Breast Cancer Initiating Cells Promote Chemoresistance by Pre-Activation of the DNA Damage Repair.

Abstract

Abstract Background: Breast tumor initiating cells (BT-ICs) display a variety of drug-resisting mechanisms including overxpression of ATP binding cassette (ABC) transporters and anti-apoptotic molecules. Unfortunately, application of P-glycoprotein inhibitors has not been successful in several clinical trials .Based on the tumor initiating cell concept, an alternative model posits that the T-ICs are naturally resistant to chemotherapy through their quiescence and their capacity for DNA repair. Gaining a better insight into the mechanisms of BT-IC resistance to chemotherapy might therefore lead to new therapeutic targets and better anticancer strategies.Material and Methods: We generated large numbers of BT-IC-enriched cells (SK-3rd) by in vivo passage of breast cancer cells SKBR3 in NOD/SCID mice treated with epirubicin, then enriched BT-ICs again by isolating spherical clusters of self-replicating cells (“mammospheres”) from suspension cultures. Under chemotherapy pressure, compared the apoptosis between BT-IC and non BT-IC by MTT, Annexin V, DNA ladder and hochest 33342 staining, and analyzed the cell cycles by Flow cytometry. We tested the DNA repair capacity after chemo-induced DNA damage between BT-IC and non BT-IC by single cell gel electrophoresis and tested the expression of DNA damage checkpoint protein by immunoblot.Results:SK-3rd formed 40-fold more mammospheres than SKBR3 (16.7%±2.6% vs 0.5%±0.2%; P<0.001). Compared with non BT-IC, BT-IC treated with epirubicin showed more viability as assessed by MTT assay. After treated with 0.3ug/ml Epirubicin for 24 hours, withdrew the drug, and then repaired for 12, 24 hours respectively, analyzed the Annexin V positive cells in BT-IC were less than n non BT-IC(17.27%±2.3% vs 56%±6.7%; P=0.026), hochest staining also showed the similar results.SK-3rd mammospheric cells treated with or without Epirubicin arrested in G0/1 stage. Breast T-IC can repair chemo-induced DNA damage more efficiently than non BT-IC. Treated with or without Epirubicin, Sk-3rd mammospheric cells can pre-activated phosphorylated DNA chk2checkpoint (Thr68),but not in non BT-IC.Dicussion: The discovery of cancer initiating cells in solid tumors has changed our view of carcinogenesis and chemotherapy. In our study, we found that, BT-IC was more resistant than non BT-IC under the same chemotherapy pressure Although initiating cells can self-renew, they are generally quiescent, spending most of their time in G0. BT-IC treated with or without Epirubicin activated DNA checkpoint phosphorylated chk2 and arrested in G0/1 stage, which implied that BT-IC can target itself to arrest in G0/1 stage and pre-active chk2. When treated with DNA damage, BT-IC has enough time and quickly responses to repair damaged DNA, and at last contributes to the failure of chemotherapy. The new insight into the mechanisms of initiating -cell chemoresistance might allow the development of new strategies to improve targeted therapies in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1121.