// Upal Basu-Roy 1,* , Eugenia Han 2,* , Kirk Rattanakorn 1 , Abhilash Gadi 1 , Narendra Verma 1 , Giulia Maurizi 1 , Preethi H. Gunaratne 3 , Cristian Coarfa 4 , Oran D. Kennedy 5 , Michael J. Garabedian 1,2 , Claudio Basilico 1,2 and Alka Mansukhani 1,2 1 Department of Microbiology, NYU School of Medicine, New York, NY, USA 2 Perlmutter Cancer Center, Langone Medical Center, New York, NY, USA 3 Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA 4 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA 5 Department of Orthopaedic Surgery, NYU School of Medicine, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Alka Mansukhani, email: // Keywords : osteosarcoma, thiazolidinediones, cancer stem cells, osteoblast lineage, adipocyte Received : July 15, 2016 Accepted : August 01, 2016 Published : August 12, 2016 Abstract Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma.
Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.
Abstract Introduction Propranolol was recently discovered to be an effective treatment for infantile haemangiomas, and varying doses and monitoring regimens have been proposed. Adverse events, although uncommon, have been reported. Materials and methods This was a retrospective chart review of infants with haemangiomas who were started on propranolol at a dose of 3 milligrams per kilogram per day on an outpatient basis. After a baseline cardiac evaluation including an electrocardiogram and an echocardiogram, treatment was initiated during 6 hours of observation. Results A total of 15 patients were identified; however, only 13 returned for at least one follow-up visit. This cohort was followed up for a median of 2.8 months with a range from 0.2 to 10.0. No hypotension, hypoglycaemia, bronchospasm, or clinically significant bradycardia occurred during treatment. All patients had clinical improvement of their haemangiomas. Conclusions This study suggests that initiating treatment during outpatient observation may be a reasonable alternative to inpatient admission. In addition, expensive testing may not be necessary during pre-treatment screening when the physical examination is normal.
The repressive Hippo pathway has a profound tumour suppressive role in cancer by restraining the growth-promoting function of the transcriptional coactivator, YAP. We previously showed that the stem cell transcription factor Sox2 maintains cancer stem cells (CSCs) in osteosarcomas. We now report that in these tumours, Sox2 antagonizes the Hippo pathway by direct repression of two Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), leading to exaggerated YAP function. Repression of Nf2, WWC1 and high YAP expression marks the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression. YAP depletion sharply reduces CSCs and tumorigenicity of osteosarcomas. Thus, Sox2 interferes with the tumour-suppressive Hippo pathway to maintain CSCs in osteosarcomas. This Sox2-Hippo axis is conserved in other Sox2-dependent cancers such as glioblastomas. Disruption of YAP transcriptional activity could be a therapeutic strategy for Sox2-dependent tumours. Transcriptional regulators Sox2 and YAP maintain expression of stemness genes in normal and cancerous cells. Here the authors show that, in osteosarcomas, Sox2 activates YAP by directly repressing transcription of its upstream negative regulators Nf2 and WWC1, promoting cancer cell stemness.
Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation.This study investigated changes in lung NOS expression and activity in a mouse model of ventilator-induced lung injury.C57BL/6J (wild-type [WT]) and iNOS-deficient (iNOS(-/-)) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay.iNOS mRNA and protein expression was absent in iNOS(-/-) mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS(-/-) mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS(-/-) mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS(-/-) mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS(-/-) mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine.Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury.
Primary neuroendocrine carcinomas (NEC) are rare tumors in children and young adults, resulting in a lack of standardized treatment approach. To refine the molecular taxonomy of these rare tumors, we performed whole exome sequencing in a pediatric patient with mediastinal NEC. We identified a somatic mutation in HRAS gene and LOH regions in NF2, MYO18B, and RUX3 genes. In addition, a germline heterozygous somatic variant in BRCA2 with LOH at that same position in the tumor tissue was also found. Our data provide valuable insight into the genomic landscape of this tumor, prompting further investigation of therapeutic targets.
ABSTRACT Background The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. Conclusion ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.
