A Selective ALDH1A3 Inhibitor Impairs Mesothelioma 3-D Multicellular Spheroid Growth and Neutrophil Recruitment.

Aldehyde dehydrogenase 1A3 (ALDH1A3), one of the three members of the aldehyde dehydrogenase 1A subfamily, has been associated with increased progression and drug resistance in various types of solid tumours. Recently, it has been reported that high ALDH1A3 expression is prognostic of poor survival in patients with malignant pleural mesothelioma (MPM), an asbestos-associated chemoresistant cancer. We treated MPM cells, cultured as multicellular spheroids, with NR6, a potent and highly selective ALDH1A3 inhibitor. Here we report that NR6 treatment caused the accumulation of toxic aldehydes, induced DNA damage, CDKN2A expression and cell growth arrest. We observed that, in CDKN2A proficient cells, NR6 treatment induced IL6 expression, but abolished CXCL8 expression and IL-8 release, preventing both neutrophil recruitment and generation of neutrophil extracellular traps (NETs). Furthermore, we demonstrate that in response to ALDH1A3 inhibition, CDKN2A loss skewed cell fate from senescence to apoptosis. Dissecting the role of ALDH1A3 isoform in MPM cells and tumour microenvironment can open new fronts in the treatment of this cancer.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes' recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Unsymmetric Cisplatin-Based Pt(IV) Conjugates Containing a PARP-1 Inhibitor Pharmacophore Tested on Malignant Pleural Mesothelioma Cell Lines.

Cisplatin is widely employed as a first-line chemotherapeutic agent for many solid tumors, including malignant pleural mesothelioma (MPM). However, its clinical use is limited by heavy side effects and acquired resistance, the latter being mainly related to enhanced DNA repair. Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPis) have been carried out, with the hope that such combinations might lead to improved therapeutic efficacy against tumors. Here, the synthesis and efficacy in reducing MPM cell viability of four cisplatin-based Pt(IV) prodrugs containing the PARPi 3-aminobenzamide (3-ABA) fragment are described. The most promising conjugate is more effective than cisplatin or cisplatin/3-ABA combination, administered in equimolar doses, in inhibiting PARP-1 activity and inducing apoptosis in BRCA1/2 wild type MPM cells, grown as monolayer or as multicellular spheroids.

Specific low-frequency electromagnetic fields induce expression of active KDM6B associated with functional changes in U937 cells.

In this study, we investigated the effects of specific low-frequency electromagnetic field sequences on U937 cells, an in vitro model of human monocyte/macrophage differentiation. U937 cells were exposed to electromagnetic stimulation by means of the SynthéXer system using two similar sequences, XR-BC31 and XR-BC31/F. Each sequence was a time series of 29 wave segments, equal to a total duration of 77 min. Here, we report that exposure (4 d, once a day) of U937 cells to the XR-BC31 setting, but not to the XR-BC31/F, resulted in increased expression of the histone demethylase KDM6B along with a global reduction in histone H3 lysine 27 tri-methylation (H3K27me3). Furthermore, exposure to the XR-BC31 sequence induced differentiation of U937 cells towards a macrophage-like phenotype displaying a KDM6B dependent increase in expression and secretion of the anti-inflammatory interleukins (ILs), IL-10 and IL-4. Importantly, all the observed changes were highly dependent on the nature of the sequence. Our results open a new way of interpretation for the effects of low-frequency electromagnetic fields observed in vivo. Indeed, it is conceivable that a specific low-frequency electromagnetic fields treatment may cause the reprogramming of H3K27me3 and cell differentiation.

Agonist activation of estrogen receptor beta (ERß) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity.

BACKGROUND: Estrogen receptor (ER) ß acts as a tumor suppressor in malignant mesotheliomas. METHODS: Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ERß agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo. RESULTS: KB9520 showed significant anti-proliferative effect in ERß positive human malignant pleural mesothelioma cells in vitro. Selective activation of ERß with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells. CONCLUSIONS: Together, the data presented suggest that selective targeting of ERß may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.

A New Vista of Aldehyde Dehydrogenase 1A3 (ALDH1A3): New Specific Inhibitors and Activity-Based Probes Targeting ALDH1A3 Dependent Pathways in Glioblastoma, Mesothelioma and Other Cancers.

Aldehyde dehydrogenases of the subfamily 1A (ALDH1A) are enzymes necessary for the oxidation of all-trans or 9-cis retinal to retinoic acid (RA). Retinoic acid and its derivatives are important for normal development and maintenance of epithelia, reproduction, memory, and immune function in adults. Moreover, in recent years, it has been demonstrated that ALDH1A members are also expressed and functional in several human cancers where their role is not limited to the synthesis of RA. Here, we review the current knowledge about ALDH1A3, one of the 1A isoforms, in cancers with an emphasis on two of the deadliest tumors that affect humans: glioblastoma multiforme and mesothelioma. In both tumors, ALDH1A3 is considered a negative prognostic factor, and its level correlates with excessive proliferation, chemoresistance, and invasiveness. We also review the recent attempts to develop both ALDH1A3-selective inhibitors for cancer therapy and ALDH1A3-specific fluorescent substrates for fluorescence-guided tumor resection.

PARP1 inhibition affects pleural mesothelioma cell viability and uncouples AKT/mTOR axis via SIRT1.

Malignant Pleural Mesothelioma (MMe) is a rare but increasingly prevalent, highly aggressive cancer with poor prognosis. The aetiology of MMe is essentially a function of previous exposure to asbestos fibres, which are considered to be an early-stage carcinogen. Asbestos is toxic to human mesothelial cells (HMCs), that activate the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) to repair DNA. The targeting of PARP1 is showing considerable potential for delivering selective tumour cell kill while sparing normal cells, and offers a scientifically rational clinical application. We investigated PARP1 expression in normal mesothelial and MMe tissues samples. Immunohistochemical analysis revealed low PARP1 staining in peritumoural mesothelium. As opposite, a progressive increase in epithelioid and in the most aggressive sarcomatoid MMe tissues was evident. In MMe cell lines, we correlated increased PARP1 expression to sensitivity to its inhibitor CO-338 and demonstrated that CO-338 significantly reduced cell viability as single agent and was synergistic with cis-platin. Interestingly, we described a new correlation between PARP1 and the AKT/mTOR axis regulated by SIRT1. SIRT1 has a role in the modulation of AKT activation and PARP1 has been described to be a gatekeeper for SIRT1 activity by limiting NAD+ availability. Here, we firstly demonstrate an inverse correlation between AKT acetylation and phosphorylation modulated by SIRT1 in MMe cells treated with CO-338. In conclusion, this study demonstrates that PARP1 overexpression defines increased responsiveness to its inhibition, then these results imply that a substantial fraction of patients could be candidates for therapy with PARP inhibitors.

Melanoma Cells Inhibit iNKT Cell Functions via PGE2 and IDO1.

Invariant natural killer T (iNKT) cells are a distinct group of immune cells known for their immunoregulatory and cytotoxic activities, which are crucial in immune surveillance against tumors. They have been extensively investigated as a potential target for adoptive cell immunotherapy. Despite the initial promise of iNKT cell-based immunotherapy as a treatment for melanoma patients, its effective utilization has unfortunately yielded inconsistent outcomes. The primary cause of this failure is the immunosuppressive tumor microenvironment (TME). In this study, we specifically directed our attention towards melanoma cells, as their roles within the TME remain partially understood and require further elucidation. Methods: We conducted co-culture experiments involving melanoma cell lines and iNKT cells. Results: We demonstrated that melanoma cell lines had a significant impact on the proliferation and functions of iNKT cells. Our findings revealed that co-culture with melanoma cell lines led to a significant impairment in the expression of the NKG2D receptor and cytolytic granules in iNKT cells. Moreover, we observed a strong impairment of their cytotoxic capability induced by the presence of melanoma cells. Furthermore, through the use of selective inhibitors targeting IDO1 and COX-2, we successfully demonstrated that the melanoma cell line's ability to impair iNKT cell activation and functions was attributed to the up-regulation of IDO1 expression and PGE2 production.

Benefits and Challenges of Inhibiting EZH2 in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer that is mainly associated with prior exposure to asbestos fibers. Despite being a rare cancer, its global rate is increasing and the prognosis remains extremely poor. Over the last two decades, despite the constant research of new therapeutic options, the combination chemotherapy with cisplatin and pemetrexed has remained the only first-line therapy for MPM. The recent approval of immune checkpoint blockade (ICB)-based immunotherapy has opened new promising avenues of research. However, MPM is still a fatal cancer with no effective treatments. Enhancer of zeste homolog 2 (EZH2) is a histone methyl transferase that exerts pro-oncogenic and immunomodulatory activities in a variety of tumors. Accordingly, a growing number of studies indicate that EZH2 is also an oncogenic driver in MPM, but its effects on tumor microenvironments are still largely unexplored. This review describes the state-of-the-art of EZH2 in MPM biology and discusses its potential use both as a diagnostic and therapeutic target. We highlight current gaps of knowledge, the filling of which will likely favor the entry of EZH2 inhibitors within the treatment options for MPM patients.

Epigenetic Insights on PARP-1 Activity in Cancer Therapy.

The regulation of chromatin state and histone protein eviction have been proven essential during transcription and DNA repair. Poly(ADP-ribose) (PAR) polymerase 1 (PARP-1) and poly(ADP-ribosyl)ation (PARylation) are crucial mediators of these processes by affecting DNA/histone epigenetic events. DNA methylation/hydroxymethylation patterns and histone modifications are established by mutual coordination between all epigenetic modifiers. This review will focus on histones and DNA/histone epigenetic machinery that are direct targets of PARP-1 activity by covalent and non-covalent PARylation. The effects of these modifications on the activity/recruitment of epigenetic enzymes at DNA damage sites or gene regulatory regions will be outlined. Furthermore, based on the achievements made to the present, we will discuss the potential application of epigenetic-based therapy as a novel strategy for boosting the success of PARP inhibitors, improving cell sensitivity or overcoming drug resistance.

The Strange Case: The Unsymmetric Cisplatin-Based Pt(IV) Prodrug [Pt(CH3COO)Cl2(NH3)2(OH)] Exhibits Higher Cytotoxic Activity with respect to Its Symmetric Congeners due to Carrier-Mediated Cellular Uptake.

The biological behavior of the axially unsymmetric antitumor prodrug (OC-6-44)-acetatodiamminedichloridohydroxidoplatinum(IV), 2, was deeply investigated and compared with that of analogous symmetric Pt(IV) complexes, namely, dihydroxido 1 and diacetato 3, which have a similar structure. The complexes were tested on a panel of human tumor cell lines. Complex 2 showed an anomalous higher cytotoxicity (similar to that of cisplatin) with respect to their analogues 1 and 3. Their reduction potentials, reduction kinetics, lipophilicity, and membrane affinity are compared. Cellular uptake and DNA platination of Pt(IV) complexes were deeply investigated in the sensitive A2780 human ovarian cancer cell line and in the corresponding resistant A2780cisR subline. The unexpected activity of 2 appears to be related to its peculiar cellular accumulation and not to a different rate of reduction or a different efficacy in DNA platination and/or efficiency in apoptosis induction. Although the exact mechanism of cell uptake is not fully deciphered, a series of naïve experiments indicates an energy-dependent, carrier-mediated transport: the organic cation transporters (OCTs) are the likely proteins involved.

Corrigendum: CDKN2A determines mesothelioma cell fate to EZH2 inhibition.

[This corrects the article DOI: 10.3389/fonc.2021.678447.].

Malignant pleural mesothelioma: Germline variants in DNA repair genes may steer tailored treatment.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a tumour associated with asbestos exposure. Approximately, 10% of patients with MPM carry a germline pathogenic variant (PV), mostly in DNA repair genes, suggesting the occurrence of inherited predispositions. AIM: This article aimed to 1) search for new predisposing genes and assess the prevalence of PVs in DNA repair genes, by next-generation sequencing (NGS) analysis of germline DNA from 113 unselected patients with MPM and 2) evaluate whether these patients could be sensitive to tailored treatments. METHODS: NGS was performed using a custom panel of 107 cancer-predisposing genes. To investigate the response to selected drugs in conditions of DNA repair insufficiency, we created a three-dimensional-MPM cell model that had a defect in ataxia telangiectasia mutated (ATM), the master regulator of DNA repair. RESULTS: We identified PVs in approximately 7% of patients with MPM (8/113) and a new PV in BAP1 in a further patient with familial MPM. Most of these PVs were in genes involved or supposedly involved in DNA repair (BRCA1, BRIP1, CHEK2, SLX4, FLCN and BAP1). In vitro studies showed apoptosis induction in ATM-silenced/inhibited MPM spheroids treated with an enhancer of zeste homologue 2 inhibitor (tazemetostat). CONCLUSIONS: Overall these data suggest that patients with MPM and DNA repair insufficiency may benefit from this treatment, which induces synthetic lethality.

Expression and clinical implications of estrogen receptors in thoracic malignancies: a narrative review.

Thoracic malignancies represent a significant global health burden with incidence and mortality increasing year by year. Thoracic cancer prognosis and treatment options depend on several factors, including the type and size of the tumor, its location, and the overall health status of patients. Gender represents an important prognostic variable in thoracic malignancies. One of the greatest biological differences between women and men is the presence of female sex hormones, and an increasing number of studies suggest that estrogens may play either a causative or a protective role in thoracic malignancies. Over the past 60 years since the discovery of the first nuclear estrogen receptor (ER) isoform α and the almost 20 years since the discovery of the second estrogen receptor, ERß, different mechanisms governing estrogen action have been identified and characterized. This literature review reports the published data regarding the expression and function of ERs in different thoracic malignancies and discuss sex disparity in clinical outcomes. From this analysis emerges that further efforts are warranted to better elucidate the role of sex hormones in thoracic malignancies, and to reduce disparities in care between genders. Understanding the mechanisms by which gender-related differences can affect and interfere with the onset and evolution of thoracic malignancies and impact on response to therapies could help to improve the knowledge needed to develop increasingly personalized and targeted treatments.

CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition.

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis.

Transglutaminase 2 maintains a colorectal cancer stem phenotype by regulating epithelial-mesenchymal transition.

Transglutaminase 2 (TG2), a multifunctional protein, is reported in regulating the cancer stem cell (CSC) phenotype in various cancers. Our previous work suggested the link between TG2 and Epithelial-Mesenchymal Transition (EMT) in colorectal cancer (CRC). Here we demonstrate the importance of TG2 in CSC development in human CRC cell lines HCT116 and SW620. CRC spheroid cells showed increased CSC characteristics over their monolayer cells with increased expression of CD44 and over expression of Oct3/4, Sox2 and Nanog. They also showed increased EMT and invasiveness, and enhanced expression of TG2. TG2 inhibition by its selective inhibitor 1-155 reduced both spheroid formation and invasive potential of the spheroid cells. ß-catenin, a mediator of stem cell maintenance, was overexpressed in the spheroid cells and could be attenuated by TG2 inhibition. Spheroid cells possessed increased angiogenesis stimulating ability via overexpression of Vascular Endothelial Growth Factor (VEGF). Increased VEGF was present in the culture media from spheroid cells when compared to monolayer cultures which could be reduced by selective inhibition by 1-155. Stemness and malignancy in the colorectal spheroid cells was associated with increased TG2, EMT, ß-catenin and VEGF. Here we demonstrate that inhibiting TG2 reduces both stemness and angiogenic stimulating activity in CRC.

Targeting estrogen receptor beta (ERß) for treatment of ovarian cancer: importance of KDM6B and SIRT1 for ERß expression and functionality.

Estrogen receptor (ER) ß has growth inhibitory and chemo drug potentiating effect on ovarian cancer cells. We studied the dependence of ERß function on the presence of KDM6B and SIRT1 in human ovarian cancer cells in vitro. Activation of ERß with the subtype-selective agonist KB9520 resulted in significant inhibition of human ovarian cancer cell growth. KB9520-activated ERß had an additive effect on growth inhibition in combination with cisplatin and paclitaxel, respectively. Loss of KDM6B expression had a negative effect on ERß function as a ligand-dependent inhibitor of ovarian cancer cell growth. In contrast, loss or inhibition of SIRT1 deacetylase activity restored ligand-activated ERß functionality. Presented data suggest that selective targeting of ERß with an agonist potentiate chemotherapy efficacy for the treatment of ovarian cancer and that downregulation or inhibition of SIRT1 may further enhance its therapeutic effect.

Tissue transglutaminase (TG2) enables survival of human malignant pleural mesothelioma cells in hypoxia.

Malignant pleural mesothelioma (MPM) is an aggressive tumor linked to environmental/occupational exposure to asbestos, characterized by the presence of significant areas of hypoxia. In this study, we firstly explored the expression and the role of transglutaminase 2 (TG2) in MPM cell adaptation to hypoxia. We demonstrated that cells derived from biphasic MPM express the full-length TG2 variant at higher levels than cells derived from epithelioid MPM and normal mesothelium. We observed a significant induction of TG2 expression and activity when cells from biphasic MPM were grown as a monolayer in chronic hypoxia or packed in spheroids, where the presence of a hypoxic core was demonstrated. We described that the hypoxic induction of TG2 was HIF-2 dependent. Importantly, TGM2-v1 silencing caused a marked and significant reduction of MPM cell viability in hypoxic conditions when compared with normoxia. Notably, a TG2-selective irreversible inhibitor that reacts with the intracellular active form of TG2, but not a non-cell-permeable inhibitor, significantly compromised cell viability in MPM spheroids. Understanding the expression and function of TG2 in the adaptation to the hypoxic environment may provide useful information for novel promising therapeutic options for MPM treatment.

SIRT1 at the crossroads of AKT1 and ERß in malignant pleural mesothelioma cells.

In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects.We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1.Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERß. We further demonstrate an inhibitory feedback loop by ERß, activated by the selective agonist KB9520, on this axis both in vitro and in vivo.Our data broaden the current knowledge of ERß and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM.

KDM6B histone demethylase is an epigenetic regulator of estrogen receptor ß expression in human pleural mesothelioma.

AIM: To assess the correlation between KDM6B and estrogen receptor ß (ERß) expression in malignant pleural mesothelioma (MPM). MATERIALS & METHODS: We evaluated gene expression by in silico analysis of microarray data, real-time PCR and western blot in MPM tumors and cell lines. RESULTS & CONCLUSION: We report a strong positive correlation between the expression of KDM6B and ERß in MPM tumors and cell lines. We describe that, in hypoxia, the HIF2α-KDM6B axis induces an epithelioid morphology and ERß expression in biphasic MPM cells with estrogen receptor-negative phenotype. Reduced histone H3K27 tri-methylation confirms KDM6B activity under hypoxic conditions. Importantly, cells treated during reoxygenation with the selective ERß agonist, KB9520, maintain ERß expression and the less aggressive phenotype acquired in hypoxia.