Background:Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK–p53–MDM2 signalling loops were evaluated.Methods:Interactions of FAK–p53 or NF2–FAK were evaluated by phosphotyrosine-p53 immunoaffinity purification and tandem mass spectrometry, and p53, FAK, and NF2 immunoprecipitations. Activation and/or expression of FAK, p53, and NF2 were also evaluated in mesotheliomas. Effects of combination MDM2 and FAK inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, expression of cell cycle checkpoints, and cell cycle alterations.Results:We observed constitutive activation of FAK, a known negative regulator of p53, in each of 10 mesothelioma cell lines and each of nine mesothelioma surgical specimens, and FAK was associated with p53 in five of five mesothelioma cell lines. In four mesotheliomas with wild-type p53, FAK silencing by RNAi induced expression and phosphorylation of p53. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared with four mesothelioma cell lines with nonmutant p53. Additive effects were obtained through a coordinated reactivation of p53, by FAK knockdown/inhibition and MDM2 inhibition, as demonstrated by immunoblots, cell viability, and cell-cycle analyses, showing increased p53 expression, apoptosis, anti-proliferative effects, and cell-cycle arrest, as compared with either intervention alone. Our results also indicate that NF2 regulates the interaction of FAK–p53 and MDM2–p53.Conclusions:These findings highlight novel therapeutic opportunities in mesothelioma.

BackgroundInduced pluripotent stem cells (iPS) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). Mφ show great promise in disease pathogenesis, particularly tuberculosis. However, there is no information about human iPS-derived (hiPS) macrophages (hiPS-Mφ) in response to tuberculosis infection.MethodsIn the present study, macrophages derived from hiPS were established via embryoid body (EB) formation by using feeder-free culture conditions, and the human monocyte cell line THP-1 (THP-1-Mφ) was used as control. iPS-Mφ were characterized by using morphology, Giemsa staining, nonspecific esterase staining (α-NAE), phagocytosis, and surface phenotype. Additionally, after treatment with Bacillus Calmette-Guérin (BCG) for 24 h, cell apoptosis was detected by using an Annexin V-FITC Apoptosis Detection assay. The production of nitric oxide (NO), expression of tumor necrosis factor alpha (TNF-α), activity of apoptosis-related protein cysteine-3 (Caspase-3) and expression of B-cell lymphoma-2 (Bcl-2) were analyzed.ResultsWith respect to morphology, surface phenotype, and function, the iPS-Mφ closely resembled their counterparts generated in vitro from a human monocyte cell line. iPS-Mφ exhibited the typically morphological characteristics of macrophages, such as round, oval, fusiform and irregular characteristics. The cells were Giemsa-stained-positive, α-NAE-positive, and possessed phagocytic ability. iPS-Mφ express high levels of CD14, CD11b, CD40, CD68, and major histocompatibility complex II (MHC-II). Moreover, with regard to the apoptotic rate, the production of NO, expression of TNF-α, and activity of Caspase-3 and Bcl-2, iPS-Mφ closely resemble that of their counterparts generated in vitro from human monocyte cell line in response to BCG infection. The rate of apoptosis of BCG-treated iPS-Mφ was 37.77 ± 7.94% compared to that of the untreated group at 4.97 ± 1.60% (P < 0.01) by using Annexin V-FITC Apoptosis Detection. Additionally, the rate of apoptosis of BCG-treated THP-1-Mφ was 37.1 ± 2.84% compared to that of the untreated group at 6.19 ± 1.68% (P < 0.001). The expression of TNF-α and the production of NO were significantly increased (P < 0.001), and the activity of Caspase-3 was increased. However, the expression of Bcl-2 was inhibited (P < 0.001).ConclusionsOur results demonstrate that Mφ derived from hiPS perform the immunological function in response to Bacillus Calmette-Guérin infection by undergoing apoptosis, increasing the production of NO and expression of TNF-α. Thus, our study may help to overcome the limitations of research into certain rare diseases due to the lack of adequate supply of disease-specific primary cells.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0800-x) contains supplementary material, which is available to authorized users.

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