Tumor Biol. DOI 10.1007/s13277-014-2466-z
RESEARCH ARTICLE
LEF1 regulates glioblastoma cell proliferation, migration, invasion, and cancer stem-like cell self-renewal Xingchun Gao & Yajing Mi & Yue Ma & Weilin Jin
Received: 6 July 2014 / Accepted: 6 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common primary tumors of the central nervous system. This disease remains one of the incurable human malignancies because the molecular mechanism driving the GBM development and recurrence is still largely unknown. Here, we show that knockdown of lymphocyte enhancer factor-1 (LEF1), a major transcription factor of Wnt pathway, inhibits U251 cell migration, invasion, and proliferation. Furthermore, downregulation of LEF1 expression inhibits the self-renewal capacity of U251 GBM stemlike cells and decreases the expression level of the GBM stemlike cell (GSC) markers such as CD133 and nestin. Our findings reveal that LEF1 maintains the GBM cell proliferation, migration, and GBM stem-like cell self-renewal. Taken together, these results suggest that LEF1 may be a novel therapeutic target for GBM suppression. Keywords Glioblastoma multiforme . LEF1 . Proliferation . Invasion . Stemness Xingchun Gao and Yajing Mi contributed equally to this work. X. Gao : Y. Mi : W. Jin Institute of Basic Medicine Science, Xi’an Medical University, 1 Xin Wang Road, Xi’an 710021, China X. Gao (*) : Y. Ma : W. Jin Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, Key Lab. for Thin Film and Microfabrication Technology of Ministry of Education, School of Electronic Information and Electronic Engineering, Shanghai Jiao Tong University, Shanghai 200240, China e-mail: [email protected]
Introduction Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common central nervous system tumors and has the worst prognosis. Despite advances in effective therapies by surgery combined with radiation and/or chemotherapy, the estimated median survival of the patients with the GBM is approximately 1–2 years [1]. One of the major causes of tumor recurrence and patient lethality is the infiltrative property of GBM cells. It is difficult to remove the GBM cells with local therapeutic modalities when GBM cell infiltrates toward normal tissues [2]. Recent findings support that GBM arises from cancer stem cells which have been isolated from GBM cell lines and human brain tumors [3]. The cancer stem cells retain many of the properties of normal stem cells, such as self-renewal and differentiation [4]. More and more studies show that multiple stemness factors, such as Bmi1 and Wnt signaling, play a role in cancer stem cell development [5–7]. In particular [8], Wnt signaling is frequently activated in gliomas, governing glioma stem cell stemness and invasiveness [8, 9]. Lymphocyte enhancer factor-1 (LEF1) is a major transcription factor in Wnt signaling pathway [10] and has been reported to regulate GBM invasion as a direct target of miR-218 [5, 11]. However, the function of LEF1 has been not well understood in relation to GBM stemness. In this study, we found that short hairpin RNA (shRNA)-mediated knockdown of LEF1 inhibited U251 cell migration, invasion, and proliferation. Furthermore, we also demonstrated that LEF1 regulated stemness of GBM stem-like cells.
Materials and methods
W. Jin (*) School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China e-mail: [email protected]
Cancer stem-like cell culture, transfection, and stable cell line selection
W. Jin e-mail: [email protected]
U251 cells are purchased from the Chinese Academy of Sciences Cell Bank in 2012, and the cell line was grown in
Tumor Biol.
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (GIBCO, USA). The serum-free medium (SFM) was composed of DMEM/F12, 20 ng/ml basic fibroblast growth factor (bFGF; Sigma, USA), 20 μl/ml B27 supplement (Life Technologies), and 20 ng/ml epidermal growth factor (EGF; Sigma, USA). GBM stem-like cells (GSC) were isolated from U251 cell lines by using SFM. These cells can form neurosphere-like cell aggregates in less than 7 days [12]. The cell transfections were carried out with FuGENE HD reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions. For selection of GBM stable cell lines, U251 cell was cultured in 400 μg/ml neomycin (G418) for 21 days after transfection. The expression of LEF1 was confirmed by quantitative RT-PCR and Western blotting. The LEF1-shRNA-1# target sequence was as the follows: 5′-GCACGGAAAGAAAGACAGCTA-3′ and LEF1-shRNA-2#: 5′-CCATCAGATGTCAACTCCAAA-3′.
antibodies were used: CD133 (Abgent, USA; 1:50) and nestin (R&D, USA; 1:100). After incubation with the primary antibodies, they were rinsed and incubated for 1 h at room temperature with Alexa Fluor-labeled secondary antibodies (Molecular Probes, Leiden, The Netherlands; 1:800). After washing, the coverslips were mounted with glycerine/PBS containing 5 mg/ml Hoechst 33258 for nuclei staining. WST-1 assay U251 cells were seeded at the concentration of 2×103 cells/ well in 100 μl culture medium of 96-well tissue culture plate, each group was repeated six times. Cell proliferation reagent WST-1 was added with 10 μl/well at three preselected time points (24, 48, and 72 h); then, the cells were incubated for 2 h in humidified atmosphere (37 °C, 5 % CO2). After shaking the plates thoroughly for 1 min, the absorbance of the samples was measured using an ELISA reader at 465 nm. The reference wavelength was 610 nm.
Quantitative RT-PCR Wound healing assays Total RNA from GBM cells was isolated using TRIzol reagent (Invitrogen, CA, USA). The RNA was subsequently treated with RNase-free DNase I (Roche, Mannheim, Germany). Synthesis of complementary DNA was performed by using BcaBest RNA PCR kit from TaKaRa according to the manufacturer’s instructions. Quantitative RT-PCR was performed using an iQTM5 Multicolor Real-Time PCR Detection System (BioRad, Hercules, CA) with Realtime PCR Master Mix (SYBR Green, Toyobo, Osaka, Japan). GAPDH was chosen as the housekeeping gene control in the assay. Western blotting and immunofluorescence The total cell lysates were prepared in high KCl lysis buffer (10 mM Tris-HCl, pH 8.0; 140 mM NaCl; 300 mM KCl; 1 mM EDTA; 0.5 % Triton X-100; and 0.5 % sodium deoxycholate) with complete protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific Inc, IL, USA). The Western blotting assay had been previously described [13]. The following primary antibodies were used: α-tubulin (Santa Cruz Biotechnology, USA; 1:2,000) and LEF1 (Abgent, USA; 1:200). The immunofluorescence staining was conducted as described previously [13]. Cells on poly-L-lysine-coated glass coverslips were fixed with 4 % paraformaldehyde for 15 min at room temperature and then permeabilized by treatment with ice-cold methanol for 10 min. After being blocked by 15 % normal donkey serum for 30 min, cells were incubated at room temperature for 1 h with primary antibody diluted in antibody buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 100 mM L-lysine; 1 % BSA; and 0.04 % azide). The following
U251 cells were seeded in six-well plates and cultured until confluence. A wound was then created by manually scraping the cell monolayer with a 200-μl pipette tip. The cultures were washed twice with SFM to remove floating cells. The cells were then incubated in DMEM supplemented with 1 % FBS. Cell migration into the wound was observed at two preselected time points (0 and 24 h) in eight randomly selected microscopic fields for each condition and time point. Images were acquired with a Nikon DS-5M Camera System mounted on a phase-contrast Leitz microscope and were processed using Adobe Photoshop 7.0. The distance traveled by the cells was determined by measuring the wound width at different time points and then subtracting it from the wound width at time 0. The values were expressed as migration percentage, setting the gap width at 0 h as 0 %. In vitro migration and invasion assays Cells (5×105) were planted on the top side of polycarbonate Transwell filters (without Matrigel for Transwell assay) or plated on the top side of polycarbonate Transwell filter coated with Matrigel (for Transwell matrix penetration assay) in the top chamber of the QCM 24-well cell invasion assay (Cell Biolabs, Inc. Santiago, USA). For Transwell migration assays, cells were suspended in medium without serum and medium without serum was used in the bottom chamber. For the invasion assay, cells were suspended in medium without serum and medium supplemented with serum was used as a chemoattractant in the bottom chamber. The cells were incubated at 37 °C for 10 h (Transwell assay) or 48 h (invasion assay). The nonmigratory or noninvasive cells in the top
Tumor Biol.
chambers were removed with cotton swabs. The migrated and invaded cells on the lower membrane surface were fixed in 100 % methanol for 10 min, air-dried, then stained with 40,6diamidino-2-phenylindole (DAPI, Beyotime Institute of Biotechnology, Haimen, China), and counted under a microscope. Three independent experiments were conducted and the data were presented as the AVE±SEM. Limiting dilution assay A limiting dilution assay was performed as described previously [12, 14]. Tumor sphere cells were dissociated and plated on 96-well micro-well plates in 0.2 ml volumes of SFM. Final cell dilutions ranged from 1 to 120 cells/well in 0.2 ml volumes. Cultures were fed with 0.025 ml of SFM every 2 days until day 7; then, the percentage of wells without spheres for each cell plating density was calculated and plotted against the number of cells per well. Statistics Data were analyzed using the two-tailed Student’s t test. P
RESEARCH ARTICLE
LEF1 regulates glioblastoma cell proliferation, migration, invasion, and cancer stem-like cell self-renewal Xingchun Gao & Yajing Mi & Yue Ma & Weilin Jin
Received: 6 July 2014 / Accepted: 6 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common primary tumors of the central nervous system. This disease remains one of the incurable human malignancies because the molecular mechanism driving the GBM development and recurrence is still largely unknown. Here, we show that knockdown of lymphocyte enhancer factor-1 (LEF1), a major transcription factor of Wnt pathway, inhibits U251 cell migration, invasion, and proliferation. Furthermore, downregulation of LEF1 expression inhibits the self-renewal capacity of U251 GBM stemlike cells and decreases the expression level of the GBM stemlike cell (GSC) markers such as CD133 and nestin. Our findings reveal that LEF1 maintains the GBM cell proliferation, migration, and GBM stem-like cell self-renewal. Taken together, these results suggest that LEF1 may be a novel therapeutic target for GBM suppression. Keywords Glioblastoma multiforme . LEF1 . Proliferation . Invasion . Stemness Xingchun Gao and Yajing Mi contributed equally to this work. X. Gao : Y. Mi : W. Jin Institute of Basic Medicine Science, Xi’an Medical University, 1 Xin Wang Road, Xi’an 710021, China X. Gao (*) : Y. Ma : W. Jin Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, Key Lab. for Thin Film and Microfabrication Technology of Ministry of Education, School of Electronic Information and Electronic Engineering, Shanghai Jiao Tong University, Shanghai 200240, China e-mail: [email protected]
Introduction Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common central nervous system tumors and has the worst prognosis. Despite advances in effective therapies by surgery combined with radiation and/or chemotherapy, the estimated median survival of the patients with the GBM is approximately 1–2 years [1]. One of the major causes of tumor recurrence and patient lethality is the infiltrative property of GBM cells. It is difficult to remove the GBM cells with local therapeutic modalities when GBM cell infiltrates toward normal tissues [2]. Recent findings support that GBM arises from cancer stem cells which have been isolated from GBM cell lines and human brain tumors [3]. The cancer stem cells retain many of the properties of normal stem cells, such as self-renewal and differentiation [4]. More and more studies show that multiple stemness factors, such as Bmi1 and Wnt signaling, play a role in cancer stem cell development [5–7]. In particular [8], Wnt signaling is frequently activated in gliomas, governing glioma stem cell stemness and invasiveness [8, 9]. Lymphocyte enhancer factor-1 (LEF1) is a major transcription factor in Wnt signaling pathway [10] and has been reported to regulate GBM invasion as a direct target of miR-218 [5, 11]. However, the function of LEF1 has been not well understood in relation to GBM stemness. In this study, we found that short hairpin RNA (shRNA)-mediated knockdown of LEF1 inhibited U251 cell migration, invasion, and proliferation. Furthermore, we also demonstrated that LEF1 regulated stemness of GBM stem-like cells.
Materials and methods
W. Jin (*) School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China e-mail: [email protected]
Cancer stem-like cell culture, transfection, and stable cell line selection
W. Jin e-mail: [email protected]
U251 cells are purchased from the Chinese Academy of Sciences Cell Bank in 2012, and the cell line was grown in
Tumor Biol.
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (GIBCO, USA). The serum-free medium (SFM) was composed of DMEM/F12, 20 ng/ml basic fibroblast growth factor (bFGF; Sigma, USA), 20 μl/ml B27 supplement (Life Technologies), and 20 ng/ml epidermal growth factor (EGF; Sigma, USA). GBM stem-like cells (GSC) were isolated from U251 cell lines by using SFM. These cells can form neurosphere-like cell aggregates in less than 7 days [12]. The cell transfections were carried out with FuGENE HD reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions. For selection of GBM stable cell lines, U251 cell was cultured in 400 μg/ml neomycin (G418) for 21 days after transfection. The expression of LEF1 was confirmed by quantitative RT-PCR and Western blotting. The LEF1-shRNA-1# target sequence was as the follows: 5′-GCACGGAAAGAAAGACAGCTA-3′ and LEF1-shRNA-2#: 5′-CCATCAGATGTCAACTCCAAA-3′.
antibodies were used: CD133 (Abgent, USA; 1:50) and nestin (R&D, USA; 1:100). After incubation with the primary antibodies, they were rinsed and incubated for 1 h at room temperature with Alexa Fluor-labeled secondary antibodies (Molecular Probes, Leiden, The Netherlands; 1:800). After washing, the coverslips were mounted with glycerine/PBS containing 5 mg/ml Hoechst 33258 for nuclei staining. WST-1 assay U251 cells were seeded at the concentration of 2×103 cells/ well in 100 μl culture medium of 96-well tissue culture plate, each group was repeated six times. Cell proliferation reagent WST-1 was added with 10 μl/well at three preselected time points (24, 48, and 72 h); then, the cells were incubated for 2 h in humidified atmosphere (37 °C, 5 % CO2). After shaking the plates thoroughly for 1 min, the absorbance of the samples was measured using an ELISA reader at 465 nm. The reference wavelength was 610 nm.
Quantitative RT-PCR Wound healing assays Total RNA from GBM cells was isolated using TRIzol reagent (Invitrogen, CA, USA). The RNA was subsequently treated with RNase-free DNase I (Roche, Mannheim, Germany). Synthesis of complementary DNA was performed by using BcaBest RNA PCR kit from TaKaRa according to the manufacturer’s instructions. Quantitative RT-PCR was performed using an iQTM5 Multicolor Real-Time PCR Detection System (BioRad, Hercules, CA) with Realtime PCR Master Mix (SYBR Green, Toyobo, Osaka, Japan). GAPDH was chosen as the housekeeping gene control in the assay. Western blotting and immunofluorescence The total cell lysates were prepared in high KCl lysis buffer (10 mM Tris-HCl, pH 8.0; 140 mM NaCl; 300 mM KCl; 1 mM EDTA; 0.5 % Triton X-100; and 0.5 % sodium deoxycholate) with complete protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific Inc, IL, USA). The Western blotting assay had been previously described [13]. The following primary antibodies were used: α-tubulin (Santa Cruz Biotechnology, USA; 1:2,000) and LEF1 (Abgent, USA; 1:200). The immunofluorescence staining was conducted as described previously [13]. Cells on poly-L-lysine-coated glass coverslips were fixed with 4 % paraformaldehyde for 15 min at room temperature and then permeabilized by treatment with ice-cold methanol for 10 min. After being blocked by 15 % normal donkey serum for 30 min, cells were incubated at room temperature for 1 h with primary antibody diluted in antibody buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 100 mM L-lysine; 1 % BSA; and 0.04 % azide). The following
U251 cells were seeded in six-well plates and cultured until confluence. A wound was then created by manually scraping the cell monolayer with a 200-μl pipette tip. The cultures were washed twice with SFM to remove floating cells. The cells were then incubated in DMEM supplemented with 1 % FBS. Cell migration into the wound was observed at two preselected time points (0 and 24 h) in eight randomly selected microscopic fields for each condition and time point. Images were acquired with a Nikon DS-5M Camera System mounted on a phase-contrast Leitz microscope and were processed using Adobe Photoshop 7.0. The distance traveled by the cells was determined by measuring the wound width at different time points and then subtracting it from the wound width at time 0. The values were expressed as migration percentage, setting the gap width at 0 h as 0 %. In vitro migration and invasion assays Cells (5×105) were planted on the top side of polycarbonate Transwell filters (without Matrigel for Transwell assay) or plated on the top side of polycarbonate Transwell filter coated with Matrigel (for Transwell matrix penetration assay) in the top chamber of the QCM 24-well cell invasion assay (Cell Biolabs, Inc. Santiago, USA). For Transwell migration assays, cells were suspended in medium without serum and medium without serum was used in the bottom chamber. For the invasion assay, cells were suspended in medium without serum and medium supplemented with serum was used as a chemoattractant in the bottom chamber. The cells were incubated at 37 °C for 10 h (Transwell assay) or 48 h (invasion assay). The nonmigratory or noninvasive cells in the top
Tumor Biol.
chambers were removed with cotton swabs. The migrated and invaded cells on the lower membrane surface were fixed in 100 % methanol for 10 min, air-dried, then stained with 40,6diamidino-2-phenylindole (DAPI, Beyotime Institute of Biotechnology, Haimen, China), and counted under a microscope. Three independent experiments were conducted and the data were presented as the AVE±SEM. Limiting dilution assay A limiting dilution assay was performed as described previously [12, 14]. Tumor sphere cells were dissociated and plated on 96-well micro-well plates in 0.2 ml volumes of SFM. Final cell dilutions ranged from 1 to 120 cells/well in 0.2 ml volumes. Cultures were fed with 0.025 ml of SFM every 2 days until day 7; then, the percentage of wells without spheres for each cell plating density was calculated and plotted against the number of cells per well. Statistics Data were analyzed using the two-tailed Student’s t test. P
