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Abstract Background Malignant Pleural Mesothelioma (MPM) is a rare malignancy with a poor prognosis. Current therapies are unsatisfactory and novel cures are urgently needed. In a previous drug screening, we identified thonzonium bromide (TB) as one of the most active compounds against MPM cells. Since the biological effects of TB are poorly known, in this work we departed from some hints of previous studies and investigated several hypotheses. Moreover, we evaluated the efficacy of TB in an in vivo xenograft rodent model. Methods In vitro assessment was made on five MPM (Mero-14, Mero-25, Ren, NCI-H28, MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A). We evaluated TB ability to affect proliferation, apoptosis, mitochondrial functions and metabolism, and the mevalonate pathway. In vivo assay was carried out on MPM-xenograft NOD-SCID mice (4 mg/kg delivered intraperitoneally, twice a week for 4 weeks) and the overall survival was analysed with Kaplan-Meier curves. Results After TB treatment, we observed the suppression of ERK 1/2 phosphorylation, the increase of BAX expression and p38 phosphorylation. TB affected Ca2+ homeostasis in both mitochondrial and cytosolic compartments, it regulated the mitochondrial functioning, respiration, and ATP production as well as the mevalonate pathway. The in vivo study showed an increased overall survival for TB treated group vs. vehicle control group (P = 0.0076). Conclusions Both in vitro and in vivo results confirmed the effect of TB on MPM and unravelled novel targets with translational potential.

Mesothelioma is a type of late‐onset cancer that develops in cells covering the outer surface of organs. Although it can affect the peritoneum, heart, or testicles, it mainly targets the lining of the lungs, making pleural mesothelioma (PMe) the most common and widely studied mesothelioma type. PMe is caused by exposure to fibres of asbestos, which when inhaled leads to inflammation and scarring of the pleura. Despite the ban on asbestos by most Western countries, the incidence of PMe is on the rise, also facilitated by a lack of specific symptomatology and diagnostic methods. Therapeutic options are also limited to mainly palliative care, making this disease untreatable. Here we present an overview of biological aspects underlying PMe by listing genetic and molecular mechanisms behind its onset, aggressive nature, and fast‐paced progression. To this end, we report on the role of deubiquitinase BRCA1‐associated protein‐1 (BAP1), a tumour suppressor gene with a widely acknowledged role in the corrupted signalling and metabolism of PMe. This review aims to enhance our understanding of this devastating malignancy and propel efforts for its investigation.

Abstract Background Malignant mesothelioma (MM) is a rare tumor with a dismal prognosis. The low efficacy of current treatment options highlights the urge to identify more effective therapies aimed at improving MM patients’ survival. Bortezomib (Bor) is a specific and reversible inhibitor of the chymotrypsin-like activity of the 20S core of the proteasome, currently approved for the treatment of multiple myeloma and mantle cell lymphoma. On the other hand, Bor appears to have limited clinical effects on solid tumors, because of its low penetration and accumulation into tumor tissues following intravenous administration. These limitations could be overcome in MM through intracavitary delivery, with the advantage of increasing local drug concentration and decreasing systemic toxicity. Methods In this study, we investigated the effects of Bor on cell survival, cell cycle distribution and modulation of apoptotic and pro-survival pathways in human MM cell lines of different histotypes cultured in vitro. Further, using a mouse MM cell line that reproducibly forms ascites when intraperitoneally injected in syngeneic C57BL/6 mice, we investigated the effects of intraperitoneal Bor administration in vivo on both tumor growth and the modulation of the tumor immune microenvironment. Results We demonstrate that Bor inhibited MM cell growth and induced apoptosis. Further, Bor activated the Unfolded Protein Response, which however appeared to participate in lowering cells’ sensitivity to the drug’s cytotoxic effects. Bor also affected the expression of EGFR and ErbB2 and the activation of downstream pro-survival signaling effectors, including ERK1/2 and AKT. In vivo, Bor was able to suppress MM growth and extend mice survival. The Bor-mediated delay of tumor progression was sustained by increased activation of T lymphocytes recruited to the tumor microenvironment. Conclusions The results presented herein support the use of Bor in MM and advocate future studies aimed at defining the therapeutic potential of Bor and Bor-based combination regimens for this treatment-resistant, aggressive tumor.

Abstract YTHDF1 is a well-characterized m6A reader protein that is essential for protein translation, stem cell self-renewal, and embryonic development. YTHDF1 regulates target gene expression by diverse molecular mechanisms, such as promoting protein translation or modulating the stability of mRNA. The cellular levels of YTHDF1 are precisely regulated by a complicated transcriptional, post-transcriptional, and post-translational network. Very solid evidence supports the pivotal role of YTHDF1 in embryonic development and human cancer progression. In this review, we discuss how YTHDF1 influences both the physiological and pathological biology of the central nervous, reproductive and immune systems. Therefore we focus on some relevant aspects of the regulatory role played by YTHDF1 as gene expression, complex cell networking: stem cell self-renewal, embryonic development, and human cancers progression. We propose that YTHDF1 is a promising future cancer biomarker for detection, progression, and prognosis. Targeting YTHDF1 holds therapeutic potential, as the overexpression of YTHDF1 is associated with tumor resistance to chemotherapy and immunotherapy.

Immune-checkpoint inhibitors (ICIs) were proven effective in inducing tumor regression. However, its toxicity tends to be fatal. We sought to investigate the hospital volume/outcomes relationship. Databases were searched for studies reporting immune-checkpoint inhibitors adverse events (AEs) in patients with solid-organ malignancies. The outcomes were A) the pooled events rate (PER) of grade 5, grade 3–4, cardiac-related, and pulmonary-related AEs, and B) the assessment of the volume/outcomes relationship. One hundred and forty-seven studies met our inclusion criteria. The PER of grade 5, grade 3–4, and any-grade AEs was 2.75% (95%CI: 2.18–3.47), 26.69% (95%CI: 21.60–32.48), and 77.80% (95%CI: 70.91–83.44), respectively. The PER of pulmonary-related AEs was 4.56% (95%CI: 3.76–5.53). A higher number of annual cases per center was significantly associated with reduced grade 5 (p = 0.019), grade 3–4 (p = 0.004), and cardiac-related AEs (p = 0.035) in the meta-regression. In the current era of cancer immunotherapy, knowledge regarding the early diagnosis and management of immunotherapy-related AEs is essential. Our meta-analysis demonstrates the importance of center volume in improving outcomes and reducing the incidence of severe AEs.

Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells’ tensegrity or the mechanical stability of their structure.

Abstract In this commentary, using existing clinical trial data and FDA approvals we propose that there is currently a critical need for an appropriate balancing between the financial impact of new cancer drugs and their actual benefit for patients. By adopting “pleural mesothelioma” as our clinical model we summarize the most relevant pertinent and available literature on this topic, and use an analysis of the reliability of the trials submitted for registration and/or recently published as a case in point to raise concerns with respect to appropriate trial design, biomarker based stratification and to highlight the ongoing need for balancing the benefit/cost ratio for both patients and healthcare providers.

IntroductionDespite 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.MethodsTCGA 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.Results107 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.DiscussionThe 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.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite the recent advent of promising new targeted therapies, lung cancer diagnostic strategies still have difficulty in identifying the disease at an early stage. Therefore, the characterizations of more sensible and specific cancer biomarkers have become an important goal for clinicians. Circular RNAs are covalently close, endogenous RNAs without 5′ end caps or 3′poly (A) tails and have been characterized by high stability, abundance, and conservation as well as display cell/tissue/developmental stage-specific expressions. Numerous studies have confirmed that circRNAs act as microRNA (miRNA) sponges, RNA-binding protein, and transcriptional regulators; some circRNAs even act as translation templates that participate in multiple pathophysiological processes. Growing evidence have confirmed that circRNAs are involved in the pathogenesis of lung cancers through the regulation of proliferation and invasion, cell cycle, autophagy, apoptosis, stemness, tumor microenvironment, and chemotherapy resistance. Moreover, circRNAs have emerged as potential biomarkers for lung cancer diagnosis and prognosis and targets for developing new treatments. In this review, we will summarize recent progresses in identifying the biogenesis, biological functions, potential mechanisms, and clinical applications of these molecules for lung cancer diagnosis, prognosis, and targeted therapy.

Peripheral immune-checkpoint blockade with mAbs to programmed cell death receptor-1 (PD-1) (either nivolumab or pembrolizumab) or PD-Ligand-1 (PD-L1) (atezolizumab, durvalumab, or avelumab) alone or in combination with doublet chemotherapy represents an expanding treatment strategy for metastatic non-small cell lung cancer (mNSCLC) patients. This strategy lays on the capability of these mAbs to rescue tumor-specific cytotoxic T lymphocytes (CTLs) inactivated throughout PD-1 binding to PD-L1/2 in the tumor sites. This inhibitory interactive pathway is a physiological mechanism of prevention against dangerous overreactions and autoimmunity in case of prolonged and/or repeated CTL response to the same antigen peptides. Therefore, we have carried out a retrospective bioinformatics analysis by single-cell flow cytometry to evaluate if PD-1/PD-L1-blocking mAbs modulate the expression of specific peripheral immune cell subsets, potentially correlated with autoimmunity triggering in 28 mNSCLC patients. We recorded a treatment-related decline in CD4+ T-cell and B-cell subsets and in the neutrophil-to-lymphocyte ratio coupled with an increase in natural killer T (NKT), CD8+PD1+ T cells, and eosinophils. Treatment-related increase in autoantibodies [mainly antinuclear antibodies (ANAs) and extractable nuclear antigen (ENA) antibodies] as well as the frequency of immune-related adverse events were associated with the deregulation of specific immune subpopulations (e.g., NKT cells). Correlative biological/clinical studies with deep immune monitoring are badly needed for a better characterization of the effects produced by PD-1/PD-L1 immune-checkpoint blockade.

Lung cancer is the leading cause of cancer-related death worldwide. Therapies for lung cancer have relatively poor outcomes and need to be improved. Lung cancer immune cell infiltration associated RNA (LCIIAR) is a long noncoding RNA (lncRNA), which is overexpressed in human cancers. However, the clinical significance and functional role of LCIIAR in Lung Adenocarcinoma remain unclear. Here, we identified a novel long non-coding RNA (ENSG00000256802), termed LCIIAR (lung cancer immune cell infiltration associated lncRNA), up-regulated in lung cancer tissue and cell lines. We show that increase LCIIAR expression correlated with poor clinical stage and adverse clinical outcomes and that could also serve as an independent unfavorable prognostic factor in patients with Lung Adenocarcinima. GSEA analysis demonstrated that LCIIAR is mainly involved in the regulation of the immune response. We uncovered that elevate LCIIAR expression positively correlated with immune infiltration and immune modulator in Lung Adenocarcinoma. More importantly, we confirmed that silencing of LCIIAR expression significantly inhibits the proliferation, and migration abilities of these tumour cells. We also demonstrated that the LCIIAR/hsa-miR184/SLC16A3/CDCP1 network regulates SLC16A3/CDCP1 overexpression in and is associated with poor prognosis in this tumour. Therefore our findings revealed the critical role of LCIIAR in Lung Adenocarcinoma progression, which may also serve as a prognostic biomarker and novel therapeutic target.

Lung adenocarcinoma (LUAD) is the most common histological lung cancer, and it is the leading cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have been implicated in the initiation and progression of various cancers. LncRNA-AC087588.2 (ENSG00000274976) is a novel lncRNA that is abnormally expressed in diverse cancer types, including LUAD. However, the clinical significance, prognostic value, diagnostic value, immune role, and the potential biological function of AC087588.2 LUAD remain elusive. In this study, we found that AC087588.2 was upregulated and associated with a poor prognosis in LUAD. In addition, univariate and multivariate Cox regression analysis indicated that AC087588.2 could be an independent prognostic factor for LUAD. Functionally, the knockdown of AC087588.2 restrained LUAD cell proliferation and migration in vitro. Finally, we constructed a ceRNA network that included hsa-miR-30a-5p and four mRNAs (ANLN, POLR3G, EHBP1, and ERO1A) specific to AC087588.2 in LUAD. The Kaplan–Meier survival analysis showed that lower expression of hsa-miR-30a-5p and higher expression of ANLN, POLR3G, EHBP1, and ERO1A were associated with adverse clinical outcomes in patients with LUAD. This finding provided a comprehensive view of the AC087588.2-mediated ceRNA network in LUAD, thereby highlighting its potential role in the diagnosis and prognosis of LUAD.

Lung adenocarcinoma (LUAD) is the most common histological lung cancer, and it is the leading cause of cancer-related deaths worldwide. Long noncoding RNAs (lncRNAs) have been implicated in the initiation and progression of various cancers. LncRNA-AC099850.3 is a novel lncRNA that is abnormally expressed in diverse cancer types including LUAD. However, the clinical significance, prognostic value, diagnostic value, immune role, and potential biological function of AC099850.3 LUAD remain elusive. In this study, we found that AC099850.3 was highly expressed in LUAD and associated with an advanced tumor stage, poor prognosis, and immune infiltration. Receiver operating curve analysis revealed the significant diagnostic ability of AC099850.3 (AUC=0.888). Functionally, the knockdown of AC099850.3 restrained LUAD cell proliferation and migration in vitro. Finally, we constructed a competitive endogenous RNAs (ceRNA) network that included hsa-miR-101-3p and 4 mRNAs (ESPL1, AURKB, BUB3, and FAM83D) specific to AC099850.3 in LUAD. Kaplan–Meier survival analysis showed that a lower expression of miR-101-3p and a higher expression of ESPL1, AURKB, BUB3, and FAM83D, were associated with adverse clinical outcomes in patients with LUAD. This finding provided a comprehensive view of the AC099850.3-mediated ceRNA network in LUAD, thereby highlighting its potential role in the diagnosis and prognosis of LUAD.

Abstract Background Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the pleura, mainly related to asbestos exposure. As in other solid tumors, malignant cells exhibit high glucose uptake and glycolytic rates with increased lactic acid efflux into the interstitial space. Lactate transport into and out of cells, crucial to maintaining intracellular pH homeostasis and glycolysis, is carried out by monocarboxylate transporters (MCTs) and the chaperone basigin (CD147). We set out to examine the clinical significance of basigin, MCT1 and MCT4 in the context of MPM and to evaluate their expression in relation to the evolution of the disease. Methods We used immunohistochemistry to measure the expression of basigin, MCT1 and MCT4 in a cohort of 135 individuals with MPM compared to a series of 15 non-MPM pleura specimens. Moreover, by Kaplan–Meier and Cox analyses we evaluated whether an expression over the average of these markers could be associated with the patients’ overall survival (OS). Results We detected positive staining of basigin, MCT1, and MCT4 in most MPM specimens. In particular, MCT4 was always positive in malignant tissues but undetectable in the 4 normal pleural specimens incorporated within the tissue microarray. This was confirmed in the additional series of 15 normal pleural samples. Moreover, MCT4 expression was significantly associated with reduced OS. Conclusion In this study, the tissue expression of basigin did not prove to be exploitable as a diagnostic or prognostic marker for MPM patients. The expression of MCT1 was not informative either, being tightly correlated with that of basigin. However, the expression of MCT4 showed promise as a diagnostic/therapeutic and prognostic biomarker.

Malignant mesothelioma (MMe) is a rare neoplasm with few therapeutic options available. The landscape of effective therapy for this disease remained unchanged in the last two decades. Recently, however, the introduction of Immune Checkpoint Inhibitors (ICIs) led to small, but nevertheless, promising improvements. However, many efforts are still needed to radically improve the prognosis of MMe. In this review, we analyze all those therapeutic strategies for MMe that are still in a preclinical or early clinical phase of development. In particular, we focus on novel antiangiogenic drugs and their possible combination with immunotherapy. Furthermore, we describe also more complex strategies such as microRNA-loaded vectors, oncolytic viruses, and engineered lymphocytes.

SARS-CoV-2 is a new coronavirus that has affected the world since 2019. Interstitial pneumonia is the most common clinical presentation, but additional symptoms have been reported, including neurological manifestations. Severe forms of infection, especially in elderly patients, present as an excessive inflammatory response called “cytokine storm”, which can lead to acute respiratory distress syndrome (ARDS), multiorgan failure and death. Little is known about the relationship between symptoms and clinical outcomes or the characteristics of virus–host interactions. The aim of this narrative review is to highlight possible links between neurological involvement and respiratory damage mediated by pathological inflammatory pathways in SARS-CoV-2 infection. We will focus on neuro–immune interactions and age-related immunity decline and discuss some pathological mechanisms that contribute to negative outcomes in COVID-19 patients. Furthermore, we will describe available therapeutic strategies and their effects on COVID-19 neurological symptoms.

Peripheral-immune-checkpoint blockade (P-ICB) with mAbs to PD-1 (nivolumab and pembrolizumab) or PD-L1 (atezolizumab, durvalumab, avelumab) alone or combination with chemotherapy represents a novel active treatment for mNSCLC patients. However, this therapy can be associated to immune-related adverse events (irAEs) and high cost. Therefore, finding reliable biomarkers of response and irAEs is strongly encouraged to accurately select patients who may potentially benefit from the immuno-oncological treatment. This is a retrospective multi-institutional analysis performed on ninety-five mNSCLC patients who received real-world salvage therapy with nivolumab or atezolizumab between December 2015 and April 2020. The outcome of these patients in term of PFS and OS was evaluated in comparison with different serum levels of C-reactive protein (CRP), Erythrocyte Sedimention Rate (ESR) and Procalcitonin (PCT) by performing Kaplan–Meier and Log-rank test and multivariate analysis. We found that high baseline levels of CRP, ESR, and PCT were strongly predictive of poor outcome (P

The lack of effective therapies remains one of the main challenges for malignant pleural mesothelioma (MPM). In this perspective, drug repositioning could accelerate the identification of novel treatments. We screened 1170 FDA-approved drugs on a SV40-immortalized mesothelial (MeT-5A) and five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) cell lines. Biological assays were carried out for 41 drugs, showing the highest cytotoxicity and for whom there were a complete lack of published literature in MPM. Cytotoxicity and caspase activation were evaluated with commercially available kits and cell proliferation was assayed using MTT assay and by clonogenic activity with standard protocols. Moreover, the five most effective drugs were further evaluated on patient-derived primary MPM cell lines. The most active molecules were cephalomannine, ouabain, alexidine, thonzonium bromide, and emetine. Except for alexidine, these drugs inhibited the clonogenic ability and caspase activation in all cancer lines tested. The proliferation was inhibited also on an extended panel of cell lines, including primary MPM cells. Thus, we suggest that cephalomannine, ouabain, thonzonium bromide, and emetine could represent novel candidates to be repurposed for improving the arsenal of therapeutic weapons in the fight against MPM.

Abstract 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.

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.

An immune checkpoint blockade with mAbs to PD-1 and PD-L1 is an expanding therapeutic option for mNSCLC patients. This treatment strategy is based on the use of mAbs able to restore the anti-tumor activity of intratumoral T cells inhibited by PD-1 binding to PD-L1/2 on tumor and inflammatory cells. It has been speculated that a chronic status of systemic inflammation as well as the immunosenescence physiologically occurring in elderly patients may affect the efficacy of the treatment and the occurrence of irAEs. We performed a multi-institutional retrospective study aimed at evaluating the effects of these mAbs (nivolumab or atezolizumab) in 117 mNSCLC patients younger (90 cases) and older (27 cases) than 75 years in correlation with multiple inflammatory parameters (NLR, CRP, ESR, LDH and PCT). No differences were observed when the cohorts were compared in terms of the frequency of PFS, OS, inflammatory markers and immune-related adverse events (irAEs). Similarly, the occurrence of irAEs was strictly correlated with a prolonged OS survival in both groups. On the contrary, a negative correlation between the high baseline levels of inflammatory markers and OS could be demonstrated in the younger cohort only. Overall, PD-1/PD-L1-blocking mAbs were equally effective in young and elderly mNSCLC patients; however, the detrimental influence of a systemic inflammation at the baseline was only observed in young patients, suggesting different aging-related inflammation immunoregulative effects.

The expanding clinical application of CDK4- and CDK6-inhibiting drugs in the managements of breast cancer has raised a great interest in testing these drugs in other neoplasms. The potential of combining these drugs with other therapeutic approaches seems to be an interesting work-ground to explore. Even though a potential integration of CDK4 and CDK6 inhibitors with radiotherapy (RT) has been hypothesized, this kind of approach has not been sufficiently pursued, neither in preclinical nor in clinical studies. Similarly, the most recent discoveries focusing on autophagy, as a possible target pathway able to enhance the antitumor efficacy of CDK4 and CDK6 inhibitors is promising but needs more investigations. The aim of this review is to discuss the recent literature on the field in order to infer a rational combination strategy including cyclin-D1/CDK4-CDK6 inhibitors, RT, and/or other anticancer agents targeting G1-S phase cell cycle transition.

Background: Malignant pleural mesothelioma (MPM) is a fatal tumor with a poor prognosis. The recent developments of liquid biopsies could provide novel diagnostic and prognostic tools in oncology. However, there is limited information about the feasibility of this technique for MPMs. Here, we investigate whether cancer-specific DNA sequences can be detected in pleural fluids and plasma of MPM patients as free circulating tumor DNA (ctDNA). Methods: We performed whole-exome sequencing on 14 tumor biopsies from 14 patients, and we analyzed 20 patient-specific somatic mutations with digital droplet PCR (ddPCR) in pleural fluids and plasma, using them as cancer-specific tumor biomarkers. Results: Most of the selected mutations could be detected in pleural fluids (94%) and, noteworthy, in plasma (83%) with the use of ddPCR. Pleural fluids showed similar levels of somatically mutated ctDNA (median = 12.75%, average = 16.3%, standard deviation = 12.3) as those detected in solid biopsies (median = 21.95%; average = 22.21%; standard deviation = 9.57), and their paired difference was weakly statistically significant (p = 0.048). On the other hand, the paired difference between solid biopsies and ctDNA from plasma (median = 0.29%, average = 0.89%, standard deviation = 1.40) was highly statistically significant (p = 2.5 × 10−7), corresponding to the important drop of circulating somatically mutated DNA in the bloodstream. However, despite the tiny amount of ctDNA in plasma, varying from 5.57% down to 0.14%, the mutations were detectable at rates similar to those possible for other tumors. Conclusions: We found robust evidence that mutated DNA is spilled from MPMs, mostly into pleural fluids, proving the concept that liquid biopsies are feasible for MPM patients.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive human cancer and miRNAs can play a key role for this disease. In order to broaden the knowledge in this field, the miRNA expression was investigated in a large series of MPM to discover new pathways helpful in diagnosis, prognosis and therapy. We employed nanoString nCounter system for miRNA profiling on 105 MPM samples and 10 healthy pleura. The analysis was followed by the validation of the most significantly deregulated miRNAs by RT-qPCR in an independent sample set. We identified 63 miRNAs deregulated in a statistically significant way. MiR-185, miR-197, and miR-299 were confirmed differentially expressed, after validation study. In addition, the results of the microarray analysis corroborated previous findings concerning miR-15b-5p, miR-126-3p, and miR-145-5p. Kaplan-Meier curves were used to explore the association between miRNA expression and overall survival (OS) and identified a 2-miRNA prognostic signature (Let-7c-5p and miR-151a-5p) related to hypoxia and energy metabolism respectively. In silico analyses with DIANA-microT-CDS highlighted 5 putative targets in common between two miRNAs. With the present work we showed that the pattern of miRNAs expression is highly deregulated in MPM and that a 2-miRNA signature can be a new useful tool for prognosis in MPM.

Abstract Background Malignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge. Methods TIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment. Results We analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity. Conclusions These results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.

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.

Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer with a poor survival rate. Treatment options are limited at best and drug resistance is common. Thus, there is an urgent need to identify novel therapeutic targets in this disease in order to improve patient outcomes and survival times. MST1R (RON) is a trans-membrane receptor tyrosine kinase (RTK), which is part of the c-MET proto-oncogene family. The only ligand recognized to bind MST1R (RON) is Macrophage Stimulating 1 (MST1), also known as Macrophage Stimulating Protein (MSP) or Hepatocyte Growth Factor-Like Protein (HGFL). In this study, we demonstrate that the MST1-MST1R (RON) signaling axis is active in MPM. Targeting this pathway with a small molecule inhibitor, LCRF-0004, resulted in decreased proliferation with a concomitant increase in apoptosis. Cell cycle progression was also affected. Recombinant MST1 treatment was unable to overcome the effect of LCRF-0004 in terms of either proliferation or apoptosis. Subsequently, the effect of an additional small molecular inhibitor, BMS-777607 (which targets MST1R (RON), MET, Tyro3, and Axl) also resulted in a decreased proliferative capacity of MPM cells. In a cohort of MPM patient samples, high positivity for total MST1R by IHC was an independent predictor of favorable prognosis. Additionally, elevated expression levels of MST1 also correlated with better survival. This study also determined the efficacy of LCRF-0004 and BMS-777607 in xenograft MPM models. Both LCRF-0004 and BMS-777607 demonstrated significant anti-tumor efficacy in vitro, however BMS-777607 was far superior to LCRF-0004. The in vivo and in vitro data generated by this study indicates that a multi-TKI, targeting the MST1R/MET/TAM signaling pathways, may provide a more effective therapeutic strategy for the treatment of MPM as opposed to targeting MST1R alone.

Malignant pleural mesothelioma (MPM) is a fatal tumor lacking effective therapies. The characterization of overexpressed genes could constitute a strategy for identifying drivers of tumor progression as targets for novel therapies. Thus, we performed an integrated gene-expression analysis on RNAseq data of 85 MPM patients from TCGA dataset and reference samples from the GEO. The gene list was further refined by using published studies, a functional enrichment analysis, and the correlation between expression and patients’ overall survival. Three molecular signatures defined by 15 genes were detected. Seven genes were involved in cell adhesion and extracellular matrix organization, with the others in control of the mitotic cell division or apoptosis inhibition. Using Western blot analyses, we found that ADAMTS1, PODXL, CIT, KIF23, MAD2L1, TNNT1, and TRAF2 were overexpressed in a limited number of cell lines. On the other hand, interestingly, CTHRC1, E-selectin, SPARC, UHRF1, PRSS23, BAG2, and MDK were abundantly expressed in over 50% of the six MPM cell lines analyzed. Thus, these proteins are candidates as drivers for sustaining the tumorigenic process. More studies with small-molecule inhibitors or silencing RNAs are fully justified and need to be undertaken to better evaluate the cancer-driving role of the targets herewith identified.

Tumor-infiltrating T cell rescue by programmed cell death receptor-1 (PD-1)/PD-1 ligand-1 (PD-L1) immune checkpoint blockade is a recommended treatment for malignant diseases, including metastatic non-small-cell lung cancer (mNSCLC), malignant melanoma (MM), head and neck, kidney, and urothelial cancer. Monoclonal antibodies (mAbs) against either PD-1 or PD-L1 are active agents for these patients; however, their use may be complicated by unpredictable immune-related adverse events (irAEs), including immune-related pneumonitis (IRP). We carried out a retrospective multi-institutional statistical analysis to investigate clinical and biological parameters correlated with IRP rate on a cohort of 256 patients who received real-world treatment with PD-1/PD-L1 blocking mAbs. An independent radiological review board detected IRP in 29 patients. We did not find statistical IRP rate correlation with gender, tumor type, specific PD-1 or PD-L1 blocking mAbs, radiation therapy, inflammatory profile, or different irAEs. A higher IRP risk was detected only in mNSCLC patients who received metronomic chemotherapy +/− bevacizumab compared with other treatments prior PD-1/PD-L1 blockade. Moreover, we detected a strong correlation among the IRP rate and germinal expression of HLA-B*35 and DRB1*11, alleles associated to autoimmune diseases. Our findings may have relevant implications in predicting the IRP rate in mNSCLC patients receiving PD-1/PD-L1 blockade and need to be validated on a larger patient series.

The spread of COVID-19 is showing huge, unexplained, differences between northern and southern Italy. We hypothesized that the regional prevalence of specific class I human leukocyte antigen (HLA) alleles, which shape the anti-viral immune response, might partly underlie these differences. Through an ecological approach, we analyzed whether a set of HLA alleles (A, B, C), known to be involved in the immune response against infections, correlates with COVID-19 incidence. COVID-19 data were provided by the National Civil Protection Department, whereas HLA allele prevalence was retrieved through the Italian Bone-Marrow Donors Registry. Among all the alleles, HLA-A*25, B*08, B*44, B*15:01, B*51, C*01, and C*03 showed a positive log-linear correlation with COVID-19 incidence rate fixed on 9 April 2020 in proximity of the national outbreak peak (Pearson’s coefficients between 0.50 and 0.70, p-value < 0.0001), whereas HLA-B*14, B*18, and B*49 showed an inverse log-linear correlation (Pearson’s coefficients between −0.47 and −0.59, p-value < 0.0001). When alleles were examined simultaneously using a multiple regression model to control for confounding factors, HLA-B*44 and C*01 were still positively and independently associated with COVID-19: a growth rate of 16% (95%CI: 0.1–35%) per 1% point increase in B*44 prevalence; and of 19% (95%CI: 1–41%) per 1% point increase in C*01 prevalence. Our epidemiologic analysis, despite the limits of the ecological approach, is strongly suggestive of a permissive role of HLA-C*01 and B*44 towards SARS-CoV-2 infection, which warrants further investigation in case-control studies. This study opens a new potential avenue for the identification of sub-populations at risk, which could provide Health Services with a tool to define more targeted clinical management strategies and priorities in vaccination campaigns.

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.

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.

[This corrects the article DOI: 10.1371/journal.pone.0085935.].

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

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.

Ribonucleases are a superfamily of RNA-cleaving enzymes that can be cytotoxic since the cleavage of RNA makes its information indecipherable. Ranpirnase is a novel ribonuclease which preferentially degrades tRNA, thus leading to an inhibition of protein synthesis and, ultimately, to cytostasis and cytotoxicity. Ranpirnase has demonstrated antitumor activity both in vitro and in vivo in several tumor models, including malignant mesothelioma. A large phase II trial showed that ranpirnase has diseasemodifying activity against mesothelioma. A first phase III study demonstrated that rampirnase may be combined with doxorubicin and that such an association is more active than Ranpirnase alone against mesothelioma. At present, another large, phase III trial in combination with doxorubicin has completed enrollment and its results are awaited. In all the above studies, ranpirnase died not demonstrate conventional anticancer activity, stabilizing progressive disease and potentially prolonging patients’ survival. Finally, a better understanding of its mechanism of action, coupled with its favorable toxicity profile, especially characterized by the lack of major hematologic toxicities, makes ranpirnase an attractive drug to test in combination with other anticancer agents, in MMe as well as in other tumor types.

Camillo Porta1, Chiara Paglino1, Luciano Mutti21Medical Oncology and Laboratory of Pre-Clinical Oncology and Developmental Therapeutics, I.R.C.C.S. San Matteo University Hospital Foundation, Pavia, Italy; 2Local Health Authority 6, Piedmont, ItalyAbstract: Ribonucleases are a superfamily of enzymes which operate at the crossroads of transcription and translation, catalyzing the degradation of RNA; they can be cytotoxic because the cleavage of RNA renders indecipherable its information. Ranpirnase is a novel ribonuclease which preferentially degrades tRNA, thus leading to inhibition of protein synthesis and, ultimately, to cytostasis and cytotoxicity. Ranpirnase has demonstrated antitumor activity both in vitro and in vivo in several tumor models. The maximum tolerated dose emerging from phase I studies was 960 g/m2, with renal toxicity as the main dose-limiting toxicity. A large phase II trial showed that ranpirnase has disease-modifying activity against malignant mesothelioma. Ranpirnase proved to be superior to doxorubicin in a phase III trial, while preliminary results of another large, phase III trial, suggest that the combination of ranpirnase and doxorubicin could be more effective than doxorubicin alone. In all the above studies, ranpirnase seems to act mainly as a cytostatic rather than a cytotoxic drug, stabilizing progressive disease and potentially prolonging patients’ survival. Ranpirnase may thus find its niche in combination with doxorubicin for mesothelioma as a second-line therapy, where no standard of care presently exists.Keywords: ranpirnase, mesothelioma, ribonucleases, doxorubicin, antitumor activity

Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

Malignant pleural mesothelioma (MPM) is a poor prognosis disease lacking adequate therapy. We have previously shown that ascorbic acid administration is toxic to MPM cells. Here we evaluated a new combined therapy consisting of ascorbate/epigallocatechin-3-gallate/gemcitabine mixture (called AND, for Active Nutrients/Drug). In vitro effects of AND therapy on various MPM cell lines revealed a synergistic cytotoxic mechanism. In vivo experiments on a xenograft mouse model for MPM, obtained by REN cells injection in immunocompromised mice, showed that AND strongly reduced the size of primary tumor as well as the number and size of metastases, and prevented abdominal hemorrhage. Kaplan Meier curves and the log-rank test indicated a marked increase in the survival of AND-treated animals. Histochemical analysis of dissected tumors showed that AND induced a shift from cell proliferation to apoptosis in cancer cells. Lysates of tumors from AND-treated mice, analyzed with an antibody array, revealed decreased TIMP-1 and -2 expressions and no effects on angiogenesis regulating factors. Multiplex analysis for signaling protein phosphorylation exhibited inactivation of cell proliferation pathways. The complex of data showed that the AND treatment is synergistic in vitro on MPM cells, and blocks in vivo tumor progression and metastasization in REN-based xenografts. Hence, the AND combination is proposed as a new treatment for MPM.

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05-10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.

PI3K/AKT signalling pathway is aberrantly active and plays a critical role for cell cycle progression of human malignant pleural mesothelioma (MMe) cells. AKT is one of the important cellular targets of perifosine, a novel bio-available alkylphospholipid that has displayed significant anti-proliferative activity in vitro and in vivo in several human tumour model systems and is currently being tested in clinical trials.We tested Perifosine activity on human mesothelial cells and different mesothelioma cell lines, in order to provide evidence of its efficacy as single agent and combined therapy.We demonstrate here that perifosine, currently being evaluated as an anti-cancer agent in phase 1 and 2 clinical trials, caused a dose-dependent reduction of AKT activation, at concentrations causing MMe cell growth arrest. In this study we firstly describe that MMe cells express aside from AKT1 also AKT3 and that either the myristoylated, constitutively active, forms of the two proteins, abrogated perifosine-mediated cell growth inhibition. Moreover, we describe here a novel mechanism of perifosine that interferes, upstream of AKT, affecting EGFR and MET phosphorylation. Finally, we demonstrate a significant increase in cell toxicity when MMe cells were treated with perifosine in combination with cisplatin.This study provides a novel mechanism of action of perifosine, directly inhibiting EGFR/MET-AKT1/3 axis, providing a rationale for a novel translational approach to the treatment of MMe.

BACKGROUND: The role of estrogen and estrogen receptors in oncogenesis has been investigated in various malignancies. Recently our group identified estrogen receptor beta (ERβ) expression as an independent prognostic factor in the progression of human Malignant Pleural Mesothelioma (MMe), but the underlying mechanism by which ERβ expression in tumors determines clinical outcome remains largely unknown. This study is aimed at investigating the molecular mechanisms of ERβ action in MMe cells and disclosing the potential translational implications of these results. METHODS: We modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation. RESULTS: Our data indicate that ERβ knockdown in ER positive cells confers a more invasive phenotype, increases anchorage independent proliferation and elevates the constitutive activation of EGFR-coupled signal transduction pathways. Conversely, re-expression of ERβ in ER negative cells confers a more epithelioid phenotype, decreases their capacity for anchorage independent growth and down-modulates proliferative signal transduction pathways. We identify a physical interaction between ERβ, EGFR and caveolin 1 that results in an altered internalization and in a selective reduced activation of EGFR-coupled signaling, when ERβ is over-expressed. We also demonstrate that differential expression of ERβ influences MMe tumor cell responsiveness to the therapeutic agent: Gefitinib. CONCLUSIONS: This study describes a role for ERβ in the modulation of cell proliferation and EGFR activation and provides a rationale to facilitate the targeting of a subgroup of MMe patients who would benefit most from therapy with Gefitinib alone or in combination with Akt inhibitors.