Abstract
One of the main reasons for cancer treatment resistance is the existence of cancer stem-like cells (CSCs). Here, we elucidated the relationship between low proteasome activity cells (LPACs) and CSCs. The human colorectal cancer cell lines HCT116, SW480, DLD1, and KM12SM were engineered to stably express a green fluorescent molecule fused to the degron of ornithine decarboxylase, resulting in an accumulation of the fluorescence in LPACs. LPACs were isolated by flow cytometry. Treatment resistance (radio- and chemotherapy) and the capacity of LPACs to act as CSCs were analyzed. Microarray analysis was performed to reveal genes related to treatment resistance. The prognostic impact of potent genes was examined in 190 patients with colorectal cancer. LPACs had a significantly increased capacity for radioresistance and chemoresistance (5-fluorouracil and oxaliplatin), significantly lower reactive oxygen species activity, and significantly increased sphere formation capacity compared with non-LPACs. The number of cells in the G0-G1 phase was significantly higher among LPACs. Subcutaneous injection of as few as 20 LPACs led to tumor formation in immunologically incompetent mice. Microarray analysis revealed that the expression of EP300-interacting inhibitor of differentiation 3 (EID3) was significantly increased in LPACs. In vitro assay revealed that EID3 positively controlled cell proliferation and treatment resistance. The high expression of EID3 was an adverse prognostic indicator in patients with colorectal cancer (P = 0.0400). LPACs have characteristic treatment resistance and act as CSCs in colorectal cancer. In addition, EID3 is one of the potential regulators of treatment resistance in colorectal cancer and may be a potential therapeutic target. Clin Cancer Res; 22(21); 5277-86. ©2016 AACR.
Highlights
Colorectal cancer is one of the most common human malignancies worldwide
low proteasome activity cells (LPACs) had a significantly increased capacity for radioresistance and chemoresistance (5-fluorouracil and oxaliplatin), significantly lower reactive oxygen species activity, and significantly increased sphere formation capacity compared with nonLPACs
The number of cells in the G0–G1 phase was significantly higher among LPACs
Summary
LPACs from colorectal cancer cells have increased radioresistance and chemoresistance After stable transfection, LPACs were detected by fluorescent microscopy and flow cytometry (Fig. 1A). After chemotherapy (5-FU and L-OHP), the LPAC population was significantly increased in three of four cell lines (HCT116 5-FU P 1⁄4 0.0004, L-OHP P < 0.0001, KM12SM 5-FU P < 0.0001, L-OHP P 1⁄4 0.0010; Fig. 1C), and there was no significant difference in SW480 (5-FU P 1⁄4 0.2606, L-OHP P 1⁄4 0.1210). The subcutaneous inoculation of 104 cells from each population revealed more rapid tumorigenicity for LPACs than non-LPACs (P 1⁄4 0.0485; Fig. 2F) This indicates that CSCs are enriched in LPACs. Microarray analysis was performed to investigate the mechanism underlying treatment resistance and factors leading to cancer stemness in LPACs. Several cancer-related gene candidates were identified using microarray data from the four cell lines (HCT116, SW480, DLD1, KM12SM; Supplementary Table S1). Upregulation of EID3 in colorectal cancer cell lines conferred stronger radioresistance compared with cells transfected with empty control vector (HCT116 P 1⁄4 0.0012, KM12SM P 1⁄4 0.0014; Fig. 4B). Sensitivity to chemotherapy was significantly increased in siEID3-treated colorectal cancer cells compared with controls (HCT116 5-FU siRNA#1 P 1⁄4 0.0307, KM12SM 5-FU siRNA#1 P 1⁄4 0.0413, similar results for siRNA#2; Fig. 5C, left; HCT116 L-OHP siRNA#1 P 1⁄4 0.0546, KM12SM L-OHP siRNA#1 P 1⁄4 0.0048, similar results for siRNA#2; Fig. 5C, right)
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