We describe the impact of lifestyle related factors on the formation of micronuclei (MN) in buccal cells. It is known that consumption of alcohol and of other drugs including tobacco and other plants cause damage of the genetic material and leads to adverse health effects. Overall, 235 articles were evaluated in total. Tobacco smoking, i.e., consumption of cigarettes (75 publications), water pipes (11 publications) and crack smoking (5 publications) caused mainly positive effects, while negative results were obtained with pure marijuana (2 publications). Chewing habits (smokeless tobacco (27 publications) and betel with and without tobacco (23 publications) caused induction of MN in cells from the oral cavity (numbers indicate the number of publications). A study concerning khat chewing yielded a positive finding while reduction of MN was seen in consumers of coca leaves. Investigations concerning the effects of alcohol (11 publications), meat consumption (3 publications) and studies concerning the impact of the body mass index and body fat (4 publications) led to inconsistent results. No evidence of MN induction was observed in studies on continuous physical exercise (2 publications). Furthermore, exhaustive sporting activities also showed no indications of chromosomal damage (2 publications). A number of studies (in total 22 publications) were realized to investigate the effects of mobile phone specific radiation. The exposure was not adequately assessed in these investigations with questionnaires, furthermore, frequently inadequate stains (11 publications). The findings are controversial, and a clear negative result was obtained in a recent controlled intervention study. Overall the results indicate that the MN assay with buccal cells can be used to monitor chromosomal damage caused by smoking and chewing habits and to predict adverse health effects in the respiratory system and on the upper gastrointestinal tract. To draw firm conclusions about the effects of nutrition, mobile phone use, physical exercise, and body weight, further well-controlled trials are needed.

Reactive oxygen species (ROS)-induced aberrant oncogenic signalling has been proposed to mediate the progression and development of pleural mesothelioma (PM). In this study, we demonstrate how ROS promote oncogenic signalling, especially in the context of cell migration and immune evasion via YB-1 phosphorylation in mesothelial and PM cell models. Xanthine (X)- and xanthine oxidase (XO)-generated ROS exposure led to increased migration and a more elongated cell shape in mesothelial and PM cells in live-cell videomicroscopy analyses. These effects were associated with the enhanced phosphorylation of ERK, AKT, and YB-1 and the elevated gene expression of PD-L1 and PD-L2, which were analysed with immunoblotting and quantitative real-time RT-PCR, respectively. The pharmacological inhibition of AKT (ipatasertib), MEK (trametinib), and RSK (BI-D1870) resulted in the reversal of ROS-induced effects, with the strongest effects observed upon the inhibition of YB-1 phosphorylation by BI-D1870. The results suggest that ROS exposure has a strong impact on cell migration and immune evasion not only in PM cells but also in mesothelial cells, from which PM arises. Interfering with ROS-responsive kinase pathways, particularly YB-1 phosphorylation, could counteract pro-migratory and immune-evasive effects in PM.

This review describes the use of the micronucleus (MN) assay with exfoliated cells from the oral mucosa in occupational studies. The molecular mechanisms leading to formation of MN (chromosomal breakage and aneuploidy) are well known. In total, 222 articles have been published since the method was developed in 1982. The majority of investigations were conducted with agricultural workers, followed by petrol station attendants, painters, pathology/anatomy lab workers, and miners. Positive results were reported in the majority of studies (86%); Brazil, Italy, Mexico, and Turkey being the most productive countries. The use of this technique increased substantially in recent years, and a recent correlation analysis with data from MN studies with lymphocytes indicates that the method is useful for predicting the cancer risks of chemically and radiation-exposed workers. The methodological quality of the studies increased in recent years since standardized and validated guidelines have been published. However, major shortcomings are still the lack of adequate matching (in particular in regard to nutrition and intake of dietary supplements), the lack of chemical exposure measurements, and the use of inadequate (DNA-nonspecific) stains. The most pronounced effects were seen in metal production workers, miners, petrol station attendants, agricultural workers, and pathologists. The sampling of cells from the oral cavity is noninvasive and requires no cultivation under sterile conditions is required. The currently available data indicate that this fast and easy-to-perform procedure provides valuable information about combined effects of chemical exposures and about the efficiency of safety measurements. Therefore, it should be used in the future for the routine surveillance of workers.

The light-mediated, specific, and precise control of cell functions enabled by optogenetics has become a versatile method for investigating and combatting cancer. An increasing set of optogenetic tools enables tightly controlled regulation of ion flux across biological membranes, gene expression, gene editing, and protein-protein interactions and is being used to interrogate hallmark traits of cancer at the cellular, subcellular, and organismic level. This enables, on the one hand, the identification of critical signaling circuits required for cancer development and progression in vitro and in animal models and can flag potential intervention points for pharmacologic interference. On the other hand, optogenetics can improve the level of control in cell-based therapeutics. The current article provides a review of optogenetic tools and approaches used in the cancer research field and their multiple applications for improving our understanding of signal transduction pathways, modulating immune functions in the tumor microenvironment, facilitating drug screening, or directly attacking cancer cells. Key advantages and achievements of optogenetics in the cancer research field and remaining barriers for clinical applications are discussed.

Aim of this study was to find out if demographic factors (age and body weight) have an impact on the sensitivity of peripheral lymphocytes towards DNA damage caused by extremely low frequency electromagnetic fields (ELF-EMF), to which humans are exposed worldwide. Cells from three different groups (young normal-weight, young obese, and old normal-weight individuals) were treated with different intensities of ELF-EMF (50 Hz, 50, 200 and 1000 µT) in combination with genotoxic agents, which cause damage to the genetic material via different molecular mechanisms. BPDE (benzo[a]pyrene diol epoxide) is a metabolite of the polycyclic aromatic hydrocarbon benzo(a)pyrene (B(a)P); 4-nitroquinoline oxide (4NQO) is a UV-mimetic agent; the metal compounds NiCl2 and CrO3 induce cross-links and oxidative damage. The cells were treated with radiation in combination with the different chemicals (simultaneous and sequential exposure), subsequently, DNA damage was monitored using single-cell gel electrophoresis (SCGE) experiments. We found no evidence for induction of genetic damage by the radiation in cells from the different groups. However, a decrease of damage was observed in combined experiments with NiCl2, and an increase of DNA migration was detected in some experiments with BPDE. These findings indicate that ELF-EMF does not cause DNA damage in lymphocytes regardless of the age and body weight of the donors. Furthermore, our results show that the radiation has an impact on the extent of DNA damage caused by specific environmentally and occupationally relevant exposures and may increase the genotoxic effects of polycyclic aromatic hydrocarbons.

Background/Objective: The identification of novel non-invasive diagnostic and prognostic biomarkers is urgently needed in pleural mesothelioma (PM). While soluble mesothelin-related peptides (SMRP) are the most established circulating biomarker, their prognostic value is limited. A wide range of microRNAs (miRs) play diverse roles in regulating gene expression in PM. MiR-21 has been shown to be upregulated in mesothelioma tissue; nevertheless, the diagnostic and prognostic utility of miR-21 in the circulation and its association with survival in PM have not been extensively investigated to date. The objective of the current study was to evaluate miR-21 as a potential blood-based diagnostic and prognostic biomarker in PM. Methods: Plasma samples from PM patients (n = 94) were collected at the time of diagnosis, prior to treatment. Sex- and age-matched healthy individuals (n = 30) served as controls. MiR-21 levels were measured using quantitative RT-PCR and normalized to miR-16, and potential correlations with clinicopathological data were analyzed. Serum SMRP levels were measured in matched patients (n = 84), and a direct comparative analysis of miR-21 and SMRP was conducted. In situ hybridization (ISH) was used to confirm the presence of miR-21 in tumor cells. Results: Plasma miR-21 levels were significantly elevated in PM patients compared to healthy controls (p < 0.001), demonstrating good diagnostic performance (AUC 0.81). The localization of miR-21 in PM cells was confirmed by ISH. High miR-21 levels were associated with significantly shorter median overall survival (12.4 vs. 24.3 months, p < 0.001). Elevated SMRP levels were also associated with reduced survival (12.4 vs. 19.5 months, p = 0.032); however, SMRP did not retain independent prognostic significance in multivariable analysis. In contrast, high-circulating miR-21 was confirmed as an independent predictor for poor survival (HR 3.12, p < 0.001). Conclusions: Our findings highlight that circulating miR-21 is a potential non-invasive biomarker with both diagnostic and independent prognostic value in pleural mesothelioma and outperforms SMRP in multivariable survival analysis. Further research is warranted to validate its role in the biology of this disease and to assess its correlation with outcome and treatment responses.

Proteomic analysis resulting from the comparison of SCLC cell lines showing synergistic or additive responses to entinostat and cisplatin therapy

Background/Objectives: Malignant pleural mesothelioma (MPM) remains challenging to treat, with a poor prognosis. As controversy about clinical management continues, predictive biomarkers for patient selection to indicate the benefit of treatment modalities are urgently needed. Methods: In a retrospective analysis of 195 patients between 1994 and 2020 at the Department of Thoracic Surgery, Medical University of Vienna, Austria, the Mesothelioma Systemic Inflammation Score (MSIS)—consisting of pretreatment neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP), and fibrinogen—was tested for its prognostic and predictive significance. The prognostic impact of MSIS was subsequently validated in an independent cohort of 80 patients treated at the Department of Thoracic Surgery, Karl Landsteiner Institute for Clinical and Translational Thoracic Surgery Research, Clinic Floridsdorf, Vienna, Austria. Results: Median overall survival (OS) was 14 months for the entire cohort (95% CI: 11.4–16.6). Patients undergoing multimodality treatment including macroscopic complete resection had a longer OS (22.3 months, 95% CI: 18.6–26.0; p < 0.001). In multivariable analysis, MSIS (p < 0.001), disease stage (p = 0.001), and the type of treatment (p = 0.004) were confirmed as independent predictors for OS. Higher MSIS was associated with shorter OS (p < 0.001). Significant survival benefit of multimodality regimens including surgery was limited to patients with low MSIS. Among patients with low (≤ 2) MSIS, multimodality therapy was associated with significantly prolonged OS when compared with chemo- and/or radiotherapy alone (25.8 months [95% CI: 16.4–35.3] vs. 14.4 months [95% CI: 10.4–18.4], p < 0.001). In contrast, among patients with elevated MSIS, no survival benefit was achieved by surgery over conservative treatment (11.8 months [95% CI: 8.3–15.3] vs. 8.2 months [95% CI: 5.2–11.3], p = 0.233). The ability of MSIS to predict survival was equivalent between the baseline and the independent validation cohort (p < 0.001). Conclusions: The Mesothelioma Systemic Inflammation Score was found to be an independent prognostic score in pleural mesothelioma, predicting benefit from macroscopic complete resection as part of multimodality treatment in distinct patients.

Peritoneal membrane injury induces the activation of local fibroblasts and tissue remodelling, which ultimately can progress to fibrosis. Metastasis of colorectal cancer (CRC) to the abdominal cavity results in such peritoneal damage. Patients with colorectal cancer peritoneal metastasis (CPM) have a particularly poor prognosis, and CPM tumours are characterised by a high infiltration of fibroblasts. Here, we characterised the molecular and functional features of these fibroblasts, and investigated their interaction with other cells in the peritoneal microenvironment. Primary fibroblasts were isolated from 89 patients with different malignant and benign disorders of the peritoneum. We performed comprehensive analyses of single-cell and transcriptome profiling, secretome characterization, and functional enzymatic activity. We were able to identify a peritoneum-specific fibroblast population that increases in response to different types of damage-inducing peritoneal pathologies, including metastasis. These fibroblasts are characterised by the IGFBP2-dependent expression of CD38, which mediates extracellular non-canonical adenosinergic activity and contributes to the suppression of macrophages and T cells. Importantly, peritoneal fibroblasts promoted the growth and invasiveness of tumour cells in a xenograft mouse model of peritoneal metastasis, highlighting their pro-tumorigenic role. Their specific gene signature was associated with poor prognosis in a dataset of 51 patients suffering from colorectal peritoneal metastasis. This study revealed that the CPM is infiltrated by a peritoneal fibroblast subtype, which is absent in healthy tissue, but also observed in benign peritoneal diseases. Given the limited therapeutic options for these patients, these pro-tumorigenic peritoneal fibroblasts could represent an attractive target for inhibiting the peritoneal spread of tumour cells.

Objectives: Lung squamous cell carcinoma (LUSC) is associated with a poor prognosis and a lack of specific treatment options. The dysregulation of activin A (ActA) has been reported in various malignancies. Herein, we investigated the diagnostic and prognostic significance of ActA in LUSC. Materials and methods: ActA concentrations were measured using ELISA in plasma samples of 128 LUSC patients (stage I-IV) and 73 controls, and correlated those values with clinicopathological parameters and survival. Results: ActA plasma levels were significantly higher in therapy-naive LUSC patients compared to controls (444.1 ± 310.9 pg/mL vs 338.9 ± 145.5 pg/mL, p = 0.010). ActA levels significantly correlated with advanced stage as well as with T and N factors. High circulating ActA levels were significantly increased in metastatic disease patients compared to M0 disease. Further, patients with ActA levels above a computationally established optimal cut-off value of 443.0 pg/mL had a significantly worse median overall (OS, 17.63 vs 64.77 months, HR 0.391, 95 % CI 0.200–0.762, p < 0.001) and median disease-/progression-free survival (DFS/PFS; 11.57 vs 30.20 months, HR 0.502, 95 % CI 0.248–1.019, p = 0.020). Multivariate analysis revealed that high ActA levels were an independent prognostic factor for shorter OS (p = 0.001) and DFS/PFS (p = 0.018). A newly developed score combining CRP and ActA levels was also an independent prognostic factor for OS and DFS/PFS. Conclusion: Measurement of circulating ActA levels may help identify advanced-stage LUSC patients, and this value could serve as a prognostic parameter in LUSC. Thus, ActA may be a novel blood-based biomarker for identifying LUSC patients with distant metastasis.

Proteomic analysis resulting from the comparison of entinostat sensitive versus resistant cell lines

The cold-shock domain protein YB-1 is overexpressed in pleural mesothelioma (PM) and was shown to contribute to increased cell migration and platinum resistance. Phosphorylation of YB-1 at position serine 102 was analysed by immunohistochemistry, immunofluorescence and immunoblotting in PM tissue specimens and cell lines. Intracellular localisation experiments involved immunoblotting, transfection of fluorescent protein-tagged YB-1 and confocal imaging. YB-1 phosphorylation was inhibited with the RSK inhibitors BI-D1870 and LJH685. Effects of inhibition alone and in combination with radiation or cisplatin treatment were analysed by cell viability assays, clonogenic assays and videomicroscopy-based migration and cell fate map analyses. YB-1 phosphorylated at serine 102 is present in PM cell lines and tissue. Inhibition of phosphorylation with BI-D1870 reduced YB-1 localisation in the cell nucleus and led to reduced cell viability, clonogenicity, migration and disrupted cell division. Moreover, exposure to BI-D1870 increased the effect of radiation and cisplatin treatment with additive to synergistic effects in PM cell lines and primary cultures. The serine 102 phosphorylated form of YB-1 contributes to the malignant phenotype of PM. Inhibition of YB-1 phosphorylation warrants further exploration as part of treatment strategies for this devastating disease.

Early metastatic spread represents a challenge in fighting small cell lung cancer (SCLC). The molecular mechanisms underlying metastatic dissemination remain unclear in this devastating disease. Invasive traits were investigated in 13 SCLC cell lines using 3D-spheroid formation, sprouting assays, co-cultures and a zebrafish xenograft model. Proteomic analysis was performed to unravel metastatic drivers, which were validated by qPCR, growth factor arrays and specific inhibitors. Overall, 8 cell lines formed spheroids, and half of these displayed invasive sprouting in collagen. The ‘sprouter’ SCLC cells, which all had a YAP1-dominant subtype, showed increased migration in zebrafish larvae and penetrated endothelial cell monolayers to a higher extent, thereby mimicking intra- and extravasation. Proteomics revealed differences in adhesion properties, oncogenic pathways and receptor tyrosine kinase signalling. Sprouter cells showed higher expression levels of mesenchymal cell state markers. Stimulation with fibroblast growth factor 2 (FGF2) further induced invasive sprouting, while blocking the FGF/R axis resulted in a significant reduction of sprouting in vitro and in vivo. The FGF/R axis is a key driver of SCLC metastatic spread in the YAP1-dominant subtype. These data might facilitate the development of potential future therapies targeting FGF/R signalling to prevent SCLC progression and metastasis.

ERK1/2 mitogen‐activated protein kinases (ERK) are key regulators of basic cellular processes, including proliferation, survival, and migration. Upon phosphorylation, ERK becomes activated and a portion of it dimerizes. The importance of ERK activation in specific cellular events is generally well documented, but the role played by dimerization is largely unknown. Here, we demonstrate that impeding ERK dimerization precludes cellular movement by interfering with the molecular machinery that executes the rearrangements of the actin cytoskeleton. We also show that a constitutively dimeric ERK mutant can drive cell motility per se , demonstrating that ERK dimerization is both necessary and sufficient for inducing cellular migration. Importantly, we unveil that the scaffold protein kinase suppressor of Ras 1 (KSR1) is a critical element for endowing external agonists, acting through tyrosine kinase receptors, with the capacity to induce ERK dimerization and, subsequently, to unleash cellular motion. In agreement, clinical data disclose that high KSR1 expression levels correlate with greater metastatic potential and adverse evolution of mammary tumors. Overall, our results portray both ERK dimerization and KSR1 as essential factors for the regulation of cell motility and mammary tumor dissemination.

Despite significant advances in oncology, 1 of 108 female patients succumb to ovarian cancer (OC) each year. Improved novel treatments against this aggressive disease would be a major improvement. The growth of OC cells has been demonstrated to be highly dependent on lipids. OC cells are abundantly present in the abdominal cavity and omentum, the main sites of OC expansion. Accordingly, it has been attempted not only to block the hyperactive synthesis of fatty acids (FAs) in cancer cells, but also to disrupt lipid supply. While either strategy has yielded promising results as monotherapy, the induction of resistance pathways diminishing the anticancer effects is yet conceivable. The endogenous regulation of lipid biosynthesis in OC has been extensively studied. However, the role of stromal cells in the modulation of the effects of anti-lipogenic drugs has not yet been well documented. The present study thus examined the interaction between OC cells and associated stromal cells, when de novo FA synthesis was blocked. It has recently been revealed by the authors that when FA are provided to OC cells in monoculture, the lipid deficiency induced by pharmacological inhibition of FA synthase (FASN), the key enzyme of endogenous FA synthesis, cannot be compensated through an increased FA uptake by OC cells. In the present study, OC cells were co-cultured with adipocytes preloaded with fluorescent FA and the effects of FASN-inhibition on OC homing to adipocytes and the transcellular delivery of fluorescent FA from adipocytes to OC cells were examined. The FASN inhibitors, G28UCM and Fasnall, stimulated the spontaneous migration of A2780 OC cells in a concentration-dependent manner and stimulated the transfer of FA from adipocytes to OC cells. Similar effects were observed with all types of adipocytes tested. The models applied in the present study demonstrated that co-cultured cancer-associated adipocytes may attenuate the anticancer effects of FASN inhibitors by attracting tumor cells and by supplying the cells with FA. This lipid-mediated dependency may provide a rationale for the design of new treatment approaches for the treatment of OC.

BACKGROUND:Pleural mesothelioma (PM) is a relatively rare malignancy with limited treatment options and dismal prognosis. We have previously found elevated FGF18 expression in PM tissue specimens compared with normal mesothelium. The objective of the current study was to further explore the role of FGF18 in PM and evaluate its suitability as a circulating biomarker. METHODS:FGF18 mRNA expression was analyzed by real-time PCR in cell lines and in silico in datasets from the Cancer Genome Atlas (TCGA). Cell lines overexpressing FGF18 were generated by retroviral transduction and cell behavior was investigated by clonogenic growth and transwell assays. Plasma was collected from 40 PM patients, six patients with pleural fibrosis, and 40 healthy controls. Circulating FGF18 was measured by ELISA and correlated to clinicopathological parameters. RESULTS:FGF18 showed high mRNA expression in PM and PM-derived cell lines. PM patients with high FGF18 mRNA expression showed a trend toward longer overall survival (OS) in the TCGA dataset. In PM cells with low endogenous FGF18 expression, forced overexpression of FGF18 resulted in reduced growth but increased migration. Surprisingly, despite the high FGF18 mRNA levels observed in PM, circulating FGF18 protein was significantly lower in PM patients and patients with pleural fibrosis than in healthy controls. No significant association of circulating FGF18 with OS or other disease parameters of PM patients was observed. CONCLUSIONS:FGF18 is not a prognostic biomarker in PM. Its role in PM tumor biology and the clinical significance of decreased plasma FGF18 in PM patients warrant further investigation.

BackgroundNo targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies.MethodsExpression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids.ResultsThe highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction.ConclusionsThe current study reveals the subtype specificity of BCL-2 expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

Supplementary Figure S7. Coding variants detected in MPM cell cultures and their relation to TERT promoter status.

PDF file - 42K, Gives proof of efficient FGFR4 knock-down

PDF file - 47K, Compares the staging of the CORSA and hospital population