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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.

Various stresses of the tumor microenvironment produced by insufficient nutrients, pH, and oxygen can contribute to the generation of altered metabolic and proliferative states that promote the survival of metastatic cells. Among many cellular stress–response pathways activated under such conditions are the hypoxia-inducible factor (HIF) pathway and the unfolded protein response (UPR), which is elicited as a response to endoplasmic reticulum (ER) stress. In this study, we report the identification of a novel cancer invasion and metastasis-related gene (hereafter referred to as CIM, also called ERLEC1), which influences both of these stress–response pathways to promote metastasis. CIM was identified by comparing the gene expression profile of a highly metastatic human lung cancer cell line with its weakly metastatic parental clone. We showed that CIM is critical for metastatic properties in this system. Proteomic approaches combined with bioinformatic analyses revealed that CIM has multifaceted roles in controlling the response to hypoxia and ER stress. Specifically, CIM sequestered OS-9 from the HIF-1α complex and PHD2, permitting HIF-1α accumulation by preventing its degradation. Ectopic expression of CIM in lung cancer cells increased their tolerance to hypoxia. CIM also modulated UPR through interaction with the key ER stress protein BiP, influencing cell proliferation under ER stress conditions. Our findings shed light on how tolerance to multiple cellular stresses at a metastatic site can be evoked by an integrated mechanism involving CIM, which can function to coordinate those responses in a manner that promotes metastatic cell survival. Cancer Res; 70(23); 9949–58. ©2010 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]

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.

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.

It has been suggested that attenuation of the decatenation G2 checkpoint function, which ensures sufficient chromatid decatenation by topoisomerase II before entering into mitosis, may contribute to the acquisition of genetic instability in cancer cells. To date, however, very little information is available on this type of checkpoint defect in human cancers. In this study, we report for the first time that a proportion of human lung cancer cell lines did not properly arrest before entering mitosis in the presence of a catalytic, circular cramp-forming topoisomerase II inhibitor ICRF-193, whereas the decatenation G2 checkpoint impairment was present independently of the impaired DNA damage G2 checkpoint. In addition, the presence of decatenation G2 checkpoint dysfunction was found to be associated with diminished activation of ataxia-telangiectasia mutated in response to ICRF-193, suggesting the potential involvement of an upstream pathway sensing incompletely catenated chromatids. Interestingly, hypersensitivity to ICRF-193 was observed in cell lines with decatenation G2 checkpoint impairment and negligible activation of ataxia-telangiectasia mutated. These findings suggest the possible involvement of decatenation G2 checkpoint impairment in the development of human lung cancers, as well as the potential clinical implication of selective killing of lung cancer cells with such defects by this type of topoisomerase II inhibitor.

In this study, we report for the first time reduced expression of the let-7 microRNA in human lung cancers. Interestingly, 143 lung cancer cases that had undergone potentially curative resection could be classified into two major groups according to let-7 expression in unsupervised hierarchical analysis, showing significantly shorter survival after potentially curative resection in cases with reduced let-7 expression (P = 0.0003). Multivariate COX regression analysis showed this prognostic impact to be independent of disease stage (hazard ratio = 2.17; P = 0.009). In addition, overexpression of let-7 in A549 lung adenocarcinoma cell line inhibited lung cancer cell growth in vitro. This study represents the first report of reduced expression of let-7 and the potential clinical and biological effects of such a microRNA alteration.

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.

Genomic instability is an important factor in cancer susceptibility, but a mechanistic understanding of how it arises remains unclear. We examined hypothesized contributions of the replicative DNA polymerase δ (pol δ) subunit POLD4 to the generation of genomic instability in lung cancer. In examinations of 158 lung cancers and 5 mixtures of 10 normal lungs, cell cycle- and checkpoint-related genes generally showed mRNA expression increases in cancer, whereas POLD4 showed reduced mRNA in small cell lung cancer (SCLC). A fraction of non–small cell lung cancer patients also showed low expression comparable with that in SCLC, which was associated with poor prognosis. The lung cancer cell line ACC-LC-48 was found to have low POLD4 expression, with higher histone H3K9 methylation and lower acetylation in the POLD4 promoter, as compared with the A549 cell line with high POLD4 expression. In the absence of POLD4, pol δ exhibited impaired in vitro DNA synthesis activity. Augmenting POLD4 expression in cells where it was attenuated altered the sensitivity to the chemical carcinogen 4-nitroquinoline-1-oxide. Conversely, siRNA-mediated reduction of POLD4 in cells with abundant expression resulted in a cell cycle delay, checkpoint activation, and an elevated frequency of chromosomal gap/break formation. Overexpression of an engineered POLD4 carrying silent mutations at the siRNA target site rescued these phenotypes, firmly establishing the role of POLD4 in these effects. Furthermore, POLD4 overexpression reduced intrinsically high induction of γ-H2AX, a well-accepted marker of double-stranded DNA breaks. Together, our findings suggest that reduced expression of POLD4 plays a role in genomic instability in lung cancer. Cancer Res; 70(21); 8407–16. ©2010 AACR.

There is marked disparity with a slight overlap among prognosis-predictive signatures reported thus far for lung cancers. In this study, we aimed at linking poor prognosis with particular pathways and/or functions of the gene sets involved to better understand the underlying molecular characteristics associated with the prognosis of lung adenocarcinomas. Gene set enrichment analysis identified a gene set down-regulated by rapamycin as the most significant, whereas several others responsive to withdrawal of glucose or amino acids, which are related to signaling converging onto mammalian target of rapamycin (mTOR), were also shown to be significantly associated, in addition to those related to DNA damage response and cell cycle progression. We also used connectivity map (C-MAP) analysis, an independent bioinformatics approach, to search for Food and Drug Administration–approved drugs that potentially transform an unfavorable signature to a favorable one. Those results identified inhibitors of phosphatidylinositol 3-kinase (PI3K) and mTOR, as well as unexpected drugs such as phenothiazine antipsychotics and resveratrol as potential candidates. Experimental validation revealed that the latter unexpected agents also inhibited signaling converging onto mTOR and exhibited antitumor activities. In addition, deregulation of multiple signaling converging onto mTOR was shown to be significantly associated with sensitivity to PI-103, a dual specificity PI3K/mTOR inhibitor that is not contained in the C-MAP database, lending further support for the connection. Our results clearly show the existence of gene set–definable, intrinsic heterogeneities in lung adenocarcinomas, which seem to be related to both clinical behavior and sensitivity to agents affecting the identified pathways. [Cancer Res 2009;69(9):4027–35]

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]

DNA damage response (DDR) pathways maintain genomic stability. A 657del5 mutation of NBS1, a key DDR component, causing the rare cancer-predisposing Nijmegen breakage syndrome has been reported nearly exclusively in Slavic populations. In this study, we describe the first identification in a Japanese population of an unprecedented type of heterozygous NBS1 mutant, termed IVS11+2insT, lacking the MRE11- and ATM-binding site at the COOH terminus. Profoundly defective in crucial binding to MRE11, MDC1, BRCA1, and wild-type NBS1, the mutant caused impaired ATM phosphorylation in response to low-dose irradiation in a heterozygous state. Importantly, whereas IVS11+2insT was found in only 2 (0.09%) of 2,348 control subjects, it was identified in 2% (2 of 96) of heterozygotes with gastric cancer, 0.8% (3 of 376) of those with colorectal cancer, and 0.4% (2 of 532) of those with lung cancer, which were comparable to frequencies reported for other DDR-related genes known to confer cancer susceptibility. The presence of the heterozygous IVS11+2insT mutation seemed to be associated with an increased risk for gastrointestinal cancers, with an odds ratio of 12.6 and 95% confidence interval (95% CI) of 2.05 to 132.1 (P = 0.0001). The odds ratios separately calculated for gastric and colorectal cancers were 25.0 (95% CI, 1.78–346.0) and 9.43 (95% CI, 1.08–113.1), respectively. These findings suggest that IVS11+2insT is associated with an increased risk for the development of certain types of common cancers, warranting future investigation including detailed phenotypic characterization of age of onset and penetrance in heterozygotes, as well as screening in other ethnic groups. [Cancer Res 2007;67(23):11158–65]

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]

In nearly 50% of all lung adenocarcinomas the oncogenic driver is still unknown. We performed transcriptome sequencing on 25 pan-negative lung adenocarcinomas of never smokers leading to the identification of the novel CD74-NRG1 gene fusion. In an additional cohort of 102 adenocarcinomas we could identify and validate four additional cases bearing the CD74-NRG1 fusion gene - all of the invasive mucinous subtype. We then transduced NIH-3T3 cells with the fusion and found it localized on the cell surface in FACS experiments. As we found the identical breakpoint in CD74 in our fusion as in CD74-ROS1, we hypothesized that the functional active part needs to be NRG1. (Soda et al. 2012) The NRG1 isoform III-β3 that we found is generally not expressed in lung tissue - therefore the CD74 part leads to the expresseion of NRG1 III-β3 in the lung. As Neuregulins are known interaction partners of HER receptor family members we transduced the human adenocarcinoma cell lines H322 and H1568, that are HER receptor and KRAS wildtype, with the fusion and found phosphorylation of HER2 and HER3 in the transduced cells under starvation conditions compared to empty vector controls. (Hobbs et al. 2002) This holds true as well in the patient situation in which phospho-HER3 was only found in tumors expressing the fusion (p We could further show that the CD74-NRG1 fusion is associated with activation of the phosphoinsitide-3-kinase pathway activation and the mitogen-activated protein (MAP) kinase pathway . As functional consequence we could observe in several human adenocarcinoma cell lines increased colony forming ability in soft-agar assays showing the CD74-NRG1 fusion being a new oncogenic driver in lung adenocarcinoma. This finding may lead to new treatment options for patients harboring this fusion gene who do not benefit from any effective treatment option up to now. Citation Format: Dennis Plenker, Lynnette Fernandez-Cuesta, Hirotaka Osada, Ruping Sun, Marc Bos, Juliane Daßler, Gavin Wright, Elisabeth Brambilla, Reinhard Büttner, Sascha Ansen, Stefan Haas, Yasushi Yatabe, Roman K. Thomas. Functional characterization of recurrent CD74-NRG1 fusions in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 441. doi:10.1158/1538-7445.AM2014-441

Accumulating evidence suggests that a coordinately controlled G(2) checkpoint prevents cells with damaged DNA from entering mitosis, thus playing an important role in the maintenance of chromosomal integrity. In the study presented here, we identified a homozygous deletion of the 14-3-3epsilon gene, which resides within a previously identified, commonly deleted region at 17p13.3 in lung cancers, in two small cell lung cancer cell lines that originate from distinct metastatic sites of the same patients. The introduction of 14-3-3epsilon induced significantly restored G(2) checkpoint responses, which resulted in the reduction of mitotic cells as well as of aberrant mitotic figures in the X-ray-irradiated 14-3-3epsilon-null small cell lung cancer cell line. Interestingly, we also found that the G(2) checkpoint response is frequently impaired to various degrees in a large fraction of small cell lung cancer cell lines. These findings suggest the possible involvement of the perturbed G(2) checkpoint in the pathogenesis of this aggressive form of human lung cancers.

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

Emerging evidence, though currently sparse, suggests that “lineage-specific transcription factors” with developmental roles in normal progenitor cells of particular lineages may also confer dependency for survival to certain types of cancer cells. Accumulated evidence indicates that NKX2-1, a lineage-specific transcription factor also known as TITF1 and TTF-1, which is expressed in a major fraction of lung adenocarcinomas, plays essential roles in peripheral lung development. We previously identified NKX2-1 as a lineage-survival oncogene in lung adenocarcinoma, while other investigators reached similar conclusions through genome-wide searches for focal genomic aberrations. Previous findings including ours thus clearly indicate the requirement of sustained NKX2-1 expression for lung adenocarcinoma survival, while it remains elusive how NKX2-1 mediates survival signals. In the present study, we show that NKX2-1 induces the expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between prosurvival PI3K-AKT and proapoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation. Interestingly, ROR1 was also identified as a receptor tyrosine kinase with a “sustainer role” for the interaction of EGFR-ERBB3 and ERBB3 phosphorylation in a ROR1 kinase-independent manner, consequentially leading to PI3K activation. It was also of note that ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Secondary EGFR mutation, MET amplification, and HGF overexpression may arise in lung adenocarcinomas in patients undergoing EGFR-TKI treatment, leading to treatment resistance. The existence of such diverse mechanisms makes it difficult to predict which should be targeted to prevent expansion of resistant clones. From a clinical point of view, it is thus of particular interest that ROR1 inhibition appears to be effective for treatment of gefitinib-resistant lung adenocarcinomas with various resistance mechanisms. Taken together, the present findings identify ROR1 as an “Achilles’ heel” in lung adenocarcinomas. Future development of novel therapeutic means including ROR1-specific antibodies and small molecules that inhibit both or either of the two distinct prosurvival signal-sustaining functions is greatly anticipated for attempts to reduce the intolerable death toll from currently “hard-to-cure” lung adenocarcinomas. 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 LB-17. doi:1538-7445.AM2012-LB-17

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

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