Mol Biol Rep (2014) 41:5903–5911 DOI 10.1007/s11033-014-3465-2

Rab3a promotes brain tumor initiation and progression Jun-Kyum Kim • Seung-Yup Lee • Chang-Won Park • Suk-Hwang Park • Jinlong Yin • Jaebong Kim • Jae-Bong Park Jae-Yong Lee • Hyunggee Kim • Sung-Chan Kim

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Received: 15 April 2013 / Accepted: 14 June 2014 / Published online: 26 June 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The Rab protein family is composed of small GTP-binding proteins involved in intracellular vesicle trafficking. In particular, Rab3a which is one of four Rab3 proteins (a, b, c, and d isoforms) is associated with synaptic vesicle trafficking in normal brain. However, despite the elevated level of Rab3a in tumors, its role remains unclear. Here we report a tumorigenic role of Rab3a in brain tumors. Elevated level of Rab3a expression in human was confirmed in both glioma cell lines and glioblastoma multiforme patient specimens. Ectopic Rab3a expression in glioma cell lines and primary astrocytes promoted cell proliferation by increasing cyclin D1 expression, induced resistance to anti-cancer drug and irradiation, and

Jun-Kyum Kim and Seung-Yup Lee have contributed equally to this work. J.-K. Kim  S.-Y. Lee  J. Yin  H. Kim (&) Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea e-mail: [email protected] S.-Y. Lee Department of Ophthalmology, Soon Chun Hyang University Hospital, Seoul, Republic of Korea C.-W. Park  S.-H. Park  J. Kim  J.-B. Park  J.-Y. Lee  S.-C. Kim Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Republic of Korea J. Yin National Cancer Center, Research Institute and Hospital, Goyang, Republic of Korea J. Kim  J.-B. Park  J.-Y. Lee  S.-C. Kim (&) Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon, Republic of Korea e-mail: [email protected]

accelerated foci formation in soft agar and tumor formation in nude mice. The overexpression of Rab3a augmented the tumorsphere-forming ability of glioma cells and p53-/astrocytes and increased expression levels of various stem cell markers. Taken together, our results indicate that Rab3a is a novel oncogene involved in glioma initiation and progression. Keywords Glioblastoma multiforme  Rab3a  Cyclin D1  Gliomagenesis  Drug resistance

Introduction Despite advancement in diagnosis, therapies, and molecular understanding of malignant gliomas, brain tumors are still incurable. In glioblastoma multiforme (GBM) which is the most common and aggressive form of glioma, the median survival has remained to be \14 months for decades [1–5]. GBMs show resistance to chemotherapy and radiotherapy, which are conventional therapeutic approaches following surgical resection [5]. This phenomenon may be explained by the fact that GBMs are characterized by extensive heterogeneity at both cellular and molecular levels. Recent studies have identified glioma stem cells (GSCs) as a rare subpopulation of cancer cells with stem cell properties. These properties include cancer-initiating ability, self-renewal, aberrant differentiation, and resistance to chemotherapy and irradiation [6–8]. Thus, GSCs are considered as a primary therapeutic target to achieve complete eradication of the GBMs [9]. However, the mechanism underlying the regulation of GSCs remains poorly elucidated. Rab3a is a small GTP binding protein of the Ras gene superfamily and it is thought to regulate intracellular

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vesicle transport and secretion of neurotransmitters in endocrine cells and tumors derived from the neural tube, such as neuroblastomas, ganglioneuroblastomas, and adult nervous system neoplasms [10–12]. In human insulinoma, Rab3a shows high expression in tumor tissue but not in normal islets [13]. Besides insulinoma, Rab3a expression is higher in several other neoplasms than their normal counterparts; however, its function in neoplasms is not well understood. In the present study, we have observed elevated expression level of Rab3a in glioma cell lines, and tumor tissues derived from GBM patients and that its mRNA expression was correlated with high-grade glioma. The ectopic expression of Rab3a promoted cyclin D1 dependent glioma cell proliferation, anchorage-independent growth in soft agar assay, and resistance to anti-cancer drug and irradiation of glioma cell. Upon the injection of Rab3aoverexpressing cells to immunocompromised mice, tumor progression and initiation were enhanced. Moreover, Rab3a increased tumorsphere-forming ability and expression level of various stem cell markers. Together, our data demonstrate that Rab3a promotes glioma initiation and progression by augmenting cell proliferation and cancer stem cell properties.

Materials and methods Cell culture and conditions Mouse astrocytes were isolated from the cerebral cortices of 5-day-old Ink4a/Arf or p53 knockout mice as described previously [14]. Human GBM cell lines [A1207, A172, LN18, LN229, T98G, U138MG, U373MG, and U87MG, all of which were purchased from American Type Culture Collection (ATCC)], p53-/- astrocytes, and Ink4a/Arf-/astrocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose medium with 10 % fetal bovine serum (FBS; Hyclone), 1 % penicillin and streptomycin (Life Technologies), and 2 mM L-glutamine (Life Technologies). To determine cell growth rates, cells were plated at a density of 1 9 104 cells/well in six-well plates, and cell numbers were counted every 2 days up to 5 or 6 days using a hemacytometer. Human glioma stem cells (GSC1, GSC2, and GSC3) [15] and glioma cells were grown in suspension culture with neurobasal medium (NBE; Invitrogen) supplemented with modified N2, B27 (Invitrogen), epidermal growth factor (EGF; 20 ng/ml; R&D Systems), and basic fibroblast growth factor (bFGF; 20 ng/ml; R&D Systems). For tumorsphere formation assays, cells were seeded at a density of 4 9 102 cells/well in 12-well plates, and then maintained in suspension. For in vitro limiting dilution assays, Rab3a overexpressing- and

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control-Ink4a/Arf-/- astrocytes and U87MG cells were plated in 96-well plates with decreasing numbers of cells per well (100, 50, 25, 5) containing neurobasal medium supplemented with modified N2, B27, EGF (20 ng/ml) and bFGF (20 ng/ml). EGF and bFGF were replaced every 3 days, and the tumorsphere numbers were determined after 14 days. Extreme limiting dilution analysis was performed using software available at http://bioinf.wehi.edu. au/software/elda/. Plasmid, shRNA construction, siRNAs and retroviral infection Ink4a/Arf-/- astrocytes were infected with retrovirus produced from the PT67 amphotropic packaging cell line (Clontech) transfected with retroviral vectors (pWZL-Rab3a/ Blast). Cells plated 24 h earlier at 106 cells/10-cm dish were transduced by refeeding them with pre-filtered (0.45 lm) retroviral supernatant containing 6 lg/ml polybrene (Sigma). Cells were then subjected to blasticidin selection for 10 days. Additional Rab3a overexpressing cells, such as p53-/astrocytes and human GBM cell lines (A172-Rab3a and U87MG-Rab3a), were generated by transfecting cells with the pcDNA3.1-Rab3a-puro (?) plasmid using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Simultaneously, control cells were generated by transfecting cells with the pcDNA3.1-puro (?) plasmid. Stably transfected cells were selected in DMEM supplemented with 10 % FBS and 1 lg/ml puromycin for 7 days. LN229 cells were infected with retrovirus with Rab3a-shRNA, which were cloned into pSuperRetro-puro according to the manufacturer’s instructions (Oligoengine). The target sequence for human Rab3a-shRNA: 50 -AACTTCGACTACATGTTC AAG. Cells were transiently transfected with human cyclin D1 siRNAs (Sigma, siCyclin D1-1 sense 50 -CCACAGAU GUGAAGUUCAU-30 , antisense 50 -AUGAACUUCACAUCUGUGG-30 ; siCyclin D1-2 sense 50 -UAAGAAUAGG CAUUAACA-30 , antisense 50 -UGUUAAUGCCUAUU CUUAC-30 ) or control siRNA at a final concentration 30nM using ScreenFect A (Wako) according to the manufacturer’s instruction. Two days after transfection, cells were trypsinized and harvested for immunoblot and cell cycle analyses. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA was isolated from cells using the TRIzol reagent (GibcoBRL) according to the manufacturer’s instructions. Three micrograms of DNase I-treated RNA was converted to cDNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. For quantitative real-time RT-PCR, experiments were conducted using the Takara SYBR Premix Ex

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Taq and CFX096 (Bio-Rad). The quantitative expression level of Rab3a mRNA, and human and mouse CD133, Nestin, and Sox2 mRNAs was calculated using the standard 2-DDCt method as described previously [16]. Information regarding PCR parameters used in RT-PCR will be provided upon request.

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biotinylated secondary antibody for 20 min. The samples were then visualized using the DAB/Ni substrate. Slides were counterstained with hematoxylin for 1 min and rinsed with abundant H2O before dehydration and mounting. Soft agar and subcutaneous implantation assays for tumorigenicity

Fluorescence-activated cell sorting (FACS) analysis For cell cycle analysis, cells were dissociated with TrypinEDTA (sigma), fixed overnight in cold 70 % ethanol at 20 °C. After washed with PBS, cells were incubated in 500 ll RNase A solution (250 lg/ml) for 30 min at 37 °C, then stained with propidium iodide at a final concentration of 5 lg/ml, and subjected to FACS analysis. The fluorescent intensity (10,000 cells) was measured using FACS Calibur (BD Biosciences). Immunoblot analysis Whole cell extracts were prepared using RIPA lysis buffer [150 mM NaCl, 1 % NP-40, 0.1 % sodium dodecyl sulfate (SDS), 50 mM Tris, pH 7.4] containing 1 mM b-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor (Roche). Extracts were quantitated using the Bradford assay reagent (BioRad) according to the manufacturer’s instructions. Protein in the extracts (30–100 lg) was separated using a 4–12 % gradient or 10 % SDS–polyacrylamide gel electrophoresis (PAGE) NuPAGE gel (Invitrogen), and then was transferred to a polyvinylidene fluoride membrane (Millipore). Membranes were blocked with 5 % non-fat milk and incubated with anti-Rab3a (K-15, Santa Cruz Biotechnology), anti-cyclin D1 (H-295, Santa Cruz Biotechnology), anti-a-tubulin (Sigma), and anti-b-actin (Sigma). Membranes were then incubated with horseradish peroxidaseconjugated anti-secondary IgG (Pierce) antibody and visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce). Immunohistochemistry Human GBM samples were collected from patients who had provided standard procedure consent. Five-micrometer-thick sections of normal and tumor tissues were used for immunohistochemical studies. Immunohistochemistry experiments were performed according to the methods provided in the Vectastain ABC Kit (Vector). In brief, paraffin sections were deparaffinized, and hydrated tissue sections were steamed for 30 min in 0.1 % citric acid and blocked in 10 % serum (or 0.1 % BSA) for 45–60 min at room temperature. Samples were subsequently incubated with anti-Rab3a for 1 h at room temperature and with

To assess anchorage-independent growth, U87MG and A172 cells (transfected with pcDNA3.1-puro, pcDNA3.1Rab3a-puro) were cultured (2 9 104 cells) in soft agar dishes (1.6 and 0.7 % bottom and top agar, respectively) for 3 weeks. HeLa and human BJ fibroblasts were used as positive and negative controls, respectively. For the subcutaneous implantation assay, cells (1 9 106) were subcutaneously transplanted into nude mice (BALB/c nu/nu). Subcutaneous tumors were grossly visible at the injection sites after 15 days (in the mouse cell lines) and 5 weeks (in the human U87MG cell line). The mice were kept under observation for 2–3 months. All mouse experiments were approved by the animal care committee at the College of Life Science and Biotechnology, Korea University, and were performed in accordance with government and institutional guidelines and regulations. Cell death analysis Cells grown in DMEM supplemented with 1 % penicillin/ streptomycin and 10 % FBS were treated with doxorubicin (1 lM; Sigma) or staurosporine (1 lM; Sigma) for 3 days. For fluorescence-activated cell sorting (FACS) analysis, cell pellets were centrifuged at 1,500 rpm for 5 min, and then stained with Annexin V-FITC (BD BiosciencesPharmingen) and propidium iodide (PI, Sigma). Briefly, a cell suspension (100 ll of 1 9 Binding Buffer) stained with 10 ll Annexin V-FITC and 20 ll PI (50 lg/ml) was gently mixed and incubated for 15 min at room temperature in the dark. Next, 200 ll of 1 9 Annexin binding buffer was added and cells were immediately analyzed by flow cytometry (BD Biosciences). For irradiation assay, 137 Cs c-ray irradiation at a dose rate of 2.04 Gy/min for a total dose of 20 Gy was conducted using IBL 437C (CIS biointernational). Prior to measuring the cell viability after irradiation, a defined number of irradiated cells were cultured at 37 °C in 5 % CO2 for 7 days. Subsequently, all cells were collected, centrifuged and resuspended in PBS. Then the cell viability was determined using trypan blue exclusion assay in combination with cell counting. Statistics Data were statistically analyzed using a two-tailed Student’s t test. The level of statistical significance stated in

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Fig. 1 Rab3a expression in human glioma cell lines and GBM patient specimens. a Immunoblot analysis showing increased Rab3a expression in human glioma cell lines. Normal human astrocyte (NHA) cells were used as a control. Tubulin was used as a loading control. b Immunohistochemistry showing increased Rab3a

expression in human GBM compared with normal brain tissues (magnification: left 9 200; right 9 400). c Quantitative RT-PCR shows an increased Rab3a mRNA level in grade IV glioma patient samples compared with that in grade I (*p \ 0.05, **p \ 0.01)

the text was based on p values. A p value \0.05 was considered statistically significant.

glioma samples (10/20 samples) (Fig. 1c). These data indicate that Rab3a is likely to be associated with glioma progression and a possible diagnostic marker for highgrade gliomas.

Results Rab3a promotes cell proliferation Rab3a is highly expressed in human glioma cell lines and GBM specimens Prior to investigating the role of Rab3a in gliomagenesis, we analyzed whether Rab3a expression is correlated with gliomas. We have found that Rab3a was upregulated in A1207, A172, LN18, LN229, T98G, U138MG, and U373MG cells compared to normal human astrocytes (NHA) (Fig. 1a). We then examined Rab3a expression in GBM specimens derived from glioma patients by comparing them with normal (non-tumor) tissues. As shown in Fig. 1b, Rab3a was highly expressed in GBM specimens compared to normal tissues, and its expression was detected in both cytoplasm and nuclear and peri-nuclear regions. Next, we isolated RNA from glioma patient tissues (Grade I–Grade IV) and detected Rab3a mRNA levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess whether the Rab3a expression level is correlated with glioma grade. Indeed, the Rab3a transcripts showed a high expression pattern in Grade IV

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In order to examine the tumorigenic role of Rab3a in gliomagenesis, we established Rab3a-overexpressing GBM cell lines (A172-Rab3a and U87MG-Rab3a) (Fig. 2a). Because accelerated cell proliferation is one of the hallmarks of aggressive tumor cells [17], we examined cell proliferation of Rab3a-overexpressing cells and found that ectopic Rab3a expression promoted cell proliferation in A172 cells (Fig. 2b) and U87MG cells (Fig. 2c). Although the loss of p53 and Ink4a/Arf tumor suppressors frequently occurs in human GBM [18], primary astrocytes derived from p53-/and Ink4a/Arf-/- mice are non-tumorigenic cells. However, these astrocytes were easily transformed by transducing various oncogenes, such as a constitutively active mutant of epidermal growth factor receptor (EGFRvIII) [14], inhibitor of differentiation 3 (Id3) [19], Id4 [20], and oncogenic Ras (H-RasV12) [21]. To investigate the direct role of Rab3a in gliomagenesis, we established Rab3a-overexpressing p53-/-astrocytes and Ink4a/Arf-/-astrocytes (Fig. 2d) and examined their proliferation. p53-/- astrocytes (Fig. 2e) and

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Ink4a/Arf-/-astrocytes (Fig. 2f) exhibited accelerated cell proliferation upon ectopic expression of Rab3a compared with each control. Cell proliferation is mainly executed by positive regulation of cyclins and cyclin-dependent kinases, and overexpression of cyclin D1 is frequently associated with many types of malignancies [22]. We found that cyclin D1 expression was induced by Rab3a in A172 cells, U87MG cells, p53-/-astrocytes, and Ink4a/Arf-/- astrocytes (Fig. 2a, d). We also established Rab3a-depleted cells (LN229-shRab3a) using a short-hairpin RNA (shRNA) against Rab3a and found that cyclin D1 expression was decreased in LN229-shRab3a compared to the control (Fig. 3a). To interrogate the role of cyclin D1 in increased proliferation of Rab3a-overexpressing cells, we generated cyclin D1-depleted A172 and U87MG glioma cells using two individual cyclin D1-specific short interference RNAs (Fig. 3b, c). Fluorescence-activated cell sorting (FACS) analysis revealed that cyclin D1 depletion markedly reduced cell population of S and G2/M phases (Fig. 3d, e). These results indicate that Rab3a-driven cell proliferation is primarily accelerated by altered expression of cyclin D1 in human glioma cell lines and primary astrocytes derived from p53-/- and Ink4a/Arf-/- mice.

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Fig. 2 Rab3a promotes cell proliferation. a, d Expression of Rab3a and cyclin D1 in Rab3aoverexpressing glioma cells (a), p53-/-astrocytes, and Ink4a/ Arf-/-astrocytes (d). Cell proliferation was accelerated by Rab3a in A172 cells (b), U87MG cells (c), p53-/-astrocytes (e), and Ink4a/ Arf-/-astrocytes (f) (*p \ 0.05, **p \ 0.01)

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Rab3a promotes transformation, anti-cancer drug resistance, and tumorigenesis Since anchorage-independent growth is one of the characteristics of transformed cells, we performed a soft agar assay which allows observation of foci formed by transformed cells undergoing anchorage-independent growth. Rab3a significantly induced anchorage-independent growth of U87MG cells and Ink4a/Arf-/- astrocytes under the soft-agar culture conditions. Consistent with this result, knockdown of Rab3a with the shRab3a construct decreased the foci-forming ability of LN229 cells (Fig. 4a). In addition, Rab3a markedly promoted tumor formation of U87MG cells and p53-/- astrocytes in subcutaneously injected nude mice (Fig. 4b, c). To verify whether drug resistance is acquired by Rab3a, we investigated the cell viability of Rab3a-overexpressing U87MG cells after the treatment of doxorubicin (1 lM) or staurosporine (1 lM) for 72 h. FACS analysis of Annexin V/PI staining showed enhanced viability of Rab3a-overexpressing U87MG cells compared to the control (Fig. 4d). We also tested the cell viability of Rab3a-overexpressing U87MG cells 7 days after 20 Gy irradiations. Rab3a-overexpressing U87MG

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Fig. 3 Rab3a promotes cell proliferation by increasing cyclin D1. a Expression of Rab3a and cyclin D1 in LN229 transduced with Rab3a-specific shRNAi constructs (shRab-1 and -2) and a scrambled control (Scram). b, c The depletion of cyclin D1 in Rab3a-

overexpressing A172 (b) and U87MG (c) glioma cells by transfecting two individual cyclin D1-specific siRNAs. d, e Cell cycle analysis of controls and cyclin D1-depleted Rab3a-overexpressing A172 (d) and U87MG (e) glioma cells was examined by FACS

cells were more resistant to irradiation than U87MG control cells (Fig. 4e). Taken together, our results suggest that Rab3a accelerates resistance to anti-cancer drug and irradiation and tumor initiation capacity in gliomagenesis.

on the cell types examined, Rab3a increased CD133 stem cell marker expression in Ink4a/Arf-/- astrocytes, and CD133 and SOX2 expression in U87MG cells (Fig. 5e, f). Rab3a expression level was higher in undifferentiated GSCs derived from GBM patients compared to that in normal human astrocytes (NHAs) (Fig. 5g). Marked decrease in Rab3a expression level was observed as GSCs underwent differentiation in DMEM supplemented with 5 % FBS [23] (Fig. 5h). Together, these results indicate that Rab3a promotes self-renewal of glioma cells and that it is involved in maintenance of the undifferentiated state of glioma stem cells.

Rab3a is highly expressed in glioma stem cell lines and promotes self-renewal of glioma cells Having that the tumor initiation capacity and drug/irradiation resistance phenotype are well known characteristics of glioma stem cells (GSCs), our results prompted us to extend our investigation to figure out whether ectopic Rab3a expression allows human glioma cell lines and mouse astrocytes to acquire GSC properties. Therefore, we examined the tumorsphere-forming ability of Rab3a-overexpressing cells which is a common indicator of GSC selfrenewal [7]. As shown in Fig. 5a, b, ectopic expression of Rab3a significantly elevated the tumorsphere-forming ability of U87MG cells and Ink4a/Arf-/- astrocytes under neural stem cell culture conditions (DMEM/F12 medium supplemented with EGF and bFGF) [23]. We also validated the effect of Rab3a on tumorsphere formation using in vitro limiting dilution assay, and found that Rab3a promotes tumorsphere formation in Ink4a/Arf-/- astrocytes and U87MG cells (Fig. 5c, d). Although its activity depended

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Discussion In this study, we have identified a novel oncogenic role of Rab3a. Rab3a accelerates cell proliferation by increasing cyclin D1 expression, enhances anti-cancer drug resistance, and increases tumorigenicity and self-renewal of glioma cells. Rab GTPase is a master regulator that controls intracellular vesicle trafficking in both exocytic and endocytic pathways. Among 60 RAB genes encoded by the human genome, most Rab proteins are constitutively expressed in

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Fig. 4 Rab3a induces tumorigenicity and resistance to anti-cancer drug and irradiation. a Increased foci formation in Rab3a-overexpressing cells (U87MG cells and Ink4a/ Arf-/-astrocytes) and decreased foci formation in LN229shRab3a cells compared with the control in soft-agar culture conditions. HeLa cells and human BJ fibroblast cells were used as a positive and negative control. b, c Elevated tumor formation of Rab3aoverexpressing U87MG (b) and p53-/-astrocytes (c) in nude mice. d Annexin V/PI-mediated FACS cell death analysis of Rab3a-overexpressing U87MG cells after treatment with doxorubicin (1 lM) or staurosporine (1 lM) for 72 h (**p \ 0.01). e Cell viability of Rab3a-overexpressing U87MG and control cells at 7 day after 20 Gy irradiations (**p \ 0.01)

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all mammalian cells, although some Rab proteins have shown to be differentially expressed in several cell types [24]. Recent studies have reported multiple links between Rab GTPase and human diseases such as breast cancer, hepatocellular carcinomas, ovarian cancer, and Griscelli syndrome type 2. For example, Rab25 is overexpressed in ovarian cancer and it drives cancer progression and Rab27A promotes invasiveness and the metastatic potential through secretion of insulin-like growth factor II in breast cancer cells [24, 25]. Moreover, high expression of Rab5a

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and its involvement in EGF signaling has been reported in hepatocellular carcinoma [26]. In our study, we found that Rab3a promoted cell growth by increasing the expression level of cyclin D1. Cyclin D1 is a member of the cyclin family that is highly expressed in various tumors and its role in tumorigenesis and drug resistance has been known. Studies have found that cyclin D1 is upregulated by Rab27a in breast cancer cells [26] and this raises the possibility that cyclin D1 may be an effector molecule in cell cycle regulation, drug resistance, and oncogenic

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Fig. 5 Rab3a expression is associated with glioma stem cell characteristics. a, b Tumorsphere formation of Rab3a-overexpressing Ink4a/Arf-/- astrocytes (a) and U87MG cells (b) in low density seeding and neural stem cell culture conditions for 14 days (400 cells per 12-well plate or one cell per square millimeter) (**p \ 0.01). The graph shows the number of spheres ([10 lm) formed from these cells with representative photos showing tumorspheres. c, d Tumorsphereforming ability of controls and Rab3a-overexpressing Ink4a/Arf-/astrocytes (c) and U87MG cells (d) was examined by an in vitro limiting dilution assay (**p \ 0.01). e, f Expression of stem cell

markers (CD133, Nestin and Sox2) in controls and Rab3a-overexpressing Ink4a/Arf-/- astrocytes (e) and U87MG cells (f). g Immunoblot analysis showing expression levels of Rab3a in human glioma stem cells derived from patients with glioma. Normal human astrocytes (NHA) cells were used as a control. f Immunoblot analysis showing decreased Rab3a expression in human glioma stem cells grown under differentiation culture conditions (FBS: NBE medium supplemented with 5 % FBS) compared with neural stem cell culture conditions (NBE: NBE medium supplemented with EGF and bFGF)

transformation that functions under the regulation of oncogenic Rab proteins, including Rab3a. Interestingly, Rab3a expression was upregulated in GSCs and downregulated under the differentiation culture conditions, implying a plausible link between Rab3a and cancer stemness. Subsequent studies will focus on the identification of the stemness-associated signal that is regulated by Rab3a. This approach will be able to elucidate the mechanism underlying the drug resistance and glioma stem cell generation regulated by Rab3a. Identification of intracellular function and modification of Rab3a will further allow us to validate the mechanism underlying Rab3a-driven tumorigenesis and cancer

stemness. It is generally understood that GDP-Rab3a dissociates from vesicles by forming a complex with guanine nucleotide dissociation inhibitor (GDI). This process is followed by reattachment of GDP-Rab3a to the vesicles and conversion into GTP-Rab3a [27]. In this process, Rab3a not only interacts with multiple Rab-associated proteins but also transports proteins in the form of vesicles. These may be a clue to the identification of the mechanism that is responsible for the Rab3a-associated tumorigenic and stemness signal.

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Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) Grants funded by the Korea

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government (MSIP) (No. NRF-2009-0070325 to S.C. Kim and No. 2013M2A2A7042530 to H. Kim) and Hallym University Research Fund (HRF-G-2012-4, to S.C. Kim). 15.

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