阻断EGFR和mTOR信号通路对C6胶质瘤及其干细胞作用研究
摘要
背景：胶质瘤耐药问题至今仍是胶质瘤治疗中亟待解决的难题,现已发现在胶质瘤中存在少量的胶质瘤干细胞,在肿瘤迁徙和侵袭性中发挥关键作用,是胶质瘤复发和治疗抗性的根源。以肿瘤干细胞作为研究对象,对阐明脑肿瘤发生发展机制有重要的价值。表皮生长因子受体(EGFR)是一种具酪氨酸激酶活性的跨膜受体,哺乳动物雷帕霉素靶蛋白(mTOR)是胞浆中一种丝/苏氨酸蛋白激酶,细胞中EGFR及mTOR两信号通路对于细胞增殖,细胞周期都有重要作用。将两者联合应用于胶质瘤及胶质瘤干细胞的作用如何却鲜有报道。
目的：在细胞水平研究埃罗替尼(Erlotinib)和雷帕霉素(Rapamycin)单独及联合用药对脑胶质瘤C6细胞及其肿瘤干细胞(GSCs)的增殖、周期的作用,和细胞内EGFR和mTOR信号通路中基因及蛋白表达情况,探讨及验证埃罗替尼、雷帕霉素联合抑制胶质瘤细胞增殖、细胞周期及转移的可能机制,为胶质瘤靶向治疗提供新的研究切入点。
方法：采用无血清培养基培养C6细胞,获取C6胶质瘤干细胞(GSCs),细胞免疫荧光染色检测CD133和nestln鉴定胶质瘤干细胞；采用CCK-8、RT-PCR和Western blot技术检测经埃罗替尼、雷帕霉素干预后,肿瘤细胞、胶质痛干细胞的细胞增殖和细胞周期与EGFR和mTOR信号通路相关基因蛋白EGFR、Akt、p-Akt、mTOR、p-mTOR、rps6k、p-rps6k表达变化的关系.
结果：胶质瘤干细胞表达CD133、Nestin:埃罗替尼、雷帕霉素对C6及C6胶质瘤干细胞均呈剂量-时间依赖性抗增殖作用(P<0.05),两者联合用药抗增殖效果更加明显。埃罗替尼与雷帕毒霉素能够增加GL期细胞比例,减少S期比例,但C6胶质瘤干细胞对埃罗替尼、雷帕霉素的敏感性低于C6细胞(P<0.05)；用药组与空白组相比,细胞中EGFR和mTOR通路中相关蛋白表达明显下调(P<0.05),联合用药组效果更明显。
结论：胶质瘤C6细胞系中存在一定的肿瘤干细胞；埃罗替尼与雷帕霉素通过阻断EGFR和mTOR抑制C6、C6GSCs生长增殖、细胞周期。
Backgroud:Glioma drug resistance are still problems to be solved in glioma treatment, there is a small amount of glioma stem cells in glioma. Cancer stem cells play a key role in tumor migration and aggressive, is the root of glioma recurrence and treatment resistance. cancer stem cells for the research object has important value to elucidate the mechanism of brain development. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor, Mammals rapamycin target protein (mTOR) is a kind of silk cytoplasm/threonine protein kinase, EGFR and mTOR signaling pathway has important role of cell proliferation and cell cycle. Applies both glioma and the role of glioma stem cells rarely has been reported.
Objective:Using Erlotinib and Rapamycin alone and combination Observe proliferation and cycle in glioma C6cells and cancer stem cells (GSCs) at a cellular level and gene and protein expression in EGFR and mTOR signaling pathways.Discuss and testthe possible mechanisms ofproliferation, cycle and metastasis of Erlotinib and Rapamycin combined inhibition of glioma cell forproviding a new research direction in gliomas targeted therapy.
Methods:C6cells were cultured by serum free medium, and the tumor stem cells (GSCs) were obtained. Cell immunofluorescence staining was used for detection CD133and nestin which expressed by tumor stem cells; CCK-8. RT-PCR and Western blot were used to observe the relationship of tumor cell proliferation, cell cycle and EGFR,mTOR signal pathway related genes, and its expressions of protein (EGFR, Akt, p-Akt, mTOR, p-mTOR, rps6k, p-rps6k after intervention of Erlotinib and Rapamycin.
Results:The expressions of CD133and Nestin were detected in glioma stem cells; Erlotinib and Rapamycin had the dose-time dependence antiproliferative effect in C6and C6GSCs (P<0.05). Erlotinib and Rapamycin can increase the ratios of cells in G1phase and reduce the ratios of cells in S phase, the sensitivity to Erlotinib and Rapamycin in GSCswas lower than in C6cells(P<0.05); Protein expressions of EGFR and mTOR pathway in treatment group were lower than those in the control group(P<0.05), the efficacy is significantly in combined treatment.
Conclusion:Tumor stem cells exist in C6glioma cell lines; Erlotinib and Rapamycin can inhibit the growth and proliferation of C6and C6GSCs by means of blocking the EGFR and mTOR signaling pathway.
目的：在细胞水平研究埃罗替尼(Erlotinib)和雷帕霉素(Rapamycin)单独及联合用药对脑胶质瘤C6细胞及其肿瘤干细胞(GSCs)的增殖、周期的作用,和细胞内EGFR和mTOR信号通路中基因及蛋白表达情况,探讨及验证埃罗替尼、雷帕霉素联合抑制胶质瘤细胞增殖、细胞周期及转移的可能机制,为胶质瘤靶向治疗提供新的研究切入点。
方法：采用无血清培养基培养C6细胞,获取C6胶质瘤干细胞(GSCs),细胞免疫荧光染色检测CD133和nestln鉴定胶质瘤干细胞；采用CCK-8、RT-PCR和Western blot技术检测经埃罗替尼、雷帕霉素干预后,肿瘤细胞、胶质痛干细胞的细胞增殖和细胞周期与EGFR和mTOR信号通路相关基因蛋白EGFR、Akt、p-Akt、mTOR、p-mTOR、rps6k、p-rps6k表达变化的关系.
结果：胶质瘤干细胞表达CD133、Nestin:埃罗替尼、雷帕霉素对C6及C6胶质瘤干细胞均呈剂量-时间依赖性抗增殖作用(P<0.05),两者联合用药抗增殖效果更加明显。埃罗替尼与雷帕毒霉素能够增加GL期细胞比例,减少S期比例,但C6胶质瘤干细胞对埃罗替尼、雷帕霉素的敏感性低于C6细胞(P<0.05)；用药组与空白组相比,细胞中EGFR和mTOR通路中相关蛋白表达明显下调(P<0.05),联合用药组效果更明显。
结论：胶质瘤C6细胞系中存在一定的肿瘤干细胞；埃罗替尼与雷帕霉素通过阻断EGFR和mTOR抑制C6、C6GSCs生长增殖、细胞周期。
Backgroud:Glioma drug resistance are still problems to be solved in glioma treatment, there is a small amount of glioma stem cells in glioma. Cancer stem cells play a key role in tumor migration and aggressive, is the root of glioma recurrence and treatment resistance. cancer stem cells for the research object has important value to elucidate the mechanism of brain development. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor, Mammals rapamycin target protein (mTOR) is a kind of silk cytoplasm/threonine protein kinase, EGFR and mTOR signaling pathway has important role of cell proliferation and cell cycle. Applies both glioma and the role of glioma stem cells rarely has been reported.
Objective:Using Erlotinib and Rapamycin alone and combination Observe proliferation and cycle in glioma C6cells and cancer stem cells (GSCs) at a cellular level and gene and protein expression in EGFR and mTOR signaling pathways.Discuss and testthe possible mechanisms ofproliferation, cycle and metastasis of Erlotinib and Rapamycin combined inhibition of glioma cell forproviding a new research direction in gliomas targeted therapy.
Methods:C6cells were cultured by serum free medium, and the tumor stem cells (GSCs) were obtained. Cell immunofluorescence staining was used for detection CD133and nestin which expressed by tumor stem cells; CCK-8. RT-PCR and Western blot were used to observe the relationship of tumor cell proliferation, cell cycle and EGFR,mTOR signal pathway related genes, and its expressions of protein (EGFR, Akt, p-Akt, mTOR, p-mTOR, rps6k, p-rps6k after intervention of Erlotinib and Rapamycin.
Results:The expressions of CD133and Nestin were detected in glioma stem cells; Erlotinib and Rapamycin had the dose-time dependence antiproliferative effect in C6and C6GSCs (P<0.05). Erlotinib and Rapamycin can increase the ratios of cells in G1phase and reduce the ratios of cells in S phase, the sensitivity to Erlotinib and Rapamycin in GSCswas lower than in C6cells(P<0.05); Protein expressions of EGFR and mTOR pathway in treatment group were lower than those in the control group(P<0.05), the efficacy is significantly in combined treatment.
Conclusion:Tumor stem cells exist in C6glioma cell lines; Erlotinib and Rapamycin can inhibit the growth and proliferation of C6and C6GSCs by means of blocking the EGFR and mTOR signaling pathway.
引文
[1]王忠诚神经外科学.湖北科学技术出版社,2005.548
[2]Jemal A,Siegal R,Xu J,Ward E:Cancer statistics,2010.CA Cancer J Clin 2010,60(5):277-300.
[3]Central Brain Tumor Registry of the United States(CBTRUS):2010 CBTRUSstatistical. report:primary brain and central nervous system tumors diagnosed in the United States in 2004-2006.
[4]Stupp R,Roila F. Malignant glioma:ESMO clinical recommendations for diagnosis,treatment and follow-up.Ann Oncol.2008;19 Suppl2:83-85.
[5]Rainov NO, Ren H.Gene therapy for human malignant brain tumors. Cancer J,2003,9:180-188.
[6]Singh SK,Clarke ID,Terasaki M,et al. Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63:5821-5828.
[7]Singh SK, Hawkins C,Clarke ID, et al. Identification of human brain tumour initiating cells[J].Nature,2004,432(7015):396-401.
[8]Altanerova V, Altaner C. Cancer stem cells[J]. Neoplasma,2005,52(6): 435-440.
[9]BELDA-INIESTA C,DE CASTRO CARPERIO J,SERENOM,et al.Epidermalgrowth factor receptorand glioblastoma multiforme:molecular basis fora newapproach[J].Clin Transl Oncol,2008,10(2):73-77.
[10]Halatsch ME, Low S, Hielscher T, et al. Epidermal growth factor receptorpathway geneexpressionsandbiological response ofglioblastoma multiforme celllines to erlotinib. Anticancer Res 2008,28(6):3725-3728.
[11]Abraham R T, Gibbons J J. The mammalian target of rapamycin signalingpathway:twists and turns in the road to cancer therapy[J]. Clin Cancer Res 2007,13(11):3109-3114.
[12]I. Nakano, H.I. Kornblum. Brain tumor stem cells.Pediatr.Res.59 (2006) 54R-58R. [13] T.C. Johannessen. R. Bjerkvig. B.B. Tysnes. DNA repair and cancer stem-like Cells-potential partners in glioma drug resistance?. Cancer Treat Rev.34 (2008)558-567.
[14]Makino S. The role of tumor stem cells in regrowth of the tumor Following drastic applications[J].Acta Unio Int Contra Cancrum, 1959,15(1):196-198.
[15]Dontu G,Al-Hajj M, Abdallah WM,et al. Stem cells in normal breast Development and breast cancer[J]. Cell Prolif,2003,36(1):59-72.
[16]Passegue E, Jamieson CH, Ailles LE, et al. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics[J]. Proc Natl Acad Sci USA,2003,100(S1):11842-11849.
[17]黄强,董军,朱玉德等.人脑胶质瘤组织中分离和培养干细胞.中华肿瘤杂志,2006,5(25)：331-333.
[18]Peterson DL, Sheridan PJ, Brown WE.Animal models for brain tumors: Historical perspeetives and future diretions [J]. J Neurosurg,1994,80(5): 865-876.
[19]Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells [J]. Nature,2004,432(7015):396-401.
[20]Abraham R T, Gibbons J J. The mammalian target of rapamycin signaling pathway:twists and turns in the road to cancer therapy[J]. Clin Cancer Res 2007, 13(11):3109-3114.
[21]Olson W C, Heston W D, Rajasekaran A K. Clinical trials of cancer therapies targeting prostate-specific membrane antigen[J]. Rev Recent Clin Trials,2007, 2(3):182-190.
[22]Wang S S, Guan Z Z, Xiang Y Q, et al. Significance of EGFR and p-ERK expression in nasopharyngeal carcinoma[J]. Zhong hua Zhong Liu Za Zhi,2006, 28(1):28-31.
[23]Tao Q, Chan A T. Nasopharyngeal carcinoma:molecular pathogenesis and therapeutic developments[J]. Expert Rev Mol Med,2007,9(12):1-24.
[24]Gupta K, Salunke P. Molecular markers of glioma:an update on recent progress and perspectives[J]. J Cancer Res Clin Oncol.2012,138(12):1971-1981.
[25]贾践博,侯现增,刘滨,等.EGFR和HER2在胶质瘤复发前后表达的变化[J].山东大学学报：医学版,2011,49(12)：107-110.
[26]Bishayee S.Role of conformational alteration in the epidermal growth factor receptor(EGFR) function.Biochem Pharmacol,2000,60(8):1217-1223.
[27]Bishayee A, Beguinot L, Bishayee S.Phosphorylation of tyrosine 992,1068, and 1086 is required for conformational change of the human epidermal growth factor receptor C Terminal tail.Mol Biol Cell,1999,10(3):525-536.
[28]Cohen S.Isolation of amouse submaxillary gland protein accelerating incisor Eruption and eyelid opening in the new-born animals, J Biol Chem 1962,237:1555-1562.
[29]Salomon DS, Brandt R, Ciardiello F, et al.Epidermal growth factor related peptides and their receptors in human malignancies.Crit Rev Oncol Hematol 1995,19:183-232.
[30]Le Tourneau C, Faivre S, Siu L L. Molecular targeted therapy of head and neck cancer:review and clinical development challenges[J]. Eur J Cancer,2007,43(17): 2457-2466.
[31]Servidei T, Riccardi A, Mozzetti S, et al. Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib[J]. Int J Cancer,2008,123(12):2939-2949.
[32]De Luca A, Carotenuto A, Rachiglio A, et al. The role of the EGFR signaling in tumor microenvironment[J]. J Cell Physiol,2008,214(3):559-567.
[33]Bernier J. Drug Insight:cetuximab in the treatment of recurrent and metastatic squamous cell carcinoma of the head and neck[J]. Nat Clin Pract Oncol,2008, 5(12):705-713.
[34]Kim DH, Sarbassov DD, A li SM, et al. Gbetal a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR[J].Mol Cell,2003,11(4):895-904.
[35]Schm idt A. Bickle M, Beck T, et al. The yeast phosphatidy linositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchang factor ROM2[J].Cell,1997,88(4):531-542.
[36]Vermorken J B, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer[J]. N Engl J Med,2008,359(11):1116-1127.
[37]Eng C P, Sehgal S N, Vezina C. Activity of rapamycin (AY-22,989) against transplanted tumors [J]. J Antibiot(Tokyo),1984,37(10):1231-1237.
[38]Abraham R T. Identification of TOR signaling complexes:more TORC or the cell growth engine[J]. Cell,2002,111(1):9-12.
[39]Moon SH,Kim DK,Cha Y,et al.PI3K/Akt and Stat3 signaling regulated by PTEN control of the cancer stem cell population, proliferation and senescence in a glioblastoma cell line[J].Int J Oncol,2013,42(3):921-928.
[40]季晓燕,黄强,董军,等.脑肿瘤干细胞体外分化的形态、标志物及细胞增殖动力学特征[J].中华医学杂志,2006,86(23)：1604-1609.
[41]Iwadate Y,Namba H, Sakiyama, et al.Interleukin-12mediated induction of systemic Immunity in the periphery and recruitment of activated T cells into the brain produce limited antitumor effects compared with interleukin-2.Int J Mol Med,2002,10(6):741-747.
[42]Sebastian S, Settleman J, Reshkin S J, et al. The Complexity of targeing EGFR signalling in cancer:from expression to turnover[J]. Biochim Biophys Acta,2006, 1766(1):120-139.
[43]Fan Q W, Cheng C,Knight Z A,et al.EGFR Signals to mTOR Through PKC and Independently of Akt in Glioma[J].Sci Siqnal,2009,2(55):ra4.
[2]Jemal A,Siegal R,Xu J,Ward E:Cancer statistics,2010.CA Cancer J Clin 2010,60(5):277-300.
[3]Central Brain Tumor Registry of the United States(CBTRUS):2010 CBTRUSstatistical. report:primary brain and central nervous system tumors diagnosed in the United States in 2004-2006.
[4]Stupp R,Roila F. Malignant glioma:ESMO clinical recommendations for diagnosis,treatment and follow-up.Ann Oncol.2008;19 Suppl2:83-85.
[5]Rainov NO, Ren H.Gene therapy for human malignant brain tumors. Cancer J,2003,9:180-188.
[6]Singh SK,Clarke ID,Terasaki M,et al. Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63:5821-5828.
[7]Singh SK, Hawkins C,Clarke ID, et al. Identification of human brain tumour initiating cells[J].Nature,2004,432(7015):396-401.
[8]Altanerova V, Altaner C. Cancer stem cells[J]. Neoplasma,2005,52(6): 435-440.
[9]BELDA-INIESTA C,DE CASTRO CARPERIO J,SERENOM,et al.Epidermalgrowth factor receptorand glioblastoma multiforme:molecular basis fora newapproach[J].Clin Transl Oncol,2008,10(2):73-77.
[10]Halatsch ME, Low S, Hielscher T, et al. Epidermal growth factor receptorpathway geneexpressionsandbiological response ofglioblastoma multiforme celllines to erlotinib. Anticancer Res 2008,28(6):3725-3728.
[11]Abraham R T, Gibbons J J. The mammalian target of rapamycin signalingpathway:twists and turns in the road to cancer therapy[J]. Clin Cancer Res 2007,13(11):3109-3114.
[12]I. Nakano, H.I. Kornblum. Brain tumor stem cells.Pediatr.Res.59 (2006) 54R-58R. [13] T.C. Johannessen. R. Bjerkvig. B.B. Tysnes. DNA repair and cancer stem-like Cells-potential partners in glioma drug resistance?. Cancer Treat Rev.34 (2008)558-567.
[14]Makino S. The role of tumor stem cells in regrowth of the tumor Following drastic applications[J].Acta Unio Int Contra Cancrum, 1959,15(1):196-198.
[15]Dontu G,Al-Hajj M, Abdallah WM,et al. Stem cells in normal breast Development and breast cancer[J]. Cell Prolif,2003,36(1):59-72.
[16]Passegue E, Jamieson CH, Ailles LE, et al. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics[J]. Proc Natl Acad Sci USA,2003,100(S1):11842-11849.
[17]黄强,董军,朱玉德等.人脑胶质瘤组织中分离和培养干细胞.中华肿瘤杂志,2006,5(25)：331-333.
[18]Peterson DL, Sheridan PJ, Brown WE.Animal models for brain tumors: Historical perspeetives and future diretions [J]. J Neurosurg,1994,80(5): 865-876.
[19]Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells [J]. Nature,2004,432(7015):396-401.
[20]Abraham R T, Gibbons J J. The mammalian target of rapamycin signaling pathway:twists and turns in the road to cancer therapy[J]. Clin Cancer Res 2007, 13(11):3109-3114.
[21]Olson W C, Heston W D, Rajasekaran A K. Clinical trials of cancer therapies targeting prostate-specific membrane antigen[J]. Rev Recent Clin Trials,2007, 2(3):182-190.
[22]Wang S S, Guan Z Z, Xiang Y Q, et al. Significance of EGFR and p-ERK expression in nasopharyngeal carcinoma[J]. Zhong hua Zhong Liu Za Zhi,2006, 28(1):28-31.
[23]Tao Q, Chan A T. Nasopharyngeal carcinoma:molecular pathogenesis and therapeutic developments[J]. Expert Rev Mol Med,2007,9(12):1-24.
[24]Gupta K, Salunke P. Molecular markers of glioma:an update on recent progress and perspectives[J]. J Cancer Res Clin Oncol.2012,138(12):1971-1981.
[25]贾践博,侯现增,刘滨,等.EGFR和HER2在胶质瘤复发前后表达的变化[J].山东大学学报：医学版,2011,49(12)：107-110.
[26]Bishayee S.Role of conformational alteration in the epidermal growth factor receptor(EGFR) function.Biochem Pharmacol,2000,60(8):1217-1223.
[27]Bishayee A, Beguinot L, Bishayee S.Phosphorylation of tyrosine 992,1068, and 1086 is required for conformational change of the human epidermal growth factor receptor C Terminal tail.Mol Biol Cell,1999,10(3):525-536.
[28]Cohen S.Isolation of amouse submaxillary gland protein accelerating incisor Eruption and eyelid opening in the new-born animals, J Biol Chem 1962,237:1555-1562.
[29]Salomon DS, Brandt R, Ciardiello F, et al.Epidermal growth factor related peptides and their receptors in human malignancies.Crit Rev Oncol Hematol 1995,19:183-232.
[30]Le Tourneau C, Faivre S, Siu L L. Molecular targeted therapy of head and neck cancer:review and clinical development challenges[J]. Eur J Cancer,2007,43(17): 2457-2466.
[31]Servidei T, Riccardi A, Mozzetti S, et al. Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib[J]. Int J Cancer,2008,123(12):2939-2949.
[32]De Luca A, Carotenuto A, Rachiglio A, et al. The role of the EGFR signaling in tumor microenvironment[J]. J Cell Physiol,2008,214(3):559-567.
[33]Bernier J. Drug Insight:cetuximab in the treatment of recurrent and metastatic squamous cell carcinoma of the head and neck[J]. Nat Clin Pract Oncol,2008, 5(12):705-713.
[34]Kim DH, Sarbassov DD, A li SM, et al. Gbetal a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR[J].Mol Cell,2003,11(4):895-904.
[35]Schm idt A. Bickle M, Beck T, et al. The yeast phosphatidy linositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchang factor ROM2[J].Cell,1997,88(4):531-542.
[36]Vermorken J B, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer[J]. N Engl J Med,2008,359(11):1116-1127.
[37]Eng C P, Sehgal S N, Vezina C. Activity of rapamycin (AY-22,989) against transplanted tumors [J]. J Antibiot(Tokyo),1984,37(10):1231-1237.
[38]Abraham R T. Identification of TOR signaling complexes:more TORC or the cell growth engine[J]. Cell,2002,111(1):9-12.
[39]Moon SH,Kim DK,Cha Y,et al.PI3K/Akt and Stat3 signaling regulated by PTEN control of the cancer stem cell population, proliferation and senescence in a glioblastoma cell line[J].Int J Oncol,2013,42(3):921-928.
[40]季晓燕,黄强,董军,等.脑肿瘤干细胞体外分化的形态、标志物及细胞增殖动力学特征[J].中华医学杂志,2006,86(23)：1604-1609.
[41]Iwadate Y,Namba H, Sakiyama, et al.Interleukin-12mediated induction of systemic Immunity in the periphery and recruitment of activated T cells into the brain produce limited antitumor effects compared with interleukin-2.Int J Mol Med,2002,10(6):741-747.
[42]Sebastian S, Settleman J, Reshkin S J, et al. The Complexity of targeing EGFR signalling in cancer:from expression to turnover[J]. Biochim Biophys Acta,2006, 1766(1):120-139.
[43]Fan Q W, Cheng C,Knight Z A,et al.EGFR Signals to mTOR Through PKC and Independently of Akt in Glioma[J].Sci Siqnal,2009,2(55):ra4.