Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes.

Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.

Use of Human Pluripotent Stem Cell Derived-Cardiomyocytes to Study Drug-Induced Cardiotoxicity.

Drug-induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug-induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug-induced cardiotoxicty using doxorubicin as an example. We present a workflow that explains procedure for editing hPSC using the CRISPR/Cas9 system and for differentiation of hPSC into cardiomyocytes. We also report protocols to study drug effect on ROS production, intracellular calcium concentration, formation of DNA double strand breaks, gene expression and electrophysiological properties of hPSC-CMs. © 2017 by John Wiley & Sons, Inc.

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients.Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.

Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes.

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.

Redox regulation of p53, redox effectors regulated by p53: a subtle balance.

SIGNIFICANCE: Reactive oxygen species (ROS), generated by cells as side products of biological reactions, function as secondary messengers by impacting a host of cellular networks involved in maintaining normal homeostatic growth as well as pathological disease states. Redox-sensitive proteins, such as the tumor suppressor protein p53, are susceptible to ROS-dependent modifications, which could impact their activities and/or biological functions. RECENT ADVANCES: p53 is a transcription factor that controls a wide variety of target genes and regulates numerous cellular functions in response to stresses that lead to genomic instability. Thus, redox modifications of p53 could impact cell fate signaling and could have profound effects on pathways fundamental to maintaining cell and tissue integrity. CRITICAL ISSUES: Recent studies present evidence that ROS function upstream of p53 in some model systems, while in others ROS production could be a downstream effect of p53 activation. FUTURE DIRECTIONS: In this review, we describe how ROS production regulates p53 activity and how p53 can, in turn, influence cellular ROS production.

The airways, a novel route for delivering monoclonal antibodies to treat lung tumors.

PURPOSE: Lung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited, with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer. We have shown that cetuximab, a chimeric anticancer anti-EGFR mAb, is suitable for airway delivery as it resists the physical constraints of aerosolization, and have evaluated the aerosol delivery of cetuximab in vivo. METHODS: We developed an animal model of lung tumor sensitive to cetuximab by injecting Balb/c Nude mice intratracheally with A431 cells plus 10 mM EDTA and analyzed the distribution, pharmacokinetics and antitumor efficacy of cetuximab aerosolized into the respiratory tract. RESULTS: Aerosolized IgG accumulated durably in the lungs and the tumor, but passed poorly and slowly into the systemic circulation. Aerosolized cetuximab also limited the growth of the mouse tumor. Thus, administering anticancer mAbs via the airways is effective and may limit systemic side effects. CONCLUSION: Delivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.

Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization.

PURPOSE: Despite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine. MATERIALS AND METHODS: Cetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+, a mesh nebulizer AeronebPro and an ultrasonic nebulizer SYST'AM LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells. RESULTS: The aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab. CONCLUSIONS: Altogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.