Proteomics of the radioresistant phenotype in head-and-neck cancer: Gp96 as a novel prediction marker and sensitizing target for radiotherapy.
OBJECTIVE
Radiotherapy is an integral part of the treatment modality for head-neck cancer (HNC), but in some cases the disease is radioresistant. We designed this study to identify molecules that may be involved in this resistance.


METHODS
Two radioresistant sublines were established by fractionated irradiation of the HNC cell lines, to determine differentially proteins between parental and radioresistant cells. Proteomic analysis and reverse-transcription polymerase chain reaction were used to identify and confirm the differential proteins. The siRNA knockdown experiments were applied to examine cellular functions of a radioresistant gene, with investigation of the alterations in colonogenic survival, cell cycle status, and reactive oxygen species levels. Xenografted mouse tumors were studied to validate the results.


RESULTS
IN all, 64 proteins were identified as being potentially associated with radioresistance, which are involved in several cellular pathways, including regulation of stimulus response, cell apoptosis, and glycolysis. Six genes were confirmed to be differentially expressed in both radioresistant sublines, with Gp96, Grp78, HSP60, Rab40B, and GDF-15 upregulated, and annexin V downregulated. Gp96 was further investigated for its functions in response to radiation. Gp96-siRNA transfectants displayed a radiation-induced growth delay, reduction in colonogenic survival, increased cellular reactive oxygen species levels, and increased proportion of the cells in the G2/M phase. Xenograft mice administered Gp96-siRNA showed significantly enhanced growth suppression in comparison with radiation treatment alone (p = 0.009).


CONCLUSIONS
We identified 64 proteins and verified 6 genes that are potentially involved in the radioresistant phenotype. We further demonstrated that Gp96 knockdown enhances radiosensitivity both in cells and in vivo, which may lead to a better prognosis of HNC treatment.
Lin
Ting-Yang
TY
Department of Medical Biotechnology, Chang Gung University, Taoyuan, Taiwan.
Chang
Joseph Tung-Chieh
JT
Wang
Hung-Ming
HM
Chan
Shih-Hsuan
SH
Chiu
Chi-Ching
CC
Lin
Chien-Yu
CY
Fan
Kang-Hsing
KH
Liao
Chun-Ta
CT
Chen
I-How
IH
Liu
Tsan Z
TZ
Li
Hsiao-Fang
HF
Cheng
Ann-Joy
AJ
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D045744:Cell Line, Tumor; D018834:Chaperonin 60 / Q000032:analysis; D000091342:Endoplasmic Reticulum Chaperone BiP; D016195:G2 Phase; D055785:Gene Knockdown Techniques; D055436:Growth Differentiation Factor 15 / Q000032:analysis; D006258:Head and Neck Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000532:radiotherapy*; D006360:Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis*; D010641:Phenotype; D040901:Proteomics / Q000379:methods*; D034741:RNA, Small Interfering / Q000627:therapeutic use; D011836:Radiation Tolerance / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014183:Transplantation, Heterologous; D014410:Tumor Stem Cell Assay
D017304:Annexin A5; D018834:Chaperonin 60; D000091342:Endoplasmic Reticulum Chaperone BiP; D055436:Growth Differentiation Factor 15; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C053387:endoplasmin