Futoshi Ishiguro

LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

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

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes

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.

Effect of selective lymph node dissection based on patterns of lobe-specific lymph node metastases on patient outcome in patients with resectable non-small cell lung cancer: A large-scale retrospective cohort study applying a propensity score

OBJECTIVE Lobectomy with systematic complete mediastinal lymph node dissection is standard surgical treatment for localized non-small cell lung cancer. However, selective mediastinal lymph node dissection based on lobe-specific metastases (selective dissection) has often been performed. This study was designed to evaluate the validity of the selective lymph node dissection. METHODS From 1995 through 2003, 625 patients in our hospital had surgery for complete mediastinal lymph node dissection and 147 for selective dissection. We evaluated whether selective dissection adversely affected overall survival. To minimize possible biases due to confounding by treatment indication, we performed a retrospective cohort analysis by applying a propensity score. The propensity score was calculated by logistic regression based on 15 factors available that were potentially associated with treatment indication. Patients were divided into 4 groups according to quartile, and comparison between selective dissection and complete mediastinal lymph node dissection was made using propensity score quartile-stratified Cox proportional hazard models. RESULTS Comparison of baseline characteristics between patients having selective dissection and patients having complete mediastinal lymph node dissection according to propensity score quartile supported comparability of the 2 groups. The 5-year overall survival rates were 76.0% for selective dissection versus 71.9% for complete mediastinal lymph node dissection. The 5-year survival probabilities stratified by propensity score quartile consistently showed no marked difference. In multivariate models, there was no significant difference between the 2 groups (hazard ratio = 1.17, P = .500) as also seen in the analysis without propensity score (hazard ratio = 1.06; 95% confidence interval, 0.68-1.64; P = .810). Therefore, selective dissection showed no significant impact on poor survival compared with complete mediastinal lymph node dissection. CONCLUSIONS Selective lymph node dissection did not worsen the survival of patients with non-small cell lung cancer.

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 p21WAF1/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.

Prognostic Impact of Tumor Size Eliminating the Ground Glass Opacity Component: Modified Clinical T Descriptors of the Tumor, Node, Metastasis Classification of Lung Cancer

Introduction: The presence of ground glass opacity (GGO) on high-resolution computed tomography (HRCT) is well known to be pathologically closely associated with adenocarcinoma in situ. Recently, measuring the tumor diameter including areas of GGO on HRCT has been reported to possibly overestimate the T status. The purpose of this study was to evaluate the significance of the tumor size measured eliminating the area of GGO on HRCT as a prognostic factor and to propose a refined TNM classification based on modified T descriptors. Methods: Four hundred seventy-five patients with clinical T1a-T2bN0M0 non-small-cell lung cancer underwent surgical resection. All tumors were reclassified based on the diameter measured eliminating the GGO area on HRCT according to the seventh TNM classification of lung cancer. We defined this new classification as modified T descriptors categorizing into five groups: mTis, mT1a, mT1b, mT2a, and mT2b. The overall survival rates of the patients in the current and modified staging groups were evaluated. Results: The 5-year survival rates were 88% and 82% in the patients with T1a and T1b tumors and 90% and 75% in the patients with mT1a and mT1b tumors, respectively. The differences in the survival rate of the patients classified by using mT1a and the other modified T descriptors were more clearly separated statistically than those of the patients classified by using the current T1a and other T descriptors. Conclusion: The modified T descriptors of the tumor size measured eliminating the GGO component on HRCT more clearly classified the prognoses of patients with early lung cancer than did the current T classification.

Pulmonary metastasectomy for osteogenic and soft tissue sarcoma: who really benefits from surgical treatment?

OBJECTIVES Surgical resection is widely accepted as a beneficial treatment of pulmonary metastases originating from osteogenic and soft tissue sarcomas despite adequate validation. The factors associated with the selection of patients who receive pulmonary metastasectomy (PM) are controversial and not well known. In this study, we aimed to identify the prognostic factors associated with survival after treatment with PM and to disclose the candidates who profit from PM being performed on patients with osteogenic and soft tissue sarcomas. METHODS We retrospectively reviewed the variables and survival outcomes in 52 consecutive patients who underwent PM to treat lung metastases originating from osteogenic and soft tissue malignancies from April 1996 to January 2011. Prognostic factors associated with overall survival after the first PM were evaluated using univariate and multivariate analyses. RESULTS Fifty-eight PM procedures were performed in 52 patients as the first PM including 6 bilateral diseases. Wedge resection was the most frequently performed PM procedure (84%), and video-assisted thoracic surgery was introduced in 34 (59%). The median follow-up of the patients was 33 months and the 5-year survival rate after the first PM was 50.9%. Forty-eight (92%) patients underwent complete resection during the first PM. Thirty-three patients (62%) experienced relapse after the first PM. Among those patients, 20 received redo surgeries for pulmonary relapse, and the 5-year survival rate in this group was 49.7%. According to univariate analyses, the use of complete resection, the number of metastatic nodules (one or two) and the length of the disease-free interval prior to the first PM were each found to be significant favourable factors. According to a multivariate analysis, the use of complete resection and the number of metastatic nodules were both found to be independent prognostic factors associated with overall survival. Although our cohort included 15 patients with poor prognostic factors (29%), 5 patients who underwent redo surgery survived >22 months. CONCLUSIONS The survival of those patients with one or two pulmonary nodules and those who underwent complete resection was favourable following the treatment of osteogenic and soft tissue sarcomas with PM. Redo surgery may also provide some survival benefit in patients with poor prognostic factors.

Multilocular thymic cyst associated with thymoma: a clinicopathologic study of 20 cases with an emphasis on the pathogenesis of cyst formation.

Multilocular thymic cysts (MTCs) are considered to be acquired lesions associated with various inflammatory conditions and/or malignant tumors. MTCs associated with thymomas are rare, with only 11 cases having been reported. On reviewing 110 consecutive patients with thymomas, we found 20 cases of MTCs. The patients included 18 men and 2 women aged 32 to 65 years (median 52 y). Eleven of the patients were symptomatic, and 6 presented with symptoms associated with inflammation. Computed tomography images were available for 11 patients, and cystic lesions were identified in 4 patients. The histologic subtypes of thymoma observed were: 3 tumors of type AB, 4 tumors of type B1, 9 tumors of type B2, and 4 tumors of type B3. In addition, 2 tumors were in advanced stages. Multilocular cystic structures accompanied by acute and chronic inflammation were observed in the remnant thymic tissues. Immunohistochemically, CK13 was diffusely expressed in the inner surface cells lining the cysts, whereas CK5/6 and p63 were primarily expressed in the basal cells of the cysts. D2-40 was weakly expressed in a small number of basal epithelial cells. The immunohistochemical profiles of the cysts were similar to those of Hassall corpuscles of normal thymi. A clinical follow-up showed that 15 patients continued to be alive without any evidence of disease, 1 patient with tumor recurrence continued to be alive, and 3 patients had died of other diseases. Our results suggest that MTCs associated with thymomas are not as uncommon as thought and may develop from the promotion of differentiation of increased numbers of epithelial cells into Hassall corpuscles by inflammatory processes. Our data also suggest a better clinical behavior for patients with thymomas accompanied by MTCs than patients with thymomas unaccompanied by those cysts, although further investigation is needed.

Subcategorization of Resectable Non-Small Cell Lung Cancer Involving Neighboring Structures

BACKGROUND Although the prognoses of patients with resectable lung cancer involving neighboring structures vary, the current tumor-nodes-metastasis (TNM) classification system does not elucidate criteria for tumor subcategorization. METHODS We studied 196 consecutive patients who underwent resection of non-small cell lung cancer involving neighboring structures at the Aichi Cancer Center Hospital and were diagnosed as pathologic T3 diseases using the current staging system. Tumors were divided into six groups based on the involved neighboring structures: parietal or mediastinal pleura, subpleural soft tissue, ribs, main bronchus, pericardium, and diaphragm. RESULTS The overall 5-year survival rate was 39.8%. The survival rates for the six groups were: pleura (n = 62), 54.8%; subpleural soft tissue (n = 50), 30.0%; rib (n = 25), 24.0%; main bronchus (n = 33), 48.5%; pericardium (n = 14), 21.4%; and diaphragm (n = 12), 33.3%. The combined pleura and bronchus groups (n = 95) demonstrated significantly better survival outcome than the other groups (n = 101): 52.6% and 27.7%, respectively (p = 0.0002). Furthermore, among 108 patients with pT3N0 (stage IIB) disease, the prognostic difference between the pleura and bronchus groups (n = 50) and the other groups (n = 58) was significant: 64.0% and 25.9%, respectively (p < 0.0001). Similar results were confirmed in patients with complete resection (n = 159). CONCLUSIONS Subcategorization of resectable lung cancer involving neighboring structures resulted in tumor groups infiltrating pleura or main bronchus, and those involving subpleural structures, pericardium, or diaphragm.

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

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

Activated Leukocyte Cell-Adhesion Molecule (ALCAM) Promotes Malignant Phenotypes of Malignant Mesothelioma

Introduction: Cell-adhesion molecules play important roles involving the malignant phenotypes of human cancer cells. However, detailed characteristics of aberrant expression status of cell-adhesion molecules in malignant mesothelioma (MM) cells and their possible biological roles for MM malignancy remain poorly understood. Methods: DNA microarray analysis was employed to identify aberrantly expressing genes using 20 MM cell lines. Activated leukocyte cell-adhesion molecule (ALCAM) expression in MM cell lines was analyzed with quantitative reverse transcription-polymerase chain reaction and Western blot analyses in 47 primary MM specimens with immunohistochemistry. ALCAM knockdown in MM cell lines was performed with lentivirus-mediated short hairpin RNA (shRNA) transduction. Purified soluble ALCAM (sALCAM) protein was used for in vitro experiments, whereas MM cell lines infected with the sALCAM-expressing lentivirus were tested for tumorigenicity in vivo. Results: ALCAM, a member of the immunoglobulin superfamily, was detected as one of the most highly upregulated genes among 103 cell-adhesion molecules with microarray analysis. Elevated expression levels of ALCAM messenger RNA and protein were detected in all 20 cell lines. Positive staining of ALCAM was detected in 26 of 47 MM specimens (55%) with immunohistochemistry. ALCAM knockdown with shRNA suppressed cell migration and invasion of MM cell lines. Purified sALCAM protein impaired the migration and invasion of MM cells in vitro, and the infection of sALCAM-expressing virus into MM cells significantly prolonged survival periods of MM-transplanted nude mice in vivo. Conclusion: Our study suggests that overexpression of ALCAM contributes to tumor progression in MM and that ALCAM might be a potential therapeutic target of MM.