Abstract
Sphingosine-1-phosphate (S1P), the backbone of most sphingolipids, activating S1P receptors (S1PRs) and the downstream G protein signaling has been implicated in chemoresistance. In this study we investigated the role of S1PR2 internalization in 5-fluorouracil (5-FU) resistance in human colorectal cancer (CRC). Clinical data of randomly selected 60 CRC specimens showed the correlation between S1PR2 internalization and increased intracellular uracil (P < 0.001). Then we explored the regulatory mechanisms in CRC model of villin-S1PR2−/− mice and CRC cell lines. We showed that co-administration of S1P promoted S1PR2 internalization from plasma membrane (PM) to endoplasmic reticulum (ER), thus blunted 5-FU efficacy against colorectal tumors in WT mice, compared to that in S1PR2−/− mice. In HCT116 and HT-29 cells, application of S1P (10 μM) empowered S1PR2 to internalize from PM to ER, thus inducing 5-FU resistance, whereas the specific S1PR2 inhibitor JTE-013 (10 μM) effectively inhibited S1P-induced S1PR2 internalization. Using Mag-Fluo-AM-labeling [Ca2+]ER and LC-ESI-MS/MS, we revealed that internalized S1PR2 triggered elevating [Ca2+]ER levels to activate PERK-eLF2α-ATF4 signaling in HCT116 cells. The activated ATF4 upregulated RNASET2-mediated uracil generation, which impaired exogenous 5-FU uptake to blunt 5-FU therapy. Overall, this study reveals a previously unrecognized mechanism of 5-FU resistance resulted from S1PR2 internalization-upregulated uracil generation in colorectal cancer, and provides the novel insight into the significance of S1PR2 localization in predicting the benefit of CRC patients from 5-FU-based chemotherapy.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (91629303/81673449/81872884/81973350) and the Beijing Natural Science Foundation and Scientific Research Program of Municipal Commission of Education (KZ201710025020/KZ201810025033). In addition, YHZ wants to thank, in particular, the inimitable support and care from Wen-yu Wang. Hope to spend the rest of my life with you.
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XJQ conceived the project. YHZ performed the experiments of molecular mechanisms and conducted animal studies. SBW implemented experiments of pharmaceutical analysis. SHW provided clinical samples and pathological analysis. SXC performed statistical analysis. YHZ wrote the manuscript, which was edited by all authors.
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Zhang, Yh., Cui, Sx., Wan, Sb. et al. Increased S1P induces S1PR2 internalization to blunt the sensitivity of colorectal cancer to 5-fluorouracil via promoting intracellular uracil generation. Acta Pharmacol Sin 42, 460–469 (2021). https://doi.org/10.1038/s41401-020-0460-0
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DOI: https://doi.org/10.1038/s41401-020-0460-0
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