Abstract 1981: Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype
Authors: Ishida Kaoru , Nishizuka Satoshi S. , Kume Kohei , Nukatsuka Mamoru , Sato Kei , Endo Fumitaka , Katagiri Hirokatsu , Kobunai Takashi , Takechi Teiji , Koeda Keisuke , Wakabayashi Go 
Published: May 2015

Abstract
Background: Post-operative adjuvant chemotherapy for gastric cancer is a treatment for suppressing the growth of invisible cancer cells, but substantial numbers of patients experience recurrence despite of this therapy. We attempted to clarify the mechanism of cancer relapse after chemotherapy using 5-FU tolerant human gastric cancer cell line, MKN45.
Methods: Through a stepwise dose escalation of 5-fluorouracil (5-FU) for one year, a 5-FU-tolerant cell subpopulation, MKN45T, was established from a human gastric cancer cell line, MKN45. Nucleotide variation was screened with a panel of 46 cancer-associated genes, and western blot analyses were performed to examine whether known proteins were involved in the acquisition of the drug-tolerant phenotype. For molecular profiling, cancer cell subpopulations emerging as colonies in the presence of anticancer drugs including 5-FU, cisplatin, and docetaxel, “reverse-phase” protein arrays (RPPAs) were produced with the drug-tolerant 480 colonies. Subcutaneous or orthotopic xenografts of MKN45 and MKN45T to immunodeficient mice were performed to examine tumorigenicity.
Results: Western blot analysis revealed that most of protein expression levels were not visibly different between MKN45 and MKN45T, except that p53 showed slight reduction in MKN45T. Analysis of 46 cancer-associated genes revealed that all gene variations were identical in both MKN45 and MKN45T, suggesting that gene mutations had a limited effect on the acquisition of the drug-tolerant phenotype. RPPA analysis revealed that colonies from MKN45T exhibited a protein level increase in a 5-FU concentration-dependent manner in Atg5, Atg7, and PI3K, suggesting a functional association between drug-tolerance and autophagy/glucose metabolism. An orthotopic xenograft to the stomach demonstrated tumors in the stomach (9 out of 9, 100%), and lymph node and liver metastases only in MKN45T at a high frequency (6 out of 9, 67%). No difference in tumorigenicity was observed when MKN45 and MKN45T were transplanted subcutaneously. The orthotopic xenograft of MKN45T followed by administration of 5-FU (30mg/kg/day) for five post-xenograft days suppressed metastasis in 4 out of 5 mice (80%) at 42 post-xenograft days, indicating that chemotherapy is particularly effective if it is performed before initiating tumor formation.
Conclusion: Chemotherapy does not only reduce tumor size, but may also be selecting for tumors that are resistant to the chemotherapy. Our clinical experiences are well-explained by this observation, as relapse after chemotherapy is often chemotherapy resistant.
Citation Format: Kaoru Ishida, Satoshi S. Nishizuka, Kohei Kume, Mamoru Nukatsuka, Kei Sato, Fumitaka Endo, Hirokatsu Katagiri, Takashi Kobunai, Teiji Takechi, Keisuke Koeda, Go Wakabayashi. Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1981. doi:10.1158/1538-7445.AM2014-1981