PDF file - 2945K, A, Flow cytometry of CD44 expression in PHK26 + cells. Data represent the mean�standard deviation (SD); n = 3. B, The delineation of CNE1 and CNE2 cells after labeling with PKH26 and treated with PI in Flow cytometry. C, RT-PCR validation of c-MYC, CHK1, CHK2, and ATM RNA levels in CNE2 cells. D, Western blot analysis of c-MYC, pCHK1, and pCHK2 markers in CNE2 cells. GAPDH served as a loading control
ARTICLE ABSTRACT
Radiotherapy is the most successful nonsurgical treatment for nasopharyngeal carcinoma (NPC). Despite this, the prognosis remains poor. Although NPCs initially respond well to a full course of radiation, recurrence is frequent. The cancer stem cell (CSC) hypothesis provides a framework for explaining the discrepancy between the response of NPC to therapy and the poor survival rate. In this study, a stem cell-like subpopulation (PKH26+) was identified in NPC cell lines using a label-retention technique. PKH26+ cells were enriched for clonogenicity, sphere formation, side-population cells, and resistance to radiotherapy. Using genomic approaches, we show that the proto-oncogene c-MYC (MYC) regulates radiotolerance through transcriptional activation of CHK1 (CHEK1) and CHK2 (CHEK2) checkpoint kinases through direct binding to the CHK1 and CHK2 promoters. Overexpression of c-MYC in the PKH26+ subpopulation leads to increased expression of CHK1 and CHK2 and subsequent activation of the DNA-damage-checkpoint response, resulting in radioresistance. Furthermore, loss of CHK1 and CHK2 expression reverses radioresistance in PKH26+ (c-MYC high expression) cells in vitro and in vivo. This study elucidates the role of the c-MYC-CHK1/CHK2 axis in regulating DNA-damage-checkpoint responses and stem cell characteristics in the PKH26+ subpopulation. Furthermore, these data reveal a potential therapeutic application in reversal of radioresistance through inhibition of the c-MYC-CHK1/CHK2 pathway. Cancer Res; 73(3); 1219–31. ©2012 AACR.
