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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 celllines.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-y-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-eLF2a-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 S1 PR2 localization in predicting the benefit of CRC patients from 5-FU-based chemotherapy.