Human Mesothelioma Cell Research Articles

Sirtuin Family Members Selectively Regulate Autophagy in Osteosarcoma and Mesothelioma Cells in Response to Cellular Stress.

The class III NAD+ dependent deacetylases-sirtuins (SIRTs) link transcriptional regulation to DNA damage response and reactive oxygen species generation thereby modulating a wide range of cellular signaling pathways. Here, the contribution of SIRT1, SIRT3, and SIRT5 in the regulation of cellular fate through autophagy was investigated under diverse types of stress. The effects of sirtuins' silencing on cell survival and autophagy was followed in human osteosarcoma and mesothelioma cells exposed to DNA damage and oxidative stress. Our results suggest that the mitochondrial sirtuins SIRT3 and 5 are pro-proliferative under certain cellular stress conditions and this effect correlates with their role as positive regulators of autophagy. SIRT1 has more complex role which is cell type specific and can affect autophagy in both positive and negative ways. The mitochondrial sirtuins (SIRT3 and SIRT5) affect both early and late stages of autophagy, whereas SIRT1 acts mostly at later stages of the autophagic process. Investigation of potential crosstalk between SIRT1, SIRT3, and SIRT5 revealed several feedback loops and a significant role of SIRT5 in regulating SIRT3 and SIRT1. Results presented here support the notion that sirtuin family members play important as well as differential roles in the regulation of autophagy in osteosarcoma vs. mesothelioma cells exposed to DNA damage and oxidative stress, and this can be exploited in increasing the response of cancer cells to chemotherapy.

Anti-cancer activity of grape seed semi-polar extracts in human mesothelioma cell lines

Malignant mesothelioma is a tumor that affects pleural surface and has very poor prognosis. The standard therapeutic modalities for this cancer have yielded unsatisfactory outcomes, therefore the development of alternative and effective therapies is currently an urgent requirement. Grapevine is a plant rich of bioactive compounds, known for its therapeutic effects. Here, we describe the anti-cancer activity of grape seeds semi-polar extracts of two Italian grape varieties (Aglianico and Falanghina) in mesothelioma in vitro. Seed extracts from both varieties induced intrinsic apoptosis in a dose and time-dependent manner in three different human mesothelioma cell lines. Global metabolic analysis of this fraction revealed a higher accumulation of phenylpropanoid precursors and proanthocyanidins and expression of genes involved in the aforementioned pathways. These findings suggest that new phenolic molecules from grape seeds could be viewed as new drugs to be used alone or in combinations with standard chemotherapeutics in mesothelioma treatment.

Curcumin C3 complex\xae/Bioperine\xae has antineoplastic activity in mesothelioma: an in vitro and in vivo analysis

BackgroundA major limitation in the treatment for malignant mesothelioma is related to serious side effects caused by chemotherapeutics and to the development of cancer-resistance. Advances in cancer therapies have been reached thanks to the introduction of alternative approaches, such as the use of phytochemicals. Curcumin-C3complex®/Bioperine® is a commercially standardized extract containing a ratio-defined mixture of three curcuminoids and piperine that greatly increase its bioavailability. Interestingly, the anticancer effect of this formulation has been described in different studies and several clinical trials have been started, but to our knowledge none refers to human mesothelioma.MethodsCurcumin-C3complex®/Bioperine® anticancer effect was evaluated in vitro in different human mesothelioma cell lines analysing cell proliferation, colony-forming assay, wound healing assays, invasion assay and FACS analysis. In vivo anticancer properties were analysed in a mesothelioma xenograft mouse model in CD1 Nude mice.ResultsCurcumin-C3complex®/Bioperine® in vitro induced growth inhibition in all mesothelioma cell lines analysed in a dose- and time-depended manner and reduced self-renewal cell migration and cell invasive ability. Cell death was due to apoptosis. The analysis of the molecular signalling pathway suggested that intrinsic apoptotic pathway is activated by this treatment. This treatment in vivo delayed the growth of the ectopic tumours in a mesothelioma xenograft mouse model.ConclusionsCurcumin-C3complex®/Bioperine® treatment strongly reduces in vitro tumorigenic properties of mesothelioma cells by impairing cellular self-renewal ability, proliferative cell rate and cell migration and delays tumor growth in xenograft mouse model by reducing angiogenesis and increasing apoptosis. Considering that curcumin in vivo synergizes drug effects, its administration to treatment regimen may help to enhance drug therapeutic efficacy in mesothelioma. Our results suggest that implementation of standard pharmacological therapies with novel compounds may pave the way to develop alternative approaches to mesothelioma.

Abstract CT201: Final results of Phase II STELLAR trial: TTFields with chemotherapy in unresectable malignant pleural mesothelioma

Abstract Background Tumor Treating Fields (TTFields) are an anti-mitotic, regional treatment modality, utilizing low intensity alternating electric fields delivered non-invasively to the tumor using a portable device. In-vitro, human mesothelioma cells were highly susceptible to TTFields. TTFields plus chemotherapy have significantly extended survival in glioblastoma. Method Patients (n= 80) with unresectable, previously untreated mesothelioma were treated with continuous 150 kHz TTFields (>18h/day) in combination with pemetrexed and cisplatin or carboplatin. Inclusion criteria: ECOG PS 0-1, pathologically proven mesothelioma and at least one measurable lesion per modified RECIST criteria. The primary endpoint was overall survival (OS) and secondary endpoints were response rate, progression free survival (PFS) and toxicity. EOCG status and cancer-related pain were assessed until disease progression using a visual analog scale. This prospective, single arm study assumed an historical control with a median survival of 12.1 months (Vogelzang, JCO. 2003). The sample size provides 80% power with a two-sided alpha of 0.05 to detect an increase in median OS of 5.5 months. Result All 80 patients had a minimum follow up of 12 months. Median age was 67 (range 27-78), 84% were male and 44% (35 patients) had an ECOG PS of 1. 66% (53 patients) had epithelioid histology, similar to the Vogelzang study. Median OS was 18.2 months (95% CI 12.1-25.8) versus 12.1 months in the historical control. The 1-year survival rate was 62.2% (95% CI 50.3-72.0). Median OS for epithelioid patients was 21.2 months (95% CI 13.2-25.8). Clinical benefit (PR+SD) was seen in 97.2% of patients. ECOG score was stable during the first year of follow up. Compliance with TTFields was 68% (16.3 hours/day) during the first 3 months of therapy. 63% (50 patients) received carboplatin. Median time to deterioration in performance status was 13.1 months. Average pain score was lower compared to baseline during the first 7 months of treatment and higher later with a median time to a clinical significant 33% increase in pain of 8.4 months. No device-related serious adverse events (AEs) were reported. Expected TTFields-related dermatitis was reported in 46% (37 patients). Four patients (5%) had grade 3 dermatitis. Grade 3-4 systemic AEs were reported in >3% of patients: hematological AEs (15%) and fatigue (4%). Conclusion The study met its primary endpoint of significant extension of survival in previously untreated mesothelioma patients. Secondary efficacy endpoints were also improved compared to historical control. The study demonstrated no safety concerns for TTFields plus standard chemotherapy to the thorax. The use of the TTFields was not associated with a decrease in performance status or an increase in pain. TTFields in combination with chemotherapy are efficacious in malignant pleural mesothelioma vs chemotherapy alone in historical data. Citation Format: Giovanni Luca Ceresoli, Joachim Aerts, Jaroslaw Madrzak, Rafal Dziadziuszko, Rodryg Ramlau, Susana Cedres, Birgitta Hiddinga, Jan VanMeerbeeck, Manlio Mencoboni, David Planchard, Antonio Chella, Lucio Crino, Maciej Krzakowski, Federica Grosso. Final results of Phase II STELLAR trial: TTFields with chemotherapy in unresectable malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT201.

Abstract 2994: In vivo targeting of mesothelioma’s molecular signature with nanoparticle functionalized with nucleolin-binding peptide

Abstract Mesothelioma is a malignant tumor of the mesothelium, often associated with asbestos exposure, with an overall survival of only 8.8 months. A combination of cisplatin and pemetrexed has been used as an adjuvant treatment to surgical resection or in patients who have inoperable disease. This combination remains as the current standard of care, regardless the sub-type of mesothelioma, with only a 9% 5-year survival rate. Herein, it is hypothesized that a novel targeted treatment, based on a doxorubicin (DXR)-containing nanoparticle functionalized with the nucleolin-binding F3 peptide (named PEGASEMP), will have significant a therapeutic impact against the most aggressive subtype of mesothelioma (biphasic). The hypothesis relies on nucleolin deregulated overexpression in cancer and endothelial cells from tumor blood vessels. Human biphasic mesothelioma cells were stably transduced with luciferase-reporter gene, and orthotopically injected intrapleurally into female immunocompromised mice. Three weeks (Early Stage) and 4 weeks (Advanced Stage) after cell injection, animals were randomly allocated to different treatment groups: vehicle, PEGASEMP at 5.6 or 7 mg of DXR/kg alone (q7dx5w); cisplatin at 4.0 mg/kg alone or combined with PEGASEMP at 5.6 mg of DXR/kg (q7dx5w); and the standard of care, a combination of cisplatin at 4.0 mg/kg (q7dx5w) plus pemetrexed at 100.0 mg/kg (q2dx3x5w). Bioluminescence was monitored weekly with IVIS Spectrum In Vivo Imaging system. Differential gene expression upon PEGASEMP treatment of advanced stage tumors was also performed in comparison with non-treated animals using the GeneChip Human Transcriptome Array 2.0. In the early stage of development, PEGASEMP, at 7.0 mg/kg, inhibited tumor growth by 2,713-fold relative to standard of care, which translated into a 1223-fold decrease in tumor burden. At an advanced stage of tumor development, the combination of PEGASEMP and cisplatin enabled 107-fold tumor growth inhibition, enabling a 55-fold reduction in tumor burden, relative to standard of care. In both cases, no decrease of the body weight was observed. Furthermore, 75% (146) gene transcripts were found to be deregulated (while the remaining 25% were upregulated), involving the downregulation of cell division, including ontologies such as cell cycle or chromatin organization and assembly. Specifically, downregulation of topoisomerase 2, cyclin B1 or Ki67 transcripts, part of the described molecular signature of mesothelioma, was observed and was consistent with the mechanism of action of doxorubicin. Overall, the novel mechanism of action associated with PEGASEMP, enables a significant benefit in terms of efficacy and safety in the treatment of biphasic mesothelioma, as compared with the current standard of care, affecting the molecular signature of the disease and thus supporting future clinical evaluation. Citation Format: Nuno A. Fonseca, Vera Moura, Fabiana Colelli, Daniela Pesce, Giacomo Signorino, Laura Focareta, Alessandra Fucci, Francesco Cardile, Claudio Pisano, Sérgio Simões, João Nuno Moreira. In vivo targeting of mesothelioma’s molecular signature with nanoparticle functionalized with nucleolin-binding peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2994.

Whole-genome sequencing of human malignant mesothelioma tumours and cell lines.

Pleural mesothelioma is a cancer of serosal surfaces caused by environmental exposure to asbestos. Clinical outcome remains poor and while trials of new treatments are ongoing it remains an understudied cancer. Mesothelioma cell lines can readily be grown from primary tumour and from tumour cells shed into pleural effusion with the latter representing a particularly valuable source of DNA in clinical settings, procurable without the need for additional invasive procedures. However, it is not well understood how accurately patient-derived cultured tumour cells represent the molecular characteristics of their primary tumour. We used whole-genome sequencing of primary tumour and matched cultured cells to comprehensively characterize mutations and structural alterations. Most cases had complex rearranged genomes with evidence of chromoanagenesis and rearrangements reminiscent of chromoplexy. Many of the identified driver mutations were structural, indicating that mesothelioma is often caused by structural alterations and catastrophic genomic events, rather than point mutations. Because the majority of genomic changes detected in tumours were also displayed by the genomes of cultured tumour cells, we conclude that low-passage cultured tumour cells are generally suitable for molecular characterization of mesothelioma and may be particularly useful where tissue samples with high tumour cell content are not available. However, the subclonal compositions of the cell lines did not fully recapitulate the subclonal diversity of the primary tumours. Furthermore, longitudinal acquisition of major alterations in subclonal cell populations was observed after long-term passaging. These two factors define limitations of tumour-derived cell lines as genomic substrate for clinical purposes.

A Novel Nutrient Mixture Induces Apoptosis in Human Mesothelioma Cells (MSTO-211H) via Activation of Caspases

Background: Malignant mesothelioma is a highly aggressive and fatal cancer of older people. Consistently associated with asbestos exposure, mesothelioma is diagnosed when it is extensively metastasized and it has a dismal prognosis. Purpose of the study: Surgery, chemotherapy, and radiotherapy are the mainstay of treatment yet they are ineffective in increasing the mesothelioma patient survival. Therefore, a different approach is needed. A novel nutrient mixture containing green tea extract, ascorbic acid, lysine, and proline exhibited anti-cancer effects in various cancers. In our previous studies, the nutrient mixture was seen to block MMP secretion and invasion through MatrigelTM by mesothelioma cells. In the current study, we wanted to explore if the nutrient mixture could induce apoptosis in mesothelioma cells

196O - STELLAR: Final updated results of a phase II trial of TTFields with chemotherapy for unresectable malignant pleural mesothelioma

Background: Tumor Treating Fields (TTFields), an anti-mitotic, regional treatment approved for glioblastoma utilizes low intensity, alternating electric fields delivered non-invasively to the tumor using a portable medical device. In-vitro, human mesothelioma cells were highly susceptible to TTFields. Methods: The trial accrued 80 patients with unresectable, previously untreated mesothelioma. Patients were treated with continuous 150 kHz TTFields (>18h/day) in combination with pemetrexed and cisplatin or carboplatin. Inclusion criteria included ECOG PS of 0-1 and pathologically proven mesothelioma. The primary endpoint was overall survival (OS). A visual analog scale was used to assess EOCG performance status and cancer-related pain assessed until disease progression. The sample size provided 80% power with two-sided alpha of 0.05 to detect an increase in median OS of 5.5 months compared to historical controls (Vogelzang, JCO 2003). Results: All 80 patients had a minimum follow up of 12 months. Median age was 67 (range 27-78), 84% were male and 44% (35 patients) had an ECOG PS of 1. 66% (53 patients) had epithelioid histology, similar to the Vogelzang study. Median OS was 18.2 months (95% CI 12.1-25.8) versus 12.1 months in the historical control. Median OS for epithelioid patients was 21.2 months (95% CI 13.2-25.8). ECOG score was stable during the first year of follow up. Median time to deterioration in performance status was 13.1 months. Average score of pain was lower compared to baseline during the first 7 months of the treatment and was higher later on the study, with a median time to a clinical significant 33% increase in pain of 8.4 months. No device-related serious adverse events (AEs) were reported. Expected TTFields-related dermatitis was reported in 46% (37 patients). Four patients (5%) had grade 3 dermatitis. Conclusions: The study met primary endpoint of significant extension of overall survival in previously untreated mesothelioma patients. TTFields was not associated with a decrease in performance status or an increase in pain for the duration of TTFields use. TTFields in combination with chemotherapy is efficacious in malignant pleural mesothelioma compared to historical data.

BAP1 Status Determines the Sensitivity of Malignant Mesothelioma Cells to Gemcitabine Treatment.

Malignant mesothelioma (MMe) is a cancer with poor prognosis and resistance to standard treatments. Recent reports have highlighted the role of the BRCA1 associated protein 1 gene (BAP1) in the development of MMe. In this study, the chemosensitivity of human mesothelioma cell lines carrying BAP1 wild-type (WT), mutant and silenced was analysed. The BAP1 mutant cells were significantly less sensitive than BAP1 WT cell lines to the clinically relevant drug gemcitabine. Silencing of BAP1 significantly increased resistance of MMe cells to gemcitabine. Cell cycle analysis suggested that gemcitabine induced Sub-G1 phase accumulation of the BAP1 WT cells and increased in the S-phase in both BAP1 WT and mutant cells. Analysis of the role of BAP1 in apoptosis suggested that gemcitabine induced early apoptosis in both BAP1 WT and BAP1 mutant cells but with a much higher degree in the WT cells. Effects on the population of cells in late apoptosis, which can mark necrosis and necroptosis, could not be seen in the mutant cells, highlighting the possibility that BAP1 plays a role in several types of cell death. Significantly decreased DNA damage in the form of double-strand breaks was observed in gemcitabine-treated BAP1 mutant cells, compared to BAP1 WT cells under the same conditions. After BAP1 silencing, a significant decrease in DNA damage in the form of double-strand breaks was observed compared to cells transfected with scramble siRNA. Taken together, the results presented in this manuscript shed light on the role of BAP1 in the response of MMe cells to gemcitabine treatment and in particular in the control of the DNA damage response, therefore providing a potential route for more efficient MMe therapy.

ADAM10 mediates malignant pleural mesothelioma invasiveness

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options and treatment efficiency. Even if the latency period between asbestos exposure, the main risk factor, and mesothelioma development is very long, the local invasion of mesothelioma is very rapid leading to a mean survival of one year after diagnosis. ADAM10 (A Disintegrin And Metalloprotease) sheddase targets membrane-bound substrates and its overexpression is associated with progression in several cancers. However, nothing is known about ADAM10 implication in MPM. In this study, we demonstrated higher ADAM10 expression levels in human MPM as compared to control pleural samples and in human MPM cell line. This ADAM10 overexpression was also observed in murine MPM samples. Two mouse mesothelioma cell lines were used in this study including one primary cell line obtained by repeated asbestos fibre injections. We show, in vitro, that ADAM10 targeting through shRNA and pharmacological (GI254023X) approaches reduced drastically mesothelioma cell migration and invasion, as well as for human mesothelioma cells treated with siRNA targeting ADAM10. Moreover, ADAM10 downregulation in murine mesothelioma cells significantly impairs MPM progression in vivo after intrapleural cell injection. We also demonstrate that ADAM10 sheddase downregulation decreases the production of a soluble N-cadherin fragment through membrane N-cadherin, which stimulated mesothelioma cell migration. Taken together, we demonstrate that ADAM10 is overexpressed in MPM and takes part to MPM progression through the generation of N-cadherin fragment that stimulates mesothelioma cell migration. ADAM10 inhibition is worth considering as a therapeutic perspective in mesothelioma context.

Pyruvate Treatment Restores the Effectiveness of Chemotherapeutic Agents in Human Colon Adenocarcinoma and Pleural Mesothelioma Cells.

Emerging evidence supports the idea that a dysfunction in cell metabolism could sustain a resistant phenotype in cancer cells. As the success of chemotherapeutic agents is often questioned by the occurrence of multidrug resistance (MDR), a multiple cross-resistance towards different anti-cancer drugs represent a major obstacle to cancer treatment. The present study has clarified the involvement of the carbon metabolites in a more aggressive tumor colon adenocarcinoma phenotype and in a chemoresistant mesothelioma, and the role of pyruvate treatment in the reversion of the potentially related resistance. For the first time, we have shown that human colon adenocarcinoma cells (HT29) and its chemoresistant counterpart (HT29-dx) displayed different carbon metabolism: HT29-dx cells had a higher glucose consumption compared to HT29 cells, whereas human malignant mesothelioma (HMM) cells showed a lower glucose consumption compared to HT29 cells, accompanied by a lower pyruvate production and, consequently, a higher production of lactate. When treated with pyruvate, both HT29-dx and HMM cells exhibited a re-established accumulation of doxorubicin and a lower survival ability, a decreased activity of multidrug resistance protein 1 (MRP1) and a restored mitochondrial respiratory chain function, improving the effectiveness of the chemotherapeutic agents in these resistant cancer cells.

1807P - STELLAR: Final results of a phase II trial of TTFields with chemotherapy for first-line treatment of pleural mesothelioma

Background: Tumor Treating Fields (TTFields) are an anti-mitotic, regional treatment modality, using low intensity alternating electric fields delivered non-invasively to the tumor using a portable, medical device. In vitro, human mesothelioma cells were highly susceptible to TTFields. TTFields have been shown to extend survival of patients with glioblastoma when added to chemotherapy. Methods: The trial accrued 80 patients with unresectable, untreated mesothelioma. Patients were treated with continuous 150 kHz TTFields in combination with pemetrexed and platinum. Inclusion criteria included ECOG 0-1 and at least one measurable lesion according to modified RECIST. Patients were followed q3w (CT scan q6w) until disease progression. The primary endpoint was overall survival (OS). This single arm study assumed historical control with a median survival of 12.1 months (Vogelzang et al. 2003). The sample size provided 80% power with a two-sided alpha of 0.05 to detect an increase in median OS of 5.5 months. Results: All patients had a minimum follow up of 12 months. Median age was 67 (range 27-78), 84% were male and 56% smokers. 16% (13 patients) had metastatic disease and 44% (35 patients) had an ECOG PS of 1. 66% (53 patients) had epithelioid histology. Compliance with TTFields was 68% (16.3 hours/day) during the first 3 months of therapy. Median OS was 18.2 months (95% CI 12.1-25.8) compared to 12.1 months in historical controls. Median PFS was 7.6 months (95% CI 6.7-8.6) compared to 5.7 months in historical controls. Partial responses were seen in 40.3% of patients and clinical benefit (PR+SD) was seen in 97.2% of patients. No device-related serious adverse events (AEs) were reported. Expected TTFields-related dermatitis was reported in 46% (37 patients). Only 4 patients (5%) had grade 3 dermatitis. Conclusions: The study met its primary endpoint of significant extension of survival for previously untreated mesothelioma patients. Secondary efficacy endpoints were also improved compared to historical control. The study demonstrated no safety concerns for the combination of TTFields to the thorax with chemotherapy. These results support the addition of TTFields to chemotherapy in the first-line treatment of malignant pleural mesothelioma. Clinical trial identification: NCT02397928. Legal entity responsible for the study: Novocure. Funding: Novocure. Disclosure: J.G. Aerts: Advisory boards: BMS, MSD, Roche, AstraZeneca, Eli Lilly, Boehringer Ingelheim, Amphera; Stock owner: Amphera. R. Ramlau: Consultant: Novocure. All other authors have declared no conflicts of interest.

Berberine induces mitochondrial‑mediated apoptosis and protective autophagy in human malignant pleural mesothelioma NCI‑H2452 cells.

Increasing evidence shows that berberine has antitumor effects against a number of tumor cells. In the present study, we evaluated the effect of berberine on the proliferation of the human malignant pleural mesothelioma (MPM) cell line NCI‑H2452, and explored the therapeutic potential and underlying mechanisms of this agent. Our results showed that berberine inhibited the proliferation of NCI‑H2452 cells in a dose‑and time‑dependent manner and could induce apoptosis, possibly through a caspase‑9‑dependent intrinsic mitochondrial pathway. In addition, autophagy was induced by berberine, which was characterized by the accumulation of LC3‑II and decreased p62 expression. We used inhibitors of apoptosis and autophagy, and an inducer of autophagy, to evaluate the significance of autophagy in berberine‑induced cell death. The results demonstrated that apoptosis is the primary route through which berberine induces NCI‑H2452 cell death. Berberine‑induced autophagy may be an adaptive response to antitumor agents and have a protective role in MPM cells. Inhibition of autophagy enhanced berberine‑induced apoptosis. Therefore, inhibition of autophagy may be an effective treatment strategy in the management of MPM. In conclusion, berberine is a potent antitumor agent for treating MPM, and it induces mitochondrial‑mediated apoptosis and protective autophagy in human NCI‑H2452 MPM cells.

BAMLET kills chemotherapy-resistant mesothelioma cells, holding oleic acid in an activated cytotoxic state.

Malignant pleural mesothelioma is an aggressive cancer with poor prognosis. Here we have investigated in vitro efficacy of BAMLET and BLAGLET complexes (anti-cancer complexes consisting of oleic acid and bovine α-lactalbumin or β-lactoglobulin respectively) in killing mesothelioma cells, determined BAMLET and BLAGLET structures, and investigated possible biological mechanisms. We performed cell viability assays on 16 mesothelioma cell lines. BAMLET and BLAGLET having increasing oleic acid content inhibited human and rat mesothelioma cell line proliferation at decreasing doses. Most of the non-cancer primary human fibroblasts were more resistant to BAMLET than were human mesothelioma cells. BAMLET showed similar cytotoxicity to cisplatin-resistant, pemetrexed-resistant, vinorelbine-resistant, and parental rat mesothelioma cells, indicating the BAMLET anti-cancer mechanism may be different to drugs currently used to treat mesothelioma. Cisplatin, pemetrexed, gemcitabine, vinorelbine, and BAMLET, did not demonstrate a therapeutic window for mesothelioma compared with immortalised non-cancer mesothelial cells. We demonstrated by quantitative PCR that ATP synthase is downregulated in mesothelioma cells in response to regular dosing with BAMLET. We sought structural insight for BAMLET and BLAGLET activity by performing small angle X-ray scattering, circular dichroism, and scanning electron microscopy. Our results indicate the structural mechanism by which BAMLET and BLAGLET achieve increased cytotoxicity by holding increasing amounts of oleic acid in an active cytotoxic state encapsulated in increasingly unfolded protein. Our structural studies revealed similarity in the molecular structure of the protein components of these two complexes and in their encapsulation of the fatty acid, and differences in the microscopic structure and structural stability. BAMLET forms rounded aggregates and BLAGLET forms long fibre-like aggregates whose aggregation is more stable than that of BAMLET due to intermolecular disulphide bonds. The results reported here indicate that BAMLET and BLAGLET may be effective second-line treatment options for mesothelioma.

Adenosine diphosphate regulates MMP2 and MMP9 activity in malignant mesothelioma cells.

Although an association between cancer progression and matrix metalloproteinase (MMP) 2 and MPP9 expression has been known, the expression, nuclear localization, and physiologically controlled activation of these two MMPs have not been investigated in malignant mesothelioma cells. We examined the expression and intracellular localization of MMP2/9 in ZL55 malignant mesothelioma cells, as well as their regulation by ADP. Using real-time PCR, we showed that activation of the P2Y1 receptor by ADP increased the expression of MMP2/9 mRNAs; MMP2/9 collected from conditioned media also showed an increase in activity; and ADP induced the nuclear localization of MMP2/9. The effects of ADP on transcription of the MMPs were due to activation of c-Src, Akt, and NF-κB, while ERK1/2 phosphorylation was needed for the increase in enzymatic activity and the regulation of nuclear import. We also showed that the nuclear localization of MMP2/9 induced by ADP causes the cleavage and inactivation of poly-ADP-ribose polymerase-1. These findings may help to elucidate the mechanisms regulating MMP2/9 activation in ZL55 human epithelioid mesothelioma cells, and perhaps other cells. Therapeutic approaches that promote ADP accumulation in a tumor environment may constitute an effective means to induce anticancer activity.

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells.

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.

Abstract LB-014: Pirfenidone as a new potential therapy for malignant mesothelioma

Abstract The purpose of this study was to examine pirfenidone (PFD) as a potential new therapy for malignant pleural mesothelioma. PFD is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis (IPF). PFD reduces fibrous tissue formation e.g. by downregulating the production of fibroblasts and procollagens and inhibiting TGFß, TNFα and PDGF pathways. Since mesothelioma shares characteristics with IPF alongside with the activation of PI3K/Akt/mTOR, EGF, PDGF, HGF, VEGF, FGF and TGF-β pathways, we assessed the effect of PFD on mesothelioma cells in vitro and in vivo. In vitro studies were carried out using mesothelioma cell lines (JL-1, H2052, AB12) and primary mesothelioma cells. Cells treated with PFD and/or other agents (cisplatin, pemetrexed, MK-2206, PD98059, MG132) were subjected to cell viability assays, Transwell migration assays and 3D collagen or Matrigel growth and invasion assays. TGFβ/BMP, MAPK/Erk and Akt pathway activities were studied using luciferase reporter assays and human phospho-kinase array and validated with ELISA, RT-qPCR and Western blotting. For in vivo xenograft studies mice were injected subcutaneously with primary human mesothelioma cells expressing luciferase marker. Mice were treated with pirfenidone daily for 60 days and tumor growth was followed by bioluminescent imaging. Proliferation was studied by IHC. Differential gene expression in treated vs. untreated tumors was analyzed using RNA sequencing. We demonstrated that PFD treatment reduced significantly mesothelioma cell proliferation, migration and invasive growth in 3D in a concentration-dependent manner. The inhibitory effect of PFD increased when combined with cisplatin treatment in vitro. We also observed reduction of Akt and MAPK/Erk pathway activities and increase in BMP signaling in PFD treated tumor cells. Preliminary analyses of in vivo experiment revealed differential expression of multiple genes in pirfenidone treated vs. untreated tumors associated with a shift to more anti-fibrotic tumor microenvironment in PFD treated tumors. Expression profiles are currently under closer investigation. We identified pirfenidone as an inhibitor of mesothelioma invasive growth and showed that it has anti-fibrotic effects to tumor microenvironment. Since mesotheliomas are often inherently chemoresistant in fibrotic microenvironment, the use of pirfenidone could aid in overcoming chemoresistance by loosening ECM barrier for better drug penetration into the tumor tissue. Based on the current research we suggest that pirfenidone should be subjected to more intensive study as potential adjuvant-therapy for malignant mesotheliomas. Citation Format: Chang Li, Veronika Rezov, Emmi I. Joensuu, Ville Vartiainen, Mikko Rönty, Miao Yin, Marjukka Myllärniemi, Katri Koli. Pirfenidone as a new potential therapy for malignant mesothelioma [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 LB-014.

Experimental Model of Human Malignant Mesothelioma in Athymic Mice.

Malignant pleural mesothelioma (MPM) is a thoracic aggressive cancer caused by asbestos exposure, which is difficult to diagnose and treat. Here, we characterized an in vivo orthotopic xenograft model consisting of human mesothelioma cells (designed as H2052/484) derived from a pleural NCI-H2052 tumor injected in partially immunodeficient athymic mice. We assessed tumor formation and tumor-dependent patterns of inflammation. H2052/484 cells conserved their mesothelioma phenotype and most characteristics from the parental NCI-H2052 cells. After intra-thoracic injection of H2052/484 cells, thoracic tumors developed in nearly all mice (86%) within 14 days, faster than from parental NCI-H2052 cells. When the mice were euthanized, the pleural lavage fluid was examined for immune cell profiles. The pleural immune cell population increased with tumor development. Interestingly, the proportion of myeloid-derived suppressor cell and macrophage (especially CD206+ M2 macrophages) populations increased in the pleural fluid of mice with large mesothelioma development, as previously observed in immunocompetent mice. This reliable orthotopic model recapitulates human mesothelioma and may be used for the study of new treatment strategies.

Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients.

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859-72. ©2018 AACR.

Connective tissue growth factor-specific monoclonal antibody inhibits growth of malignant mesothelioma in an orthotopic mouse model.

Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.