Intratumor microbiota as a novel potential prognostic indicator in mesothelioma.

Introduction: Despite increased attention on immunotherapy, primarily immune checkpoint blockade, as a therapeutic approach for mesothelioma (MMe), its efficacy and tolerability remain questioned. One potential explanation for different responses to immunotherapy is the gut and intratumor microbiota; however, these remain an underexplored facet of MMe. This article highlights the cancer intratumor microbiota as a novel potential prognostic indicator in MMe. Methods: TCGA data on 86 MMe patients from cBioPortal underwent bespoke analysis. Median overall survival was used to divide patients into "Low Survivors" and "High Survivors". Comparison of these groups generated Kaplan-Meier survival analysis, differentially expressed genes (DEGs), and identification of differentially abundant microbiome signatures. Decontamination analysis refined the list of signatures, which were validated as an independent prognostic indicator through multiple linear regression modelling and Cox proportional hazards modelling. Finally, functional annotation analysis on the list of DEGs was performed to link the data together. Results: 107 genera signatures were significantly associated with patient survival (positively or negatively), whilst clinical characteristic comparison between the two groups demonstrated that epithelioid histology was more common in "High Survivors" versus biphasic in "Low Survivors". Of the 107 genera, 27 had published articles related to cancer, whilst only one (Klebsiella) had MMe-related published articles. Functional annotation analysis of the DEGs between the two groups highlighted fatty acid metabolism as the most enriched term in "High Survivors", whilst for "Low Survivors" the enriched terms primarily related to cell cycle/division. Linking these ideas and findings together is that the microbiome influences, and is influenced by, lipid metabolism. Finally, to validate the independent prognostic value of the microbiome, multiple linear regression modelling as well as Cox proportional hazards modelling were employed, with both approaches demonstrating that the microbiome was a better prognostic indicator than patient age or stage of the cancer. Discussion: The findings presented herein, alongside the very limited literature from scoping searches to validate the genera, highlight the microbiome and microbiota as a potentially rich source of fundamental analysis and prognostic value. Further in vitro studies are needed to elucidate the molecular mechanisms and functional links that may lead to altered survival.

Elimination of Off-Target Effect by Chemical Modification of 5'-End of siRNA.

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'-O-methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

Comparison of 3 Randomized Clinical Trials of Frontline Therapies for Malignant Pleural Mesothelioma.

Importance: Some recently proposed frontline therapies for malignant pleural mesothelioma (MPM) are very costly, yet their impact on quality of life and overall survival of these patients remains arguable. Given the high social toll of this aggressive occupational cancer, it is paramount to establish the real clinical benefit of these treatments. Objective: To directly compare and analyze the statistical robustness of the 3 randomized clinical trials (RCTs) of frontline therapies recommended for MPM since 2003. Design, Setting, and Participants: This comparative effectiveness study assessed the following phase 3 RCTs: the Mesothelioma Cisplatin Pemetrexed Study (MPS) of cisplatin plus pemetrexed vs cisplatin; the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) of cisplatin plus pemetrexed plus bevacizumab vs cisplatin plus pemetrexed; and the CheckMate743 (CM743) study of nivolumab plus ipilimumab vs cisplatin plus pemetrexed. Data collection dates for the RCTs ranged from April 1999 to April 2018. Data for this study were analyzed from February to October 2021. Main Outcomes and Measures: Patient selection criteria, superiority of the intervention groups, survival-inferred fragility index, and censoring patterns in each RCT. Results: A total of 1501 patients were included in the analysis (1170 men [77.9%]; range of median age for treatment groups, 60 [IQR, 19-84] to 69 [IQR, 65-75] years). A virtual comparison of overall survival in MAPS vs the CM743 study showed no statistically significant difference (hazard ratio [HR], 0.97 [95% CI, 0.79-1.20]; P = .79), and the survival-inferred fragility index in the intention-to-treat (ITT) populations was as low as 0.22% of the total sample size in MPS, -0.45% of the total sample size in MAPS, and 0.99% of the total sample size in the CM743 trial. Moreover, reverse restricted mean survival time (RMST) analysis of overall survival using RMST-difference (RMST-D) demonstrated differential censoring in the ITT population of the CM743 trial favoring the control group (0.56 [95% CI, 0.18-0.94]; P = .004) and in the nonepithelioid group (reverse RMST-D, 0.90 [95% CI, 0.001-1.79]; P = .048). Conclusions and Relevance: This comparative effectiveness study found no survival benefit in the CM743 trial over MAPS, despite the inclusion of patients with worse prognosis in the latter trial. Moreover, the statistical conclusions of all the examined trials were shown to be extremely fragile, and the findings of differential censoring in the CM743 trial and in the ITT nonepithelial subset raised additional areas of concern. These findings suggest that selection criteria, fragility, and censoring patterns may affect the original conclusions drawn for the respective trials, casting a shadow on the real benefit. This model of analysis lays a rigorous groundwork extendable to trials of all cancer treatments before their registration.

Protein disulfide isomerase A1­associated pathways in the development of stratified breast cancer therapies.

The oxidoreductase protein disulfide isomerase A1 (PDIA1) functions as a cofactor for many transcription factors including estrogen receptor α (ERα), nuclear factor (NF)­κB, nuclear factor erythroid 2­like 2 (NRF2) and regulates the protein stability of the tumor suppressor p53. Taking this into account we hypothesized that PDIA1, by differentially modulating the gene expression of a diverse subset of genes in the ERα­positive vs. the ERα­negative breast cancer cells, might modify dissimilar pathways in the two types of breast cancer. This hypothesis was investigated using RNA­seq data from PDIA1­silenced MCF­7 (ERα­positive) and MDA­MB­231 (ERα­negative) breast cancer cells treated with either interferon Î³ (IFN­Î³) or etoposide (ETO), and the obtained data were further analyzed using a variety of bioinformatic tools alongside clinical relevance assessment via Kaplan­Meier patient survival curves. The results highlighted the dual role of PDIA1 in suppressing carcinogenesis in the ERα(+) breast cancer patients by negatively regulating the response to reactive oxygen species (ROS) and promoting carcinogenesis by inducing cell cycle progression. In the ERα(­) breast cancer patients, PDIA1 prevented tumor development by modulating NF­κΒ and p53 activity and cell migration and induced breast cancer progression through control of cytokine signaling and the immune response. The findings reported in this study shed light on the differential pathways regulating carcinogenesis in ERα(+) and ERα(­) breast cancer patients and could help identify therapeutic targets selectively effective in ERα(+) vs. ERα(­) patients.

Evaluation of the Role of p53 Tumour Suppressor Posttranslational Modifications and TTC5 Cofactor in Lung Cancer.

Mutations in the p53 tumor suppressor are found in over 50% of cancers. p53 function is controlled through posttranslational modifications and cofactor interactions. In this study, we investigated the posttranslationally modified p53, including p53 acetylated at lysine 382 (K382), p53 phosphorylated at serine 46 (S46), and the p53 cofactor TTC5/STRAP (Tetratricopeptide repeat domain 5/ Stress-responsive activator of p300-TTC5) proteins in lung cancer. Immunohistochemical (IHC) analysis of lung cancer tissues from 250 patients was carried out and the results were correlated with clinicopathological features. Significant associations between total or modified p53 with a higher grade of the tumour and shorter overall survival (OS) probability were detected, suggesting that mutant and/or modified p53 acts as an oncoprotein in these patients. Acetylated at K382 p53 was predominantly nuclear in some samples and cytoplasmic in others. The localization of the K382 acetylated p53 was significantly associated with the gender and grade of the disease. The TTC5 protein levels were significantly associated with the grade, tumor size, and node involvement in a complex manner. SIRT1 expression was evaluated in 50 lung cancer patients and significant positive correlation was found with p53 S46 intensity, whereas negative TTC5 staining was associated with SIRT1 expression. Furthermore, p53 protein levels showed positive association with poor OS, whereas TTC5 protein levels showed positive association with better OS outcome. Overall, our results indicate that an analysis of p53 modified versions together with TTC5 expression, upon testing on a larger sample size of patients, could serve as useful prognostic factors or drug targets for lung cancer treatment.

Protein disulfide isomerase A1 regulates breast cancer cell immunorecognition in a manner dependent on redox state.

Oxidoreductase protein disulphide isomerases (PDI) are involved in the regulation of a variety of biological processes including the modulation of endoplasmic reticulum (ER) stress, unfolded protein response (UPR), ER­mitochondria communication and the balance between pro­survival and pro­death pathways. In the current study the role of the PDIA1 family member in breast carcinogenesis was investigated by measuring ROS generation, mitochondrial membrane disruption, ATP production and HLA­G protein levels on the surface of the cellular membrane in the presence or absence of PDIA1. The results showed that this enzyme exerted pro­apoptotic effects in estrogen receptor (ERα)­positive breast cancer MCF­7 and pro­survival in triple negative breast cancer (TNBC) MDA­MB­231 cells. ATP generation was upregulated in PDIA1­silenced MCF­7 cells and downregulated in PDIA1­silenced MDA­MB­231 cells in a manner dependent on the cellular redox status. Furthermore, MCF­7 and MDA­MB­231 cells in the presence of PDIA1 expressed higher surface levels of the non­classical human leukocyte antigen (HLA­G) under oxidative stress conditions. Evaluation of the METABRIC datasets showed that low PDIA1 and high HLA­G mRNA expression levels correlated with longer survival in both ERα­positive and ERα­negative stage 2 breast cancer patients. In addition, analysis of the PDIA1 vs. the HLA­G mRNA ratio in the subgroup of the living stage 2 breast cancer patients exhibiting low PDIA1 and high HLA­G mRNA levels revealed that the longer the survival time of the ratio was high PDIA1 and low HLA­G mRNA and occurred predominantly in ERα­positive breast cancer patients whereas in the same subgroup of the ERα­negative breast cancer mainly this ratio was low PDIA1 and high HLA­G mRNA. Taken together these results provide evidence supporting the view that PDIA1 is linked to several hallmarks of breast cancer pathways including the process of antigen processing and presentation and tumor immunorecognition.

Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells.

Acute lymphoblastic leukaemia (ALL) is the most frequent childhood cancer and, although it is highly treatable, resistance to therapy, toxicity and side effects remain challenging. The synthetic glucocorticoid (GC) dexamethasone (Dex) is commonly used to treat ALL, the main drawback of which is the development of resistance to this treatment. The aim of the present study was to investigate potential molecular circuits mediating resistance and sensitivity to GC­induced apoptosis in ALL. The leukaemia cell lines CEM­C7­14, CEM­C1­15 and MOLT4 treated with chloroquine (CLQ), thapsigargin (TG) and rotenone (ROT) were used to explore the roles of autophagy, endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and reactive oxygen species (ROS) generation in the response to GC treatment. ROS levels were associated with increased cell death and mitochondrial membrane potential in rotenone­treated CEM cells. Autophagy inhibition by CLQ exhibited the strongest cytotoxic effect in GC­resistant leukaemia. Autophagy may act as a pro­survival mechanism in GC­resistant leukaemia since increasing trends in beclin­1 and microtubule­associated protein 1 light chain 3α levels were detected in CEM­C1­15 and MOLT4 cells treated with Dex, whereas decreasing trends in these autophagy markers were observed in CEM­C7­14 cells. The intracellular protein levels of the ER stress markers glucose­regulated protein (GRP)78 and GRP94 were stimulated by Dex only in the GC­sensitive cells, suggesting a role of these chaperones in the GC­mediated ALL cell death. Increased cell surface levels of GRP94 were recorded in CEM­C7­14 cells treated with combination of Dex with TG compared with those in cells treated with TG alone, whereas decreasing trends were observed in CEM­C1­15 cells under these conditions. Taken together, the results of the present study demonstrated that autophagy may be a pro­survival mechanism in GC­resistant leukaemia, and by modulating intracellular and surface GRP94 protein levels, Dex is involved in the regulation of ER stress/UPR­dependent cell death and immune surveillance. These observations may be of clinical importance if confirmed in patients.

Telomerase inhibition, telomere attrition and proliferation arrest of cancer cells induced by phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT.

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 µM concentration but less efficiently at 1 µM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells.

Circular and linear: a tale of aptamer selection for the activation of SIRT1 to induce death in cancer cells.

It is a challenge to select the right target to treat conditions without affecting non-diseased cells. Cancer belongs to the top 10 causes of death in the world and it remains difficult to treat. Amongst cancer emerging targets, silent information regulator 1 (SIRT1) - a histone deacetylase - has shown many roles in cancer, ageing and metabolism. Here we report novel SIRT1 ligands that bind and modulate the activity of SIRT1 within cells and enhance its enzymatic activity. We developed a modified aptamer capable of binding to and forming a complex with SIRT1. Our ligands are aptamers, they can be made of DNA or RNA oligonucleotides, their binding domain can recognise a target with very high affinity and specificity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to develop circular and linear aptamers selectively binding to SIRT1. Cellular consequences of the interaction were monitored by fluorescence microscopy, cell viability assay, stability and enzymatic assays. Our results indicate that from our pool of aptamers, circular AC3 penetrates cancerous cells and is recruited to modulate the SIRT1 activity. This modulation of SIRT1 resulted in anticancer activity on different cancer cell lines. Furthermore, this modified aptamer showed no toxicity on one non-cancerous cell line and was stable in human plasma. We have demonstrated that aptamers are efficient tools for localisation of internal cell targets, and in this particular case, anticancer activity through modulation of SIRT1.

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

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

What can independent research for mesothelioma achieve to treat this orphan disease?

Introduction: Malignant pleural mesothelioma (MPM) is a rare neoplasm with a poor prognosis, as current therapies are ineffective. Despite the increased understanding of the molecular biology of mesothelioma, there is still a lack of drugs that dramatically enhance patient survival. Area Covered: This review discusses recent and complete clinical trials supported by the NIH, other U.S. Federal agencies, universities and organizations found on clinicaltrials.gov. Firstly, chemotherapy-based trials are described, followed by immunotherapy and multitargeted therapy. Then we introduce drug repositioning and the use of drug docking as tools to find new interesting molecules. Finally, we highlight potential molecular pathways that may play a role in mesothelioma biology and therapy. Expert Opinion: Numerous biases are present in the clinical trials due to a restricted number of cases, inappropriate endpoints and inaccurate stratification of patients which delay the finding of a treatment for MPM. The most crucial issue of independent research for MPM is the lack of more substantive funding to translate these findings to the clinical setting. However, this approach is not necessarily scientific given the low mutational load of mesothelioma relative to other cancers, and therefore patients need a more solid rationale to have a good chance of successful treatment.

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

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

p53 modeling as a route to mesothelioma patients stratification and novel therapeutic identification.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an orphan disease that is difficult to treat using traditional chemotherapy, an approach which has been effective in other types of cancer. Most chemotherapeutics cause DNA damage leading to cell death. Recent discoveries have highlighted a potential role for the p53 tumor suppressor in this disease. Given the pivotal role of p53 in the DNA damage response, here we investigated the predictive power of the p53 interactome model for MPM patients' stratification. METHODS: We used bioinformatics approaches including omics type analysis of data from MPM cells and from MPM patients in order to predict which pathways are crucial for patients' survival. Analysis of the PKT206 model of the p53 network was validated by microarrays from the Mero-14 MPM cell line and RNA-seq data from 71 MPM patients, whilst statistical analysis was used to identify the deregulated pathways and predict therapeutic schemes by linking the affected pathway with the patients' clinical state. RESULTS: In silico simulations demonstrated successful predictions ranging from 52 to 85% depending on the drug, algorithm or sample used for validation. Clinical outcomes of individual patients stratified in three groups and simulation comparisons identified 30 genes that correlated with survival. In patients carrying wild-type p53 either treated or not treated with chemotherapy, FEN1 and MMP2 exhibited the highest inverse correlation, whereas in untreated patients bearing mutated p53, SIAH1 negatively correlated with survival. Numerous repositioned and experimental drugs targeting FEN1 and MMP2 were identified and selected drugs tested. Epinephrine and myricetin, which target FEN1, have shown cytotoxic effect on Mero-14 cells whereas marimastat and batimastat, which target MMP2 demonstrated a modest but significant inhibitory effect on MPM cell migration. Finally, 8 genes displayed correlation with disease stage, which may have diagnostic implications. CONCLUSIONS: Clinical decisions related to MPM personalized therapy based on individual patients' genetic profile and previous chemotherapeutic treatment could be reached using computational tools and the predictions reported in this study upon further testing in animal models.

Insight into glucocorticoid receptor signalling through interactome model analysis.

Glucocorticoid hormones (GCs) are used to treat a variety of diseases because of their potent anti-inflammatory effect and their ability to induce apoptosis in lymphoid malignancies through the glucocorticoid receptor (GR). Despite ongoing research, high glucocorticoid efficacy and widespread usage in medicine, resistance, disease relapse and toxicity remain factors that need addressing. Understanding the mechanisms of glucocorticoid signalling and how resistance may arise is highly important towards improving therapy. To gain insight into this we undertook a systems biology approach, aiming to generate a Boolean model of the glucocorticoid receptor protein interaction network that encapsulates functional relationships between the GR, its target genes or genes that target GR, and the interactions between the genes that interact with the GR. This model named GEB052 consists of 52 nodes representing genes or proteins, the model input (GC) and model outputs (cell death and inflammation), connected by 241 logical interactions of activation or inhibition. 323 changes in the relationships between model constituents following in silico knockouts were uncovered, and steady-state analysis followed by cell-based microarray genome-wide model validation led to an average of 57% correct predictions, which was taken further by assessment of model predictions against patient microarray data. Lastly, semi-quantitative model analysis via microarray data superimposed onto the model with a score flow algorithm has also been performed, which demonstrated significantly higher correct prediction ratios (average of 80%), and the model has been assessed as a predictive clinical tool using published patient microarray data. In summary we present an in silico simulation of the glucocorticoid receptor interaction network, linked to downstream biological processes that can be analysed to uncover relationships between GR and its interactants. Ultimately the model provides a platform for future development both by directing laboratory research and allowing for incorporation of further components, encapsulating more interactions/genes involved in glucocorticoid receptor signalling.

Immunotherapy advances for mesothelioma treatment.

INTRODUCTION: Mesothelioma is a rare type of cancer that is strongly tied to asbestos exposure. Despite application of different modalities such as chemotherapy, radiotherapy and surgery, patient prognosis remains very poor and therapies are ineffective. Much research currently focuses on the application of novel approaches such as immunotherapy towards this disease. Areas covered: The types, stages and aetiology of mesothelioma are detailed, followed by a discussion of the current treatment options such as radiotherapy, surgery, and chemotherapy. A description of innate and adaptive immunity and the principles and justification of immunotherapy is also included. Clinical trials for different immunotherapeutic modalities are described, and lastly the article closes with an expert commentary and five-year view, the former of which is summarised below. Expert commentary: Current efforts for novel mesothelioma therapies have been limited by attempting to apply treatments from other cancers, an approach which is not based on a solid understanding of mesothelioma biology. In our view, the influence of the hostile, hypoxic microenvironment and the gene expression and metabolic changes that resultantly occur should be characterised to improve therapies. Lastly, clinical trials should focus on overall survival rather than surrogate endpoints to avoid bias and inaccurate reflections of treatment effects.

Promising investigational drug candidates in phase I and phase II clinical trials for mesothelioma.

INTRODUCTION: Malignant mesothelioma is a rare and lethal malignancy primarily affecting the pleura and peritoneum. Mesothelioma incidence is expected to increase worldwide and current treatments remain ineffective, leading to poor prognosis. Within this article potential targets to improve the quality of life of the patients and assessment of further avenues for research are discussed. Areas covered: This review highlights emerging therapies currently under investigation for malignant mesothelioma with a specific focus on phase I and phase II clinical trials. Three main areas are discussed: immunotherapy (immune checkpoint blockade and cancer vaccines, among others), multitargeted therapy (such as targeting pro-angiogenic genes) and gene therapy (such as suicide gene therapy). For each, clinical trials are described to detail the current or past investigations at phase I and II. Expert opinion: The approach of applying existing treatments from other cancers does not show significant benefit, with the most promising outcome being an increase in survival of 2.7 months following combination of chemotherapy with bevacizumab. It is our opinion that the hypoxic microenvironment, the role of the stroma, and the metabolic status of mesothelioma should all be assessed and characterised to aid in the development of new treatments to improve patient outcomes.

Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia.

Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy.

Anti-CTLA-4 therapy for malignant mesothelioma.

Immunotherapy is an emerging therapeutic strategy with a promising clinical outcome in some solid tumors, particularly metastatic melanoma. One approach to immunotherapy is immune checkpoint inhibitors, such as blockage of CTLA-4 and PD-1/PD-L1. This special report aims to describe the state of clinical trials of tremelimumab in patients with unresectable malignant mesothelioma (MM) in particular with regard to the clinical efficacy, safety and tolerability. Criticism and perspective of this treatment are also discussed. Biological and clinical considerations rule out the use of tremelimumab as single agent for MM and, more generally, the use of immune checkpoint inhibitors for MM is still largely questionable and not supported by evidences.

miR-663a regulates growth of colon cancer cells, after administration of antimicrobial peptides, by targeting CXCR4-p21 pathway.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.

Repurposing atovaquone: targeting mitochondrial complex III and OXPHOS to eradicate cancer stem cells.

Atovaquone is an FDA-approved anti-malarial drug, which first became clinically available in the year 2000. Currently, its main usage is for the treatment of pneumocystis pneumonia (PCP) and/or toxoplasmosis in immune-compromised patients. Atovaquone is a hydroxy-1,4-naphthoquinone analogue of ubiquinone, also known as Co-enzyme Q10 (CoQ10). It is a well-tolerated drug that does not cause myelo-suppression. Mechanistically, it is thought to act as a potent and selective OXPHOS inhibitor, by targeting the CoQ10-dependence of mitochondrial complex III. Here, we show for the first time that atovaquone also has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that atovaquone treatment of MCF7 breast cancer cells inhibits oxygen-consumption and metabolically induces aerobic glycolysis (the Warburg effect), as well as oxidative stress. Remarkably, atovaquone potently inhibits the propagation of MCF7-derived CSCs, with an IC-50 of 1 µM, as measured using the mammosphere assay. Atovaquone also maintains this selectivity and potency in mixed populations of CSCs and non-CSCs. Importantly, these results indicate that glycolysis itself is not sufficient to maintain the proliferation of CSCs, which is instead strictly dependent on mitochondrial function. In addition to targeting the proliferation of CSCs, atovaquone also induces apoptosis in both CD44+/CD24low/- CSC and ALDH+ CSC populations, during exposure to anchorage-independent conditions for 12 hours. However, it has no effect on oxygen consumption in normal human fibroblasts and, in this cellular context, behaves as an anti-inflammatory, consistent with the fact that it is well-tolerated in patients treated for infections. Future studies in xenograft models and human clinical trials may be warranted, as the IC-50 of atovaquone's action on CSCs (1 µM) is >50 times less than its average serum concentration in humans.