Increased S1P induces S1PR2 internalization to blunt the sensitivity of colorectal cancer to 5-fluorouracil via promoting intracellular uracil generation

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.