Mesothelioma Lines Research Articles

Diverse and divergent functions of IL-32β and IL-32γ isoforms in the regulation of malignant pleural mesothelioma cell growth and the production of VEGF-A and CXCL8

In this study, we sought to elucidate the roles of the interleukin (IL)-32β and IL-32γ in mesothelioma cell growth, and vascular endothelial growth factor (VEGF)-A and C-X-C motif chemokine ligand 8 (CXCL8) expression. IL-32 elicited a growth-promoting effect against one of the six mesotheliomas lines and exerted diverse regulatory functions in VEGF-A and CXCL8 secretion from mesotheliomas stimulated with or without IL-17A. Retroviral-mediated transduction of mesothelioma lines with IL-32γ resulted in enhanced IL-32β expression, which facilitated or suppressed the in vitro growth, and VEGF-A and CXCL8 expression. Overexpressed IL-32β-augmented growth and VEGF-A and CXCL8 production were mainly mediated through the phosphatidylinositol-3 kinase (PI3K) signaling pathway. On the other hand, overexpressed IL-32β-deceased growth was mediated through mitogen-activated protein kinase (MAPK) pathway. NCI-H2373IL-32γ tumors grew faster than NCI-H2373Neo tumors in a xenograft model, which was associated with increased vascularity. These findings indicate that IL-32 are involved in the regulation of growth and angiogenic factor production in mesotheliomas.

Comprehensive Pharmacogenomic Profiling of Malignant Pleural Mesothelioma Identifies a Subgroup Sensitive to FGFR Inhibition.

Purpose: Despite intense research, treatment options for patients with mesothelioma are limited and offer only modest survival advantage. We screened a large panel of compounds in multiple mesothelioma models and correlated sensitivity with a range of molecular features to detect biomarkers of drug response.Experimental design: We utilized a high-throughput chemical inhibitor screen in a panel of 889 cancer cell lines, including both immortalized and primary early-passage mesothelioma lines, alongside comprehensive molecular characterization using Illumina whole-exome sequencing, copy-number analysis and Affymetrix array whole transcriptome profiling. Subsequent validation was done using functional assays such as siRNA silencing and mesothelioma mouse xenograft models.Results: A subgroup of immortalized and primary MPM lines appeared highly sensitive to FGFR inhibition. None of these lines harbored genomic alterations of FGFR family members, but rather BAP1 protein loss was associated with enhanced sensitivity to FGFR inhibition. This was confirmed in an MPM mouse xenograft model and by BAP1 knockdown and overexpression in cell line models. Gene expression analyses revealed an association between BAP1 loss and increased expression of the receptors FGFR1/3 and ligands FGF9/18. BAP1 loss was associated with activation of MAPK signaling. These associations were confirmed in a cohort of MPM patient samples.Conclusions: A subgroup of mesotheliomas cell lines harbor sensitivity to FGFR inhibition. BAP1 protein loss enriches for this subgroup and could serve as a potential biomarker to select patients for FGFR inhibitor treatment. These data identify a clinically relevant MPM subgroup for consideration of FGFR therapeutics in future clinical studies. Clin Cancer Res; 24(1); 84-94. ©2017 AACR.

Malignant pleural fluid from mesothelioma has potent biological activities.

Malignant pleural effusion (MPE) affects >90% of mesothelioma patients. Research on MPE has focused on its physical impact on breathlessness; MPE is rich in growth mediators but its contribution to tumour biology has not been investigated. We aimed to examine the potential effects of MPE in promoting growth, migration and chemo-resistance of mesothelioma. Pleural fluid samples from 151 patients (56 mesothelioma, 60 metastatic pleural cancer and 35 benign) were used. Seven validated human mesothelioma cell lines and three primary cultured mesothelioma lines were employed. Pleural fluid from mesothelioma patients (diluted to 30%) consistently stimulated cell proliferation (trypan-blue cell viability assay) in five mesothelioma cell lines tested by (median) 2.23-fold over controls (all P < 0.0001). The fluid also induced cell migration by (median) 2.13-fold in six mesothelioma cell lines using scratch-wound assay. In a murine flank model of mesothelioma, tumour infused with daily instillations of pleural fluid grew significantly faster over saline controls (median 52.5 cm2 vs 28.0 cm2 at day 13, P = 0.028). Addition of MPE (diluted to 30%) to culture media significantly protected mesothelioma from cisplatin/pemetrexed-induced cell death in all three cell lines tested (median fold reduction of 1.29, 1.98 and 3.90, all P < 0.001 vs control). The growth effects of matched pleural fluid and cultured mesothelioma cells from the same patients did not differ significantly from unmatched pairs. This 'proof-of-concept' study reveals potent biological capabilities of malignant pleural fluid in mesothelioma pathobiology.

HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status.

The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

Abstract 5136: HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status

Abstract Liposarcoma (LPS) is the most common sarcoma of humans. There are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved molecular understanding of LPS to stimulate the development of effective targeted therapies. The MDM2-p53 pathway plays a prominent role in WDLPS pathogenesis, with the vast majority of human tumors harboring either MDM2 amplifications or p53 mutations. Nutlin-3, a small-molecular antagonist of MDM2, inhibits cell proliferation via blocking the interaction of MDM2-p53 in LPS with wild-type p53. We wonder whether MDM2 overexpression due to amplification or/and mutant p53 still plays a crucial oncogenic role in LPS containing p53 mutation. Herein, we developed isogenic liposarcoma lines in which the parental forms were MDM2 amplification and p53 wild-type, whereas sublines had mutation p53 expression, showed Nutlin-3 resistance. mRNA sequencing and immunoblotting showed that MDM2 and CDK4 were overexpression in the parental lines and sublines, whereas MDM2 and CDK4 expression was low in mesothelioma and GIST cell lines. HDAC inhibitor (HDACi, SAHA and LBH589) treatment resulted in the dephosphorylation and degradation of MDM2 and p53, but little affected CDK4 and JUN expression, irrespective of p53 mutational status in LPS with MDM2 amplification, and in a mesothelioma cell line JMN1B with p53 mutation, but not two mesothelioma lines containing normal MDM2 level and wild-type p53. Our findings indicate that regulation of wild-type 53 degradation by HDACi is MDM2 amplification-dependent. HDAC inhibition by SAHA and LBH589 had a substantially effect on LPS and mesothelioma proliferation and survival associated with upregulation of the PTEN and p21, inhibition of cell proliferation marker cyclin A and PCNA expression, induction of G1 or G2 phase arrest; induction of apoptosis showing an increase of caspase 3/7 activity and expression, PARP cleavage, and the sub-G1 apoptotic population. Moreover, we characterize biological functions of MDM2-p53 axis in nutlin-3-sensitive and nutlin-3-resistant LPS, showing MDM2 knockdown in the four LPS lines and p53 knockdown in the three mutant-p53 LPS lines resulted in anti-proliferative effects. Additive effects were obtained through a coordinated attack on MDM2 and p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that MDM2 and p53 knockdown, in LPS cell lines containing the p53 mutation, induced greater cell apoptosis and anti-proliferative effects, compared to either intervention alone, which is comparable to the effects seen after HDAC inhibition by SAHA and LBH589. These compelling pro-apoptotic and anti-proliferative responses indicate that HDAC inhibition warrants clinical evaluation as a novel therapeutic strategy in LPS, including nutlin-3 resistant sublines with the p53 mutation. Note: This abstract was not presented at the meeting. Citation Format: Wen-bin Ou, Hailong Li, Li Liu, Shengmei Zhou, Ye Kuang, Ziqin Yan, Zhe Jiang, Si Shi, Fanguo Meng, Qing Sheng, Haimeng Zhou, Jonathan A. Fletcher. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5136. doi:10.1158/1538-7445.AM2014-5136

Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases.

Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs.

Extracellular Signal–Regulated Kinase 5: A Potential Therapeutic Target for Malignant Mesotheliomas

Malignant mesothelioma is a devastating disease with a need for new treatment strategies. In the present study, we showed the importance of extracellular signal-regulated kinase 5 (ERK5) in malignant mesothelioma tumor growth and treatment. ERK5 as a target for malignant mesothelioma therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft severe combined immunodeficient (SCID) mouse models. We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in malignant mesothelioma cells. ERK5 silencing increased doxorubicin-induced cell death and doxorubicin retention in malignant mesothelioma cells. In addition, shERK5 malignant mesothelioma lines exhibited both attenuated colony formation on soft agar and invasion of malignant mesothelioma cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion, and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 malignant mesothelioma cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from intraperitoneal shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis-related (interleukin-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the intraperitoneal model of tumor growth. ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in patients with malignant mesothelioma.

Abstract A3: Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases.

Abstract The use of chimeric antigen receptor (CAR)-redirected CD8+ T cells in the treatment of malignancy is a novel strategy that has shown tremendous promise; however, it remains likely this strategy could be enhanced when combined with additional approaches. We hypothesized that deletion of signaling mediators that negatively regulate TCR signal transduction might enhance CAR signaling and function, since CARs utilize the same machinery that transduces TCR signals. We tested CAR activity and function in T cells that lacked one or both lymphocyte-specific isoforms of diacylglycerol kinase (dgk), proteins that metabolize the second messenger DAG and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells, and identify dgks as a potential target for improving the clinical potential of CARs. Citation Format: Matthew Riese, Liang-Chuan Wang, Anjana Ranganathan, Gary Koretzky, Steven Albelda. Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A3.

LRRN4 and UPK3B Are Markers of Primary Mesothelial Cells

BackgroundMesothelioma is a highly malignant tumor that is primarily caused by occupational or environmental exposure to asbestos fibers. Despite worldwide restrictions on asbestos usage, further cases are expected as diagnosis is typically 20–40 years after exposure. Once diagnosed there is a very poor prognosis with a median survival rate of 9 months. Considering this the development of early pre clinical diagnostic markers may help improve clinical outcomes.MethodologyMicroarray expression arrays on mesothelium and other tissues dissected from mice were used to identify candidate mesothelial lineage markers. Candidates were further tested by qRTPCR and in-situ hybridization across a mouse tissue panel. Two candidate biomarkers with the potential for secretion, uroplakin 3B (UPK3B), and leucine rich repeat neuronal 4 (LRRN4) and one commercialized mesothelioma marker, mesothelin (MSLN) were then chosen for validation across a panel of normal human primary cells, 16 established mesothelioma cell lines, 10 lung cancer lines, and a further set of 8 unrelated cancer cell lines.ConclusionsWithin the primary cell panel, LRRN4 was only detected in primary mesothelial cells, but MSLN and UPK3B were also detected in other cell types. MSLN was detected in bronchial epithelial cells and alveolar epithelial cells and UPK3B was detected in retinal pigment epithelial cells and urothelial cells. Testing the cell line panel, MSLN was detected in 15 of the 16 mesothelioma cells lines, whereas LRRN4 was only detected in 8 and UPK3B in 6. Interestingly MSLN levels appear to be upregulated in the mesothelioma lines compared to the primary mesothelial cells, while LRRN4 and UPK3B, are either lost or down-regulated. Despite the higher fraction of mesothelioma lines positive for MSLN, it was also detected at high levels in 2 lung cancer lines and 3 other unrelated cancer lines derived from papillotubular adenocarcinoma, signet ring carcinoma and transitional cell carcinoma.

An Extracellular Signal–Regulated Kinase 2 Survival Pathway Mediates Resistance of Human Mesothelioma Cells to Asbestos-Induced Injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)-linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal-regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death-related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag-immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Inhibition of Met/HGF receptor and angiogenesis by NK4 leads to suppression of tumor growth and migration in malignant pleural mesothelioma

NK4 exhibits two distinct biological actions: antagonistic inhibition of hepatocyte growth factor (HGF) through binding to the Met/HGF receptor, and antiangiogenic action through binding to perlecan. Here, the anti-tumor effect of NK4 on malignant pleural mesothelioma was investigated. Of the 7 human malignant mesothelioma cell lines (ACC-Meso-1, ACC-Meso-4, EHMES-1, EHMES-10, H28, H2052 and JMN-1B), only EHMES-10 cells formed subcutaneous tumors when implanted into mice. For EHMES-10 cells, HGF facilitated invasion of the cells in collagen gel, whereas NK4 and neutralizing anti-HGF antibody suppressed the HGF-induced invasion. In addition, NK4 but not anti-HGF antibody suppressed proliferation of EHMES-10 cells in collagen, suggesting that the suppression by NK4 was independent of the HGF-Met pathway. In the subcutaneous tumor model, recombinant adenovirus-mediated intratumoral expression of NK4 inhibited tumor growth, while the invasive characteristic of tumor cells was not observed. Analysis of Met receptor tyrosine phosphorylation, proliferation, apoptosis and blood vessels in the tumor tissues indicated that the inhibitory effect of NK4 expression might be primarily caused by the inhibition of tumor angiogenesis. In all the 7 mesothelioma lines, HGF stimulated Met tyrosine phosphorylation, and this was associated with enhanced cell migration. HGF-dependent Met activation and migration were inhibited by NK4. Since malignant pleural mesothelioma represents an aggressive neoplasm characterized by extensive invasive growth, suppression of invasive growth has therapeutic value. Thus, the simultaneous inhibition of the HGF-Met pathway and angiogenesis by NK4 for treatment of malignant pleural mesothelioma is significant, particularly to attenuate migration and invasive growth.

Abstract 1513: Pre-clinical evaluation of two oncolytic viral vectors expressing IFNβ in treatment of thoracic cancer: Vaccinia virus versus vesicular stomatitis virus

Abstract Rationale: Given recent success in delivering interferon-beta (IFNβ) using replication incompetent adenovirus in patients with malignant pleural effusions, we hypothesized that vectors that could replicate (inducing oncolysis) and express IFNβ at high level would be more effective. Both vaccinia virus (VV) and vesicular stomatitis (VSV) expressing mouse IFNβ have been constructed in a manner to improve their safety and efficacy as oncolytic vectors. In this study, we evaluated these two oncolytic vectors in different murine thoracic cancer models for clinical translation. Experimental procedures: We compared the efficacy of VVIFNβ and VSVIFNβ to kill 3 mouse lung cancer and 4 mouse mesothelioma lines in vitro over a 5 day-period assessing cell viability by MTT assay. The amount of IFNβ produced by these two vectors were measured with ELISA. The viral replication in infected mice was assessed by measuring viral titers in both immunocompetent and immunodeficient mice bearing an IFN-non-responsive AB12 tumor, following intratumoral injection of either vector at 108 pfu/mouse. To assess their anti-tumor activity, the two vectors were also given at 108 pfu/mouse intratumorally in mice bearing two lung cancer tumors on their flanks. Results: The replication of VSVIFNβ and its killing ability was limited in some of the tumor lines. This appeared to be due to an intact IFN response pathway. In contrast, VVIFNβ was able to kill all of the tumor lines. In culture, VSV replicates faster than VV, which resulted in faster cell killing (in susceptible cell lines), but with less IFNβ production. The amount of IFNβ production by VVIFNβ was comparable with AdIFNβ over a 5 day period. VVIFNβ-infected immunodeficient mice showed minimal or no toxicity when given virus intravenously, while all 5 mice infected with VSVIFNβ developed neurotoxicity within a week. In the TC-1 lung cancer model, VVIFNβ caused tumor regression, while VSVIFNβ and AdIFNβ were completely ineffective. In a second model using LKRM2, all 3 viral vectors were equivalent. The anti-tumor effect of VVIFNβ was shown to be mainly CD8 dependant, as depletion of CD8 with antibody dampened the tumor growth inhibition from 48% to 20%. Conclusions: Overall, VVIFNβ appears to be a better oncolytic vector than VSVIFNβ for a number of reasons: 1) it is safer, 2) it is able to replicate in vitro and in vivo in almost every cell line tested, 3) it induces longer and larger IFNβ production, and 4) it can potentially be given intravenously. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1513.

Abstract 1912: Significant changes in chemokine genes in tumor-associated neutrophils (TANs) using a microarray approach

Abstract Background: The role of tumor-associated neutrophils (TAN) in tumor biology is unclear. We have recently published that resident TAN can acquire a protumor phenotype (similar to M2 macrophages), largely driven by TGF-beta to become N2 neutrophils. After TGF-beta blockade, neutrophils acquire an antitumor phenotype (N1 TAN (similar to M1 macrophages). In the current study, we further evaluated the characteristics of TAN using a gene microarray approach. Methods: Balb/C mice (10-20 in each group) bearing large flank tumors of the mesothelioma line AB12, where treated with either control or SM16 (a TGF-β-R1 kinase inhibitor) chow. After one week of treatment, the tumors were harvested, and neutrophils were isolated using microbeads (CD11b) and flow cytometry (Ly6G), into two groups - control TAN (N2) and SM16 TAN (N1). Neutrophils were also isolated from bone marrow of naïve mice (BM) and from spleens harvested from tumor-bearing mice (i.e., the granulocytic fraction of myeloid derived suppressor cells (G-MDSC)). mRNA from each subgroup (4-6 samples) was isolated and subjected to microarray analysis on Illumina chips. Selected results were validated by RT-PCR and neutrophils from a second non Small Cell Lung Cancer tumor, LKR, were studied. Results: After filtering, we found 16,322 informative probes. Hierarchical clustering and PCA analysis showed that the four groups were clearly separated. We found that many more genes were up-regulated than down-regulated in the N2 TAN from untreated mice compared to either BM or G-MDSC. Many of the highly up-regulated genes in TAN were chemokines, and genes encoding for inflammatory and immune responses, especially chemokines. This includes chemoattractants for T-cells (e.g. CXCL-9, CXCL10 and CXCL16), neutrophils (eg. CXCL1, CXCL2 and CCL3), B-cells (CXCL13) and macrophages (e.g. CCL2, CXCL-10 and CCL7). N1 (SM16-treated) and N2 TAN were more similar to each other than to the other groups, suggesting an intra-tumoral polarization between them. In N1 TAN, three different chemokines attracting macrophages were up-regulated - CCL2, CCL6 and CXCL10, and only one major chemokine, the T-regs chemoattractant CCL17, was downregulated. There was no significant difference in most other chemokines. Conclusion: Our data shows the major differences between naïve neutrophils, G-MDSC and N1/N2 TAN. The patterns support the hypothesis that neutrophils enter the tumor, possibly as G-MDSC, and the change between N2 and N1 is intra-tumoral (and not by differential recruitment). Our data further suggests that TAN secrete many chemokines, initiating the recruitment of other inflammatory cells of the immune system. N1 neutrophils appear to up-regulate several macrophage attracting chemokines compared to N2 TAN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1912.

Establishment of a human malignant fibrous mesothelioma cell line and the biological characteristics compared with malignant epithelial mesothelioma cell line

Two human malignant mesothelioma cell lines, which we designated "epithelial mesothelioma cells" and "fibrous mesothelioma cells", were established from the pleural fluid containing malignant mesothelial cells of a 72-year-old Japanese man. These cell lines were separated by the colonial techniques from the initiation of the primary cultures and grew well without interruption for 12 years. They were characterized as producing hyaluronic acid. These cell lines displayed different biological characteristics, including morphology, heterotransplantability and genetics using with BAC array CGH. The epithelial mesothelioma cells were epithelial in shape and transplantable into the subcutis of nude mice, while the cells of the fibrous mesothelioma line were fibroblast-like and transplantable into the submucosa of Hamster's cheek pouches but not into the subcutis of nude mice. The mesotheliomas are classified into three types: epithelial mesothelioma, fibrous mesothelioma and mixed type. The gene copy number losses observed on 9p21.3, 9p21.2, 9p21.1, among others may be a major mechanism of malignant mesothelioma carcinogenesis. We considered and supported the combination theory for the histogenesis of malignant mesothelioma.

Receptor tyrosine kinase and phosphoinositide-3 kinase signaling in malignant mesothelioma

The phosphoinositide-3 kinase signaling pathway is implicated in the development of malignancy and promotes cell-cycle progression and resistance to apoptosis. Malignant mesothelioma tumor specimens demonstrate high levels of the phosphoinositide-3 kinase downstream mediator phosphorylated Akt. Exposure of mesothelioma cell lines to LY294002, a phosphoinositide-3 kinase inhibitor, results in apoptotic cell death and decreased phosphorylated Akt in vitro and tumor burden reduction in vivo. Phosphoinositide-3 kinase is activated by cell-surface receptor tyrosine kinases. We sought to determine which receptors are present in mesothelioma and their role in cellular survival and phosphoinositide-3 kinase signaling. Western blot analysis was performed to determine the relative expression of epidermal growth factor receptor, insulin-like growth factor receptor, and platelet-derived growth factor receptor in the mesothelioma cell lines I-45 and REN and the mesothelial line Met5a. After exposure of mesothelioma lines to kinase inhibitors, a cell viability assay was performed, cell-cycle analysis was performed to determine the percentage of apoptosis, and Western blot analysis was performed for phosphorylated Akt. Inhibition of epidermal growth factor receptor resulted in apoptotic cell death and Akt hypophosphorylation in mesothelioma cell lines. Insulin-like growth factor receptor inhibition led to apoptotic cell death without affecting Akt phosphorylation. Platelet-derived growth factor receptor inhibition did not affect cellular survival or phosphoinositide-3 kinase signaling. In malignant mesothelioma constitutive activation of phosphoinositide-3 kinase/Akt results in cellular survival and contributes to the malignant phenotype. We have demonstrated that epidermal growth factor receptor inhibition leads to apoptotic cell death through downregulation of phosphoinositide-3 kinase signaling in mesothelioma cell lines, whereas insulin-like growth factor receptor inhibition leads to apoptosis independent of phosphoinositide-3 kinase. Epidermal growth factor receptor, insulin-like growth factor receptor, and phosphoinositide-3 kinase inhibition might be clinically relevant in malignant mesothelioma.

344. Recombinant Adeno-Associated Virus 2 Suicide Vectors for the Treatment of Human Sarcomas and Mesotheliomas

Top of pageAbstract Although great efforts have been made to improve conventional therapy for sarcoma and malignant mesothelioma, the median survival time of these entities after appearance of clinical symptoms stays poor. Effective locoregional therapy using viral vectors that contain a suicide gene may be an alternative treatment strategy. For sarcoma, we previously reported the highest susceptibility for recombinant adeno-associated virus 2 (rAAV-2) vectors in human connective tissue sarcoma cells (HS-1). Now we confirm our findings in five further human sarcoma cell lines: fibrosarcoma (HT-1080), Ewing's sarcoma (RD-ES), Askin's tumor (SK-N-MC), rhabdomyosarcoma (A-204) and soft tissue sarcoma (WSKL-1). Furthermore, we found that rAAV-2 also achieved both high transduction rates and GFP expression levels in human mesothelioma (H-Messo-1, MSTO-211H and NCI-H-28). Among rAAV-2-constructs containing different promotors, after transduction, the vector with the elongation factor 1-alpha (EF1a) promotor showed the highest expression rates in both the sarcoma and mesothelioma cell lines. To ensure the use of constant vector particles number, all stocks were titrated using both the functional and our real-time PCR-based titration assay. Several new thymidine kinase (TK) gene-containing vectors under control of either the Cytomegalovirus or the elongation-factor 1 alpha (EF1a) promoter were cloned and tested. A higher expression level of the transgene was observed in the sarcoma and mesothelioma lines when using the EF1a-suicide gene-containing vectors. For the sarcomas, we were able to show a complete eradication of all rAAV-EF1a-TK/eGFP (contains a thymidine kinase/enhanced green fluorescent protein fusion protein) transduced tumor cells following exposure to ganciclovir (2.5 μg/ml) in vitro, while at this dose level >90% of mock-transduced tumor cells survived. Using mesotheliomas, a nearly complete eradication (>2 log) of transduced and GCV-treated cells could be obtained using this vector. Xenotransplantation tumor models (i.p., s.c., i.v.) for both the human sarcoma and mesothlioma cell lines were established in nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice. In proof-of-principle experiments, mice transplanted with rAAV-TK/eGFP-transduced and ganciclovir-exposed sarcoma or mesothelioma tumor cells survived >5 months while in the non-transduced group all mice had died 1 month after inoculation. Currently, further animal experiments using this suicide system are ongoing. The data shown here hold promise for further development of AAV-2-based suicide gene therapy of both soft tissue sarcoma and mesothelioma towards a future clinical application.

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 μM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 μM adriamycin with 1 and 10 μM adriamycin being less inhibitory than 0.1 μM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.

Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product.

Malignant pleural mesothelioma is resistant to conventional therapies and to apoptosis. The bcl-2 family genes are major determinants of apoptotic homeostasis. Malignant pleural mesothelioma lines and tumors rarely express the antiapoptotic Bcl-2 protein but routinely express the antiapoptotic protein Bcl-xl and the proapoptotic proteins Bax and Bak. We have previously shown pharmacologic inhibition of bcl-xl expression in malignant pleural mesothelioma can lead to apoptosis, so we sought to determine whether antisense oligonucleotides directed at bcl-xl messenger RNA would engender apoptosis, possibly through a "forced imbalance" of bcl-2 family proteins. Malignant pleural mesothelioma lines REN (epithelial) and I-45 (sarcomatous) were exposed to modified bcl-xl antissense oligonecleotides directed near the messenger RNA initiation sequence with and without a liposomal delivery system. Untreated cells and bcl-xl sense oligonucleotides were controls. Cell viability was measured by colorimetric assay, and apoptosis was evaluated with Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis. Bcl-xl protein expression after antisense oligonucleotides was downwardly regulated in both cell lines relative to sense oligonucleotides (>65%). Significant cellular killing in both the I-45 and REN cell lines was achieved with antisense oligonucleotides (compared with sense oligonucleotides) without (P =.003 and.006, respectively) and with (P =.006 and.0005, respectively) liposomal delivery. Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis demonstrated apoptosis to be the mechanism of cellular death. Use of a liposomal delivery system increased therapeutic effect and allowed lower doses of antisense oligonucleotides. Antisense oligonucleotides directed at the bcl-xl gene product engender apoptosis in mesothelioma cell lines. The therapeutic potential of inhibiting expression of this protein in mesothelioma should be evaluated.

Induction of apoptosis in malignant pleural mesothelioma cells by activation of the Fas (Apo-1/CD95) death-signal pathway

Objective: Although well characterized in several solid tumors, the effects of Fas/Fas ligand interactions in malignant pleural mesothelioma cells have not been defined. The present study was undertaken to examine the functional status of the Fas/Fas ligand pathway in malignant pleural mesothelioma cells and to determine the feasibility of targeting this death-signal pathway for molecular intervention in patients with mesotheliomas. Methods: Fas expression in primary normal human bronchial epithelial cells and 6 malignant pleural mesothelioma cell lines was quantified by means of flow cytometry. The caspase components of the Fas-mediated apoptotic pathway were evaluated by means of Western blot techniques. Soluble Fas ligand–mediated cytotoxicity and apoptosis were evaluated by means of MTS and TUNEL assays, respectively. Cisplatin (3 μg/mL) and lymphokine-activated killer cells were used to enhance mesothelioma sensitivity to soluble Fas ligand. An H2373 nude mouse xenograft model of malignant pleural mesothelioma was established to assess the in vivo effects of soluble Fas ligand. Results: Four of 6 malignant pleural mesothelioma lines exhibited high levels of Fas expression, and 2 of 4 were inherently susceptible to soluble Fas ligand–mediated cytotoxicity (soluble Fas ligand 50% inhibitory concentration, <15 ng/mL). Two soluble Fas ligand refractory cell lines (H2052 and H513) exhibited high levels of Fas receptor. Pretreatment with cisplatin resulted in a reduction of 50% inhibitory concentration from infinity to 4.17 ± 0.14 ng/mL and 10.23 ± 1.58 ng/mL, respectively. Two additional soluble Fas ligand refractory cell lines (H2595 and REN) expressed low levels of Fas. Exposure of these cells to lymphokine-activated killer cells or lymphokine-activated killer cell–conditioned medium followed by a 24-hour treatment with cisplatin resulted in a significant reduction in 50% inhibitory concentration of soluble Fas ligand and pronounced induction of apoptosis. Intraperitoneally administered soluble Fas ligand mediated regression of H2373 xenografts. Conclusion: The Fas/Fas ligand pathway in mesothelioma cells is either intrinsically intact or can be rendered functional with chemotherapeutic agents or immune effector cells. These preclinical data support further evaluation of strategies to enhance Fas-mediated apoptosis in mesotheliomas.

Resistance of pleural mesothelioma cell lines to apoptosis: relation to expression of Bcl-2 and Bax.

A failure of normal apoptosis, often due to mutant p53, may contribute to the formation of a cancer and to its resistance to therapy. Mesothelioma, an asbestos-induced tumor, is highly resistant to therapy but generally expresses wild-type p53. We asked whether mesothelioma was resistant to apoptosis and whether resistance was associated with altered expression of the antiapoptotic protein Bcl-2 or proapoptotic protein Bax. We found that three mesothelioma cell lines (1 with wild-type p53) were highly resistant to apoptosis induced by oxidant stimuli (asbestos, H2O2) or nonoxidant stimuli (calcium ionophore) compared with primary cultured mesothelial cells. By immunostaining, one of these three lines expressed Bcl-2 but only during mitosis. By immunoblotting, 3 of 14 additional mesothelioma lines (9 of 14 with wild type p53) expressed Bcl-2 but all 14 of 14 expressed the proapoptotic Bax, giving a low ratio of Bcl-2 to Bax. We conclude that mesothelioma cell lines are resistant to apoptosis and that the failure in apoptosis is not explained by Bcl-2 but by other mechanisms that counteract the proapoptotic effect of Bax.