Molecular biological analysis of 5-FU-resistant gastric cancer organoids; KHDRBS3 contributes to the attainment of features of cancer stem cell

Abstract

5-FU is one of the key drugs in the treatment of gastric cancer (GC). Much evidence has shown that cancer stem cells (CSCs) play a key role in the acquisition of drug resistance. The organoid is a novel 3D cell culture system technology that sustains stem-cell-driven formation of near-physiological, self-renewing tissues using specific niche factors in a dish. In this study, we established GC organoids (GCOs) and gradually treated them with higher concentrations of 5-FU. We successfully harvested four 5-FU-resistant GCOs, which were supported by significant changes in the expression of molecules related to 5-FU metabolism. We then performed microarray analysis using three normal gastric organoids and three pairs of 5-FU-resistant and parental GCOs. Through the comparison of expression profiles and further validation, we chose KHDRBS3 as a target gene. We found KHDRBS3 to be an independent prognostic factor in GC patients, especially in GC patients treated with 5-FU chemotherapy. We also determined that KHDRBS3 might play an important role in the acquisition of stem cell-like features, such as multi-drug resistance and organoid formation, by regulating CD44 variant expression. We found KHDRBS3, which is thought to play an important role in the acquisition of characteristics of CSCs in GC, to be a promising candidate marker for predicting therapeutic effect and prognosis in GC patients.

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Fig. 1: Drug sensitivity screening in 10 GC organoids and establishment of 5-FU-resistant GCOs.
Fig. 2: Microarray analysis of 5-FU-resistant GCOs.
Fig. 3: Immunohistochemistry and Kaplan–Meier curves for KHDRBS3 in human gastric tissues.
Fig. 4: Targeted knockout of KHDRBS3 in GCO2/5-FU and GCO4/5-FU.
Fig. 5: Overexpression of KHDRBS3 in GCO2 and GCO4.

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Acknowledgements

We thank Mr. Shinichi Norimura (Technical Center, Hiroshima University) for his excellent technical assistance. This research was carried out with the kind cooperation of the Research Center for Molecular Medicine of the Faculty of Medicine of Hiroshima University. We also thank the Analysis Center of Life Science of Hiroshima University for the use of their facilities. The R-spondin-producing cell line was a kind gift from Professor Jeffery Whitsett (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA). We would like to thank Professor Eric Fearon (University of Michigan, Ann Arbor, MI, USA) for providing collaborative research resources and comments.

Funding

The present study was supported by Grants in Aid for Scientific Research (JP15H04713, JP16K08691, JP16H06999) and for Challenging Exploratory Research (26670175, JP16K15247) from the Japan Society for the Promotion of Science.

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Correspondence to Wataru Yasui.

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Ukai, S., Honma, R., Sakamoto, N. et al. Molecular biological analysis of 5-FU-resistant gastric cancer organoids; KHDRBS3 contributes to the attainment of features of cancer stem cell. Oncogene (2020). https://doi.org/10.1038/s41388-020-01492-9

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