[Liver Herniation through a Diaphragmatic Defect Mimicking Pleural Tumor;Report of a Case].

Liver herniation is rare and sometimes difficult to differentiate from pleural or diaphragmatic tumor. A 64-year-old woman was admitted due to a mass-like shadow in the right lower lung field. Computed tomography, coronal view, showed a well-defined mass forming an acute angle with the right diaphragm, mimicking pleural tumor. Video-assisted thoracic surgery was performed, revealing herniated liver through one of the multiple diaphragmatic defects, which was repositioned into the abdominal cavity, and the diaphragmatic defect was repaired. The patient recovered well and was discharged on postoperative day 5.

Micronodular thymoma with lymphoid stroma: an immunohistochemical study of the distribution of Langerhans cells and mature dendritic cells in six patients.

AIMS: Micronodular thymoma with lymphoid stroma (MNT) is an uncommon variant of thymoma, characterized by multiple small nodules consisting of type A thymoma-like cells, which are separated by abundant B lymphocytes. The aim of the study was to elucidate the pathogenesis of the stromal lymphoid hyperplasia, which is currently unclear. METHODS AND RESULTS: We retrieved six cases of MNT, and immunohistochemically examined the number and distribution of Langerhans cells (LCs) and mature dendritic cells (DCs), and compared them with those in type A and type AB thymomas. Many LCs were present within the small tumour nests, but LCs were rarely seen in the stroma (75.5/HPF versus 6.1/HPF, P < 0.0001). In contrast, mature DCs were present mainly in the surrounding stroma rather than within the tumour nodules (63.5/HPF versus 6.0/HPF, P < 0.0001), forming clusters with mature T lymphocytes adjacent to lymphoid follicles. In large nodules, as well as in type A and type AB thymomas, a few scattered LCs and DCs were identified. All patients were still alive and well. CONCLUSIONS: Our results suggest that LCs take up tumour antigens and migrate to the stroma, where they mature and form clusters with T lymphocytes to activate them, resulting in lymphoid follicle formation. The favourable clinical behaviour may be attributable to the immune response induced by LCs.

The ABO blood group is an independent prognostic factor in patients with resected non-small cell lung cancer.

BACKGROUND: The ABO blood group is reported to be associated with the incidence and patient survival for several types of malignancies. We conducted a retrospective study to evaluate the prognostic significance of the ABO blood group in patients with resected non-small cell lung cancer (NSCLC). METHODS: A total of 333 patients (218 men and 115 women) with resected NSCLC were included in this study. In addition to age, sex, smoking status, preoperative serum carcinoembryonic antigen (CEA) level, operative procedure, histology of tumors, pathological stage (p-stage), and adjuvant therapy, the association between the ABO blood group and survival was explored. RESULTS: The 5-year overall and disease-free survival rates were 83.0% and 71.6% for blood group O, 67.2% and 62.3% for blood group A, 68.8% and 68.8% for blood group B and 69.2% and 65.3% for blood group AB, respectively. A multivariate analysis for overall survival showed the ABO blood group (group A vs. group O: HR 2.47, group AB vs. group O: HR 3.62) to be an independent significant prognostic factor, in addition to age, sex, smoking status, p-stage, and serum CEA level. A multivariate analysis for disease-free survival also showed the ABO blood group to be an independent significant prognostic factor. CONCLUSIONS: The ABO blood group is an independent prognostic factor in patients with resected NSCLC. Studies of other larger cohorts are therefore needed to confirm the relationship between the ABO blood group and the prognosis among patients with resected NSCLC.

Preoperative plasma D-dimer level is an independent prognostic factor in patients with completely resected non-small cell lung cancer.

PURPOSE: The plasma D-dimer (D-dimer) level, a marker of hypercoagulation, has been reported to be associated with survival in several types of cancers. This retrospective study was conducted to evaluate the prognostic significance of the preoperative D-dimer level in patients with completely resected non-small cell lung cancer (NSCLC). METHODS: A total of 237 completely resected NSCLC patients were included in this study. In addition to age, sex, the smoking status, etc., the association between the preoperative D-dimer level and survival was explored. RESULTS: The patients were divided into three groups according to the D-dimer level: group A (≤ 0.50 µg/ml, n = 76), group B (0.51-0.86 µg/ml, n = 79) and group C (>0.86 µg/ml, n = 82). The 5-year overall survival rate was 89.6 % (95 % confidence interval (CI) 77.7-95.3) for group A, 75.1 % (95 % CI 62.3-83.6) for group B and 60.1 % (95 % CI 46.8-71.1) for group C (P trend <0.001). A multivariate survival analysis showed that the D-dimer level (group B vs. group A HR 4.25, group C vs. group A HR 4.11) was an independent significant prognostic factor, in addition to age, sex, the pathological stage and the serum carcinoembryonic antigen level. CONCLUSIONS: The preoperative D-dimer level is an independent prognostic factor in patients with completely resected NSCLC.

TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival.

TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7-fold (P < 0.001) in non-small cell lung cancer cell lines (n = 116) compared to normal lung controls (n = 59). In addition, small cell lung cancer cell lines (n = 29) expressed TIM at levels 2.2-fold (P < 0.001) higher than non-small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.

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

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

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

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

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

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

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells.

BACKGROUND: The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear. METHODS: The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity. RESULTS: Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent. CONCLUSIONS: RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.

Type AB thymoma with brain metastasis: Report of a case.

Type AB thymomas are considered to be nonaggressive tumors, and the great majority are classified as Masaoka stage I or II. This report presents a case with Masaoka stage I and type AB thymoma, which metastasized to the brain 2 years 5 months after removal of the primary tumor. The original mediastinal lesion was adhesive but not invasive to the lung. The patient is now alive with multiple tiny pulmonary metastases 3 years after complete resection of the brain metastasis. Some reports of recurrent thymomas have suggested that the presence of peritumoral adherence to the adjacent structures might be a risk factor for recurrence in patients with such noninvasive thymomas. During the development of fibrosis which thus causes adhesion, the tumor may have an increased chance to metastasize because of the increased vessels and lymphatics.

Phase II study of carboplatin and gemcitabine as adjuvant chemotherapy in patients with completely resected non-small cell lung cancer: a report from the Central Japan Lung Study Group, CJLSG 0503 trial.

BACKGROUND: The aim of this phase II study was to evaluate the feasibility and safety of a carboplatin and gemcitabine combination regimen in the treatment of completely resected non-small cell lung cancer (NSCLC). METHODS: Patients with completely resected pathologically documented stage IB, II or IIIA NSCLC were treated with carboplatin and gemcitabine. Chemotherapy consisted of 4 cycles of carboplatin at an area under the curve of 5 (level 1) or 4 (level 2) on day 1 combined with gemcitabine 1,000 mg/m(2) on days 1 and 8 every 3 weeks. The primary endpoint of this study was the completion rate of 4 cycles. RESULTS: Twenty patients were treated, and the patient's demographics were: median age 61 years (range 51-74), gender male (n = 13, 65%)/female (n = 7, 35%), stage IB (n = 8, 40%), IIA (n = 1, 5%), IIB (n = 6, 30%), IIIA (n = 5, 25%). Seventeen patients (85%, 95% confidence interval 64.0-94.8) received the planned 4 cycles of the chemotherapy regimen at level 1 every 3 weeks. Among the 3 patients who failed to complete 4 cycles, the reasons for stopping were refusal (n = 1), thrombocytopenia (n = 1) and rash (n = 1). The main adverse effects were hematological toxicity as well as grade 3/4 neutropenia and thrombocytopenia (which occurred in 65% and 40% of the patients, respectively). CONCLUSIONS: Adjuvant chemotherapy with a carboplatin and gemcitabine combination regimen has an acceptable toxicity profile, and the majority of patients completed 4 cycles of therapy.

Thymic basaloid carcinoma with pleural dissemination that developed after a curative resection: report of a case.

Thymic basaloid carcinoma is an extremely rare tumor type, with only 10 such reports published to date in the English literature. We herein present a new case of thymic basaloid carcinoma with pleural dissemination that developed after a curative resection. A cystic tumor in the anterior mediastinum was observed in a 72-year-old man, and the tumor was completely resected via a median sternotomy with a combined resection of the adjacent structures. One year later, pleural disseminated nodules developed in the right thorax, which were resected through a right thoracotomy. The present case and the review of previous cases indicated that this rare tumor, which had previously been considered to be a low-grade malignant thymic carcinoma, may therefore have a more obstinate and aggressive malignant nature. Histopathologically, a few CD5-positive tumor cells were observed in isolation in the squamous epithelium of the inner cyst wall, thus suggesting that malignant transformation subsequently occurs in a preexisting cyst.

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

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

Preoperative evaluation of the depth of chest wall invasion and the extent of combined resections in lung cancer patients.

The optimal extent of a combined resection in patients with lung cancer invading the chest wall remains controversial. To assess whether specific preoperative findings could lead to the precise evaluation of the depth of chest wall invasion and evade en-bloc resection of the chest wall in cases of tumor invasion limited to the parietal pleura, 132 patients with resected lung cancer involving the chest wall were retrospectively surveyed for the preoperative findings, surgical procedures, pathological results, and survival. A pathological examination of the resected specimens showed that 58 tumors had invaded only to the parietal pleura (shallow invasion) and 74 had involved the soft tissue or ribs (deep invasion). A multivariate analysis showed that preoperative CT findings of obvious tumor invasion beyond the parietal pleura (p = 0.005) and complaints of chest pain (p = 0.015) were independent indicators of deep invasion. In patients with lung cancer involving the chest wall, chest pain and/or invading on chest CT suggested that an en-bloc resection was a suitable surgical procedure, because 79% of those patients had deep invasion. On the other hand, in patients without chest pain and invasion on chest CT, an extrapleural approach was recommended at first based on the fact that 63% of them had shallow invasion. In practice, an extrapleural resection was performed in 40 cases and an en-bloc resection in 10 patients with shallow invasion. There was no significant difference in the survival between the two surgical procedures. Therefore, the CT findings of obvious tumor invasion beyond the parietal pleura and/or the presence of chest pain indicate the need to perform an en-bloc resection in patients with lung cancer involving the chest wall. However, in patients without these findings, an extrapleural approach could be initially attempted for chest wall resection, because an en-bloc resection had no survival benefit for patients with shallow invasion.

Stepwise examination for differential diagnosis of primary lung cancer and breast cancer relapse presenting as a solitary pulmonary nodule in patients after mastectomy.

BACKGROUND AND OBJECTIVES: The distinction of primary lung from metastatic breast cancer is crucial in patients presenting with a solitary pulmonary nodule after mastectomy, because treatment strategies are completely different. Definitive diagnosis of these nodules, however, is often difficult. We assessed the feasibility of our diagnostic approach for these nodules and estimated the frequency of primary lung cancer occurrence in patients after mastectomy. METHODS: We evaluated solitary pulmonary nodules appearing in 48 patients after mastectomy. For histological examination, CT-guided needle aspiration biopsy (CT-NAB) or trans-bronchial lung biopsy (TBLB) was performed. Besides conventional morphopathological examination, differential diagnosis was performed by immunohistochemical examination and evaluation using a molecular marker (mammaglobin 1). RESULTS: Biopsy specimens were obtained using minimally invasive methods, namely CT-NAB and TBLB, in 91.7% of patients. From 48 patients, differential diagnosis was obtained by morphopathological methods alone in 32, and by immunohistochemical and molecular marker examination in the remaining 16. Final diagnosis was metastatic breast and primary lung cancer in 40 (83.3%) and 8 patients (16.7%), respectively. CONCLUSIONS: Our results show the clinical feasibility of our stepwise approach to the differential diagnosis of primary lung cancer and breast cancer relapse presenting as a solitary nodule in patients after mastectomy.

Nontuberculous mycobacterial disease concomitant with a pulmonary artery occlusion caused by Takayasu's arteritis.

We herein describe a surgical case of pulmonary involvement in Takayasu's arteritis with pulmonary infections of nontuberculous mycobacteria. A 24-year-old female was admitted to our hospital because of a recurrent fever, and contrast-enhanced computed tomography of the chest revealed the occlusion of the right pulmonary artery and cavitary lesions in the right lower lobe of the lung. A further examination of the neck revealed the occlusion of aortic branches, and the patient was diagnosed with Takayasu's arteritis. The cavitary lesions were diagnosed as nontuberculous mycobacteria disease according to the sputum culture result of Mycobacterium intracellulare. After antibiotic treatment for 6 months, the right pneumonectomy was performed with a good result. We should be aware of Takayasu's arteritis as a disease which can lead to the development of unexplained respiratory symptoms due to pulmonary artery involvement in young adults.

Enhancement of GLI1-transcriptional activity by beta-catenin in human cancer cells.

The Hedgehog (Hh) signaling pathway and the Wnt signaling pathway are known to play important roles in carcinogenesis and the progression of various human malignant tumors. Although a relationship between these two pathways has recently been reported, the mechanism by which beta-catenin, one of the key molecules of the Wnt signaling pathway, influences the Hh pathway has not yet been revealed in detail. To clarify the role of beta-catenin in relation to the Hh signaling pathway, we transfected GLI1 and beta-catenin expression constructs into human malignant cells, including stomach, colon, and lung cancers, and evaluated the luciferase activity of GLI-responsive reporter constructs. While exogenous GLI1 increased the luciferase activity, exogenous beta-catenin also enhanced the activity under overexpression of GLI1. However, co-transfection with T-cell factor (TCF)-4 or lymphocyte enhancer factor (LEF)-1 did not influence the activity, indicating that the enhancement of beta-catenin in relation to the Hh signaling pathway is not TCF/LEF-dependent. Our results suggest that beta-catenin might be involved in the Hh signaling pathway via enhancement of the transcriptional activity of GLI.

Krüppel-like factor 6 is frequently down-regulated and induces apoptosis in non-small cell lung cancer cells.

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

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Plakoglobin (gamma-catenin) has TCF/LEF family-dependent transcriptional activity in beta-catenin-deficient cell line.

Beta-catenin is an essential element for the transcriptional activation of target genes in the Wnt signaling cascade and is also a cell adhesion molecule that couples with cadherins. Although plakoglobin (gamma-catenin), a closely related homologue of beta-catenin, is also known to be a cell adhesion molecule, its function as a transcriptional factor has not been revealed in detail. Using a human malignant mesothelioma cell line, NCI-H28, in which we have identified a homozygous deletion of the beta-catenin gene, we studied whether plakoglobin has a T-cell factor/lymphocyte enhancer factor (TCF/LEF) family-dependent transcriptional activity. Transfection with the wild-type plakoglobin expression vector induced accumulation of plakoglobin in the nucleus. Immunoprecipitation assay with cotransfection of plakoglobin and either TCF-4 or LEF-1 detected binding of plakoglobin to TCF-4 or LEF-1. Luciferase reporter assay demonstrated transcriptional activity of the wild-type plakoglobin when transfected with TCF/LEF, although plakoglobin showed less activity than beta-catenin. Exogenous plakoglobin was also shown to promote entrance of exogenous beta-catenin into the nuclei. Furthermore, small interfering RNA directed against plakoglobin suppressed expression of endogenous plakoglobin and its transcriptional activity, suggesting that endogenous plakoglobin has a weak transcriptional activity. These results suggest that plakoglobin can activate the Wnt signaling cascade directly without interaction of beta-catenin, and that plakoglobin has multiple functions as a transcriptional activator and a cell adhesion molecule like beta-catenin.