Normal Mesothelial Cells Research Articles

Expression changes of miRNAs and EMT-related genes in human mesothelial cells induced by long-term exposure to asbestos

Objective: To investigate the effects of long-term exposure to chrysotile and crocidolite on miRNAs and epithelial mesenchymal transformation (EMT) -related gene expression in human pleural mesothelial cells. Methods: In November 2020, fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of EMT-related genes in human pleural mesothelioma cells (NCl-H2052 cells, NCl-H2452 cells) and human normal mesothelial cells (Met-5A cells). MiRNAs with abnormal expression in human pleural mesothelioma cells were screened out from the previous miRNA chip data of research group, and target genes of differentially expressed miRNAs were predicted using miRWalk database (http: //mirwalk.umm.uni-heidelberg.de). RT-qPCR was used to verify the abnormal expression of EMT-related miRNAs in cell lines. Met-5A cells were treated with 5μg/cm(2) chrysotile and crocidolite respectively for 48 h a time, once a week and a total of 10 times. Chrysotile group, crocidolite group and control group were set up. And the control group was added with the same volume of PBS. The expression changes of EMT-related genes and abnormal expression miRNAs in each group were detected by RT-qPCR. The differences among the groups were compared by one-way ANOVA, and the differences between the control group and the experimental group were compared by dunnet-t test. Results: Compared with Met-5A cells, the expression levels of Vimentin and Twist genes were increased, and the expression level of E-cadherin genes was decreased in NCl-H2052 cells and NCl-H2452 cells (P<0.001). Target genes of miRNAs with abnormal expression in miRNA chip were predicted, and the results showed four abnormally expressed miRNAs associated with EMT and verified the expression of these four miRNAs in the cell lines. Compared with Met-5A cells, the expression level of hsa-miR-155-5p was increased in NCl-H2052 cells and NCl-H2452 cells, the expression levels of hsa-miR-34b-5p, hsa-miR-34c-5p and hsa-miR-28-5p were decreased in NCl-H2052 cells and NCl-H2452 cells (P<0.001), which was consistent with the results of chip analysis. After exposure of Met-5A cells, it was found that compared with the control group, the expression levels of Vimentin and Twist genes, hsa-miR-155-5p, hsa-miR-34b-5p and hsa-miR-34c-5p in the crocidolite group were increased, while the expression level of E-cadherin gene was decreased (P<0.05). Compared with the control group, the expression levels of Vimentin, Twist and E-cadherin genes in chrysotile group were increased, while the expression levels of hsa-miR-34b-5p, hsa-miR-34c-5p and hsa-miR-28-5p were decreased (P<0.05) . Conclusion: Long-term exposure to chrysotile and crocidolite could cause Met-5A cells to produce miRNAs and EMT-related gene expression changes similar to mesothelioma cells.

Small Extracellular Vesicle-Derived Circular RNA hsa_circ_0007386 as a Biomarker for the Diagnosis of Pleural Mesothelioma.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools.

Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Abstract 7545: Epigenetic plasticity in rewiring DNMT1/UHRF1-RB1- NFkB signaling circuit regulates asbestos-induced mesothelial malignant transformation

Abstract The exact mechanisms underlying the causal relationship between asbestos exposure and malignant mesothelioma (MM) have yet to be elucidated. Widespread genomic and epigenomic alterations are being implicated in rewiring of oncogenic signaling networks which may contribute to initiation and progression of MM. In the present study, we examined the molecular profiles of mesothelial cells exposed to asbestos in-vitro and found that asbestos-induced activation of DNMT1/UHRF1 mediated epigenetic repression of RB1 in normal mesothelial cells as well as mesothelioma cells via promoter-specific methylation mechanisms. RB1, as a noncanonical transcription factor, was capable of also negatively modulating the transcriptional activities of DNMT1/UHRF1 mesothelium and mesothelioma. Functioning as one of the downstream effectors of RB1, NFkB was also involved in the transcriptional regulation of DNMT1/UHRF1 directly in a promoter-selective manner and played a pivotal role in the acute as well as chronic functional and phenotypic changes of mesothelial cells following asbestos exposure. Pharmacological interference of the dysregulated RB1-DNMT1/UHRF1- RB1-NFkB signaling circuit constitutively arrested the invasive growth of MPM cells. In-vivo studies using FVB genetic background wild-type and Bap1+/- mice further revealed involvement of epigenetically dysregulation of the RB1-DNMT1/UHRF1- RB1-NFkB signaling circuit during asbestos-induced malignant transformation of mesothelial cells. Taken together, our data are the first to describe that asbestos epigenetically rewires the RB1-DNMT1/UHRF1- RB1-NFkB signaling circuit during mesothelioma development and suggest potential novel strategies to reverse these epigenetic perturbations for MPM treatment in the clinic. Citation Format: Sichuan Xi, Xinwei Wu, David M Straughan, Eden C Payabyab, Haitao Wang, Ruihong Wang, Mary Zhang, Shamus R. Carr, Chuong D. Hoang, David S. Schrump. Epigenetic plasticity in rewiring DNMT1/UHRF1-RB1- NFkB signaling circuit regulates asbestos-induced mesothelial malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7545.

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Effects of microRNA-106b on migration and invasion of human malignant pleural mesothelioma cell NCI-H2452

Objective: To investigate the inhibitory effect of microRNA-106b in the process of migration and invasion of human malignant pleural mesothelioma cell NCI-H2452. Methods: In April 2017, the expression level of miRNA-106b in malignant pleural mesothelioma cells (NCI-H2452, MSTO-211H, NCI-H2052) and normal mesothelial cells MeT-5A was detected and analyzed. Using NCI-H2452 cells as a model, the NCI-H2452 cell model with miRNA-106b overexpression was established by transfecting miRNA-106b mimics. The expression level of miRNA-106b in the cells was detected by real-time fluorescent quantitative PCR. The effect of miRNA-106b on the migration and invasion ability of NCI-H2452 cells was analyzed. The gene expression data of malignant mesothelioma and the downstream target gene data of miRNA-106b in public databases were analyzed to screen the downstream target genes of miRNA-106b in mesothelioma cells that affect cell migration and invasion ability, and to verify the expression of this gene in NCI-H2452 cells with miRNA-106b overexpression. Results: The expression of miRNA-106b in three MPM cells was decreased compared with MeT-5A cells (P<0.001) . The expression level of miRNA-106b was significantly increased after transfection of miRNA-106b mimics (P<0.001) . The scratch migration levels of the experimental group were 28.45%±4.37%, 38.12%±4.82% and 50.06%±8.92% at 24h, 31h and 48h, respectively. Compared with the control group, the migration level decreased by 37.48%±2.65%, 49.21%±3.45% and 68.14%±3.81% (P<0.01) . The number of cell migration and invasion decreased in the experimental group compared with the control group (P<0.001) . Public databases were used to screen and analyze the possibility that TCF21 gene, as a downstream target gene, could affect the migration and invasion ability of MPM cells. The expression level of TCF21 gene was increased after transfection of miRNA-106b mimics in NCI-H2452 cells (P=0.009) . Conclusion: MiRNA-106b can inhibit the migration and invasion of NCI-H2452 cells and increase the expression of TCF21 gene.

MicroRNAs Present in Malignant Pleural Fluid Increase the Migration of Normal Mesothelial Cells In Vitro and May Help Discriminate between Benign and Malignant Effusions.

The sensitivity of pleural fluid (PF) analyses for the diagnosis of malignant pleural effusions (MPEs) is low to moderate. Knowledge about the pathobiology and molecular characteristics of this condition is limited. In this study, the crosstalk between stromal cells and tumor cells was investigated in vitro in order to reveal factors that are present in PF which can mediate MPE formation and aid in discriminating between benign and malignant etiologies. Eighteen PF samples, in different proportions, were exposed in vitro to mesothelial MeT-5A cells to determine the biological effects on these cells. Treatment of normal mesothelial MeT-5A cells with malignant PF increased cell viability, proliferation, and migration, and activated different survival-related signaling pathways. We identified differentially expressed miRNAs in PF samples that could be responsible for these changes. Consistently, bioinformatics analysis revealed an enrichment of the discovered miRNAs in migration-related processes. Notably, the abundance of three miRNAs (miR-141-3p, miR-203a-3, and miR-200c-3p) correctly classified MPEs with false-negative cytological examination results, indicating the potential of these molecules for improving diagnosis. Malignant PF produces phenotypic and functional changes in normal mesothelial cells. These changes are partly mediated by certain miRNAs, which, in turn, could serve to differentiate malignant from benign effusions.

Chronic Inflammation, Oxidative Stress and Metabolic Plasticity: Three Players Driving the Pro-Tumorigenic Microenvironment in Malignant Mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal and rare cancer, even if its incidence has continuously increased all over the world. Asbestos exposure leads to the development of mesothelioma through multiple mechanisms, including chronic inflammation, oxidative stress with reactive oxygen species (ROS) generation, and persistent aberrant signaling. Together, these processes, over the years, force normal mesothelial cells' transformation. Chronic inflammation supported by "frustrated" macrophages exposed to asbestos fibers is also boosted by the release of pro-inflammatory cytokines, chemokines, growth factors, damage-associated molecular proteins (DAMPs), and the generation of ROS. In addition, the hypoxic microenvironment influences MPM and immune cells' features, leading to a significant rewiring of metabolism and phenotypic plasticity, thereby supporting tumor aggressiveness and modulating infiltrating immune cell responses. This review provides an overview of the complex tumor-host interactions within the MPM tumor microenvironment at different levels, i.e., soluble factors, metabolic crosstalk, and oxidative stress, and explains how these players supporting tumor transformation and progression may become potential and novel therapeutic targets in MPM.

#4387 LIQUID BIOPSY IN PATIENTS TREATED WITH BIOCOMPATIBLE PERITONEAL DIALYSIS FLUIDS – ITS RELATIONSHIP WITH CYTOLOGICAL AND HISTOLOGICAL FINDINGS

Abstract Background and Aims Peritoneal fibrosis can limit long term peritoneal membrane function in peritoneal dialysis (PD) patients. It has been related to bioincompatible PD fluids and peritoneal infections. There are no prospective studies regarding correlation between effluent biomarkers and peritoneal biopsy findings. Our aim was to study the possible relation of peritoneal effluent biomarkers and peritoneal morphology alterations. Method Multicenter prospective study in a cohort of stable PD patients treated with biocompatible PD fluids who received a kidney transplant. We collected in each patient peritoneal biopsy samples and peritoneal effluent fluid. We analyzed the relation between effluent biomarkers, phenotype of mesothelial cells (MC) cultured ex vivo, and peritoneal biopsy parameters (mesothelial integrity, fibrosis and hyalinizing vasculopathy). We compared them with patient characteristics, including peritoneal transport parameters. The biomarkers tested were Collagen-1 (COL-1), Fibronectin (FN), Collagen-13 (COL-13), Interleukin-6 (IL-6), Trombospondin-1, Cadherin 13 (CDH-13), CA-125, Gremlin-1 (GREM-1), Matrix metalloproteinase 2 (MMP-2), CC chemokine ligand 18 (CCL18), Plasminogen activator inhibitor 1 (PAI-1) and Vascular endothelial growth factor A (VEGF-A). Results Forty patients were included (mean age 54.5±15 years, 64% male, 24.1% diabetics). Mean time on PD was 25.5±27 months, 62.1% were on automated PD (APD) and 17.2% had prior peritonitis episodes. A normal MC culture phenotype was observed in 78% of patients. In peritoneal biopsies samples we found partial or total mesothelium preservation in 39%, submesothelial thickness in 42% and vasculopathy in 14% of cases. Effluent PAI-1 levels were significantly lower in patients with mesothelial cell loss than in those with mesothelial preservation (21.62 vs. 38.59 pg/ml respectively; p = 0.031). Patients with peritoneal fibrosis showed significantly higher effluent CDH-13 levels (3.51 vs. 2.32 pg/ml; p = 0.048) and supernatant PAI-1 levels (1183 vs. 72.73; p = 0.000) than those without fibrosis. No other statistical differences were found in other biomarkers analysed. Conclusion Most patients treated with biocompatible peritoneal dialysis fluids showed normal mesothelial cell phenotype in peritoneal effluent culture. Lower PAI-1 effluent levels were associated with mesothelial cell loss and higher effluent CDH-13 levels were related to peritoneal fibrosis.

Expression of CD24 gene in human malignant pleural mesothelioma and its relationship with prognosis

Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.

Identification of a derivative of the alkaloid emetine as an inhibitor of the YAP-TEAD interaction and its potential as an anticancer agent.

TEAD is a transcription factor responsible for the output of the tumor suppressor Hippo pathway. The transcriptional activity of TEAD requires molecular interaction with its transcriptional coactivator, YAP. Aberrant activation of TEAD is deeply involved in tumorigenesis and is associated with poor prognosis, suggesting that inhibitors targeting the YAP-TEAD system are promising as antitumor agents. In this study, we identified NPD689, an analog of the natural product alkaloid emetine, as an inhibitor of the YAP-TEAD interaction. NPD689 suppressed the transcriptional activity of TEAD and reduced the viability of human malignant pleural mesothelioma and non-small cell lung cancer cells but not the viability of normal human mesothelial cells. Our results suggest that NPD689 is not only a new useful chemical tool for elucidating the biological role of the YAP-TEAD system but also has potential as a starting compound for developing a cancer therapeutic agent that targets the YAP-TEAD interaction.

Small RNA-Seq Transcriptome Profiling of Mesothelial and Mesothelioma Cell Lines Revealed microRNA Dysregulation after Exposure to Asbestos-like Fibers

Environmental exposure to fibers of respirable size has been identified as a risk for public health. Experimental evidence has revealed that a variety of fibers, including fluoro-edenite, can develop chronic respiratory diseases and elicit carcinogenic effects in humans. Fluoro-edenite (FE) is a silicate mineral first found in Biancavilla (Sicily, Italy) in 1997. Environmental exposure to its fibers has been correlated with a cluster of malignant pleural mesotheliomas. This neoplasm represents a public health problem due to its long latency and to its aggression not alerted by specific symptoms. Having several biomarkers providing us with data on the health state of those exposed to FE fibers or allowing an early diagnosis on malignant pleural mesothelioma, still asymptomatic patients, would be a remarkable goal. To these purposes, we reported the miRNA transcriptome in human normal mesothelial cell line (MeT-5A) and in the human malignant mesothelioma cell line (JU77) exposed and not exposed to FE fibers. The results showed a difference in the number of deregulated miRNAs between tumor and nontumor samples both exposed and not exposed to FE fibers. As a matter of fact, the effect of exposure to FE fibers is more evident in the expression of miRNA in the tumor samples than in the nontumor samples. In the present paper, several pathways involved in the pathogenesis of malignant pleural mesothelioma have been analyzed. We especially noticed the involvement of pathways that have important functions in inflammatory processes, angiogenesis, apoptosis, and necrosis. Besides this amount of data, further studies will be designed for the selection of the most significant miRNAs to test and validate their diagnostic potential, alone or in combination with other protein biomarkers, in high-risk individuals' liquid biopsy to have a noninvasive tool of diagnosis for this neoplasm.

Inflammation as a chemoprevention target in asbestos-induced malignant mesothelioma.

Malignant mesothelioma (MM) is an incurable cancer of the serosal lining that is often caused by exposure to asbestos. Therefore, novel agents for the prevention and treatment of this disease are urgently needed. Asbestos induces the release of pro-inflammatory cytokines such as IL-1β and IL-6, which play a role in MM development. IL-6 is a component of the JAK-STAT3 pathway that contributes to inflammation-associated tumorigenesis. Glycoprotein 130 (gp130), the signal transducer of this signaling axis, is an attractive drug target because of its role in promoting neoplasia via the activation of downstream STAT3 signaling. The anticancer drug, SC144, inhibits the interaction of gp130 with the IL-6 receptor (IL6R), effectively blunting signaling from this inflammatory axis. To test whether the inflammation-related release of IL-6 plays a role in the formation of MM, we evaluated the ability of SC144 to inhibit asbestos-induced carcinogenesis in a mouse model. The ability of sulindac and anakinra, an IL6R antagonist/positive control, to inhibit MM formation in this model was tested in parallel. Asbestos-exposed Nf2+/-;Cdkn2a+/- mice treated with SC144, sulindac or anakinra showed significantly prolonged survival compared to asbestos-exposed vehicle-treated mice. STAT3 activity was markedly decreased in MM specimens from SC144-treated mice. Furthermore, SC144 inhibited STAT3 activation by IL-6 in cultured normal mesothelial cells, and in vitro treatment of MM cells with SC144 markedly decreased the expression of STAT3 target genes. The emerging availability of newer, more potent SC144 analogs showing improved pharmacokinetic properties holds promise for future trials, benefitting individuals at high risk of this disease.

Analysis of More than 16,000 Human Tumor and Normal Tissues Identifies Uroplakin 3B as a Useful Diagnostic Marker for Mesothelioma and Normal Mesothelial Cells.

Uroplakin 3B (Upk3b) is involved in stabilizing and strengthening the urothelial cell layer of the bladder. Based on RNA expression studies, Upk3b is expressed in a limited number of normal and tumor tissues. The potential use of Upk3b as a diagnostic or prognostic marker in tumor diagnosis has not yet been extensively investigated. A tissue microarray containing 17,693 samples from 151 different tumor types/subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. In normal tissues, Upk3b expression was largely limited to mesothelial cells, urothelial umbrella cells, and amnion cells. In tumor tissues, Upk3b was detectable in only 17 of 151 (11.3%) of tumor types. Upk3b expression was most frequent in mesotheliomas (82.1% of epithelioid and 30.8% of biphasic) and in urothelial tumors of the urinary bladder, where the positivity rate decreased from 61.9% in pTaG2 (low grade) to 58.0% in pTaG3 (high grade) and 14.6% in pT2-4 cancers. Among pT2-4 urothelial carcinomas, Upk3b staining was unrelated to tumor stage, lymph node status, and patient prognosis. Less commonly, Upk3b expression was also seen in Brenner tumors of the ovary (10.8%), as well as in four other subtypes of ovarian cancer (0.9–10.6%). Four additional tumor entities showed a weak to moderate Upk3b positivity in less than 5% of cases. In summary, Upk3b immunohistochemistry is a useful diagnostic tool for the distinction of mesotheliomas from other thoracic tumors and the visualization of normal mesothelial and umbrella cells.

Synthetic Secoisolariciresinol Diglucoside (LGM2605) Prevents Asbestos-Induced Inflammation and Genotoxic Cell Damage in Human Mesothelial Cells.

Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm2). Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release (IL-1β, IL-18, IL-6, TNFα, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1β and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL ± 0.2 pg/mL and 5.4 pg/mL ± 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high (289.1 pg/mL ± 2.9 pg/mL and 511.3 pg/mL ± 10.2 pg/mL, respectively), while remaining undetectable with LGM2605 pretreatment. HMGB1 (a key inflammatory mediator and initiator of malignant transformation) release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells.

Artichoke phytocomplex modulates serum microRNAs in patients exposed to asbestos: a first step of a phase II clinical trial

BackgroundMalignant pleural mesothelioma is a highly aggressive tumor associated with asbestos exposure. There are few effective treatment options for mesothelioma, and patients have a very poor prognosis. Mesothelioma has the potential to represent an appropriate disease to prevent because of its strong association with asbestos exposure and the long latency from exposure to the disease on-set.MethodsIn the present study, we tested biological activity and toxicity of an artichoke freeze-dried extract (AWPC) as potential complementary preventive/early stage treatment agent for mesothelioma. This phase II clinical study then was conducted in 18 male-patients with evidence of radiographic characteristics related to asbestos exposure such as asbestosis or benign pleural disease as surrogate disease for mesothelioma clinical model.ResultsWe investigate AWPC biological activity assessing its effect on mesothelin serum level, a glycoprotein with low expression in normal mesothelial cells and high expression in mesothelioma and asbestos related diseases. We also assess the AWPC effect on circulating miRNAs, as novel biomarkers of both cancer risk and response to therapeutic targets. While we found a small and not significant effect of AWPC on mesothelin serum levels, we observed that AWPC intake modulated 11 serum miRNAs related to gene-pathways connected to mesothelioma etiology and development. In terms of toxicity, we also did not observe any severe adverse effects associated to AWPC treatment, only gastro-intestinal symptoms were reported by five study participants.ConclusionsWe observed an interesting AWPC effect on miRNAs which targets modulate mesothelioma development. New and much larger clinical studies based on follow-up of workers exposed to asbestos are needed to corroborate the role of AWPC in prevention and early treatment of mesothelioma.Trial registrationClinicalTrials.gov, NCT02076672. Registered 03/03/2014.

Abstract 97: Mechanisms of action and regulation of the progranulin/EphA2 axis in mesothelioma

Abstract Mesothelioma is an aggressive tumor with very poor prognosis. The limited availability of therapeutic options for mesothelioma patients suggests the need for a better understanding of mesothelioma biology and the identification of novel therapeutic targets. Progranulin is a pluripotent growth factor whose dysregulation is implicated in several human pathologies including cancer. The pro-tumorigenic activity of progranulin depends on the activation of its functional receptor EphA2. Indeed, in bladder cancer, progranulin promotes EphA2 phosphorylation at Ser897 and triggers EphA2 oncogenic action resulting in increased tumor cell migration, invasion and in vivo tumor growth. Both progranulin and EphA2 are expressed in mesothelioma, with progranulin promoting tumor angiogenesis in a VEGF-independent manner and EphA2 being often mutated or overexpressed. However, in mesothelioma, the potential functional interaction between progranulin and EphA2 is unexplored. Using a panel of mesothelioma cell lines covering all mesothelioma subtypes, we observed increased levels of phosphorylated EphA2 at Ser897 when compared to immortalized normal mesothelial cells, and the levels of phosphorylation positively correlated with progranulin expression. Progranulin-dependent activation of EphA2 in mesothelioma cells promoted the activation of the MAPK and AKT pathways and ERK1/2-dependent EphA2 phosphorylation at Ser897. Transient depletion of progranulin by siRNA approaches as well as GRN gene knock-out by CRISP/Cas9 strategies in mesothelioma cells reduced the activation of the AKT signaling pathway and inhibited the migratory and invasive capabilities of progranulin-depleted cells when compared to parental cells, suggesting that the progranulin/EphA2 axis is oncogenic in mesothelioma. However, EphA2 genetic ablation resulted in an increased basal activation of AKT and MAPK and in progranulin-evoked EphA2-independet AKT/MAPK activation, indicating that alternative membrane receptors might compensate for EphA2 loss and sustain progranulin action in mesothelioma. Using several pharmacological and genetic approaches, we uncovered a role for EGFR and FAK in compensating for EphA2 loss by triggering progranulin-dependent oncogenic signaling in mesothelioma, highlighting a complex functional cross-talk between EphA2, EGFR and FAK in mediating progranulin-dependent tumorigenesis. Thus, these results suggest the need for combinatorial therapeutic approaches to blunt progranulin oncogenic action in mesothelioma. Citation Format: Elisa Ventura, Simone Buraschi, Stephen J. Williams, Antonino Belfiore, Renato V. Iozzo, Antonio Giordano, Andrea Morrione. Mechanisms of action and regulation of the progranulin/EphA2 axis in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 97.

Aquaporin-6 May Increase the Resistance to Oxidative Stress of Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleural surface and is associated with previous asbestos exposure. The chemotherapy drug is one of the main treatments, but the median survival ranges from 8 to 14 months from diagnosis. The redox homeostasis of tumor cells should be carefully considered since elevated levels of ROS favor cancer cell progression (proliferation and migration), while a further elevation leads to ferroptosis. This study aims to analyze the functioning/role of aquaporins (AQPs) as a hydrogen peroxide (H2O2) channel in epithelial and biphasic MPM cell lines, as well as their possible involvement in chemotherapy drug resistance. Results show that AQP-3, -5, -6, -9, and -11 were expressed at mRNA and protein levels. AQP-6 was localized in the plasma membrane and intracellular structures. Compared to normal mesothelial cells, the water permeability of mesothelioma cells is not reduced by exogenous oxidative stress, but it is considerably increased by heat stress, making these cells resistant to ferroptosis. Functional experiments performed in mesothelioma cells silenced for aquaporin-6 revealed that it is responsible, at least in part, for the increase in H2O2 efflux caused by heat stress. Moreover, mesothelioma cells knocked down for AQP-6 showed a reduced proliferation compared to mock cells. Current findings suggest the major role of AQP-6 in providing mesothelioma cells with the ability to resist oxidative stress that underlies their resistance to chemotherapy drugs.

Dysregulation of microRNAs and tRNA-derived ncRNAs in mesothelial and mesothelioma cell lines after asbestiform fiber exposure

Experimental evidence demonstrated that fluoro-edenite (FE) can develop chronic respiratory diseases and elicit carcinogenic effects. Environmental exposure to FE fibers is correlated with malignant pleural mesothelioma (MPM). An early diagnosis of MPM, and a comprehensive health monitoring of the patients exposed to FE fibers are two clinical issues that may be solved by the identification of specific biomarkers. We reported the microRNA (miRNA) and transfer RNA-derived non coding RNA (tRNA-derived ncRNA) transcriptome in human normal mesothelial and malignant mesothelioma cell lines exposed or not exposed to several concentration FE fibers. Furthermore, an interactive mesothelioma-based network was derived by using NetME tool. In untreated condition, the expression of miRNAs and tRNA-derived ncRNAs in tumor cells was significantly different with respect to non-tumor samples. Moreover, interesting and significant changes were found after the exposure of both cells lines to FE fibers. The network-based pathway analysis showed several signaling and metabolic pathways potentially involved in the pathogenesis of MPM. From papers analyzed by NetME, it is clear that many miRNAs can positively or negatively influence various pathways involved in MPM. For the first time, the analysis of tRNA-derived ncRNAs molecules in the context of mesothelioma has been made by using in vitro systems. Further studies will be designed to test and validate their diagnostic potential in high-risk individuals' liquid biopsies.

Pharmacological reactivation of RBL2/p130 could be an effective antitumoral strategy for malignant pleural mesothelioma

Malignant mesothelioma (MM) is an aggressive tumor developing from the mesothelium covering the body cavities. The most common MM type affects the pleura surrounding the lungs. MM is mainly associated with asbestos exposure. At present, no curative modalities are effective against MM exist. The molecular mechanisms underlying MM development seem to depend mostly on the inactivation of tumor suppressors, among which also the dysfunction of the retinoblastoma (RB) protein pathway that is a hallmark of cancers. We previously demonstrated that RBL2/p130 is a direct AKT target and its reactivation following AKT inhibition mediates apoptosis in MM.So, we hypothesize that concomitant inhibition of CDK and AKT activity, both converging on the reactivation of RBL2/p130 tumor suppressor role, synergize in counteracting MM.We first assessed, through an MTS assay, the effect of three new generation CDK inhibitors (CDKi), palbociclib, ribociclib, abemaciclib, on the cell viability of MM (NCI‐H28, IST‐MES TWO, NCI‐H2452, MMB, MSTO‐211H, NCI‐H2052) and immortalized human normal mesothelial (MET‐5A) cells at 72h. Then, to verify whether CDKi exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment of all the MM All the MM and mesothelial cell lines. Finally, we also examined, by MTS, the possible synergistic effects of abemaciclib in combination with an AKT inhibitor (AKT1/2 VIII).Our preliminary data show that ribociclib and palbociclib only moderately reduce viability of the MPM cells, whereas abemaciclib shows cytotoxic effects at very low doses in all MM cell lines. Abemaciclib treatment induces a dose‐dependent cytotoxic effect in all the MM cell lines without meaningfully affecting the normal mesothelial cells. Interestingly, abemaciclib also induced a cytotoxic effect in the most aggressive histotype, the sarcomatoid NCI‐H2052 cells. To verify whether abemaciclib exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment with IC50 of abemaciclib and found that the CDKi dramatically reduced colony formation in all the MM cell lines. We also examined, by MTS, the possible synergistic effects of abemaciclib in combination with a kinase AKT inhibitor (iAKT1/2 VIII), so we treated the MM cell lines for 72 h with the two agents, both alone and in combination, at five different concentrations, in a constant ratio. The cell viability data were evaluated through the Chou–Talalay method and showed a strong synergism revealing a combination index (CI) values &lt; 1 for all cell.Although reactivating oncosoppressor genes is difficult to achieve because they are hardly ‘druggable’, our preliminary findings will provide the rationale for pharmacological strategies able to unleash RBL2/p130 tumor suppressor potential. The pharmacological reactivation of RBL2/p130 could be an effective strategy against MM, which can be rapidly implemented in the clinical practice.