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// Jian He 1, * , Mingju Sun 1, * , Enyou Li 2, * , Yingyong Hou 5, * , Matthew J. Shepard 3, 6 , Di Chen 1 , Karel Pacak 7 , Changsong Wang 4 , Lei Guo 2 , Zhengping Zhuang 3 and Yang Liu 1 1 Scientific Research Center for Translational Medicine, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 2 Department of Anesthesiology, First Affiliated Hospital of Harbin Medical University, Harbin, China 3 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 4 Department of Critical Care Medicine, The Third Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China 5 Department of Pathology, School of Basic Medical Sciences & Zhongshan Hospital, Fudan University, Shanghai, China 6 Department of Neurologic Surgery, University of Virginia Health System, Charlottesville, Virginia, USA 7 Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA * These authors have contributed equally to this work Correspondence to: Zhengping Zhuang, email: zhuangp@ninds.nih.gov Yang Liu, email: yliuqq@dicp.ac.cn Keywords: cardiac myxomas; PRKAR1A; somatic mutation Received: July 14, 2017 Accepted: September 20, 2017 Published: October 19, 2017 ABSTRACT Background: Cardiac myxomas are benign tumors that commonly arise within the left atria. Familial cardiac myxomas are a part of Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome caused by germline mutations in PRKAR1A . Seven percent of cardiac myxomas are associated with CNC. To date, the genetic basis of isolated cardiac myxomas (ICM), however, has not been fully elucidated. Methods: We investigated the genetic profile of ICM using whole exome sequencing (WES). Suspected mutations were confirmed using targeted sanger sequencing. To further examine the presence of PRKAR1A mutations in ICM, we performed targeted sequencing in an additional 61 ICM specimens. Results: 87.5% (7/8) of ICM harbored mutations in PRKAR1A . Three of the 8 ICM harbored biallelic somatic mutations of PRKAR1A , including c.607_610del:p.Leu203fs (pathogenic) + c.C896G:p.Ser299X (pathogenic), c.952delT:p.Leu318fs (pathogenic) + c.769-2 A>G (pathogenic) and c.178-1 G>C (pathogenic) + c. 550+1 G>C (pathogenic). Four of 8 tumors harbored monoallelic PRKAR1A mutations, including c.523_524insG:p.Tyr175_Val176delinsX (pathogenic), c.C920A:p.Ser307X (pathogenic), c.30delG:p.Glu10fs (pathogenic) and c.C289T:p.Arg97X (pathogenic). No identical variants were observed across the 8 ICM samples. Interestingly, none of these variants have been previously described in familial cardiac myxomas. In order to confirm our findings, directed sequencing of 61 ICM specimens was subsequently performed. Sixty-four percent (39/61) of ICMs tumors contained inactivating PRKAR1A mutations. Conclusion: Our findings suggest that loss-of-function mutations of PRKAR1A may play a vital role in the formation of isolated cardiac myxomas.

// Chao Zhang 1,2,* , Chunzhang Yang 3,* , Michael J. Feldman 4 , Herui Wang 4 , Ying Pang 2 , Dominic M. Maggio 4 , Dongwang Zhu 4 , Cody L. Nesvick 4 , Pauline Dmitriev 4 , Petra Bullova 2,5 , Prashant Chittiboina 4 , Roscoe O. Brady 4 , Karel Pacak 2 and Zhengping Zhuang 4 1 Department of Orthopedics, Xinqiao Hospital, The Third Military Medical University, Chongqing, China 2 Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA 3 Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA 4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 5 Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia * These authors have contributed equally to this work Correspondence to: Karel Pacak, email: // Zhengping Zhuang, email: // Keywords : HDACi, Hsp90, SAHA, HIF, hypoxia Received : December 14, 2016 Accepted : April 10, 2017 Published : May 23, 2017 Abstract Histone deacetylase inhibitors (HDACis) are a potent class of tumor-suppressive agents traditionally believed to exert their effects through loosening tightly-wound chromatin resulting in de-inhibition of various tumor suppressive genes. Recent literature however has shown altered intratumoral hypoxia signaling with HDACi administration not attributable to changes in chromatin structure. We sought to determine the precise mechanism of HDACi-mediated hypoxia signaling attenuation using vorinostat (SAHA), an FDA-approved class I/IIb/IV HDACi. Through an in-vitro and in-vivo approach utilizing cell lines for hepatocellular carcinoma (HCC), osteosarcoma (OS), and glioblastoma (GBM), we demonstrate that SAHA potently inhibits HIF-a nuclear translocation via direct acetylation of its associated chaperone, heat shock protein 90 (Hsp90). In the presence of SAHA we found elevated levels of acetyl-Hsp90, decreased interaction between acetyl-Hsp90 and HIF-a, decreased nuclear/cytoplasmic HIF-α expression, absent HIF-α association with its nuclear karyopharyin Importin, and markedly decreased HIF-a transcriptional activity. These changes were associated with downregulation of downstream hypoxia molecules such as endothelin 1, erythropoietin, glucose transporter 1, and vascular endothelial growth factor. Findings were replicated in an in-vivo Hep3B HRE-Luc expressing xenograft, and were associated with significant decreases in xenograft tumor size. Altogether, this study highlights a novel mechanism of action of an important class of chemotherapeutic.

Metabolomics has shown significant potential in identifying small molecules specific to tumor phenotypes. In this study we analyzed resected tissue metabolites using capillary electrophoresis-mass spectrometry and found that tissue hypotaurine levels strongly and positively correlated with glioma grade. In vitro studies were conducted to show that hypotaurine activates hypoxia signaling through the competitive inhibition of prolyl hydroxylase domain-2. This leads to the activation of hypoxia signaling as well as to the enhancement of glioma cell proliferation and invasion. In contrast, taurine, the oxidation metabolite of hypotaurine, decreased intracellular hypotaurine and resulted in glioma cell growth arrest. Lastly, a glioblastoma xenograft mice model was supplemented with taurine feed and exhibited impaired tumor growth. Taken together, these findings suggest that hypotaurine is an aberrantly produced oncometabolite, mediating tumor molecular pathophysiology and progression. The hypotaurine metabolic pathway may provide a potentially new target for glioblastoma diagnosis and therapy.

The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 μM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.

// Zhen-Yu Zhang 1, * , Aden Ka-Yin Chan 2, * , Xiao-Jie Ding 1 , Zhi-Yong Qin 1 , Christopher S. Hong 3 , Ling-Chao Chen 1 , Xin Zhang 1 , Fang-Ping Zhao 4 , Yin Wang 5 , Yang Wang 1 , Liang-Fu Zhou 1 , Zhengping Zhuang 3 , Ho-Keung Ng 2 , Hai Yan 6 , Yu Yao 1 , Ying Mao 1 1 Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China 2 Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 3 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 4 Genetron Health, Inc., Chaoyang District, Beijing, China 5 Department of Neuropathology, Huashan Hospital, Fudan University, Shanghai, China 6 Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, The Pediatric Brain Tumor Foundation Institute, Durham, North Carolina, USA * These authors have contributed equally to this work Correspondence to: Yu Yao, e-mail: yu_yao@fudan.edu.cn Hai Yan, e-mail: hai.yan@duke.edu Ho-Keung Ng, e-mail: hkng@cuhk.edu.hk Keywords: TERT promoter, IDH, gliomas, radiation therapy, chemotherapy Received: March 29, 2015 Accepted: June 26, 2015 Published: July 09, 2015 ABSTRACT IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase ( TERT ) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations ( p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/ TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/ TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup.

Previously, we conducted a randomized phase III trial of TPF (docetaxel, cisplatin, and 5-fluorouracil) induction chemotherapy in surgically managed locally advanced oral squamous cell carcinoma (OSCC) and found no improvement in overall survival. This study reports long-term follow-up results from our initial trial. All patients had clinical stage III or IVA locally advanced OSCC. In the experimental group, patients received two cycles of TPF induction chemotherapy (75mg/m2 docetaxel d1, 75mg/m2 cisplatin d1, and 750mg/m2/day 5-fluorouracil d1-5) followed by radical surgery and post-operative radiotherapy; in the control group, patients received upfront radical surgery and post-operative radiotherapy. The primary endpoint was overall survival. Among 256 enrolled patients with a median follow-up of 70 months, estimated 5-year overall survival, disease-free survival, locoregional recurrence-free survival, and distant metastasis-free survival rates were 61.1%, 52.7%, 55.2%, and 60.4%, respectively. There were no significant differences in survival rates between experimental and control groups. However, patients with favorable pathologic responses had improved outcomes compared to those with unfavorable pathologic responses and to those in the control group. Although TPF induction chemotherapy did not improve long-term survival compared to surgery upfront in patients with stage III and IVA OSCC, a favorable pathologic response after induction chemotherapy may be used as a major endpoint and prognosticator in future studies. Furthermore, the negative results observed in this trial may be represent type II error from an underpowered study. Future larger scale phase III trials are warranted to investigate whether a significant benefit exists for TPF induction chemotherapy in surgically managed OSCC.

Background Cardiac myxomas are benign tumors that commonly arise within the left atria. Familial cardiac myxomas are a part of Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome caused by germline mutations in PRKAR1A. Seven percent of cardiac myxomas are associated with CNC. To date, the genetic basis of isolated cardiac myxomas (ICM), however, has not been fully elucidated. Methods We investigated the genetic profile of ICM using whole exome sequencing (WES). Suspected mutations were confirmed using targeted sanger sequencing. To further examine the presence of PRKAR1A mutations in ICM, we performed targeted sequencing in an additional 61 ICM specimens. Results 87.5% (7/8) of ICM harbored mutations in PRKAR1A. Three of the 8 ICM harbored biallelic somatic mutations of PRKAR1A, including c.607_610del:p.Leu203fs (pathogenic) + c.C896G:p.Ser299X (pathogenic), c.952delT:p.Leu318fs (pathogenic) + c.769-2 A>G (pathogenic) and c.178-1 G>C (pathogenic) + c. 550+1 G>C (pathogenic). Four of 8 tumors harbored monoallelic PRKAR1A mutations, including c.523_524insG:p.Tyr175_Val176delinsX (pathogenic), c.C920A:p.Ser307X (pathogenic), c.30delG:p.Glu10fs (pathogenic) and c.C289T:p.Arg97X (pathogenic). No identical variants were observed across the 8 ICM samples. Interestingly, none of these variants have been previously described in familial cardiac myxomas. In order to confirm our findings, directed sequencing of 61 ICM specimens was subsequently performed. Sixty-four percent (39/61) of ICMs tumors contained inactivating PRKAR1A mutations. Conclusion Our findings suggest that loss-of-function mutations of PRKAR1A may play a vital role in the formation of isolated cardiac myxomas.

von Hippel-Lindau (VHL) disease is caused by mutations in the VHL gene and demonstrates marked phenotypic variability. Genotype-phenotype correlations in Chinese VHL patients have been unclear. To establish genotype-phenotype correlations in Chinese VHL patients, we collected VHL mutations and phenotypes of 291 patients with VHL disease from 115 unrelated families. Genotype-phenotype correlations at mutation type level, mutation region level, and mutation codon level were analyzed by Kaplan-Meier curves and Cox regression models. We found missense mutations conferred an increased risk of pheochromocytoma developments, but a decreased risk of central nervous system hemangioblastomas (CHBs) and pancreatic lesions. Patients with VHL deletions were more prone to developing retinal angiomas. Renal cell carcinomas were more frequent in nonsense, frameshift or splice-site mutations. Mutations in Exon 2 conferred a higher risk and earlier diagnostic age of CHBs than mutations in other exons (HR = 1.684, 95% CI 1.082-2.620, p = 0.021; 27.0 ± 9.7 years versus 32.8 ± 11.7 years, p = 0.024), while patients with mutations in Exon 3 were more prone to developing pheochromocytomas (HR = 2.760, 95% CI 1.419-5.370, p = 0.003). Mutations at codon 80 or codon167 conferred significantly higher risks of pheochromocytomas than other mutations (HR = 4.678, 95% CI 1.392-15.724, p = 0.013; HR = 4.683, 95% CI 2.515-8.719, p < 0.001 respectively). In conclusion, VHL mutation types, mutation regions and mutation codons can act as phenotypic predictors of VHL disease. Mutation regions and mutation codons may aid in directed surveillance and monitoring of VHL patients.

// Ying Pang 1 , Chunzhang Yang 2 , Jan Schovanek 3 , Herui Wang 4 , Petra Bullova 5 , Veronika Caisova 1 , Garima Gupta 1 , Katherine I. Wolf 1 , Gregg L. Semenza 6 , Zhengping Zhuang 4 and Karel Pacak 1 1 Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA 2 Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA 3 Department of Internal Medicine III-Nephrology, Rheumatology, and Endocrinology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic 4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 5 Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovak Republic 6 McKusick-Nathans Institute of Genetic Medicine and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Karel Pacak, email: // Keywords : anthracyclines, paraganglioma, pheochromocytoma, metastatic, hypoxia-inducible factors Received : January 13, 2017 Accepted : March 03, 2017 Published : March 15, 2017 Abstract Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare, neuroendocrine tumors derived from adrenal or extra-adrenal chromaffin cells, respectively. Metastases are discovered in 3-36% of patients at the time of diagnosis. Currently, only suboptimal treatment options exist. Therefore, new therapeutic compounds targeting metastatic PHEOs/PGLs are urgently needed. Here, we investigated if anthracyclines were able to suppress the progression of metastatic PHEO. We explored their effects on experimental mouse PHEO tumor cells using in vitro and in vivo models, and demonstrated that anthracyclines, particularly idarubicin (IDA), suppressed hypoxia signaling by preventing the binding of hypoxia-inducible factor 1 and 2 (HIF-1 and HIF-2) to the hypoxia response element (HRE) sites on DNA. This resulted in reduced transcriptional activation of HIF target genes, including erythropoietin ( EPO ), phosphoglycerate kinase 1 ( PGK1 ), endothelin 1 ( EDN1 ), glucose transporter 1 ( GLUT1 ), lactate dehydrogenase A ( LDHA ), and vascular endothelial growth factor ( VEGFA ), which consequently inhibited the growth of metastatic PHEO. Additionally, IDA downregulated hypoxia signaling by interfering with the transcriptional activation of HIF1A and HIF2A . Furthermore, our animal model demonstrated the dose-dependent suppressive effect of IDA on metastatic PHEO growth in vivo . Our results indicate that anthracyclines are prospective candidates for inclusion in metastatic PHEO/PGL therapy, especially in patients with gene mutations involved in the hypoxia signaling pathway.

Nasopharyngeal carcinoma (NPC), while uncommon worldwide, is a major health problem in China. Although local radiation and surgery provide good control of NPC, better treatments that permit reductions in radiation dosing are needed. Inhibition of protein phosphatase 2A (PP2A), a ubiquitous multifunctional enzyme with critical roles in cell cycle regulation and DNA-damage response, reportedly sensitizes cancer cells to radiation and chemotherapy. We studied PP2A inhibition with LB100, a small molecule currently in a Phase I clinical trial, on radiosensitization of two human nasopharyngeal cell lines: CNE1, which is reportedly radioresistant, and CNE2. In both cell lines, LB100 exposure increased intracellular p-Plk1, TCTP, and Cdk1 and decreased p53, changes associated with cell cycle arrest, mitotic catastrophe and radio-inhibition of cell proliferation. Mice bearing subcutaneous xenografts of either cell line were administered 1.5 mg/kg LB100 daily for three days and a single dose of 20 Gy radiation (day 3), which produced marked and prolonged tumor mass regression (dose enhancement factors of 2.98 and 2.27 for CNE1 and CNE2 xenografts, respectively). Treatment with either LB100 or radiation alone only transiently inhibited xenograft growth. Our results support further exploration of PP2A inhibition as part of radiotherapy regimens for NPC and potentially other solid tumors.

// Akio Soeda 1 , Justin Lathia 2 , Brian J. Williams 3 , Qiulian Wu 2 , Joseph Gallagher 2 , Andreas Androutsellis-Theotokis 4 , Amber J. Giles 5 , Chunzhang Yang 5 , Zhengping Zhuang 5 , Mark R. Gilbert 5 , Jeremy N. Rich 2 and Deric M. Park 5 1 Department of Neurosurgery, Gifu University, Gifu, Japan 2 Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic Foundation, Cleveland, OH, USA 3 Department of Neurosurgery, University of Louisville, Louisville, KY, USA 4 Division of Stem Cell Biology, Technische Universitat Dresden, Dresden, Germany 5 Neuro-Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA Correspondence to: Deric M. Park, email: deric.park@nih.gov Keywords: glioma, cancer stem cell, p38 MAPK, EGFR, quiescence Received: July 10, 2016 Accepted: March 19, 2017 Published: March 31, 2017 ABSTRACT EGFR pathway is upregulated in malignant gliomas, and its downstream signaling is important for self-renewal of glioma cancer stem-like cells (GSC). p38 mitogen-activated protein kinase (MAPK) signaling, a stress-activated signaling cascade with suppressive and permissive effects on tumorigenesis, can promote internalization and ubiquitin ligase mediated degradation of EGFR. In this study, we investigated the role of p38 MAPK signaling on the self-renewal of GSCs with the hypothesis that inhibition may lead to enhanced self-renewal capacity by retention of EGFR. Inhibition of p38 MAPK pathway led to increase in EGFR expression but surprisingly, reduced proliferation. Additional functional evaluation revealed that p38 inhibition was associated with decrease in cell death and maintenance of undifferentiated state. Further probing the effect of p38 inhibition demonstrated attenuation of EGFR downstream signaling activity in spite of prolonged surface expression of the receptor. In vitro observations were confirmed in xenograft in vivo experiments. These data suggest that p38 MAPK control of EGFR signaling activity may alter GSC cell cycle state by regulating quiescence and passage into transit amplifying state.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.