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Malignant mesothelioma (MM) frequently exhibits Hippo signaling pathway inactivation (HPI) mainly due to NF2 and/or LATS2 mutations, which leads to the activation of YAP transcriptional co-activator. Here, we show antitumor effects of statin on MM cells with HPI, through the interplay of the mevalonate and Hippo signaling pathways. Statin attenuated proliferation and migration of MM cells harboring NF2 mutation by accelerating YAP phosphorylation/inactivation. CD44 expression was decreased by statin, in parallel with YAP phosphorylation/inactivation. Importantly, we discovered that YAP/TEAD activated CD44 transcription by binding to the CD44 promoter at TEAD-binding sites. On the other hand, CD44 regulated Merlin phosphorylation according to cell density and sequentially promoted YAP transcriptional co-activator, suggesting that CD44 plays two pivotal functional roles as an upstream suppressor of the Hippo pathway and one of downstream targets regulated by YAP/TEAD. Moreover, the YAP/CD44 axis conferred cancer stem cell (CSC)-like properties in MM cells leading to chemoresistance, which was blocked by statin. Together, our findings suggest that YAP mediates CD44 up-regulation at the transcriptional level, conferring CSC-like properties in MM cells, and statin represents a potential therapeutic option against MM by inactivating YAP.

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

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.

Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4–Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.

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.

SummaryWe and others previously identified NKX2-1, also known as TITF1 and TTF-1, as a lineage-survival oncogene in lung adenocarcinomas. Here 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 pro-apoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation, as well as kinase activity-independent sustainment of the EGFR-ERBB3 association, ERBB3 phosphorylation, and consequential PI3K activation. Notably, ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines, irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Our findings thus identify ROR1 as an “Achilles' heel” in lung adenocarcinoma, warranting future development of therapeutic strategies for this devastating cancer.

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2) -tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells.

Introduction Mesenchymal status is related to "inherent resistance" to gefitinib or erlotinib in non-small cell lung cancer without epidermal growth factor receptor ( EGFR ) mutations. In addition, a recent report showed that the epithelial to mesenchymal transition (EMT) plays a role in acquired resistance to gefitinib in A549 cells, which harbor a KRAS mutation. However, recent clinical studies revealed that gefitinib or erlotinib are highly effective in the treatment of non-small cell lung cancer with EGFR mutations. Methods We developed resistant cells (HCC4006ER) from erlotinib-sensitive HCC4006 cells harboring an EGFR deletion mutation by chronic exposure to increasing concentrations of erlotinib. Acquired resistance mechanisms of HCC4006ER cells were analyzed. Results Neither known resistance mechanisms nor novel molecules that may confer erlotinib resistance were identified using candidate or comprehensive analyses. In addition, HCC4006ER cells lost dependency for EGFR. However, we found that HCC4006ER cells acquired a mesenchymal phenotype and exhibited down-regulation of E-cadherin expression (2.7 × 10 −3 times compared with parental cells). We also found that the histone deacetylase inhibitor, MS-275, restored E-cadherin expression and moderate sensitivity to erlotinib in HCC4006ER cells, on the other hand, transforming growth factor beta, an inducer of EMT, led to moderate erlotinib resistance in HCC4006 parental cells. Conclusions This is the first report of a relationship between EMT and erlotinib acquired resistance in an erlotinib 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 gefitinib or erlotinib.

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.

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. (Cancer Sci 2011; 102: 9–17)

Purpose: In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy for lung cancer patients, acquired resistance develops almost inevitably and this limits the improvement in patient outcomes. EGFR T790M mutation and MET amplification are the two main mechanisms underlying this resistance, but the relationship between these two mechanisms is unclear. In this study, we explored their relationship using in vitro models and autopsy specimens. Experimental Design: Erlotinib-resistant HCC827 (HCC827ER) cells were developed by chronic exposure to erlotinib at increasing concentrations. HCC827EPR cells were also developed by chronic exposure to erlotinib in the presence of PHA-665,752 (a MET TKI). The erlotinib-resistant mechanisms of these cells were analyzed. In addition, 33 autopsy tumor samples from 6 lung adenocarcinoma patients harboring multiple gefitinib-refractory tumors were analyzed. Results: HCC827ER developed MET amplification, and clinically relevant resistance occurred at ≥4-fold MET gene copy number gain (CNG). By contrast, HCC827EPR developed T790M without MET CNG. Of six patients harboring gefitinib-refractory tumors, three exhibited T790M only, one exhibited MET amplification only, and the other two exhibited T790M and/or MET amplification depending on the lesion sites. In these gefitinib-refractory tumors, T790M developed in 93% (14 of 15) of tumors without MET gene CNGs, in 80% (4 of 5) of tumors with moderate MET gene CNGs ( Conclusions: These results indicate a reciprocal and complementary relationship between T790M and MET amplification and the necessity of concurrent inhibition of both for further improving patient outcomes. Clin Cancer Res; 16(22); 5489–98. ©2010 AACR.

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.

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]

Small-cell lung cancer (SCLC) is a highly aggressive disease that exhibits rapid growth and genetic instability. We found earlier frequent overexpression of the miR-17-92 microRNA cluster, and showed that SCLC cells were addicted to continued expressions of miR-17-5p and miR-20a, major components of this microRNA cluster. In this study, we identified the frequent presence of constitutively phosphorylated H2AX (gamma-H2AX), which reflects continuing DNA damage, preferentially in SCLC. Knockdown of RB induced gamma-H2AX foci formation in non-small cell lung cancer (NSCLC) cells with wild-type RB, in association with growth inhibition and reactive oxygen species (ROS) generation, which was canceled by overexpression of miR-17-92. Conversely, induction of gamma-H2AX was observed in a miR-17-92-overexpressing SCLC cell line with miR-20a antisense oligonucleotides. These findings suggest that miR-17-92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability. Therefore, miR-17-92 may be an excellent therapeutic target candidate to elicit excessive DNA damage in combination with DNA-damaging chemotherapeutics.

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.

目的．microRNA（miRNA）は約22塩基のnon-coding RNAで標的遺伝子の発現を抑制し，発生·分化の制御など種々の生物学的機能を果たしている．我々は肺癌に於けるmiRNAの異常と，肺癌の発症·進展への関与を検討した．研究計画．癌に関連する可能性が考えられるmiRNA群を選択し，正常肺·肺癌検体におけるmiRNAの発現を比較検討した．また，miRNAやアンチセンスオリゴを細胞内導入し，miRNAの機能を検討した．結果．let-7ファミリーが肺癌で高頻度に発現低下し，その発現低下が予後不良因子であることが判明した．また，miR-17-92クラスターの過剰発現が見出された．このmiR-17-92クラスターの強制発現で増殖促進が見られ，一方，アンチセンスオリゴによる発現抑制が，miR-17-92クラスターを強発現している肺癌細胞株特異的に増殖抑制·細胞死誘導を引き起こした．結論．let-7ファミリーが癌抑制遺伝子として，また，miR-17-92クラスターが癌遺伝子として機能しており，これらmiRNAの異常が，肺癌の発症·進展に深く関与することが示唆された．他のmiRNAの癌への関与も報告され，今後miRNA発現解析の癌の診断·予後予測への応用や，miRNAを用いた治療·miRNAを標的とする治療などの開発が期待される．

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. © 2008 Wiley-Liss, Inc.

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.

Although widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone, NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. By applying the signature to datasets of human cancer cases, we could demonstrate significant associations with a subset of cases with poor prognosis not only for the two datasets of cancers of the lung but also for cancers of the breast. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant downregulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach in the search for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis.

We previously established a highly metastatic subline, LNM35, from the NCI-H460 lung cancer cell line, and demonstrated upregulation of a novel gene, CLCP1 (CUB, LCCL-homology, coagulation factor V/VIII homology domains protein), in LNM35 and lung cancer specimens. In this study, we focused on the potential roles of that gene in cancer metastasis. First, we established stable LNM35 RNAi clones, in which CLCP1 expression was suppressed by RNAi, and found that their motility was significantly reduced, although growth rates were not changed. Next, in vitro selection of a phage display library demonstrated that a phage clone displaying a peptide similar to a sequence within the Sema domain of semaphorin 4B (SEMA4B) interacted with LNM35. Immunoprecipitation experiments confirmed interaction of CLCP1 with SEMA4B, regulation of CLCP1 protein by ubiquitination and proteasome degradation enhanced in the presence of SEMA4B. These results are the first to indicate that CLCP1 plays a role in cell motility, whereas they also showed that at least one of its ligands is SEMA4B and that their interaction mediates proteasome degradation by CLCP1. Although the physiological role of the interaction between CLCP1 and SEMA4B remains to be investigated, this novel gene may become a target of therapy to inhibit metastasis of lung cancers.

MicroRNAs (miRNAs) encoding small non-coding RNAs have been recognized as a very large gene family present in most organisms. The precise biological effects of miRNAs are yet to be elucidated in detail, partly because each miRNA is believed to negatively regulate the expression of hundreds of target genes. Nevertheless, recent findings indicate that carcinogenic processes are associated with alterations in the expression of several miRNAs, suggesting that some function as oncogenes or tumor suppressor genes. The present review focuses on recent findings in this exciting new area of research, with special emphasis on the involvement of miRNAs in cancer development and progression. Further studies are clearly warranted to elucidate the molecular and biological roles of miRNAs, which may ultimately provide both a better understanding of disease development, as well as a foundation for novel strategies for cancer diagnosis and therapy.

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

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.

Emerging evidence suggests that microRNA, which are well-conserved, abundant and small regulatory RNA, may be involved in the pathogenesis of human cancers. We recently reported that expression of let-7 was frequently reduced in lung cancers, and that reduced let-7 expression was significantly associated with shorter patient survival. Two members of the double-stranded RNA-specific endonuclease family, Dicer and Drosha, convert precursor forms of microRNA into their mature forms using a stepwise process. In the present study, we examined expression levels of these genes in 67 non-small cell lung cancer cases, and found for the first time that Dicer expression levels were reduced in a fraction of lung cancers with a significant prognostic impact on the survival of surgically treated cases. It should be noted that multivariate COX regression analysis showed that the prognostic impact of Dicer (P=0.001) appears to be independent of disease stage (P=0.001), while logistic regression analysis demonstrated that the higher incidence of reduced Dicer expression in poorly differentiated tumors remained significant even after correction for other parameters (P=0.02). Given the fundamental and multiple biological roles of Dicer in various cellular processes, our results suggest the involvement of reduced Dicer expression in the development of lung cancers, thus warranting further investigations of the underlying mechanisms, which can be expected to enhance understanding of the molecular pathogenesis of this fatal cancer.

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.

Purpose Recently, several expression-profiling experiments have shown that adenocarcinoma can be classified into subgroups that also reflect patient survival. In this study, we examined the expression patterns of 44 genes selected by these studies to test whether their expression patterns were relevant to prognosis in our cohort as well, and to create a prognostic model applicable to clinical practice. Patients and Methods Expression levels were determined in 85 adenocarcinoma patients by quantitative reverse transcriptase polymerase chain reaction. Cluster analysis was performed, and a prognostic model was created by the proportional hazards model using a stepwise method. Results Hierarchical clustering divided the cases into three major groups, and group B, comprising 21 cases, had significantly poor survival (P = .0297). Next, we tried to identify a smaller number of genes of particular predictive value, and eight genes (PTK7, CIT, SCNN1A, PGES, ERO1L, ZWINT, and two ESTs) were selected. We then calculated a risk index that was defined as a linear combination of gene expression values weighted by their estimated regression coefficients. The risk index was a significant independent prognostic factor (P = .0021) by multivariate analysis. Furthermore, the robustness of this model was confirmed using an independent set of 21 patients (P = .0085). Conclusion By analyzing a reasonably small number of genes, patients with adenocarcinoma could be stratified according to their prognosis. The prognostic model could be applicable to future decisions concerning treatment.

Lung cancer has become the leading cause of cancer death in many economically well-developed countries. Recent molecular biological studies have revealed that overt lung cancers frequently develop through sequential morphological steps, with the accumulation of multiple genetic and epigenetic alterations affecting both tumor suppressor genes and dominant oncogenes. Cell cycle progression needs to be properly regulated, while cells have built-in complex and minute mechanisms such as cell cycle checkpoints to maintain genomic integrity. Genes in the p16INK4A-RB and p14ARF-p53 pathways appear to be a major target for genetic alterations involved in the pathogenesis of lung cancer. Several oncogenes are also known to be altered in lung cancer, leading to the stimulation of autocrine/paracrine loops and activation of multiple signaling pathways. It is widely acknowledged that carcinogens in cigarette smoke are deeply involved in these multiple genetic alterations, mainly through the formation of DNA adducts. A current understanding of the molecular mechanisms of lung cancer pathogenesis and progression is presented in relation to cigarette smoking, an absolute major risk factor for lung cancer development, by reviewing genetic alterations of various tumor suppressor genes and oncogenes thus far identified in lung cancer, with brief summaries of their functions and regulation.

Karyotype and fluorescence in situ hybridization analyses have demonstrated the frequent presence of an altered static state of the number of chromosomes (ie, aneuploidy) in lung cancer, but it has not been directly established whether aneuploidy is in fact associated with a persistent increase in the rate of chromosomal losses and gains (ie, chromosome instability, or CIN). The study presented here used a panel of 10 lung cancer cell lines to provide for the first time direct evidence that CIN is a common feature in lung cancer cell lines in association with the presence of significant aneuploidy. In addition, we found that the CIN phenotype correlates well with the presence of p53 mutations. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactivation of p53 itself is unlikely to directly induce CIN in lung cancer cells. Interestingly, however, significant CIN could be induced in conjunction with the generation of aneuploid populations when the mitotic spindle formation was transiently abrogated in p53-inactivated cells. These results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cycle under certain forms of mitotic stresses, which would result in the induction of the CIN phenotype in conjunction with the generation of aneuploidy.

This study established and characterized low-metastatic revertant and parental clones of a highly metastatic human lung cancer cell line, NCI-H460-LNM35 (hereafter referred to as LNM35). Expression-profiling analysis revealed that up-regulation of various proinflammatory cytokines and angiogenic chemotactic chemokines was present in LNM35. Further, while COX-2 itself is known to be inducible in inflammation, COX-2 expression levels correlated well with the capabilities of these clones for not only in vitro motility and invasion but also in vivo metastasis, and COX-2 inhibitors were shown for the first time to reduce lung cancer metastasis in vivo. These findings suggest that lung cancer cells may mimic inflammatory cells in the process of metastasis.

A previous study of ours unexpectedly found that in contrast to frequent reductions in non-small cell lung cancer, high expression of the p27 KIP1 cyclin-dependent kinase (CDK) inhibitor was retained in virtually all small cell lung cancers (SCLCs), suggesting the possibility of high expression of nonfunctional p27 KIP1 in this virulent tumor. The study presented here, however, shows that p27 KIP1 in SCLC biochemically functions as a CDK inhibitor, clearly showing induction apparently associated with G 1 /G 0 arrest and efficient binding to and inhibition of the cyclin E-CDK2 complex. Interestingly, induction of p27 KIP1 seems to confer on SCLC cells the ability to survive under culture conditions unfavorable for cell growth such as a lack of nutrients and hypoxia. Subsequent experiments manipulating p27 KIP1 levels by using a sense p27 KIP1 expression construct or an antisense oligonucleotide supported this notion. These observations suggest that high expression of p27 KIP1 in vivo may favor the survival of SCLC by preventing apoptosis in a microenvironment unfavorable for cell proliferation.

The mitotic checkpoint is thought to be essential for ensuring accurate chromosome segregation by implementing mitotic delay in response to a spindle defect. To date, however, very little data has become available on the defects of the mitotic checkpoint in human cancer cells. In the present study, impaired mitotic checkpoint was found in four (44%) of nine human lung cancer cell lines. To our knowledge, this is the first demonstration of frequent impairment of the mitotic checkpoint in this leading cause of cancer deaths. As an initial step towards elucidation of the underlying mechanism, we further undertook a search for mutations in a key component of the mitotic checkpoint, known as hsMAD2, and its immediate downstream molecule, p55CDC. No such mutations were found, however, in either 21 lung cancer cell lines or 25 primary lung cancer cases, although we could identify silent polymorphisms and the transcribed and processed hsMAD2 pseudogene that was subsequently mapped at 14q21-q23. The present observations appear to warrant further investigations, such as search for alterations in other components, to better understand the molecular pathogenesis of this fatal disease, and warn against potential misinterpretation when performing mutational analyses for other cancer types based on cDNA templates.

Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-beta) superfamily involved in a range of biological activities. Two highly homologous molecules, Smad2 and Smad3, have so far been identified as receptor-activated Smads for TGF-beta signaling and have become the focus of intensive studies. However, no definite differences in regulation or function have been established between these TGF-beta signaling molecules. In the present study, we show that the expression of Smad3, but not its close relative, Smad2, is down-regulated by TGF-beta mediated signals themselves in human lung epithelial cells. This down-regulation of Smad3 by TGF-beta treatment did not appear to result from shortening of the half-life of Smad3 mRNA. Constitutive expression of Smad3 in the presence of TGF-beta induced apoptotic cell death, with an adverse effect on the cell growth of human lung epithelial cells. Apoptotic cell death could also be induced by forced expression of Smad2 in the presence of TGF-beta, but less efficiently than by that of Smad3. These findings clearly define the distinctions between Smad2 and Smad3 for the first time in that a qualitative difference was observed with regard to the regulation of their expression in response to TGF-beta, while Smad2 and Smad3 appeared to have quantitatively different capabilities regarding the induction of apoptotic cell death in human lung epithelial cells.

The chromosome region 17p13 is known to be frequently deleted in lung cancers. We recently showed the presence of an independent, commonly deleted region at 17p13.3, suggesting that in addition to p53 at 17p13.1 an as-yet-unidentified tumor suppressor gene may reside in this telomeric region. Interestingly, the chromosomal location of a recently isolated novel myc antagonist gene, termed ROX/Mnt, coincides exactly with the centromeric border of the commonly deleted region at 17p13.3 in lung cancers. In conjunction with the generally acknowledged roles of myc genes in the pathogenesis of lung cancers, these findings led us to investigate whether ROX/Mnt is altered in lung cancers. Despite an extensive search for alterations in 52 lung cancer specimens. somatic mutations of ROX/Mnt could not be identified. We conclude that ROX/Mnt itself is not a frequent target for 17p13.3 deletions in lung cancers and that further explorations are required to identify the putative tumor suppressor gene at 17p13.3.

The insulin-like growth factor 2 (IGF2) gene is regulated in a complex manner, involving developmentally regulated use of four different promoters as well as transcriptional repression of the maternal allele due to genomic imprinting. It has been well documented that the liver is an exceptional organ in which overall transcription from the four IGF2 promoters is markedly imbalanced towards preferential paternal expression only in fetal life, this being relaxed during the postnatal period, resulting in biallelic expression thereafter. We previously reported a marked allelic-expression imbalance in the overall transcription of IGF2 in hepatocellular carcinoma (HCC), leading to preferential expression nonrandomly from the paternal allele. The study presented here, using 18 HCC specimens taken directly from patients, showed that this molecular change often reflects promoter switching from the adult P1 promoter to the fetal P2, P3, and P4 promoters. Interestingly, however, we found that restoration of allele-specific expression of the P1 promoter nonrandomly from the paternal allele was also frequent in HCC, suggesting retention of an imprint for paternal expression from the P1 promoter of IGF2 in adult normal liver and altered availability of its modifying factor or factors in HCC. Further studies of the molecular mechanisms involved in the fluctuation of promoter usage and genomic imprinting of IGF2 are warranted to gain an insight into the biology of the liver in terms of development and oncogenesis. Mol. Carcinog. 18:193–198, 1997. © 1997 Wiley-Liss, Inc.

Background Ras-Association Family1A (RASSF1A) is a well-established tumor suppressor. Ten RASSF homologues comprise this family, and each member is considered a tumor suppressor. RASSF3 is one of the RASSF family members, but its function has not yet been clarified. Recently, we found that RASSF3 interacts with MDM2 and facilitates its ubiquitination, which induces apoptosis through p53 stabilization. However, the role of RASSF3 in human malignancies remains largely unknown. Patients and methods Ninety-five non-small cell lung cancer (NSCLC) patients from Nagoya University Hospital and 45 NSCLC patients from Aichi Cancer Center Hospital underwent pulmonary resection at each hospital, and lung cancer and corresponding non-cancerous lung tissues were collected. The expression levels of RASSF3 were analyzed using quantitative real-time reverse transcription PCR. We performed statistical analysis to investigate the correlation with RASSF3 expression and the clinicopathological characteristics. We also transfected RASSF3-siRNA into NSCLC cells, and performed motility assays to evaluate the influence on migration ability. Results RASSF3 expression levels were downregulated in 125 of a total 140 NSCLCs. In a multivariate logistic regression analysis, the low RASSF3 expression group below the median value was independently correlated with progressive phenotypes (lymph node metastasis and pleural invasion), non-adenocarcinoma histology and wild-type epidermal growth factor receptor (EGFR) status. In motility assays, RASSF3-knockdown NSCLC cells increased the migration rate compared to the control cells. Conclusions We found that the expression levels of RASSF3 were frequently downregulated in NSCLCs. Downregulation of RASSF3 strongly correlated with the progressive phenotypes of NSCLCs and EGFR wild-type status. In vitro studies also suggested that RASSF3 downregulation increases migration ability of lung cancer cells. Together, our findings indicate RASSF3 is a candidate tumor suppressor gene of NSCLCs.

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.

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.

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.

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.

Hippo and TGF-β converge on CTGF to promote malignant mesothelioma., Malignant mesothelioma (MM) is an incurable malignancy that is caused by exposure to asbestos and is accompanied by severe fibrosis. Because MM is usually diagnosed at an advanced stage and clinical identification of early lesions is difficult, its molecular pathogenesis has not been completely elucidated. Nearly 75% of MM cases have inactivating mutations in the NF2 (neurofibromatosis type 2; Merlin) gene or in downstream signaling molecules of the Hippo signaling cascade, which negatively regulates the transcription factor Yes-associated protein (YAP). In this study, we demonstrate a functional interaction between the Hippo and TGF-β pathways in regulating connective tissue growth factor (CTGF). Expression of CTGF in MM cells was induced by the formation of a YAP–TEAD4–Smad3–p300 complex on the CTGF promoter. Knocking down CTGF expression in MM cells prolonged the survival of xenografted mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. We further suggest that CTGF may influence the malignancy of mesothelioma because of the different histological expression patterns observed in human MM tissues. These data suggest that CTGF is an important modulator of MM growth and pathology and represents a novel therapeutic target for this disease.

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.

Phenotypic and genotypic findings were correlated and compared for 35 specimens taken from 34 patients with three specific types of low-grade peripheral T-cell lymphoma: lymphoepithelioid (LeL), angioimmunoblastic (AILD), and T-zone (TzL) lymphoma. Frozen sections were stained by the double immunoenzymatic method using a combination of the monoclonal antibody Ki-67 for proliferating nuclei and those against lymphocyte surface antigens. Data were correlated by observing clonal rearrangements in the genes of the T-cell receptor beta chain (TCR beta). Of the 35 specimens studied, 32 (91%) were of predominantly CD4+ helper cell proliferation, and 21 (60%) showed the TCR beta gene rearrangement. There were 15 cases of AILD and TzL with predominantly helper cell proliferation, which contained a minimum of 21% CD4+Ki-67+ cells based on the total number of cells present in the specimen. Of these, 13 (87%) showed TCR beta rearrangement. In eight cases, containing a maximum of 20% CD4+Ki-67+ cells, only one (13%) showed any rearrangement. In addition, TCR beta rearrangement was observed in five of the nine cases of LeL, including two cases with only 12% CD4+Ki-67+ cells. For each of the three types, the proportion of CD4+ cells among the Ki-67+ population showed a relatively good correlation with the clonal TCR beta gene rearrangement. Moreover, there was a significant difference (P less than 0.05) in survival curves between groups with and without TCR beta rearrangement, although no obvious plateau was seen. These results suggest that the paucity of tumor cells in these lesions may account for the absence of a detectable band of rearrangements in some patients with one of these three specific types of low-grade peripheral T-cell lymphoma.

A case of non-Hodgkin's lymphoma showed a phenotypic and genotypic cell lineage switch twice during nine years of his clinical history; first, T-cell type, pleomorphic small cell lymphoma developed, followed by B-cell type, diffuse centroblastic/centrocytic lymphoma, and finally T-zone lymphoma without follicles again developed, from which AST-1 cultured cell line was established. Karyotype analysis demonstrated a shared abnormal chromosome, der(1)t(1;?)(p36;?), among the first relapsed B-cell tumor, the second relapsed T-cell tumor and AST-1 cell line. Furthermore, T-cell receptor (TCR) gamma gene rearrangement bands of the same size were observed in the first relapsed B-cell tumor and the second relapsed T-cell tumor as well as AST-1 cell line. These results suggested that both relapsed tumors of different cell lineages are derived from a common malignant clone, presumably a committed lymphoid stem cell. A unique translocation, t(2;14)(q37;q11.2), which may involve TCR delta/alpha gene complex, was observed in the second relapsed tumor and AST-1 cells. To attempt to isolate the breakpoint of this translocation, the configuration of TCR delta/alpha gene complex was studied. The result showed that two rearrangements of TCR alpha gene detected with J alpha probes were the products of the normal TCR rearrangement process, and were not involved in the translocation at this region. This patient, together with the AST-1 cell line, provided us a unique opportunity to study the development and clonal evolution of malignant lymphoma.

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]

Malignant mesothelioma (MM) is a highly aggressive neoplasm, which is associated with asbestos exposure. The dysregulated phosphatidylinositol 3-kinase (PI3K)-AKT pathway plays an important role in cell proliferation, survival and motility in various cancers. In this study, we analyzed the activation status and underlying mechanisms of this pathway in MM cells using 21 cell lines. AKT activation was observed in 13 (62%) of the 21 MM cell lines under serum-starved conditions. Two cell lines, ACC-MESO-1 and Y-MESO-25, showed no expression of PTEN protein, while 7 other cell lines showed low expression of PTEN mRNA and protein compared to expression levels in an immortalized normal mesothelial cell line, MeT-5A. We found that PTEN inactivation in the ACC-MESO-1 and Y-MESO-25 lines was due to a 39.4-kb deletion including PTEN exon 2, and to a 7.7-kb deletion including exon 1, respectively. Re-expression of PTEN in these cells reduced the activity of colony formation in vitro. In contrast, no mutation of PIK3CA or LKB1 was found in any of the MM cell lines. These findings suggest that AKT is frequently activated in MM cells, in part due to the downregulation of PTEN. Thus, the PI3K-AKT signaling pathway is a potential therapeutic target for MM.

The clinical, prognostic, phenotypic, and genotypic findings of 30 patients with large cell anaplastic lymphoma (Ki-1-positive large cell lymphoma) were analyzed. There were 13 male and 17 female patients (male-female ratio, 0.8) whose ages ranged from 3 to 81 years of age (mean, 28 years of age; 67% of the patients younger than 30 years of age). The 5-year survival rate was 52%; this was better than that of other types of high-grade peripheral T-cell lymphoma. Histologic examination showed distinctive morphologic features such as tumor cell pleomorphism, sinus infiltration, fibrosis, partial lymph node involvement, sparing of B-cell regions, and occasional plasma cell infiltrates. Eighty percent of the cases were of T-cell phenotype, and others expressed neither B-cell nor T-cell markers. The tumors were frequently positive for a histocompatibility antigen (HLA-DR), CD25 (the interleukin-2 receptor), and epithelial membrane antigen. Rearrangements of the T-cell receptor beta gene were observed in nine of 13 cases (69%). These findings indicated that many of the tumors had the phenotype and genotype of activated T-cells. This study also showed that large cell anaplastic lymphoma has a survival figure intermediate between Hodgkin's disease and low-grade peripheral T-cell lymphoma.

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.

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]