Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China.
Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China.
Acta Pharmacol Sin. 2021 Mar;42(3):460-469. doi: 10.1038/s41401-020-0460-0. Epub 2020 Jul 9.
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 [Ca] and LC-ESI-MS/MS, we revealed that internalized S1PR2 triggered elevating [Ca] 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.
鞘氨醇-1-磷酸（S1P）是大多数鞘脂类的骨干，激活 S1P 受体（S1PRs）和下游 G 蛋白信号转导与化疗耐药性有关。在这项研究中，我们研究了 S1PR2 内化在人结直肠癌（CRC）中 5-氟尿嘧啶（5-FU）耐药中的作用。随机选择的 60 个 CRC 标本的临床数据显示，S1PR2 内化与细胞内尿嘧啶增加之间存在相关性（P<0.001）。然后，我们在 villin-S1PR2 小鼠和 CRC 细胞系模型中探索了调节机制。我们表明，S1P 的共同给药促进了 S1PR2 从质膜（PM）到内质网（ER）的内化，从而削弱了 WT 小鼠中 S1PR2 对结直肠肿瘤的 5-FU 疗效，而不是 S1PR2 小鼠。在 HCT116 和 HT-29 细胞中，应用 S1P（10μM）使 S1PR2 从 PM 内化到 ER，从而诱导 5-FU 耐药，而特异性 S1PR2 抑制剂 JTE-013（10μM）有效抑制了 S1P 诱导的 S1PR2 内化。使用 Mag-Fluo-AM 标记[Ca]和 LC-ESI-MS/MS，我们揭示了内化的 S1PR2 触发了[Ca]水平的升高，从而激活了 HCT116 细胞中的 PERK-eLF2α-ATF4 信号通路。激活的 ATF4 上调了 RNASET2 介导的尿嘧啶生成，从而损害了外源性 5-FU 的摄取，从而削弱了 5-FU 治疗。总的来说，这项研究揭示了一种以前未被认识的结直肠癌 5-FU 耐药机制，即 S1PR2 内化上调尿嘧啶生成，为 S1PR2 定位在预测 CRC 患者从基于 5-FU 的化疗中获益的意义提供了新的见解。
