title: Combined treatment of Nimotuzumab and rapamycin is effective against temozolomide-resistant human gliomas regardless of the EGFR mutation status.
abstract: BACKGROUND
  The treatment of glioblastoma multiforme (GBM) is an unmet clinical need. The 5-year survival rate of patients with GBM is less than 3%. Temozolomide (TMZ) remains the standard first-line treatment regimen for gliomas despite the fact that more than 90% of recurrent gliomas do not respond to TMZ after repeated exposure. We have also independently shown that many of the Asian-derived glioma cell lines and primary cells derived from Singaporean high-grade glioma patients are indeed resistant to TMZ. This issue highlights the need to develop new effective anti-cancer treatment strategies. In a recent study, wild-type epidermal growth factor receptor (wtEGFR) has been shown to phosphorylate a truncated EGFR (known as EGFRvIII), leading to the phosphorylation of STAT proteins and progression in gliomagenesis. Despite the fact that combination of EGFR targeting drugs and rapamycin has been used before, the effect of mono-treatment of Nimotuzumab, rapamycin and combination therapy in human glioma expressing different types of EGFR is not well-studied. Herein, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an mTOR inhibitor (rapamycin) in Caucasian patient-derived human glioma cell lines, Asian patient-derived human glioma cell lines, primary glioma cells derived from the Mayo GBM xenografts, and primary short-term glioma culture derived from high-grade glioma patients.
  
  
  METHODS
  The combination effect of Nimotuzumab and rapamycin was examined in a series of primary human glioma cell lines and glioma cell lines. The cell viability was compared to TMZ treatment alone. Endogenous expressions of EGFR in various GBM cells were determined by western blotting.
  
  
  RESULTS
  The results showed that combination of Nimotuzumab with rapamycin significantly enhanced the therapeutic efficacy of human glioma cells compared to single treatment. More importantly, many of the Asian patient-derived glioma cell lines and primary cells derived from Singaporean high-grade gliomas, which showed resistance to TMZ, were susceptible to the combined treatments.
  
  
  CONCLUSIONS
  In conclusion, our results strongly suggest that combination usage of Nimotuzumab and rapamycin exert higher cytotoxic activities than TMZ. Our data suggest that this combination may provide an alternative treatment for TMZ-resistant gliomas regardless of the EGFR status.
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. nmocqq@nccs.com.sg.
authors.forename: Dawn Q
authors.initials: DQ
authors.lastname: Chong
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. toh.xin.yi.87@gmail.com.
authors.forename: Xin Y
authors.initials: XY
authors.lastname: Toh
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. ncmiho@gmail.com.
authors.forename: Ivy A W
authors.initials: IA
authors.lastname: Ho
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. ncmskc@gmail.com.
authors.forename: Kian C
authors.initials: KC
authors.lastname: Sia
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. ncmjpn@nccs.com.sg.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Newman
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. ncmyya@gmail.com.
authors.forename: Yulyana
authors.initials: Y
authors.lastname: Yulyana
authors.affiliation: National Neuroscience Institute, Singapore, 308433, Singapore. Wai_Hoe_Ng@nni.com.sg.
authors.forename: Wai-Hoe
authors.initials: WH
authors.lastname: Ng
authors.affiliation: Department of Pathology, Singapore General Hospital, Singapore, 169608, Singapore. lai.siang.hui@sgh.com.sg.
authors.forename: Siang H
authors.initials: SH
authors.lastname: Lai
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. dmshmf@gmail.com.
authors.forename: Mac M F
authors.initials: MM
authors.lastname: Ho
authors.affiliation: Division of Neurosurgery, National University Hospital, Singapore, 119074, Singapore. dinesh_nivedh@nuhs.edu.sg.
authors.forename: Nivedh
authors.initials: N
authors.lastname: Dinesh
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. tham.c.k.@nccs.com.sg.
authors.forename: Chee K
authors.initials: CK
authors.lastname: Tham
authors.affiliation: National Cancer Centre, 11 Hospital Drive, Singapore, 169610, Singapore. cmrlyp@nccs.com.sg.|Department of Physiology, National University of Singapore, Singapore, 117597, Singapore. cmrlyp@nccs.com.sg.|Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, 169547, Singapore. cmrlyp@nccs.com.sg.
authors.forename: Paula Y P
authors.initials: PY
authors.lastname: Lam
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003606:Dacarbazine / Q000008:administration & dosage / Q000031:analogs & derivatives; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics; D005909:Glioblastoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D006801:Humans; D009154:Mutation; D020123:Sirolimus / Q000008:administration & dosage*; D000077204:Temozolomide
chemical_list: D061067:Antibodies, Monoclonal, Humanized; C501466:nimotuzumab; D003606:Dacarbazine; C512478:EGFR protein, human; D066246:ErbB Receptors; D020123:Sirolimus; D000077204:Temozolomide