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Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation. Cancer Res; 71(3); 873–83. ©2011 AACR.

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

MicroRNAs (miRNAs) are small noncoding RNAs, thought to be involved in physiologic and developmental processes by negatively regulating expression of target genes. We have previously reported frequent down-regulation of the let-7 miRNA family in lung cancers and, in the present study, assessed alteration in a panel of 19 lung cancer cell lines. As a result, we found for the first time that the miR-17-92 cluster, which comprises seven miRNAs and resides in intron 3 of the C13orf25 gene at 13q31.3, is markedly overexpressed in lung cancers, especially with small-cell lung cancer histology. Southern blot analysis revealed the presence of increased gene copy numbers of the miRNA cluster in a fraction of lung cancer cell lines with overexpression. In addition, we were able to show predominant localization of C13orf25 transcripts within the nucleus and introduction of the expression construct of the miR-17-92 cluster, but not the putative open reading frame of C13orf25, enhancing lung cancer cell growth. These findings clearly suggest that marked overexpression of the miR-17-92 cluster with occasional gene amplification may play a role in the development of lung cancers, especially in their most aggressive form, small-cell lung cancer, and that the C13orf25 gene may well be serving as a vehicle in this regard.

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcriptional factor, which has been suggested to be a candidate tumor suppressor gene in prostate cancer and astrocytic glioma. Because KLF6 is located at chromosome 10p15, where non-small cell lung cancers (NSCLCs) also exhibit frequent allelic loss, we hypothesized that the inactivation of KLF6 is also involved in the development of NSCLC. To determine this, we performed mutational analysis for 105 NSCLCs, including 9 cell lines and 96 primary tumors, and Northern blot analysis for 74 NSCLCs, including the 9 cell lines and 65 primary tumors. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in the 9 cell lines and in 55 (85%) of the 65 primary tumors compared with normal lung tissue. Treatment of two cell lines expressing KLF6 at low levels with 5-azacytidine did not induce KLF6 expression, suggesting that KLF6 down-regulation is not due to promoter hypermethylation. We also performed loss of heterozygosity (LOH) analysis using the laser capture microdissection technique, and found that 21 of 62 (34%) informative samples had LOH in the KLF6 gene locus. Comparing the LOH status with mRNA expression of KLF6, we found that 14 of the 14 (100%) samples with LOH showed KLF6 down-regulation, and that even 23 of 31 (74%) samples without LOH also showed this down-regulation. We also studied the expression of the WAF1 gene, a possible downstream gene of KLF6, and detected simultaneous down-regulation of WAF1 and KLF6 mRNA in 6 of 9 (67%) cell lines and 48 of the 55 (87%) primary tumors, although there was not a significant association between loss of KLF6 and WAF1 expression. Furthermore, colony formation assay of two NSCLC cell lines (NCI-H1299 and NCI-H2009) induced a markedly reduced colony formation by KLF6 transfection, and Annexin V staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that KLF6 induced apoptosis. Our present studies demonstrated that KLF6 is frequently down-regulated in NSCLC and suppresses tumor growth via induction of apoptosis in NSCLC, which may suggest that KLF6 is a tumor suppressor for NSCLC.

The prognosis of patients with malignant mesothelioma (MM) remains very poor because of highly invasive tumor characteristics and therapeutic resistance of this tumor. Recent studies have shown that MM frequently shows Hippo signaling pathway inactivation, which leads to activation of YAP1 transcriptional co-activator. Although substantial data from clinical trials and epidemiological studies showed promising results for the use of statins in many cancers, few studies have argued about the underlying tumor-suppressive mechanisms of statins. Here, we show that statins act as an inhibitor of YAP1-dependent MM cell growth via mevalonate pathway. In cell culture, statins attenuate migration and invasion of H290 malignant mesothelioma cells with homozygous deletion of NF2, which encodes Merlin, an upstream regulator of Hippo pathway. In contrast, geranylgeranyl diphosphate (GGPP), a down-stream activator of mevalonate pathway, completely rescues H290 cell growth inhibition by statins. We also found that statins accelerate YAP phosphorylation/inactivation, which results in the downregulation of Hippo-pathway targeting gene transcriptions including CTGF, CYR-61 and ANKRD1. Moreover, among 11 MM cell lines, we demonstrated that five out of the six cell lines, which showed significant cell growth inhibition with statins, harbor NF2 and/or LATS2 mutations. Finally, we found that phospho-YAP/YAP ratio was increased in statin-sensitive cells, but not in statin-resistant cells. Together, our findings suggest that mevalonate pathway plays crucial roles in YAP-dependent MM cells by Hippo pathway inactivation, and therefore represents a potential therapeutic target for MM. Citation Format: Kosuke Tanaka, Taketo Kato, Akihiro Matsushita, Hiromi Furuta, Yuko Murakami, Hirotaka Osada, Yoshitaka Sekido. Statin inhibits malignant mesothelioma cell growth by inactivating YAP1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1046.

Lung cancers with neuroendocrine (NE) features are often very aggressive but the underlying molecular mechanisms remain elusive. The transcription factor ASH1/ASCL1 is a master regulator of pulmonary NE cell development that is involved in the pathogenesis of lung cancers with NE features (NE-lung cancers). Here we report the definition of the microRNA miR-375 as a key downstream effector of ASH1 function in NE-lung cancer cells. miR-375 was markedly induced by ASH1 in lung cancer cells where it was sufficient to induce NE differentiation. miR-375 upregulation was a prerequisite for ASH1-mediated induction of NE features. The transcriptional coactivator YAP1 was determined to be a direct target of miR-375. YAP1 showed a negative correlation with miR-375 in a panel of lung cancer cell lines and growth inhibitory activities in NE-lung cancer cells. Our results elucidate an ASH1 effector axis in NE-lung cancers that is functionally pivotal in controlling NE features and the alleviation from YAP1-mediated growth inhibition. Cancer Res; 71(19); 6165–73. ©2011 AACR.

The proneural basic-helix-loop-helix protein achaete-scute homologue 1 (ASH1) is expressed in a very limited spectrum of normal and cancerous cells in a lineage-specific manner, including normal pulmonary neuroendocrine cells and lung cancer cells with neuroendocrine features. Our previous results indicated that ASH1 may play a crucial role in the growth and survival of lung cancers with neuroendocrine features, which prompted us to investigate the molecular function of ASH1 in relation to its involvement in carcinogenic processes. Herein, we report for the first time that ASH1 functions as a dual transcription factor by activating neuroendocrine differentiation markers and also repressing putative tumor suppressors. This protein was found to inactivate DKK1 and DKK3, negative regulators of Wnt/β-catenin signaling, E-cadherin, and integrin β1 through ASH1-mediated deacetylation and repressive trimethylation of lysine 27 (H3K27me3) of histone H3 in the promoter regions of DKK1 and E-cadherin. In addition, ASH1-transduced A549 adenocarcinoma cells exhibited markedly altered morphology characteristics compared with lung cancer cells with neuroendocrine features both in vitro and in vivo and also grew faster in vivo. Our results provide important clues for a better understanding of the molecular and cellular biological roles of ASH1 in the process of carcinogenesis of lung cancers with neuroendocrine features and warrant future investigations to shed light on the lineage-specific dependency of this transcription factor with dual functions. [Cancer Res 2008;68(6):1647–55]

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. [Cancer Res 2007;67(13):6007–11]

Malignant mesothelioma (MM) is a highly aggressive neoplasm which is associated with asbestos exposure and around 50% of MM tumors have a mutation of the NF2 tumor suppressor gene. NF2 encodes merlin, a member of the ezrin/radixin/moesin protein family, and merlin is an upstream regulator of the Hippo signaling cascade. Activation of the Hippo pathway induces the activation of serine/threonine kinases LATS1/2, which phosphorylate and inactivate a transcriptional coactivator, YAP. The Hippo pathway activation negatively regulates tissue growth and its inactivation is involved in carcinogenesis. We previously found that, besides NF2 mutation, a subset of MM tumors harbor LATS2 mutation or YAP oncogene amplification. However, there are yet other MM cases that don't have any of these gene mutations, leading us to hypothesize that other components of this pathway might be altered. Recently, the LIM protein Jub in Droshophila was shown to activate Yoki (ortholog of human YAP) through suppression of the Hippo pathway. To determine whether the human ortholog of Jub, Ajuba, is involved in the Hippo signaling downregulation in MM, we analyzed 20 MM cell lines for the alteration of Ajuba. We found that the expression of Ajuba was significantly downregulated in 6 of 20 MM cell lines compared to an immortalized normal mesothelial cell line, Met-5A. Interestingly, three of the 6 cell lines with low Ajuba expression showed YAP activation, whereas Met-5A cells showed high Ajuba expression and YAP inactivation, both of which suggested that Ajuba might act as a tumor suppressor instead of oncogenic protein in mesothelial cells. We infected Ajuba-expressing lentivirus into these MM cell lines and found that exogenous Ajuba induced YAP phosphorylation and inhibited MM cell proliferation. Since the CCND1 and connective tissue growth factor (CTGF) genes are well-known transcriptional targets of YAP, we conducted a dual-luciferase reporter assay using these promoter regions to see if Ajuba affects their transcriptional activities. The dual-luciferase assay demonstrated that Ajuba suppressed the promoter activity of these genes and that the suppression of them was attenuated by LATS2 knockdown. These results indicated that Ajuba negatively regulates YAP through activation of LATS2. Our results suggest that the inactivation of Ajuba is one of the mechanisms for constitutive activation of YAP, which results in deregulation of MM cell proliferation. Citation Format: Ichidai Tanaka, Hirotaka Osada, Makiko Fujii, Yoshitaka Sekido. A LIM protein ajuba suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2013-4313

Functional and morphological heterogeneity characterizes aggressive glioblastoma and contributes to invasion, metastasis and drug resistance. The molecular mechanisms underlying this heterogeneity are poorly understood. Recent studies have revealed that tumors contain a minor population of tumor-initiating cells, called cancer stem cells (CSCs). The CSCs are considered able to aberrantly differentiate into diverse cell types. Such stem-like cancer cells have been well characterized in glioblastoma multiforme, and are referred to as glioma stem-like cells (GSCs). Using GSCs as a model, we investigated the roles of micro-RNA (miR) in GSCs differentiation. miR-microarray showed that 19 and 48 miRs were commonly upregulated and downregulated in two GSC lines during the differentiation, respectively. Among those miRs, quantitative-PCR analysis showed that miR-1275 was consistently downregulated during the GSC differentiation, while the expression of its target, oligodendrocyte specific protein (OSP/CLDN11), a marker of oligodendrocyte, was upregulated. Compellingly, inhibition of miR-1275 using specific antisense oligonucleotide increased the expression of OSP in GSC. Epigenetic analysis revealed that level of histone H3 lysine 27 trimethylation(H3K27me3) was enriched in the promoter of pri-miR-1275 during the GSC differentiation. Treatment of 3-Dezaneplanocin-A, an inhibitor of H3K27me3, impaired the GSC differentiation in parallel with sustained miR-1275 expression. In general, astrocytic component is the most prevalent in glioblastomas, while a subset of cases also contain an oligodendroglial component in the tumors. Our results illuminated the epigenetic regulatory pathways of miR-1275 closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity formation of glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3135. doi:1538-7445.AM2012-3135

Background: Lung cancer is the leading cause of human cancer death worldwide. 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. However, the existence and characteristics of CIMP in lung adenocarcinomas (AdCas) from the global point of view has remained elusive. The aim of this study is to clarify the significance of DNA methylation, especially the clinical impact of CIMP, in AdCas. Methods: Using genome-wide microarray analysis, DNA methylation profiling of 41 AdCas were initially examined. DNA methylation status of six CIMP markers newly identified by microarray analysis was further estimated in a validation set of 87 AdCas by bisulfate pyrosequencing. A total of 128 AdCas were examined for the significance of CIMP. In addition, responsiveness to DNA methylation inhibitor was examined in AdCa cell lines with different CIMP status. Results: Consensus clustering analysis based on DNA methylation showed that AdCas were classified into three subgroups, clusters 1, 2, and 3 (average of 767, 486, and 319 methylated genes in each subgroup, respectively; P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5003. doi:1538-7445.AM2012-5003

Malignant mesothelioma (MM) arises from the serosal cells of the pleural, peritoneal and pericardial cavities with poor prognosis because of its frequent diagnosis in advanced stages. The primary cause of this disease has often been linked to asbestos exposure, and the number of patients worldwide is expected to peak in the next two decades. There is no known curative therapy for MM and further understanding in molecular mechanism is needed to identify new candidates for targeted treatment. We found that the TGF-β pathway is associated with oncogenic mutations and loss of the tumor suppressor NF2, and induces pro-oncogenic biological activities. Loss of NF2 is one of the genetic lesions which have been related to the growth of human MM. The NF2 gene is responsible for neurofibromatosis type II syndrome and encodes Merlin which negatively regulates YAP function. Nearly 75% MM cases have inactivating mutations in the NF2 gene or in downstream signaling molecules of Hippo signaling cascade. We examined a functional interaction between the Hippo and TGF-β pathways and found that endothelin-1 and connective tissue growth factor (CTGF) are the only proteins affected by these two pathways in MM cells. The CTGF promoter contains both a YAP/TEAD-binding site and a consensus Smad-binding site adjacent to each other. The expression of CTGF induced by TGF-β in MM cell lines was greatly affected by the existence of YAP, by its binding with Smad3, forming a complex on CTGF promoter lesion. Knocking down CTGF expression in MM cells prolonged the survival of mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. This study elucidates a novel molecular mechanism induced by the TGF-β pathway, which may be applicable to malignancies with defective NF2 and of mesenchymal origin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3082. doi:1538-7445.AM2012-3082

Introduction: Almost all patients with lung cancer harboring a mutation in the epidermal growth factor receptor (EGFR) gene become refractory to the EGFR-tyrosine kinase inhibitors (TKIs), after showing an initial dramatic response. Secondary mutation of the EGFR gene (T790M) and MET gene amplification are the two main molecular mechanisms underlying this resistances, however about 30 percents of acquired resistance mechanisms are currently unknown. The purpose of the present study was to investigate novel acquired resistance mechanisms by analyzing in vitro acquired resistance models to EGFR-TKIs. Methods: We developed erlotinib resistant cells (HCC4006ER) from erlotinib-sensitive HCC4006 cells (IC50: 51nM) harboring an EGFR deletion mutation via chronic exposure to increasing concentrations of erlotinib. Erlotinib resistance mechanisms were analyzed using candidate or comprehensive approaches. Results: HCC4006ER cells were >200 times more resistant to erlotinib (IC50: >10uM) compared with the parent HCC4006 cells, although the resistant cells preserved EGFR exon19 deletion mutation. At first, we denied the involvement of known resistance mechanisms (secondary mutation of EGFR, MET activation by either MET gene amplification or HGF, activation of IGF-IR, downregulation of PTEN, or mutation of KRAS) in HCC4006ER cells. Next, we identified that HCC4006ER cells lost their dependency to EGFR; siRNA-mediated EGFR knockdown suppressed the survival of the parental cells effectively, but not of the resistant cells. However, comprehensive analyses (phospho-receptor tyrosine kinase array, phospho-kinase array, and gene expression assay) failed to identify novel candidate molecules that may confer erlotinib resistance in HCC4006ER cells. On the other hand, we found that HCC4006ER cells acquired a mesenchymal-like feature in their appearance and molecular markers and exhibited increased expression of a gene set that is related to the transforming growth factor beta (TGFbeta) signaling pathway. We also found that TGFbeta treatment led to moderate erlotinib resistance in HCC4006 parental cells. Conclusions: Recent reports have shown that the EMT plays a role in acquired resistance to EGFR-TKIs, although these previous reports utilized cell lines (A549 or H1650) that are not sensitive to these drugs originally. This is the first report of a relationship between EMT and EGFR-TKI acquired resistance in a TKI sensitive EGFR-mutant lung cancer cell line. Our results indicate that it would be important to consider the influence of EMT in the development of treatments against acquired resistance to EGFR-TKIs. 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 718. doi:10.1158/1538-7445.AM2011-718

Lung cancers with neuroendocrine (NE) features are often very aggressive, though the underlying molecular mechanisms have not been clearly elucidated. We previously reported that the basic helix-loop-helix protein, achaete-scute homologue 1 (ASH1/ASCL1), a master regulator of pulmonary neuroendocrine cell development, is crucially involved in the pathogenesis of lung cancers with neuroendocrine features (NE-lung cancers). In the present study, we searched for miRNAs regulated by ASH1 in order to gain insight into the involvement of miRNAs downstream of ASH1 in NE-lung cancer pathogenesis. We consequently found that introduction of ASH1 into A549 cells markedly induced miR-375 and down-regulated miR-200b. Interestingly, these two miRNAs inversely affected cell-cell interaction and cell motility, and they together conferred looser cell attachment and a more motile feature. miR-375 was found to be directly transactivated by ASH1. Global gene expression analysis using miR-375-introduced A549 cells and the subsequent pathway analysis using the Ingenuity IPA software revealed altered gene expressions in several cancer-related pathways (e.g., cellular movement and morphology). Interestingly, we also found that miR-375 alone can induce NE differentiation markers in A549 cells and that miR-375 is required to elicit ASH1-indued NE features. In accordance with these findings in vitro, clear induction of NE features as well as enhanced tumor growth was observed in xenografts of A549 cells stably expressing miR-375. We further found that a transcriptional co-activator YAP1 is most significantly repressed by miR-375 through recognition of its two miR-375 binding sites. A positive correlation between ASH1 and miR-375 as well as a negative correlation between miR-375 and YAP1 were observed in lung cancers, indicating the ASH1-miR-375-YAP1 regulatory pathway is indeed functionally implemented in lung cancer cells. In addition, we found that growth-regulatory functions of YAP1 is cell-lineage dependent in lung cancer cell lines. In conclusion, the present study identified the ASH1-miR-375 axis, providing clues for a better understanding of the molecular and cellular biological roles of ASH1-centered microRNA regulatory networks in the pathogenesis of NE-lung cancers. 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 3992. doi:10.1158/1538-7445.AM2011-3992

Background and aims: While the connection between chronic inflammation and tumorigenesis was suspected in hepatocellular carcinomas (HCCs), key factors linking these two processes were not completely understood. Ample evidence shows that accumulation of genetic and epigenetic abnormalities may be fundamental mechanisms underlying tumor formation. The aim of this study is to examine whether viral infection affects epigenetic profile in chronic hepatitis/liver cirrhosis (CH/LC) and HCC tissues. Methods: To evaluate the direct effects of viral infection, we examined DNA methyaltion changes in hepatitis C virus (HCV) infected cells in vitro. Genome-wide DNA methylation status was examined in liver tissues repopulated with human hepatocytes (humanized liver) in uPA/SCID mice, which were infected with hepatitis B virus (HBV-mice, 13 mice) or HCV-mice (14 mice), using methylated CpG island amplification-microarrays (MCAM, 6,157 CpG islands). Genome-wide DNA methylation status was also examined in 37 HCCs: 19 and 18 were associated with HCV and HBV infection, respectively (HCC-HBV and HCC-HCV). Further, quantitative pyrosequencing analysis was performed to examine the methylation status of selected genes. Result: MCAM analysis in humanized mouse liver revealed that larger number of genes were methylated in HCV-mice than HBV-mice (287 genes versus 157 genes, P=0.034, respectively). DNA methylation target genes in HBV-mice were also methylated in HCV-mice, while both HBV-mice and HCV-mice had own specific targets. Association between aberrant DNA methylation and infection period was more strongly observed in HBV-mice (r=0.769) than HCV-mice (r=0.175). Interestingly, both HBV and HCV affected DNA methylation not only in infected humanized liver (hepatocytes) but also surrounding mouse tissues (non-infected cells). In vitro analysis of HCV infection did not affect DNA methylaion in cultured cells for more than a month. Thses data suggest that HCV infection may affect DNA methylaiton via inflammatory responses. Consistently, various cytokines from mouse tissues were upregulated after HBV and HCV infections. In clinical samples, most of the DNA methylation target genes in HCC-HBV were also methylated in HCC-HCV, as was found in mouse model. In addition, many DNA methylation target genes in human HCCs were also identified in mouse model with MCAM analysis. Conclusion: We developed mouse model to assess DNA methylaton profiling after hepatitis viral infection. Although HBV protein has been known to induce aberrant DNA methylation, our data sugget that viral infection is a strong inducer of aberrant DNA methylation via inflammatory responses. 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 4811. doi:10.1158/1538-7445.AM2011-4811

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

Introduction: Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers, including lung cancer, and its contribution to increased proliferation through up-regulating cell cycle accelerators such as cyclin A/E is well demonstrated in breast and gastric cancers. Nevertheless, very little is known about its role in survival of cancer cells. In addition, functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. Matrials and methods: 18 human non-small cell lung cancer cell lines and an immortalized normal human bronchial epithelial cell (HBEC4) lines were used. EpCAM expression was measured by quantitative real-time PCR (qRT-PCR) and FACS. RNA interference (RNAi)-mediated gene silencing for EpCAM was done in three non-small cell lung cancer cell and HBEC4 lines. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. Apoptosis was evaluated by annexin V/7-AAD staining. FACS with PI staining was done to examine apoptosis and cell cycle. p27kip1 expression was evaluated by western blot analysis. In vitro invasion assay was done by using transwell chambers layered with matrigel and invading cells were stained and counted. Results: We analyzed the expression of EpCAM in a panel of lung cancer cell and HBEC4 lines and found that most of lung cancer cell lines expressed EpCAM. EpCAM knockdown suppressed proliferation and clonogenic growth of two EpCAM-expressing lung cancer cell lines in anchorage dependent and independent conditions. EpCAM knockdown suppressed invasiveness in a highly invasive line but not in a lowly invasive cell line. In addition, EpCAM knockdown induced massive apoptosis in both cell lines as well as another EpCAM-expressing lung cancer cell line but to a much lesser extent in HBEC4 line. EpCAM knockdown caused lung cancer cells to have increased responsiveness to contact inhibition in part through up-regulating p27kip1 cyclin dependent kinase inhibitor. Conclusion: These results demonstrate that EpCAM substantially contributes to the pathogenesis of lung cancer, especially in its survival, and the development of EpCAM-targeted therapy for lung cancer may have promise. 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 1657. doi:10.1158/1538-7445.AM2011-1657

Background: Epithelial-to-mesenchymal transition (EMT), an essential process during embryonic development and in wound healing, has been shown to be a key event in tumor migration, invasion and metastasis. EMT program is controlled by several master regulators including Twist, ZEB1, SIP1, Snail, Slug, and Goosecoid which are called master EMT genes1),2). ZEB1 protein binds E-boxes within the promoter region of E-cadherin gene, leading to its transcriptional repression. Also, it directly represses many other genes encoding components of the epithelial junctional complex and cell polarity factors. ZEB1 was shown to promote metastasis in colorectal cancer and breast cancer3), and the association between ZEB1 and tumor progression has been studied in several human cancers. Purpose: To evaluate the association between ZEB1 expression and mesenchymal phenotype in lung cancer, and to test the effect of ZEB1 knockdown with RNA interference on the growth of lung cancer cells. Methods: We analyzed the expression of E-cadherin (epithelial marker), Vimentin (mesenchymal marker), and four master EMT genes (Snail, Slug, ZEB1, SIP1) in 19 NSCLC cell lines and 32 NSCLC tumor tissues. Transient knockdown of ZEB1 with RNA interference was done in three NSCLC cell lines with high expression of ZEB1: H157, H1299, and H460. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin and Vimentin were done. Cellular proliferation was measured by WST-1 assay and clonogenic growth was measured by liquid (anchorage-dependent) and soft agar (anchorage-independent) colony formation assays. Apoptosis was evaluated by FACS and western blot of cleaved caspase-3. Results: In cell lines, of four master EMT genes, only ZEB1 expression significantly correlated with both E-cadherin and Vimentin expression. Most EGFR mutant lines showed epithelial phenotype (ratio of Vimentin to E-cadherin (RVE) < 1.0). ZEB1 expression was also inversely correlated with miR-200c and miR-205. In tumor tissues, ZEB1 and Snail expression correlated with the ratio of Vimentin to E-cadherin, and ZEB1 expression highly significantly correlated with Vimentin expression. After ZEB1 knockdown E-cadherin protein was reexpressed in H460 but not in H1299 and H157 cells. ZEB1 knockdown suppressed cell proliferation and liquid colony formation significantly in H1299 and modestly in H157 and H460. Notably, ZEB1 knockdown dramatically suppressed growth of soft agar in the all three cell lines studied. FACS and western blot of cleaved caspase-3 showed apoptosis induction by ZEB1 knockdown in H460, suggesting that growth inhibitory effect of ZEB1 knockdown in lung cancer was caused in part by apoptosis. Conclusions: ZEB1 has the dominant role in maintaining mesencymal phenotype in NSCLC and removal of ZEB1 in lung cancer cell lines induces inhibition of soft agar growth. These results suggest that ZEB1 could be a therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2295.

Background and Objective: Malignant pleural mesothelioma (MPM) is a highly aggressive disease, usually caused by asbestos exposure. The underlying molecular mechanism contributing to aggressive phenotype of MPM remains to be fully elucidated. Epithelial-to-mesenchymal transition (EMT), an early developmental program, causes a wide variety of human epithelial cancers to have the ability to invade surrounding tissues and metastasize to distant sites. MPM cells usually exhibit mesenchymal characteristics probably because of their mesodermal origin. Such characteristics include fibroblast-like morphology and expression of master EMT-inducing genes. Thus, EMT may contribute to aggressive phenotype of MPM. Among EMT-inducing genes, ZEB1, a repressor of E-cadherin, has emerged as a key player in the progression of several epithelial cancers. To explore the potential of ZEB1 as a therapeutic target for MPM, we examined the effect of ZEB1 knockdown on growth of MPM cell lines. In addition, we evaluated the effect of ZEB1 knockdown on the levels of IL-6, which is known to induce growth of mesothelioma cells. Materials and Methods: 18 human MPM cell lines and one non-tumorigenic mesothelial cell line were used. Transient and stable knockdown of ZEB1 were done in two MPM cell lines. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin, and Vimentin were done. Soluble IL-6 in cellular supernatants was measured by enzyme-linked immunosorbent assay. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. FACS with PI staining was done to examine apoptosis and cell cycle. Apoptosis was also evaluated by western blot of cleaved caspase-3. Results: The majority of MPM cells expressed higher levels of ZEB1 than a non-tumorigenic mesothelial cell. We performed ZEB1 knockdown experiments in two MPM cells, ACC-MESO-1(MESO-1) and H2052, which express high and moderate levels of ZEB1. Transient knockdown of ZEB1 caused MESO-1 but not H2052 cells to reexpress E-cadherin. Transient ZEB1 knockdown suppressed cell proliferation and liquid colony formation in the two lines. Most importantly, the ZEB1 knockdown dramatically suppressed soft agar colony formation in the two lines. We did not see apoptosis or cell cycle arrest in either of the two lines. Stable knockdown of ZEB1 induced morphologic changes suggestive of mesenchymal-to-epithelial transition (MET) in MESO-1 cells but not in H2052 cells. ZEB1 stable knockdown resulted in a decreased IL-6 production in MESO-1 cells, suggesting that growth inhibition by ZEB1 knockdown may in part due to decreased IL-6 production. Conclusion: These results suggest that ZEB1 serves as an attractive therapeutic target for MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2302.

Purpose: Epidermal growth factor receptor (EGFR)-activating mutations, such as deletion in exon 19 and L858R point mutation in exon 21,were found in non-small cell lung cancer and associated with favorable response to the reversible EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib. However, almost all NSCLC patients with EGFR mutations who show response to reversible EGFR-TKI ultimately develop resistance to these agents. Recently, several mechanisms were reported to induce acquired resistance to reversible EGFR-TKI for non-small cell lung cancer with EGFR-activating mutations. Of these, the secondary T790M mutation in EGFR is the most frequent cause of acquired resistance to the reversible EGFR-TKI. Irreversible EGFR-TKIs are expected to overcome the reversible EGFR-TKI resistance of lung cancer harboring T790M mutation in EGFR. However, it is clear that resistance may also develop to this class of inhibitors. We showed previously that hepatocyte growth factor (HGF) induced gefitinib resistance of lung cancer harboring EGFR-activating mutations. Here, we investigated whether HGF induced resistance to the irreversible EGFR-TKI, CL-387,785, in lung cancer cells (H1975) harboring both L858R activating mutation and T790M secondary mutation in EGFR. Experimental Design: CL-387,785 sensitivity and signal transduction in H1975 cells were examined in the presence or absence of HGF or HGF-producing fibroblasts with or without HGF-MET inhibitors. Results: HGF induces resistance to CL-387,785 in H1975 cells. Western blotting and small interfering RNA analyses indicated that HGF-induced resistance was mediated by the MET/phosphoinositide 3-kinase/Akt signaling pathway independent of EGFR, ErbB2, ErbB3, and ErbB4. Resistance of H1975 cells to CL-387,785 was also induced by coculture with high-level HGF-producing lung fibroblasts. The resistance was abrogated by treatment with anti-HGF neutralizing antibody, HGF antagonist NK4, or MET-TKI. Conclusions: We showed HGF-mediated resistance as a novel mechanism of resistance to irreversible EGFR-TKIs. Our findings provide a novel insight into the involvement of HGF-MET mediated signaling in de novo and acquired resistance to irreversible EGFR-TKIs in lung cancer harboring T790M mutation in EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 629.