Jpn J Clin Oncol 2012;42(3)196–201 doi:10.1093/jjco/hyr195 Advance Access Publication 4 January 2012 Down-regulation of Nedd4L is Associated with the Aggressive Progression and Worse Prognosis of Malignant Glioma Shiming He1,†, Jianping Deng1,†, Gang Li1, Boliang Wang2, Yizhan Cao2 and Yanyang Tu2,* 1Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University and 2Department of Emergency, Tangdu Hospital, Fourth Military Medical University, Xi’an, China Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 *For reprints and all correspondence: Yanyang Tu, Department of Emergency, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China. E-mail: [email protected] †These authors contributed equally to this work. Received September 6, 2011; accepted December 10, 2011 Objective: Human neural precursor cell-expressed developmentally down-regulated 4 like (Nedd4L), a ubiquitin protein ligase, is expressed by various cancer cells and might have an oncogenic property. Its expression pattern in glioma tissues is unknown. Therefore, the aim of this study was to investigate whether Nedd4L is present in glioma and to evaluate the correlation of Nedd4L expression with the progression and prognosis of the disease. Methods: Immunohistochemistry and western blot were used to investigate the expression of Nedd4L protein in 128 patients with gliomas. Results: Immunohistochemistry showed a strong-to-weak range of Nedd4L staining with increasing pathologic grade of glioma (P , 0.001), which was in line with the results from western blot analysis. In addition, a non-parametric analysis revealed that the attenuated Nedd4L expression was significantly correlated with a large tumor diameter (P ¼ 0.02), low Karnofsky performance score (P ¼ 0.008), frequent intra-tumor necrosis (P ¼ 0.01) and worse overall survival (P ¼ 0.009). Furthermore, multivariate analysis showed that Nedd4L expression (P ¼ 0.02) and intra-tumor necrosis (P ¼ 0.03) were two important independent prognostic factors identified by the Cox proportional hazards model. Conclusions: Our results provide convincing evidence for the first time that the expression of Nedd4L is down-regulated in human gliomas. The glioma patients with lower Nedd4L expression have a worse prognosis. Key words: neural precursor cell-expressed developmentally down-regulated 4 like – glioma – immunohistochemistry – western blot analysis – expression INTRODUCTION surgical resection, radiation therapy and chemotherapy (2). Glioma, as the most common primary brain tumors in adult Numerous studies have showed that age, performance status, humans, continues to be the cause of a disproportionate level histologic grade and tumor necrosis are important prognostic of morbidity and mortality across a wide range of indivi- factors for gliomas. However, these clinical parameters do duals. The key features of this disease are local invasive not fully account for the observed variation in survival rates growth and strong angiogenesis. Gliomas are histologically and the prognosis of both high- and low-grade tumors classified into four grades (Grades I–IV), according to the remains heterogeneous (3). Therefore, additional indicators World Health Organization (WHO) guidelines (1). Grade IV are needed to more accurately determine the prognosis of glioma, glioblastoma, has the worst prognosis even after patients with gliomas. # The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Jpn J Clin Oncol 2012;42(3) 197 The neural precursor cell-expressed developmentally radiotherapy prior to surgery. The clinicopathological fea- down-regulated 4-like (Nedd4L) gene encodes a ubiquitin tures and the treatment strategies of all the patients are indi- ligase that targets the epithelial sodium channel (ENaC) for catedinTable1. Paraffin and snap-frozen sections of degradation (4). It participates in the regulation of plasma non-neoplastic brain tissues from 10 patients with intractable volume and blood pressure by controlling expression of epilepsy were also included as controls. A 5-year follow-up ENaC which is a heteromultimeric protein complex. The was performed, and all patients had a complete follow-up ubiquitination of ENaC is catalyzed by Nedd4L, initiating until death. The overall survival time was calculated from ubiquitin-mediated endocytosis and lysosomal targeting of the date of the initial surgical operation to death. Patients, ENaC for its removal from the luminal cell membrane in who died of diseases not directly related to their gliomas, or renal collecting ducts (5). Recent studies have demonstrated due to unexpected events, were excluded from this study. In that Nedd4L targets a broad range of molecules along with addition, 20 glioma specimens [5 pilocytic astrocytomas ENaC and is thus thought to be involved in various biologic- (WHO I), 3 diffuse astrocytomas (WHO II), 3 anaplasia Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 al properties other than regulating ion channels (6). Human astrocytomas (WHO III) and 9 primary glioblastomas (WHO Nedd4L is a homologue of the mouse Nedd4-2, which was IV)] were snap frozen in liquid nitrogen and stored at identified as a novel protein which could inhibit transforming 2808C following surgery for analysis by western blot growth factor b (TGF-b) signaling in TGF-b receptors and analysis. Smadslevels(7). It has been reported that TGF-b signaling is involved in a variety of biological processes, including regulation of cell growth, angiogenesis, immune response IMMUNOHISTOCHEMISTRY ASSAY and modulation of extracellular matrix (8). In gliomas, previ- Formalin-fixed, paraffin-embedded, sectioned tissues (4 mm ous studies have indicated that TGF-b signaling plays an thick) were immunostained using the Labelled Streptavidin important role in the pathobiology of invasion of the tumor Biotin 2 System (BioGenex; San Ramon, CA, USA). (9). Therefore, investigation into the TGF-b system in Following peroxidase blocking with 0.3% H2O2/methanol glioma is needed in order to learn whether there are candi- for 30 min, specimens were blocked with phosphate-buffered date prognostic markers in this system can be manipulated saline (PBS) containing 5% normal horse serum (Vector for survival prediction of gliomas. Nedd4L has been Laboratories Inc., Burlingame, CA, USA). All incubations detected in various cancer cells and might have an oncogenic with a monoclonal antibody against human Nedd4L property. However, its expression pattern in glioma tissues is (HPA024618, Sigma-Aldrich, St Louis, MO, USA) at 1:10 unknown. Therefore, the aim of this study was to investigate dilution were carried out overnight at 48C. Then, the whether Nedd4L is present in glioma and to evaluate the correlation of Nedd4L expression with the progression and prognosis of the disease. Table 1. Clinicopathological features of 128 patients with gliomas Features WHO I WHO II WHO III WHO IV PATIENTS AND METHODS Case no. 18 14 38 58 PATIENTS AND TISSUE SAMPLES Mean age (years) 38.6 45.9 53.1 54.2 This study was approved by the Research Ethics Committee Gender of Tangdu Hospital, Fourth Military Medical University, Male 12 6 25 33 China. Written informed consent was obtained from all of Female 6 8 13 25 the patients. All specimens were handled and made anonymous according to the ethical and legal standards. KPS A total of 128 formalin-fixed, paraffin-embedded speci- .80 15 11 9 15 mens of gliomas resected between 2000 and 2005 were ,80 3 3 29 43 retrieved from the archives of the Pathology Department of Surgery Tangdu Hospital, Fourth Military Medical University, China. Gross total resection 18 14 28 38 All the slides were re-evaluated according to the WHO clas- Partial resection 0 0 9 15 sifications (1) by two pathologists, with differences resolved by careful discussion. A total of 76 males and 52 females Biopsy 0 0 1 5 (1.46:1) were enrolled in this study, and the median age was Adjuvant treatment 52 years (range, 12–71). Thirty-two of the 128 gliomas were Radiotherapy 0 0 30 12 classified as low-grade gliomas [18 pilocytic astrocytomas Chemotherapy 0 1 0 6 (WHO I) and 14 diffuse astrocytomas (WHO II)] and 96 Radiotherapy and 00 5 28 were classified as high-grade gliomas [38 anaplasia astrocy- chemotherapy combination tomas (WHO III) and 58 primary glioblastomas (WHO IV)]. None of the patients had received chemotherapy or KPS, Karnofsky performance score. 198 Down-regulation of Nedd4L in glioma specimens were briefly washed in PBS and incubated at Nedd4L as an optical densitometry ratio that was scored as room temperature with the anti-rabbit antibody and avidin– the densitometry of Nedd4L relative to the densitometry of biotin peroxidase (Vector Laboratories Inc.). The specimens b-actin. were then washed in PBS and color-developed by diamino- benzidine solution (Dako Corporation, Carpinteria, CA, STATISTICAL ANALYSIS USA). After washing with water, specimens were counter- stained with Meyer’s hematoxylin (Sigma Chemical Co., St All computations were carried out using the software of Louis, MO, USA). Non-neoplastic brain tissues were used as SPSS version 13.0 for Windows (SPSS Inc., IL, USA). Data control tissues and non-immune IgG was also used as a were expressed as means + standard deviation. The amount negative control antibody for immunohistochemical staining. of Nedd4L in positively expressed tissue samples was pre- Assessment