Down-Regulation of Nedd4l Is Associated with the Aggressive Progression and Worse Prognosis of Malignant Glioma

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 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 of immunohistochemical staining was evalu- sented as a percentage (opacity density value of Nedd4L ated by two independent pathologists. The scores of the two divided by the opacity density of the internal reference from Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 pathologists were compared and any discrepant scores were b-actin as control). Statistical differences between the levels trained through re-examining the stainings by both patholo- of Nedd4L expression in pathological grades were evaluated gists to achieve a consensus score. Expression of Nedd4L by the non-parametric Kruskal–Wallis test, and those differ- was compared between malignant glioma tissues and non- ences in the bi-nominal clinical categories were evaluated by neoplastic brain tissues. The number of positive-staining the non-parametric Mann–Whitney test. A life table was cal- cells showing immunoreactivity on the cytoplasm for culated according to the Kaplan–Meier method. Hazard Nedd4L in 10 representative microscopic fields was counted ratios for the time-to-event endpoint were estimated using and the percentage of positive cells was calculated. The the multivariate Cox regression analysis in a forward step- percentage scoring of immunoreactive tumor cells was as wise method to evaluate the effect of multiple independent follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (.50%). prognostic factors on survival outcome. Differences were The staining intensity was visually scored and stratified as considered statistically significant when P value was ,0.05. follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactivity score (IRS) was obtained for each case by multiplying the percentage and the intensity score. RESULTS

DOWN-REGULATION OF NEDD4L PROTEIN IN HUMAN GLIOMA WESTERN BLOT ANALYSIS TISSUES Twenty glioma and 10 non-neoplastic brain tissues were The protein expression of Nedd4L was detected by immuno- homogenized in lysis buffer [PBS, 1% Nonidet P-40, 0.5% histochemical staining in 10 normal brain tissues and 128 sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), glioma specimens. In all gliomas, 59.38% (76/128) had 100 mg/ml aprotinin, 100 mg/ml phenylmethylsulfonyl fluor- weak staining and 40.62% (52/128) had moderate-to-strong ide (PMSF) and sodium orthovanadate] at 48C throughout all staining. Immunoreactivity was also measured in six procedures and sonicated for 70 s, and then add 300 mg (60.00%) non-neoplastic brain samples, all of which had PMSF per gram of tissue and incubate on ice for 30 min, fol- strong staining. Reduced expression of Nedd4L protein with lowed by centrifugation at 15 000 rpm for 20 min at 48C. cytoplasmic localization was observed in gliomas of differ- The protein content was determined according to the ent pathological grades when compared with normal brain Bradford method (Bradford 1976), with bovine serum tissue (Fig. 1). The IRS of Nedd4L was 8.22 + 0.15 in non- albumin used as a standard. Equal amounts of protein were neoplastic brain tissues, 6.16 + 0.21 in pilocytic astrocyto- separated electrophoretically on 7.5% SDS–polyacrylamide mas (WHO I), 4.28 + 0.16 in diffuse astrocytomas (WHO gels and transferred onto polyvinylidene difluoride mem- II), 3.35 + 0.28 in anaplasia astrocytomas (WHO III) and branes (Roche, Basel, Switzerland). The membranes were 2.25 + 0.18 in glioblastomas (WHO IV). The median IRS probed with an anti-Nedd4L rabbit monoclonal antibody value of Nedd4L in all the glioma samples was 4.01 + 0.19, (1:250 dilution, HPA024618, Sigma-Aldrich). The expres- which was significantly lower than that in non-neoplastic sion level of Nedd4L was determined by incubating the brain tissues (P , 0.001). Based on these results, we deter- membranes with horseradish peroxidase-conjugated anti- mined that gliomas with an IRS , 4 were more malignant rabbit immunoglobulin G (1:3000 dilution) and enhanced and divided the glioma specimens into two categories (high chemiluminescence reagent (Pierce, Minneapolis, MN, score, IRS 4; low score, IRS , 4) for the survival ana- USA), according to the manufacturer’s suggested protocols. lysis. Consistent with the IRS results, we found that the The membranes were stripped and reprobed with an Nedd4L expression level was gradually decreased with anti-b-actin mouse monoclonal antibody (1:1000 dilution; tumor grades as assessed by western blot analysis (Fig. 2). Sigma, St Louis, MO, USA) as a loading control. The Nedd4L/b-actin ratio was 1.21 + 0.35 in non-neoplastic Densitometry was performed using ImageJ software (http brain specimens and significantly decreased in gliomas with ://rsb.info.nih.gov/ij/) from National Institutes of Health increasing pathological grade (P , 0.001). The Nedd4L/ (NIH, Bethesda, MD, USA). We evaluated the expression of b-actin ratio was 0.66 + 0.28, 0.38 + 0.13, 0.26 + 0.11 and Jpn J Clin Oncol 2012;42(3) 199

Figure 1. Nedd4L protein expression as investigated by immunohistochemistry (400). Nedd4L expression was observed in glioma specimens and non- neoplastic brain tissues. The immunohistochemical stainings of Nedd4L protein were gradually decreased with tumor grades. Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 CORRELATION OF NEDD4L EXPRESSION WITH OVERALL SURVIVAL IN PATIENTS WITH GLIOMAS Spearman’s rank correlation analysis showed that Nedd4L expression was signiﬁcantly correlated with the overall survival of glioma patients (r ¼ 0.86, P ¼ 0.01). In addition, the Kruskal–Wallis test indicated that patients with IRS 4 for Nedd4L had longer overall survival than those patients with IRS , 4 of Nedd4L (20.1 + 0.9 vs. 11.5 + 0.4 months, respectively, P , 0.01). Survival curves for the two categories according to their pathological grades are shown in Fig. 3. In multivariate analysis, the Cox proportional hazards model involving the IRS score of Nedd4L expression and various clinical parameters identiﬁed two prognostic vari- ables including intra-tumor necrosis (P ¼ 0.03) and Nedd4L expression (P ¼ 0.02). Statistical values of the expression of Nedd4L and other clinical parameters derived from the Cox stepwise proportional hazards model are indicated in Table 2.

DISCUSSION

Figure 2. Nedd4L protein expression investigated by western blot analysis. To the best of our knowledge, this is the first study to dem- The expression levels of Nedd4L protein were gradually decreased with onstrate the correlation between Nedd4L and glioma. Our tumor grades. ‘N’ refers to non-neoplastic brain tissues; ‘Ca’ refers to data from immunohistochemistry assay provided evidence glioma tissues; ‘CaI’–‘CaIV’ refer to glioma tissues with WHO grade I–IV. that the Nedd4L protein expression is significantly reduced in glioma specimens than in their non-neoplastic counterpart, non-neoplastic brain tissues. In addition, reduced Nedd4L 0.13 + 0.05 in pilocytic astrocytomas, diffuse astrocytomas, expression was correlated with increasing pathological grade anaplasia astrocytomas and glioblastomas, respectively. of gliomas, underlining a connection between down- regulation of Nedd4L and development of gliomas. This finding was consistent with the results of western blot analysis. CORRELATION OF NEDD4L EXPRESSION WITH Ubiquitination serves multiple cellular functions, includ- CLINICOPATHOLOGICAL FEATURES OF GLIOMAS ing proteasomal degradation and the control of stability, Nedd4L expression was much lower in more malignant function and intracellular localization of a wide variety of gliomas than in less malignant gliomas (P , 0.001). The proteins (10). Nedd4L is a member of the HECT class of E3 attenuated Nedd4L expression was significantly correlated ubiquitin ligases. Among various modulating proteins, with large tumor diameter (P ¼ 0.02), low Karnofsky per- Nedd4L binds the PY motif of ENaC COOH terminals and formance score (KPS) (P ¼ 0.008) and frequent intra-tumor catalyzes ubiquitination of the NH(2) terminus of the protein necrosis (P ¼ 0.01). In addition, no statistically significant for subsequent degradation. Both evolutionarily conserved correlation of Nedd4L with age at diagnosis and gender of and evolutionarily new C2 domains of human Nedd4L, a patients was found (both P . 0.05). cryptic splice variant resulting in a disrupted isoform 200 Down-regulation of Nedd4L in glioma Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021

Figure 3. Cumulative survival curves of the patients. Survival rates of (A) all, (B) WHO grade I, (C) WHO grade II, (D) WHO grade III and (E) WHO grade IV glioma patients according to the expression of Nedd4L protein.

Table 2. Cox’s multivariate analysis TGF-b-activated Smads and restricts the amplitude and dur- ation of TGF-b gene responses, and in mouse embryonic Parameters Risk ratio 95% confidence interval P value stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is pro- Age 0.89 0.58–1.65 0.71 vided by SGK1, which phosphorylates Nedd4L to prevent Gender 1.02 0.66–1.83 0.33 binding of Smad2/3 (12). Previously identified as a regulator Largest tumor diameter 1.71 0.90–2.36 0.09 of renal sodium channels, Nedd4L also plays a broader role as a general modulator of Smad turnover during TGF-b KPS 1.99 1.28–2.95 0.06 signal pathway, which represents essential regulators of cell Intra-tumor necrosis 3.01 2.17–4.82 0.03 proliferation and differentiation during embryogenesis (13). Extent of resection 1.29 0.89–2.13 0.11 Recent studies found that TGF-b signal pathway deregula- Type of adjuvant treatment 1.37 1.02–2.24 0.11 tion is a characteristic of various cancers (14–16). In Nedd4L expression 3.42 2.39–5.02 0.02 gliomas, high biological activity of TGF-b-Smad pathway characterizes the malignant phenotype of the tumor and confers poor prognosis to patients. Accordingly, TGF-b has become a novel target for the experimental treatment of product formed by a frame-shift mutation, were reported pre- these tumors (17). Pen˜uelas et al. (18) indicated that TGF-b viously (11). Nedd4L is the human homologue of mouse induces the self-renewal capacity of glioma-initiating cells, Nedd4-2, which has been identified as a novel negative but not of normal human neuroprogenitors, through the factor in TGF-b signaling. TGF-b induces phosphorylation Smad-dependent induction of LIF and the subsequent activa- of the transcription factors Smad2 and Smad3 at the C ter- tion of the JAK-STAT pathway. Lu et al. (19) also demon- minus as well as at an interdomain linker region. strated that TGF-b1 promotes cell migration and TGF-b-induced linker phosphorylation marks the activated invasiveness of glioma cells through stimulation of Smad proteins for proteasome-mediated destruction. Gao ADAM17. As Nedd4L could inhibit TGF-b signal pathway et al. have demonstrated that Nedd4L is responsible for this in TGF-b receptors and Smads levels, the decrease in step. Through its WW domain, Nedd4L specifically recog- Nedd4L in gliomas detected in the present study might asso- nizes a TGF-b-induced phosphoThr-ProTyr motif in the ciate with TGF-b signal inducing tumor growth. linker region, resulting in Smad2/3 polyubiquitination and Of particular interest in this study was the observation that degradation. Nedd4L is not interchangeable with Smurf1, a an inverse correlation between low Nedd4L expression and ubiquitin ligase that targets BMP-activated, linker- overall survival was demonstrated by Spearman’s rank cor- phosphorylated Smad1. Nedd4L limits the half-life of relation analysis. Patients with abundant Nedd4L expression Jpn J Clin Oncol 2012;42(3) 201 had better overall survival. In addition, Nedd4L expression 6. Jin HS, Hong KW, Lim JE, Hwang SY, Lee SH, Shin C, et al. Genetic and intra-tumor necrosis were two important independent variations in the sodium balance-regulating genes ENaC, NEDD4L, NDFIP2 and USP2 influence blood pressure and hypertension. Kidney prognostic factors identified by the Cox multivariate ana- Blood Press Res 2010;33:15–23. lysis. Age, gender, KPS, largest tumor diameter, extent of re- 7. Takeuchi T, Adachi Y, Nagayama T, Furihata M. Nedd4L modulates the section and type of adjuvant treatment did not reach transcription of metalloproteinase-1 and -13 genes to increase the invasive activity of gallbladder cancer. Int J Exp Pathol 2011;92: statistical significance associated with survival. 79–86. In conclusion, our results provide convincing evidence for 8. Javelaud D, Alexaki VI, Dennler S, Mohammad KS, Guise TA, the first time that the expression of Nedd4L is down- Mauviel A. TGF-fbetag/SMAD/GLI2 signaling axis in cancer progression and metastasis. Cancer Res 2011;71:5606–10. regulated in human gliomas. The glioma patients with lower 9. Strelau J, Schmeer C, Peterziel H, Sackmann T, Herold-Mende C, Nedd4L expression have a worse prognosis. Steiner H, et al. Expression and putative functions of GDF-15, a member of the TGF-beta superfamily, in human glioma and

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