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Non-LPACs were observed after each treatment, 5-FU, L-OHP and radiation (8Gy), at 0 hour, 24 hours, 48 hours and 72 hours. Non-LPACs never changed to LPACs in each assay. Bar: 100μm.

No significant differences were found between the two groups regarding clinicopathological factors, such as age, gender, tumor location, depth of tumor invasion, lymph node metastasis, histological grade, Dukes' stage, and vessel invasion.

Several cancer-related gene candidates were identified using microarray data from the four cell lines (HCT116, SW480, DLD1, KM12SM)

Purpose: 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.Experimental Design: 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.Results: 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).Conclusions: 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.

Described markers expression, CD133, CD166, Lgr5 (Alexa Fluor® 647 channel, BD Biosciences) and EpHB2 (PE channel, R&D systems, Minneapolis, MN, USA), were compared between LPACs and non-LPACs. The LPACs clearly showed up-regulation of several CSC markers including CD133, CD166, Lgr5 and EpHB2. Especially, Lgr5 was enriched in all cell lines (HCT116; 0.7% (non-LPACs) vs 18.1% (LPACs), SW480; 0.2% vs 28.1% and DLD1; 2.3% vs 45.2%).

EID3 was identified by microarray analysis. The expression of EID3 was confirmed by PCR and western blotting. (A) The mRNA expression of EID3 was significantly enriched in LPACs of HCT116, KM12SM and DLD1 compared to non-LPACs, except SW480 which just missed statistical significance (p=0.0555). (B) Western blotting was also performed for checking the expression of EID3 protein in LPACs and non-LPACs using EID3 antibody (Abcam, Cambridge, UK, ab124447), but the evaluation was difficult, because expression of EID3 is low in humans as per previous report (31, 32). * p

(A) The efficiency of over-expression was validated by expression of mRNA. The expression of EID3 was significantly higher in cells with EID3 　expression vector than cells with control vector. (B) The efficiency of down-regulation was confirmed by mRNA. The expression of EID3 was significantly lower in cells with double-stranded RNA duplexes targeting human EID3 than cells with negative control siRNA. * p