increase in the gene dosage of TTF-1 in the analysis of 214 patients with non-small-cell lung cancer, including 174 adenocarcinomas, showing a tendency of higher frequency of increased gene copies at metastatic sites than at primary sites (P=0.07, by two-sided Fisher's exact test). These findings strongly suggest that in addition to the development and maintenance of TRU lineages in normal lung, sustained TTF-1 expression may be crucial for the survival of a subset of adenocarcinomas that express TTF-1, providing credence for the lineage-specific dependency model.

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.

Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) most commonly arises from chronic inflammation due to viral infection, as a result of genetic and epigenetic abnormalities. A global picture of epigenetic changes in HCC is lacking. We used methylated CpG island amplification microarrays (MCAMs) to study 6458 CpG islands in HCC and adjacent preneoplastic tissues [chronic hepatitis (CH) or liver cirrhosis (LC)] in comparison with normal liver tissues where neither viral infection nor hepatitis has existed. MCAM identified 719 (11%) prominent genes of hypermethylation in HCCs. HCCs arising from LC had significantly more methylation than those arising from CH (1249 genes or 19% versus 444 genes or 7%, P < 0.05). There were four patterns of aberrant methylation: Type I (4%, e.g. matrix metalloproteinase 14) shows a substantially high methylation level in adjacent tissue and does not increase further in cancer. Type II (55%, e.g. RASSF1A) shows progressively increasing methylation from adjacent tissue to HCC. Type III (4%, e.g. GNA14) shows decreased methylation in adjacent tissue but either similar or increased methylation in HCC. Type IV (37%, e.g. CDKN2A) shows low levels of methylation in normal tissue and adjacent tissue but high levels in HCC. These DNA methylation changes were confirmed by quantitative pyrosequencing methylation analysis in representative 24 genes and were analyzed for correlation with clinicopathological parameters in 38 patients. Intriguingly, methylation in the Type IV genes is characteristic of moderately/poorly differentiated cancer. Our global epigenome analysis reveals distinct patterns of methylation that are probably to represent different pathophysiologic processes in HCCs.

mRNA expression of RRM1, ERCC1 and ERCC2 is not associated with chemosensitivity to cisplatin, carboplatin and gemcitabine in human lung cancer cell lines.

BACKGROUND AND OBJECTIVE: Expression of genes involved in DNA repair and/or DNA synthesis, including ribonucleotide reductase M1 (RRM1) and excision repair cross-complementation 1 (ERCC1) has been reported to be associated with chemosensitivity to platinum agents and gemcitabine. The aim of this study was to test whether similar associations would be seen between mRNA expression for the RRM1, ERCC1 and ERCC2 genes and in vitro chemosensitivity in lung cancer. METHODS: Using a panel of 20 lung cancer cell lines, including 15 NSCLC and 5 small cell lung cancers (SCLC), the mRNA expression levels for the RRM1, ERCC1 and ERCC2 genes were examined by quantitative real-time reverse transcription PCR. The in vitro cytotoxicity of cisplatin, carboplatin and gemcitabine was assessed using a tetrazolium-based colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay). RESULTS: Significantly, higher RRM1 mRNA expression was found in SCLC compared with NSCLC. However, there were no correlations between mRNA expression of the ERCC1, ERCC2 and RRM1 genes and chemosensitivity to cisplatin, carboplatin or gemcitabine. CONCLUSIONS: These in vitro results suggest that further studies are needed to evaluate the expression of the RRM1, ERCC1 and ERCC2 genes as predictive biomarkers for sensitivity to platinum agents and gemcitabine.

Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas.

AIM: The aim of the present study was to examine DNA methylation and histone modification changes in hepatocellular carcinomas (HCC). METHODS: DNA methylation in the P16, RASSF1a, progesterone receptor (PGR) and estrogen receptor alpha (ERalpha) promoters was determined by quantitative bisulfite-pyrosequencing technique in HCC patients. Histone H3-lysine (K) 4, H3-K9 and H3-K27 modifications in all these four genes were examined by chromatin immunoprecipitation (ChIP) assay in HCC cell lines. Expression of two DNA methyltransferases (DNMT1 and DNMT3b) and three histone methyltransferases (SUV39H1, G9a and EZH2) in HCC patients was measured by real-time polymerase chain reaction. RESULTS: Aberrant DNA methylation was detected in all the HCC. Patients with DNA methylation in the RASSF1a, PGR andERalpha promoters in cancers also had substantial DNA methylation in their non-cancerous liver tissues, whereas DNA methylation in the P16 promoter was cancer specific. Epigenetic states in HCC cell lines showed that silencing of P16 and RASSF1a depended on DNA methylation and histone H3-K9 methylation. However, silencing of the PGR and ERalpha genes was more closely related to H3-K27 methylation rather than DNA methylation. Consistent with the alteration of histone status, higher expression of G9a and EZH2 was found in HCC than in non-cancerous liver tissues (P < 0.01). CONCLUSION: These data suggest that multiple epigenetic silencing mechanisms are inappropriately active in HCC cells.

ASH1 gene is a specific therapeutic target for lung cancers with neuroendocrine features.

Lung cancers with neuroendocrine features are usually aggressive, although the underlying molecular mechanisms largely remain to be determined. The basic helix-loop-helix protein, achaete-scute complex-like 1/achaete-scute homologue 1 (ASH1), is expressed in normal fetal pulmonary neuroendocrine cells and lung cancers with neuroendocrine elements and is suggested to be involved in lung carcinogenesis. In the present study, we show inhibition of ASH1 expression by plasmid-based RNA interference (RNAi) to significantly suppress growth of lung cancer cells with ASH1 expression through G2-M cell cycle arrest and accumulation of sub-G1 populations, possibly linked to cleavage of caspase-9 and caspase-7. However, lung cancer cell lines without ASH1 expression and immortalized normal BEAS2B bronchial epithelial cells were not affected. The RNAi-resistant mutant ASH1 clearly induced rescue from G2-M arrest, suggesting a target-specific effect of RNAi. An ASH1-RNAi adenovirus was also established and significantly inhibited not only in vitro cell proliferation but also in vivo xenograft growth of ASH1-positive NCI-H460 cells. Elevated levels of apoptosis were also observed in NCI-H460 xenografts with the ASH1-RNAi adenovirus. The present study therefore suggests that ASH1 plays a crucial role in lung cancer development and may be an effective therapeutic target in lung cancers with neuroendocrine features.