Viability Of Mesothelioma Cells Research Articles

Abstract 2769: Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3

Abstract Malignant mesothelioma (MM) is an aggressive tumor of mesothelial tissues that has poor prognosis and limited therapeutic options. Pass et al. (N Engl J Med 2012) identified the extracellular matrix protein fibulin-3 as an accurate biomarker of MM progression, which can be detected in the tumor parenchyma and in pleural effusions. Here, we report a systematic study of the mechanisms of fibulin-3 in MM and its value as a molecular target. Fibulin-3 activated canonical NFkB signaling and regulated the expression of NFkB-downstream genes, including the proteases MMP9 and MMP13, and the proinvasive proteins TNC and MLCK. Fibulin-3 knockdown downregulated NFkB signaling, reduced MM cell viability and potentiated the cytotoxicity of cisplatin in vitro. We next developed a function-blocking antibody ("mAb428.2") against an epitope of fibulin-3 required for NFkB activation (Nandhu et al., Clin Cancer Res 2017). This antibody blocked fibulin-3 signaling in vitro and reduced the expression of this protein, as well as NFkB-regulated MMP9, in subcutaneous (sc) MM tumors implanted in nude mice. Next, we treated mice carrying sc or intrapleural models of MM with mAb428.2 or control IgG1 (8x 30 mg/kg, q24h) delivered IV. Our anti-fibulin-3 antibody caused a significant reduction of tumor burden and extension of overall survival in both models. Overall, downregulation and inhibition of fibulin-3 proved a successful approach to enhance MM therapy. Given the value of this protein as a MM biomarker, we expect that detection of fibulin-3 may serve to identify patients with the highest chance to respond to combination therapies including anti-fibulin-3 approaches, achieving maximum therapeutic against these aggressive tumors. Citation Format: Mohan Sobhana Nandhu, Chandra Goparaju, Sharon L. Longo, Harvey I. Pass, Mariano S. Viapiano. Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2769.

Synergistic effect of targeting dishevelled-3 and the epidermal growth factor receptor-tyrosine kinase inhibitor on mesothelioma cells in vitro.

It was previously revealed that Wnt signaling is activated in mesothelioma cells. Although epidermal growth factor receptor (EGFR) is expressed in mesothelioma cells, EGFR-tyrosine kinase inhibitors (TKIs) are not effective for mesothelioma treatment. However, in non-small cell lung cancer, the blocking of Wnt signaling has been identified to enhance the anticancer effect of EGFR-TKIs. To confirm the anticancer effect of blocking Wnt signaling in combination with EGFR-TKI treatment in mesothelioma, the present study evaluated the effect of simultaneous suppression of human dishevelled-3 (Dvl-3) expression with Dvl-3 small interfering RNA (siRNA) and of EGFR inhibition with gefitinib on mesothelioma cell viability. Mesothelioma cell lines with and without β-catenin gene expression were transfected with Dvl-3 siRNA and were cultured with gefitinib, and cell viability, colony formation and cell cycle analyses were performed. Dvl-3 siRNA downregulated the expression of Dvl-3 in mesothelioma cells. The combination of Dvl-3 siRNA with gefitinib acted synergistically to induce concomitant suppression of cell viability and colony formation, suggesting that inhibition of Wnt signaling by downregulating Dvl-3 with siRNA and inhibiting EGFR with gefitinib leads to significant antitumor effects.

FTY720 inhibits mesothelioma growth in vitro and in a syngeneic mouse model

BackgroundMalignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet.MethodsCell viability and anchorage–independent growth were evaluated in a panel of MM cell lines and human mesothelial cells (HM) upon FTY720 treatment to assess in vitro anti-tumor efficacy. The mechanism of action of FTY720 in MM was assessed by measuring the activity of phosphatase protein 2A (PP2A)—a major target of FTY720. The binding of the endogenous inhibitor SET to PP2A in presence of FTY720 was evaluated by immunoblotting and immunoprecipitation. Signaling and activation of programmed cell death were evaluated by immunoblotting and flow cytometry. A syngeneic mouse model was used to evaluate anti-tumor efficacy and toxicity profile of FTY720 in vivo.ResultsWe show that FTY720 significantly suppressed MM cell viability and anchorage–independent growth without affecting normal HM cells. FTY720 inhibited the phosphatase activity of PP2A by displacement of SET protein, which appeared overexpressed in MM, as compared to HM cells. FTY720 promoted AKT dephosphorylation and Bcl-2 degradation, leading to induction of programmed cell death, as demonstrated by caspase-3 and PARP activation, as well as by cytochrome c and AIF intracellular translocation. Moreover, FTY720 administration in vivo effectively reduced tumor burden in mice without apparent toxicity.ConclusionsOur preclinical data indicate that FTY720 is a potentially promising therapeutic agent for MM treatment.

Abstract 2514: CSPG4 as a target of antibody-based immunotherapy for malignant mesothelioma

Abstract Malignant mesothelioma (MM) is an aggressive tumor, resistant to conventional therapies, with median survival of 1 year from diagnosis. The lack of effective therapies available prompted us to implement antibody-based immunotherapy against novel targets and to investigate possible synergies with chemotherapy on MM. CSPG4 is involved in the onset and progression of melanoma and other tumors, as well as in neo-angiogenesis. CSPG4-specific monoclonal antibodies (mAb) have shown anti-tumor effects in both preclinical and clinical trials of melanoma. We hypothesized that targeting CSPG4 with a CSPG4-specific mAb would have therapeutic efficacy against MM. We found that chondroitin sulphate proteoglycan 4 (CSPG4) was aberrantly expressed in 6 out of 8 MM cell lines and in 24 out of 40 MM biopsies, with minimal expression in surrounding non-malignant cells. The expression of CSPG4 in MM was higher upon engagement of extracellular matrix components (ECM) and was associated with increased MM cell viability and motility. Silencing of CSPG4 expression by siRNAs supported this functional significance of CSPG4 in MM. Consistently, the CSPG4-specific mAb TP41.2 inhibited MM cell attachment to the ECM, resulting in decreased phosphorylation of FAK and AKT, decreased expression of cyclin D1 and apoptosis. Moreover, TP41.2 significantly reduced MM cell motility, migration and invasiveness, and inhibited MM growth in soft agar. TP41.2 prevented the outgrowth of human MM tumors in SCID mice and inhibited the growth of established MM xenografts, resulting in significantly extended survival of tumor-bearing mice. These results represent a novel approach for CSPG4 mAb-based immunotherapy of MM. Combinatorial strategies with chemotherapeutic agents and with mAbs targeting c-Met related receptor are ongoing, aimed at translating CSPG4-specific mAb into clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2514. doi:1538-7445.AM2012-2514

Abstract LB-28: Regulation of FOXM1 by NADPH oxidases in malignant mesothelioma cells

Abstract Malignant mesothelioma (MM) is linked to the occupational exposure to asbestos with an estimated median survival of less than two years; effective therapy is lacking. Due to long latency periods, incidence is expected to rise in regions where asbestos use has been banned, as well as where worker protection is lacking. MM cells exhibit changes in oxidant and energy metabolism, with elevated expression of the NADPH oxidase NOX4. We hypothesize that NOX4 produces reactive oxygen species (ROS) that promote uncontrolled growth through redox-dependent signaling to core cell cycle factors such as FOXM1 and FOXO3a, transcription factors involved in responses in oxidative stress and cell cycle progression. Targeted microarrays were used to assess the expression levels of 96 genes involving redox metabolism in four human MM cell lines and one control mesothelial line (LP9) immortalized with hTERT. Gene expression profiles from MM cell lines indicate tumorigenesis commonly results in elevated expression of NOX activity and adaptation to constitutive production of ROS, including up-regulation of antioxidant enzymes and cytoprotective factors. mRNA for FOXM1, a transcription factor that regulates the G2/M transition in the cell cycle, was up-regulated in all four MM cell lines, and FOXM1 continued to be expressed in MM cells, but not LP9 cells, in the absence of growth factors. Inhibitors of NOX activity markedly reduced expression of FOXM1, which correlated with dose-dependent effects on MM cell proliferation and viability. In the HM MM cell line, thiostrepton (TS), a thiazole antibiotic that inhibits the expression of FOXM1 via an unknown mechanism, increased the rate of oxidation of DCF, hyper-activated ERK1/2, and showed dose-dependent effects on MM cell viability in vitro and MM tumor growth in Fox Chase SCID mice. N-acetyl-L-cysteine blocked activation of ERK1/2 and cell death by TS, indicating the cytotoxic effects of TS in MM cells may be dependent on induction of intolerable levels of oxidative stress. In support of this possibility, inhibitors of p38 MAPK accentuated the cytotoxic effects of TS on cell viability. Our studies indicate that altered oxidant metabolism that supports expression of cell cycle factors such as FOXM1 is a common property of MM cells, and suggest that either inhibiting or accentuating the production of ROS in MM cells may be useful therapeutic approaches in the treatment of MM. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-28.

Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells

Malignant mesothelioma is an increasingly common tumor with an almost 100% mortality rate. It is refractory to conventional treatment. We have previously shown with SSH and microarray that the mRNA expression level of proteasome is higher in epithelioid mesothelioma cell lines than in sarcomatoid ones. This study evaluates the differential apoptotic effect of proteasome inhibitors on both of these mesothelioma sub-lines. Proteasome inhibitors show substantial anti-tumor activity in some tumor cells in vitro and in vivo, but the effects on mesothelioma cells has not been studied. The viability of mesothelioma cells was reduced in a dose- and time-dependent manner by the proteasome inhibitors tested; PSI was effective with a low dose, but higher concentrations were needed for calpain inhibitor I. The epithelioid mesothelioma cells are more sensitive to the inhibitors than the sarcomatoid ones, their IC 50 after 24 h of treatment with PSI being 4 and 16 μm, respectively. Other mesothelioma cell lines show similar sensitivity. PSI seemed to decrease mesothelioma viability by inducing apoptosis, as verified by cell morphology, Western blotting analysis of caspase 3 cleavage, and flow-cytometric analysis. In conclusion, PSI, a representative agent that reduces viability and induces apoptosis of mesothelioma cells, might be useful in the treatment of patients with mesothelioma, especially of epithelioid phenotype.

Lovastatin induces apoptosis in malignant mesothelioma cells.

Malignant mesothelioma causes profound morbidity and nearly universal mortality that is refractory to conventional treatment with aggressive surgery, radiotherapy, or chemotherapy. We report that pharmacologic concentrations of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, induced apoptosis in human malignant mesothelioma cell lines. Mesothelioma cell viability was decreased in a dose-dependent manner by lovastatin (5 to 30 microM). These effects were not reversed by exogenous growth factors or cholesterol, but were reversed by addition of 100 microM mevalonate, confirming that lovastatin affected mesothelioma viability by inhibiting mevalonate synthesis. Lovastatin appeared to decrease mesothelioma viability by inducing apoptosis, as indicated by morphologic changes, histologic evidence of nuclear condensation and degeneration, and flow-cytometric analysis of DNA content. Lovastatin's effects on cell viability were partially reversed in the presence of farnesol, and treatment of mesothelioma cells with a specific farnesyl-protein transferase (FTP) inhibitor decreased cell viability and induced morphologic changes indistinguishable from those caused by lovastatin. In addition, lovastatin-treated cells showed translocation of ras guanosine triphosphate (GTP)-binding proteins from membrane to cytosolic fractions on Western blots, suggesting that lovastatin's effects on mesothelioma were mediated in part by disrupting acylation of GTP-binding proteins. Thus, lovastatin is a commercially available and clinically well-tolerated agent that reduces viability and induces apoptosis of mesothelioma cells, and may provide the basis for adjunctive treatments of patients with mesothelioma.