Tumor Biol. DOI 10.1007/s13277-014-2915-8
RESEARCH ARTICLE
Cyr61 promotes growth of glioblastoma in vitro and in vivo Gang Cheng & Hangyu Zhang & Leiming Zhang & Jianning Zhang
Received: 26 October 2014 / Accepted: 27 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The molecular regulation of the growth of glioblastoma (GBM) is not completely understood. Here, we show that the Cyr61 levels were significantly higher in GBM than in the adjacent non-tumor tissues. Overexpression of Cyr61 enhanced the viability of GBM cells, while inhibition of Cyr61 decreased the viability of GBM cells, in vitro and in vivo. Further analyses revealed that Cyr61 seemed to activate PI3K/Akt/mTor signaling pathway to increase cell growth in GBM cells. Taken together, our findings suggest a potential role of Cyr61 in GBM growth and highlight Cyr61 as a novel target for GBM therapy. Keywords Glioblastoma (GBM) . Cyr61 . PI3K . Akt . mTor
Introduction Glioblastoma multiforme (GBM) is one of the most malignant tumors with extremely poor prognosis despite combined therapy including surgery and radioand chemotherapy [1–3]. Since the GBM localizes in the central nervous system, its growth in a relative limited space appears to be very malignant [1–3]. The tumorigenesis of GBM has been extensively studied in the past decade, whereas our understanding is still limited. Gang Cheng and Hangyu Zhang are equal contribution. G. Cheng : L. Zhang : J. Zhang (*) Neurosurgery Institute, Navy General Hospital of PLA, Beijing 100048, China e-mail: [email protected] H. Zhang Neurosurgery Institute, Chinese PLA General Hospital, Beijing 100853, China
Cysteine-rich protein 61 (Cyr61) belongs to growth factorinducible, immediate-early genes and has been shown to mediate cell adhesion, stimulate chemostasis, augment growth factor-induced DNA synthesis, foster cell survival, and enhance angiogenesis [4–6]. Cyr61 binds to integrin to modulate the expression of the genes that encode the extracellularrelated kinase/mitogen-activated protein kinase (ERK/ MAPK) and phosphatidylinositol 3-kinase (PI3K), which subsequently stimulate the cells to divide in response to growth factors, resulting in increase in cell growth [4–7]. Upregulation of Cyr61 has also been detected in breast, gastric, prostate cancer [8–11], and GBM [12, 13]. However, how Cyr61 may regulate GBM growth has not been clarified. Here, we show that the Cyr61 levels were significantly higher in GBM than in the adjacent non-tumor tissues. Overexpression of Cyr61 enhanced the viability of GBM cells, while inhibition of Cyr61 decreased the viability of GBM cells, in vitro and in vivo. Further analyses revealed that Cyr61 seemed to activate PI3K/Akt/mTor signaling pathway to increase cell growth in GBM cells. Taken together, our findings suggest a potential role of Cyr61 in GBM growth and highlight Cyr61 as a novel target for GBM therapy.
Materials and methods Specimens from patients A total of 28 resected specimens (paired GBM tissue and the adjacent healthy tissue) were collected for this study. All specimens had been histologically and clinically diagnosed at Neurosurgery Institute, Navy General Hospital of PLA of China from 2008 to 2013. For the use of these clinical materials for research purposes, prior patient's consents and approval from the Institutional Research Ethics Committee were obtained.
Tumor Biol.
Cell line culture
Western blot
The human glioblastoma cell line A172 was established by Dr. Giard [14] and was cultured in 1:1 mixture of DMEM and Ham’s F12 medium supplemented with L-glutamine and 5 % FBS. To trace the glioblastoma cells in vivo, we infected the cells with a recombinant lentivirus expressing luciferase under the control of CMV promoter at MOI 100 and resulted in nearly 100 % infection efficiency. These luciferase-carrying A172 cells were used in our study. LY294002 and rapamycin were all purchased from Sigma (USA). LY294002 is a specific inhibitor for Akt phosphorylation. Rapamycin is a specific mTor inhibitor.
Protein was extracted from the GBM or the adjacent healthy tissue from the same patient, or from the cultured cells, with RIPA buffer. Primary antibodies were rabbit anti-Cyr61 (Santa Cruz, USA), anti-phosphorylated Akt (p-Akt; Cell signaling, USA), anti-phosphorylated mTor (p-mTor; Cell Signaling), and anti-α-tubulin (Cell Signaling). Secondary antibody is HRP-conjugated anti-rabbit (Jackson Labs). Figure images were representative from five repeats. α-Tubulin was used as a protein loading control.
Plasmid transfection Cyr61 construct was generated by sub-cloning PCR-amplified full-length human Cyr61 complementary DNA (cDNA) into pcDNA3.1-EGFP. For depletion of Cyr61, corresponding human short hairpin small interference RNA (shRNA) sequence was cloned into pcDNA3.1-EGFP to generate the shRNA construct. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen, USA), according to the instructions of the manufacturer. The luciferase-carrying A172 cells transfected with Cyr61 were termed as A172-Cyr61. The luciferase-carrying A172 cells transfected with shCyr61 were termed as A172-shCyr61. The luciferase-carrying A172 cells transfected with null plasmids were used as controls (A172null).
Cytostatic activity assay For assay of cytostatic activity, cells were seeded into 96-well plate at 5000 cells per well in conditioned media and subjected to a Cell Proliferation Kit (MTT, Roche, USA), according to the instruction from the manufacturer.
RNA was extracted from tissue with Trizol (Invitrogen) or cells with RNeasy kit (Qiagen, Hilden, Germany). cDNA was then synthesized by reserve transcription (Qiagen). Quantitative real-time PCR (RT-qPCR) was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were first normalized against α-tubulin and then compared to controls. Mouse manipulations All mouse experiments were approved by the general principles contained in the Guide for the Care and Use of Laboratory Animals published by Navy General Hospital of PLA of China. Ten-week-old male nude mice were used for experiments. For induction of intracranial tumor, luciferase-carrying A172 cells of 105 was stereotactically implanted into the brains of 10 weeks of age male nude mice as has been previously described [2]. One month after, the animals were examined of tumor growth by luciferin assay. Imaging of glioblastoma by bioluminescence Bioluminescence was measured with the IVIS imaging system (Xenogen Corp., Alameda, CA, USA). All of the images
A
B 8
Fold change in Cyr61 protein in GBM (vs NT)
Fig. 1 Higher Cyr61 levels were detected in GBM. a, b Western blot for Cyr61 was performed on a paired GBM tissue and the adjacent non-tumor healthy tissue (NT) from the same patient. Values of Cyr61 in GBM were first normalized against α-tubulin and then compared to NT, shown by quantification (a) and by representative images (b). αTubulin was also used as a protein loading control. *p
RESEARCH ARTICLE
Cyr61 promotes growth of glioblastoma in vitro and in vivo Gang Cheng & Hangyu Zhang & Leiming Zhang & Jianning Zhang
Received: 26 October 2014 / Accepted: 27 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The molecular regulation of the growth of glioblastoma (GBM) is not completely understood. Here, we show that the Cyr61 levels were significantly higher in GBM than in the adjacent non-tumor tissues. Overexpression of Cyr61 enhanced the viability of GBM cells, while inhibition of Cyr61 decreased the viability of GBM cells, in vitro and in vivo. Further analyses revealed that Cyr61 seemed to activate PI3K/Akt/mTor signaling pathway to increase cell growth in GBM cells. Taken together, our findings suggest a potential role of Cyr61 in GBM growth and highlight Cyr61 as a novel target for GBM therapy. Keywords Glioblastoma (GBM) . Cyr61 . PI3K . Akt . mTor
Introduction Glioblastoma multiforme (GBM) is one of the most malignant tumors with extremely poor prognosis despite combined therapy including surgery and radioand chemotherapy [1–3]. Since the GBM localizes in the central nervous system, its growth in a relative limited space appears to be very malignant [1–3]. The tumorigenesis of GBM has been extensively studied in the past decade, whereas our understanding is still limited. Gang Cheng and Hangyu Zhang are equal contribution. G. Cheng : L. Zhang : J. Zhang (*) Neurosurgery Institute, Navy General Hospital of PLA, Beijing 100048, China e-mail: [email protected] H. Zhang Neurosurgery Institute, Chinese PLA General Hospital, Beijing 100853, China
Cysteine-rich protein 61 (Cyr61) belongs to growth factorinducible, immediate-early genes and has been shown to mediate cell adhesion, stimulate chemostasis, augment growth factor-induced DNA synthesis, foster cell survival, and enhance angiogenesis [4–6]. Cyr61 binds to integrin to modulate the expression of the genes that encode the extracellularrelated kinase/mitogen-activated protein kinase (ERK/ MAPK) and phosphatidylinositol 3-kinase (PI3K), which subsequently stimulate the cells to divide in response to growth factors, resulting in increase in cell growth [4–7]. Upregulation of Cyr61 has also been detected in breast, gastric, prostate cancer [8–11], and GBM [12, 13]. However, how Cyr61 may regulate GBM growth has not been clarified. Here, we show that the Cyr61 levels were significantly higher in GBM than in the adjacent non-tumor tissues. Overexpression of Cyr61 enhanced the viability of GBM cells, while inhibition of Cyr61 decreased the viability of GBM cells, in vitro and in vivo. Further analyses revealed that Cyr61 seemed to activate PI3K/Akt/mTor signaling pathway to increase cell growth in GBM cells. Taken together, our findings suggest a potential role of Cyr61 in GBM growth and highlight Cyr61 as a novel target for GBM therapy.
Materials and methods Specimens from patients A total of 28 resected specimens (paired GBM tissue and the adjacent healthy tissue) were collected for this study. All specimens had been histologically and clinically diagnosed at Neurosurgery Institute, Navy General Hospital of PLA of China from 2008 to 2013. For the use of these clinical materials for research purposes, prior patient's consents and approval from the Institutional Research Ethics Committee were obtained.
Tumor Biol.
Cell line culture
Western blot
The human glioblastoma cell line A172 was established by Dr. Giard [14] and was cultured in 1:1 mixture of DMEM and Ham’s F12 medium supplemented with L-glutamine and 5 % FBS. To trace the glioblastoma cells in vivo, we infected the cells with a recombinant lentivirus expressing luciferase under the control of CMV promoter at MOI 100 and resulted in nearly 100 % infection efficiency. These luciferase-carrying A172 cells were used in our study. LY294002 and rapamycin were all purchased from Sigma (USA). LY294002 is a specific inhibitor for Akt phosphorylation. Rapamycin is a specific mTor inhibitor.
Protein was extracted from the GBM or the adjacent healthy tissue from the same patient, or from the cultured cells, with RIPA buffer. Primary antibodies were rabbit anti-Cyr61 (Santa Cruz, USA), anti-phosphorylated Akt (p-Akt; Cell signaling, USA), anti-phosphorylated mTor (p-mTor; Cell Signaling), and anti-α-tubulin (Cell Signaling). Secondary antibody is HRP-conjugated anti-rabbit (Jackson Labs). Figure images were representative from five repeats. α-Tubulin was used as a protein loading control.
Plasmid transfection Cyr61 construct was generated by sub-cloning PCR-amplified full-length human Cyr61 complementary DNA (cDNA) into pcDNA3.1-EGFP. For depletion of Cyr61, corresponding human short hairpin small interference RNA (shRNA) sequence was cloned into pcDNA3.1-EGFP to generate the shRNA construct. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen, USA), according to the instructions of the manufacturer. The luciferase-carrying A172 cells transfected with Cyr61 were termed as A172-Cyr61. The luciferase-carrying A172 cells transfected with shCyr61 were termed as A172-shCyr61. The luciferase-carrying A172 cells transfected with null plasmids were used as controls (A172null).
Cytostatic activity assay For assay of cytostatic activity, cells were seeded into 96-well plate at 5000 cells per well in conditioned media and subjected to a Cell Proliferation Kit (MTT, Roche, USA), according to the instruction from the manufacturer.
RNA was extracted from tissue with Trizol (Invitrogen) or cells with RNeasy kit (Qiagen, Hilden, Germany). cDNA was then synthesized by reserve transcription (Qiagen). Quantitative real-time PCR (RT-qPCR) was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were first normalized against α-tubulin and then compared to controls. Mouse manipulations All mouse experiments were approved by the general principles contained in the Guide for the Care and Use of Laboratory Animals published by Navy General Hospital of PLA of China. Ten-week-old male nude mice were used for experiments. For induction of intracranial tumor, luciferase-carrying A172 cells of 105 was stereotactically implanted into the brains of 10 weeks of age male nude mice as has been previously described [2]. One month after, the animals were examined of tumor growth by luciferin assay. Imaging of glioblastoma by bioluminescence Bioluminescence was measured with the IVIS imaging system (Xenogen Corp., Alameda, CA, USA). All of the images
A
B 8
Fold change in Cyr61 protein in GBM (vs NT)
Fig. 1 Higher Cyr61 levels were detected in GBM. a, b Western blot for Cyr61 was performed on a paired GBM tissue and the adjacent non-tumor healthy tissue (NT) from the same patient. Values of Cyr61 in GBM were first normalized against α-tubulin and then compared to NT, shown by quantification (a) and by representative images (b). αTubulin was also used as a protein loading control. *p
