Elevated expression of Notch-1 and EGFR induced apoptosis in glioblastoma multiforme patients

Clinical Neurology and Neurosurgery 131 (2015) 54–58

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Elevated expression of Notch-1 and EGFR induced apoptosis in glioblastoma multiforme patients Zhen-yi Xing ∗ , Lai-guang Sun, Wu-jun Guo Department of Neurosurgery, Xinxiang Central Hospital, Xinxiang 453700, PR China

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Article history: Received 3 December 2014 Received in revised form 17 January 2015 Accepted 19 January 2015 Available online 28 January 2015 Keywords: Glioblastoma multiforme Notch-1 EGFR Apoptosis

a b s t r a c t Objective: The Notch signaling pathway has been well recognized as important adjuster in glioma tumorigenesis and could regulate the glioma cell proliferation through downstream factors such as epidermal growth factor receptor (EGFR). Our current study was aim to investigate the clinical association between Notch-1 gene and EGFR gene as well as cell survival rate in human glioblastoma multiforme (GBM) samples. Patients and methods: Samples from 90 patients with GBMs and 20 normal brain tissues were analyzed in our study. Western blot and immunohistochemistry was used to detect Notch and EGFR protein expression. RT-PCR was used to detect Notch and EGFR mRNA expression. Apoptosis was detected with flow cytometry. Results: Results demonstrated that the Notch and EGFR gene mRNA and protein levels were dramatically higher in GBM tissues compared to normal brain. Further analysis found these increased mRNA levels were only associated with patient survival period, but not related to patient age, gender and tumor size. A positive correlation was observed between Notch and EGFR protein expression. The positive correlations were also exhibited between Notch-1, EGFR gene expression and apoptosis percentage. Conclusion: Our study verified both Notch-1 and EGFR involved in GBM tumorigenesis and may provide important information for GBM clinical treatment and prognosis. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Glioblastoma multiforme (GBM) is one of the most aggressive primary brain tumors in humans, with a median survival of 12–15 months [1]. Currently, the general treatment strategies for GBM are surgical resection combined with radiation therapy and additional chemotherapy [2]. Even with these aggressive treatments, tumors recurred in a majority of patients with no specific reasons [3]. Hence, development of new therapies is necessary to overcome the universal treatment resistance in GBM. Previous studies reported Notch-1 signal acted as transcription regulator in many different kinds of human cancers including glioma [4–9]. Notch family included four homologous members from Notch-1 to Notch-4 with different unique biological functions [10]. Basically, all these members could stimulate their down stream gene transcription such as by releasing a nucleus activator

∗ Corresponding author at: Department of Neurosurgery, Xinxiang Central Hospital, No. 56, Jinsui Avenue, 453700 Xinxiang, PR China. Tel.: +86 15637359217. E-mail address: [email protected] (Z.-y. Xing). http://dx.doi.org/10.1016/j.clineuro.2015.01.018 0303-8467/© 2015 Elsevier B.V. All rights reserved.

named Notch intracellular domain (NICD) [11]. Some researches found the Notch receptor expression was significantly changed in GBM and thus subsequently impact on gliomas cell proliferation and survival [12]. Further investigation found the highly possible corresponding pathways or molecules involved in the Notch signal regulation in GBM may be Akt-mTOR and EGFR [13]. Since the increasing evidences of Notch-1 in GBM tumorigenesis, this pathway and its down stream genes were considered as potentially therapeutic target or prognosis biomarkers in GBM [14,15]. EGFR, as the most important down stream gene of Notch signal, was reported over expressed in almost 40–50% of GBM [16]. EGFR inhibition could induce G2/M arrest in glioma cells [17] and enhance cell sensitivity to cisplatin [18]. Exploration of the EGFR as a promising therapeutic target was conducted in current study [19]. Because both Notch-1 and EGFR signaling could affect the invasiveness of glioblastoma tumor cells, we thus try to explore the association between Notch-1 and EGFR expression and clinical progression of GBM, also examined the correlation between expression of Notch-1 and EGFR in GBM and tumor apoptosis, in order to shed new light on GBM therapy.

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2. Materials and methods 2.1. Patients and tissue samples Ninety resected GBMs tissue samples were collected from July 2010 to July 2013 in the Xinxiang Central Hospital Affiliated to Xinxiang Medical University. Adjacent normal tissue samples were collected at same time. Histopathological diagnosis of all tissue samples were performed by three professional neuropathologists. This study approval was obtained from the Ethics Committee of the Xinxiang Central Hospital Affiliated to Xinxiang Medical University. Specimens were handled and made anonymous according to ethical and legal standards. Written informed consent was obtained from all patients. There were 46 males and 44 females with a mean age of 41 years (range 29–68 years). None of the patients had received chemical therapy or radiotherapy prior to surgery. 2.2. Western blotting All procedures were conducted with manufacture introduction (Abcam, USA). Equal amounts of proteins (50 ␮g) obtained from GBM and non-malignant brain tissues for western blot analysis. Load protein into the wells of the SDS-PAGE gel, along with molecular weight markers. The proteins were then electro-transferred onto nitrocellulose membranes. Block the membrane for 1 h at room temperature or overnight at 4 ◦ C using 5% blocking solution. Then, the membranes were incubated with mouse monoclonal anti-Notch-1 (cat no. ab3196), rabbit monoclonal anti-EGFR (cat no. ab51037) antibody at 4 ◦ C overnight. After washing the membrane for three times with TBST (137 mM Sodium Chloride, 20 mM Tris, 0.1% Tween-20, pH 7.6), Membranes were next incubated with secondary antibody, mouse anti-IgG (Abcam, USA), diluted at an appropriate dilution in 1% bovine serum albumin (BSA), for 2 h at room temperature. Protein bands were detected by the enhanced chemiluminescence method on Kodak Biomax light film. 2.3. Immunohistochemical (IHC) analysis BioModule IHC staining kits (Life science, USA) were used in this study. All tissue slides were treated with Peroxo-Block (Life science, USA) for 2 min. Slides were washed 3 times with PBS and then incubated with anti-Notch polyclonal antibody (diluted 1:300; Sigma) or mouse anti-EGFR monoclonal antibody (diluted 1:100; Santa Cruz Biotechnology, Inc., USA) at 4 ◦ C overnight and washed twice with PBS prior to incubation with a secondary antibody (Santa Cruz Biotechnology, Inc., USA) at room temperature for 30 min. Next, the slides were incubated with HPR polymer conjugate and incubated in a humidified chamber for 10 min. Tissue sections were counterstained with hematoxylin. Apply 4 drops of mounting medium to the slide and dry at room temperature overnight. The slides were examined using a light microscope. 2.4. Real-time PCR Total RNA was extracted with the TRIzol Reagent (Invitrogen, USA) and stored in −80 ◦ C. The qRT-PCR was performance with two step method as manufactory instruction (Takara, Dalian, China). Real-time PCR was tested using the ABI 7900 fast sequence detection system (Applied Biosystems, CA, USA) with SYBR green fluorescent dye. The cycling parameters were as follows: 95 ◦ C for 10 min followed by 40 cycles at 95 ◦ C for 15 s, 60 ◦ C for 1 min and an elongation step at 72 ◦ C for 30 s. The human ␤-actin transcript was used as an internal reference to control for variations in the total mRNA quantity of each sample. Each RNA sample was analyzed in triplicate and the following primers were used:

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1. Human Notch-1 primers: forward 5 -GAGGCGTGGCAGACTATGC-3 ; reverse 5 -CTTGTACTCCGTCAGCGTGA-3 . 2. Human EGFR primers: forward 5 -TGTAACCAGAGAGCGGGATGT-3 ; reverse 5 -TTTTGGCATAACTAAGGCCGAA-3 . 3. Human ␤-actin primers: forward 5 -CACTCTTCCAGCCTTCCTTC3 ; reverse 5 -GGATGTCCACGTCACACTTC-3 . 2.5. Measurement of apoptosis by flow cytometry Apoptosis kits were purchased from Life science (Life science, USA). Single cell suspensions were firstly collected from GBM and control tissue. Harvest cells were washed in cold PBS and then treated with annexin-binding buffer. Suspensions cells were then fixed in 70% cold ethanol, treated with 10 g/l RNase. Add 5 ␮l Alexa Fluor 488 annexin V and 1 ␮l 100 ␮g/ml PI working solution to each 100 ␮l of cell suspension. After incubation for 15 min at room temperature, add 400 ␮l annexin-binding buffer, mix gently and keep samples on ice for further test. In this experiment, cells with early apoptotic signals (stained with Annexin V) and cells with late death signals (stained with PI) were quantified and analyzed using the CellQuest software. Each assay was performed in triplicate. 2.6. Statistical analysis SPSS 13.0 was used for all data statistical analyze. The difference between patients and control samples was determined by Student’s t-test. Association study was performed by Pearson correlation analysis. Numerical data are represented as the mean ± standard deviation. Each experiment was repeated at least twice or was performed in triplicates. P value <0.05 was adopted as significant. 3. Results 3.1. Enhanced expression level of Notch-1 and EGFR in glioma tissue samples The immunohistochemisty results demonstrated that the levels of phosphorylated Notch-1 and EGFR were elevated in gliomas tissue compared to the adjacent normal tissue (Fig. 1(A)). Also, as western blot results shown in Fig. 1(B), enhanced activation of Notch-1 and EGFR signaling is demonstrated in glioma tumor tissues, but the EGFR levels were only elevated in some patients not all tumor specimens compared with the adjacent normal tissues. In addition, as shown in Fig. 1(C), increased Notch-1 and EGFR mRNA levels were founded in glioma tissues (P < 0.05). 3.2. Increased apoptosis percentage in glioma multiforme tissues 1 × 106 suspension cells from tumor tissue and normal tissues were stained with Annexin V-FITC and PI to take apoptotic analysis. FACS results were exhibited in Fig. 2(A). FACS analysis identified A significantly higher apoptotic percentage was exterminated in GBM tissues than normal brain tissues (Fig. 2(B); P < 0.05). 3.3. Association between Notch-1 and EGFR expression and clinical characteristics of GBM Expression level of Notch-1 and EGFR mRNA was higher in the survival ≥1 year patients than the <1 year (P < 0.05), but no same association was observed in patient gender, age and tumor size (Table 1).

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Fig. 1. Activation of Notch-1 and EGFR in tumor tissues. (A) Notch-1 and EGFR expression were detected in glioblastoma multiforme using immunohistochemistry. (B) The protein expression levels of Notch and EGFR in gliomas. Four representative samples results were performance in glioma patient tissues and adjacent normal brain tissues of the same patients. ␤-Actin was used as the internal control. (C) Relative mRNA levels of Notch-1 and EGFR in gliomas. Data were collected from triplicate experiments and were shown as the mean ± S.D. * indicated P < 0.05, respectively, in comparison with normal brain tissue.

Fig. 2. Elevated apoptosis cells in GBM patients’ specimen with Annexin-V and PT staining. (A) Representative flow cytometry output figures in Cellquest software. (B) Percentages of apoptotic cells in GBM and normal tissues. GBM, glioblastoma multiforme.

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Fig. 3. Correlation between Notch-1, EGFR and apoptosis. The scar plots represented (A) the positive correlation between Notch-1 and EGFR (r = 0.714, P < 0.05), (B) the positive correlation between Notch-1 and apoptosis percentage (r = 0.661, P < 0.05), and (C) the positive correlation between EGFR and apoptosis percentage (r = 0.762, P < 0.05).

Table 1 Correlation between Notch-1/EGFR expression and clinical characteristics of GBM. Variables Gender Male Female Age (years) ≥40 <40 Tumor size (cm) ≥4 <4 Survival rate (years) ≥1 <1 *

n

Notch-1

EGFR

46 44

44.56 ± 7.32 42.75 ± 8.75

36.21 ± 6.32 35.78 ± 7.13

53 37

41.56 ± 5.89 43.75 ± 7.45

34.21 ± 7.05 36.78 ± 6.45

60 30

43.56 ± 6.35 43.75 ± 5.60

37.21 ± 8.56 34.78 ± 6.34

57 33

46.56 ± 6.76* 41.75 ± 5.97

39.21 ± 8.12* 32.78 ± 6.02

Compared with short survival rate. P < 0.05.

3.4. Correlation between Notch-1 and EGFR expression and apoptosis percentage Statistical analysis revealed a positive correlation between Notch-1 and EGFR mRNA levels (P < 0.05, Fig. 3(A)). A positive correlation was identified between Notch-1 and apoptosis (P < 0.05, Fig. 3(B)) and between EGFR and apoptosis (P < 0.05, Fig. 3(C)). 4. Discussion This present study demonstrated the significantly over expression of Notch-1 and EGFR protein and mRNA in GBM tissues than in the adjacent normal brain tissues. Further clinical feature analysis found the association between over expression of Notch-1 and EGFR with patient survival. Over expression of Notch-1 and EGFR protein was higher in the survival ≥1 year group than the <1 year. But no such association was established with patient gender, age and tumor size. Next, we analyzed the correlations between Notch1, EGFR and apoptosis. A positive correlation between Notch-1 and EGFR was observed. A positive correlation between Notch-1 and apoptosis and between EGFR and apoptosis was also observed. These data suggested the critical role of Notch-1 and EGFR in the induction of cell apoptosis in glioma. Thus, assessment of Notch1 and EGFR co-expression may provide useful information for the diagnosis, therapy and prognosis of GBM.

Notch-1 was recognized as a central actor in maintenance the balance among neuron cell proliferation, cell apoptosis and cell differentiation. The growth, invasion and self-renewability of glioma cell thus inevitably involved in Notch-1 mediated signaling pathway [20]. Several types of evidences indicated that The aberrant Notch signaling pathway not only restricted activated its downstream target genes such as HES, c-myc, cyclin D1, but also interacted with other signaling pathway, such as p53, Ras, NF␬B, Wnt, Shh, TGF-␤, PI3 K and EGFR [21–24]. The Notch pathway is intimately coupled to signaling through EGFR, or downstream targets, in both normal development and in the onset and maintenance of cancer [25,24]. EGFR, as an essential growth factor, participates many transcriptional regulations and also provide major contribution to GBM biogenesis. Transcription level of Notch signaling mediator genes is dramatically enhanced in the molecular subset of GBM with EGFR amplification [26]. Previous study reported that Notch activation induces apoptosis in neural progenitor cells through a p53dependent pathway which also involved in EGFR [27]. Also, another report demonstrates that EGFR was observed over expressed in GBM and Notch-1 could regulate transcription of the EGFR through TP53 [13]. Consistently, a study found that the Notch-1 receptor promotes survival of glioblastoma cells through regulation of Mcl1 protein, which also mediated by EGFR in transcriptional level. Author further verified that inhibition of the Notch-1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation [28]. We measured the expression of EGFR and found that Notch-1 expression positive correlation with the EGFR expression. Together, our results revealed the crosstalk existing between Notch and EGFR pathway and might contribute to the effect on cell survival and proliferation in gliomas, also, affected the expression of protein relevant to cell invasion and apoptosis. 5. Conclusion EGFR and Notch-1 were over expressed in GBM tumor tissues compared to adjacent GBM normal tissue, indicating inhibition of these two molecular pathway as a potential strategy in GBM therapy. Our results collectively support a key role of EGFR and Notch-1 signaling in survival of Glioblastoma cancer and provide

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rational evidence of the prospective improvement of survival of GBM patients by targeting EGFR and Notch-1. References [1] Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359: 492–5072. [2] Stupp R, Mason WP, van den Bent MJ. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–96. [3] Shankar A, Kumar S, Iskander AS, Varma NR, Janic B, de Carvalho A, et al. Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo. Chin J Cancer 2014;33: 148–58. [4] Zhao N, Guo Y, Zhang M, Lin L, Zheng Z. Akt-mTOR signaling is involved in Notch-1-mediated glioma cell survival and proliferation. Oncol Rep 2010;23: 1443–7. [5] Jiang L, Wu J, Chen Q, Hu X, Li W, Hu G. Notch1 expression is upregulated in glioma and is associated with tumor progression. J Clin Neurosci 2011;18:387–90. [6] Chigurupati S, Venkataraman R, Barrera D. Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness. Cancer Res 2010;70:418–27. [7] Xu P, Qiu M, Zhang Z. The oncogenic roles of Notch1 in astrocytic gliomas in vitro and in vivo. J Neurooncol 2010;97:41–51. [8] Sahlgren C, Gustafsson MV, Jin S, Poellinger L, Lendahl U. Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proc Natl Acad Sci U S A 2008;105:6392–7. [9] Leong KG, Niessen K, Kulic I. Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of Ecadherin. J Exp Med 2007;204:2935–48. [10] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2009;137:216–33. [11] Fortini ME. Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 2009;16:633–47. [12] Cenciare li C, Marei HE, Zonfrillo M, Pierimarchi P, Paldino E, Casalbore P, et al. PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment. Mol Cancer 2014;8(13):247. [13] Purow BW, Sundaresan TK, Burdick MJ, Kefas BA, Comeau LD, Hawkinson MP, et al. Notch-1 regulates transcription of the epidermal growth factor receptor through p53. Carcinogenesis 2008;29:918–25.

[14] Purow BW, Haque RM, Noel MW, Su Q, Burdick MJ, Lee J, et al. Expression of Notch-1 and its ligands, delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res 2005;65:2353–63. [15] Dufraine J, Funahashi Y, Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms. Oncogene 2008;27:5132–7. [16] Halatsch ME, Schmidt U, Behnke-Mursch J, Unterberg A, Wirtz CR. Epidermal growth factor receptor inhibition for the treatment of glioblastoma multiforme and other malignant brain tumors. Cancer Treat 2006;32:74–89. [17] Kapoor GS, Christie A, O’Rourke DM. EGFR inhibition in glioblastoma cells induces G2/M arrest and is independent of p53. Cancer Biol Ther 2007;6: 571–9. [18] Zhang Y, Xing X, Zhan H, Li Q, Fan Y, Zhan L, et al. EGFR inhibitor enhances cisplatin sensitivity of human glioma cells. J Huazhong Univ Sci Technol Med Sci 2011;31:773–8. [19] Jun HJ, Bronson RT, Charest A. Inhibition of EGFR induces a c-MET-driven stem cell population in glioblastoma. Stem Cell 2014;32:338–48. [20] Zhou ZD, Kumari U, Xiao ZC, Tan EK. Notch as a molecular switch in neural stem cells. IUBMB Life 2010;62:618–23. [21] Xu P, Zhang A, Jiang R, Qiu M, Kang C, Jia Z, et al. The different role of Notch1 and Notch2 in astrocytic gliomas. PLOS ONE 2013;8:e53654. [22] Niimi H, Pardali K, Vanlandewijck M, Heldin CH, Moustakas A. Notch signaling is necessary for epithelial growth arrest by TGF-beta. J Cell Biol 2007;176:695–707. [23] McKenzie G, Ward G, Stallwood Y, Briend E, Papadia S. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals. BMC Cell Biol 2006;7:10. [24] Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P. Notch mediates TGF alphainduced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565–76. [25] Stockhausen MT, Sjolund J, Axelson H. Regulation of the Notch target gene Hes1 by TGFalpha induced Ras/MAPK signaling in human neuroblastoma cells. Exp Cell Res 2005;310:218–28. [26] Brennan C, Momota H, Hambardzumyan D, Ozawa T, Tandon A, Pedraza A, et al. Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS ONE 2009;4:e7752. [27] Yang X, Klein R, Tian X, Cheng HT, Kopan R, Shen J. Notch activation induces apoptosis in neural progenitor cells through a p53-dependent pathway. Dev Biol 2004;269:81–94. [28] Fassl A, Tagscherer KE, Richter J, Berriel Diaz M, Alcantara Llaguno SR, Campos B, et al. Notch 1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1. Oncogene 2012;31: 4678–708.