依维莫司通过p70S6K/PI3K/MAPK信号通路抑制厄达替尼耐药的膀胱癌细胞生物学行为

doi:10.12122/j.issn.1673-4254.2026.08.14

摘要/Abstract

摘要：

目的探讨依维莫司通过调控p70S6K/PI3K/MAPK信号通路对膀胱癌耐药细胞RT-112-RS生物学行为的影响。方法采用梯度法筛选厄达替尼浓度，选取5 μmol/L作为终浓度并持续诱导16周以建立厄达替尼耐药的RT-112-RS细胞模型。设对照组（RT-112-RS常规培养）、PDGF组（+PDGF-BB）、PDGF+0.01 μmol/L依维莫司组、PDGF+0.1 μmol/L依维莫司组及PDGF+1 μmol/L依维莫司组。CCK-8法测定细胞毒性并计算IC50；划痕和Transwell实验评估迁移与侵袭能力；RT-qPCR检测0.001~1 μmol/L依维莫司处理下相关基因表达；流式细胞术分析凋亡率；Western blotting检测mTORC1/p70S6K、PI3K/Akt及Raf1/MEK/ERK通路关键蛋白磷酸化水平。结果依维莫司在0.001~0.1 μmol/L范围内呈剂量依赖性抑制RT-112-RS增殖（P<0.05）。相比RT-112细胞，RT-112-RS迁移与侵袭能力增强（819.83±71.39 vs 1579±136.30，P<0.05）。1 μmol/L依维莫司显著抑制p70S6K磷酸化（P<0.05），但未进一步增强抗增殖效应，并特异性激活Raf1/MEK/ERK通路，对PI3K/Akt（Thr308）或mTORC2/Akt（Ser473）无明显影响。MEK抑制剂U-0126可逆转依维莫司诱导的ERK1/2活化（5.52±0.40 vs 0.74±0.07，P<0.05），PI3K抑制剂LY294002则减弱ERK1/2激活（7.58±1.54 vs 11.2±0.24，P<0.05）。结论依维莫司通过抑制p70S6K活性抑制RT-112-RS增殖。而高浓度时可能因减弱负反馈抑制而激活PI3K-Raf1/MEK/ERK通路，从而部分抵消其抗增殖作用。

关键词: 膀胱癌, 依维莫司, mTOR信号通路, ERK信号通路

Abstract:

Objective To investigate the regulatory effect of everolimus on biological behaviors of rdafitinib-resistant bladder cancer cells and the underlying mechanism. Methods An erdafitinib-resistant RT-112-RS cell model was established by continuous induction of RT-112 cells with erdafitinib at the optimized concentration (5 μmol/L) for 16 weeks. The resistant cells were treated with PDGF-BB alone or in combination with 0.01, 0.1, or 1 μmol/L everolimus, and the cytotoxicity (IC₅₀) of everolimus was determined with CCK-8 assay. The changes in cell migration and invasion were evaluated using scratch wound healing assay and Transwell assays. The expressions of the genes of interest in the cells treated with everolimus (0.001- 1 μmol/L) were detected using RT-qPCR, and cell apoptosis was analyzed with flow cytometry. Western blotting was performed to assess phosphorylation levels of the key proteins in the mTORC1/p70S6K, PI3K/Akt, and Raf1/MEK/ERK pathways. Results Everolimus within the concentration range of 0.001-0.1 μmol/L dose-dependently inhibited RT-112-RS cell proliferation. RT-112-RS cells showed significantly enhanced migration and invasion compared with their parental RT-112 cells. Treatment with 1 μmol/L everolimus significantly suppressed p70S6K phosphorylation but did not result in an enhanced inhibition of cell proliferation effect. Everolimus at 1 μmol/L significantly activated the Raf1/MEK/ERK pathway without affecting PI3K/Akt (Thr308) or mTORC2/Akt (Ser473) phosphorylation. Treatment of the cells with the MEK inhibitor U-0126 obviously reversed everolimus-induced ERK1/2 activation, while the PI3K inhibitor LY294002 only partially reduced ERK1/2 activation in everolimus-treated cells. Conclusion Everolimus inhibits RT-112-RS cell proliferation by suppressing p70S6K activity, and at high concentrations, everolimus can result in activation of the PI3K-Raf1/MEK/ERK pathway possibly by relieving negative feedback inhibition to partially offset its inhibitory effect on cell proliferation.

Key words: bladder cancer, everolimus, mTOR signaling pathway, ERK signaling pathway

吴浩松, 蒋易, 张晓林, 汪盛. 依维莫司通过p70S6K/PI3K/MAPK信号通路抑制厄达替尼耐药的膀胱癌细胞生物学行为[J]. 南方医科大学学报, 2026, 46(8): 1870-1877.

Haosong WU, Yi JIANG, Xiaolin ZHANG, Sheng WANG. Everolimus inhibits malignant phenotype of erdafitinib-resistant bladder cancer cells through the p70S6K/PI3K/MAPK signaling pathway[J]. Journal of Southern Medical University, 2026, 46(8): 1870-1877.

图/表 6

图1 RT-112和RT-112-RS在含有厄达替尼培养基中的迁移和侵袭能力

Fig.1 Migration and invasion abilities of RT-112 and erdafitinib-resistant RT-112-RS cells in the medium containing erdafitinib. A: Migration ability of RT-112 and RT-112-RS cells determined by Transwell assay. B: Migration ability of RT-112 cells treated with erdafitinib. C: Migration ability of RT-112 and RT-112-RS cells determined by scratch assay. D: IC50 values of erdafitinib in RT-112 and RT-112-RS cells determined by CCK-8 assay. E: CCK-8 assay for assessing cell proliferation (Original magnification: ×40). *P<0.05, **P<0.01, ***P<0.001 vs RT-112; #P<0.05, ##P<0.01, ###P<0.001 vs RT-112+1 μmol/L erdafitinib (Mean±SD, n=6).

图2 依维莫司对 p-p70S6K和细胞凋亡的影响

Fig.2 Effect of PDGF-BB alone or in combination with everolimus on p-p70S6K expression and apoptosis in RT-112-RS cells. A: Everolimus significantly inhibits phosphorylation of p70S6K detected by Western blotting. B: Detection of p70S6k mRNA. C: Analysis of apoptosis of RT-112-RS cells following treatment. *P<0.05, **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01 vs PDGF-BB (Mean±SD, n=3).

图3 Raf1-MEK-ERK通路蛋白表达分析

Fig.3 Analysis of expressions of Raf1-MEK-ERK pathway proteins using Western blotting in RT-112-RS cells following treatment with PDGF-BB alone or in combination with everolimus. A: p-Akt (Ser473)/Akt expressions. B: p-Akt (Thr308)/Akt expressions. C: p-Raf1/Raf1 expressions. D: p-ERK1/2/ERK1/2 expressions. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01 vs PDGF-BB (Mean±SD, n=3).

图4 依维莫司和U-0126对 RT-112- RS中ERK和p70S6K磷酸化的影响

Fig.4 Effects of PDGF-BB, everolimus and U-0126 on phosphorylation of ERK and p70S6K in RT-112-RS cells. A: p-ERK1/2/ERK1/2. B: p-p70S6K/p70S6K. ***P<0.001 vs control; ##P<0.01, ###P<0.001 vs PDGF-BB (Mean±SD, n=3).

图5 依维莫司和LY294002对RT-112- RS中p70S6K/PI3K/ERK磷酸化的影响

Fig.5 Effects of everolimus and LY294002 on phosphorylation of p70S6K/PI3K/ERK in RT-112-RS cells. A: p-AKT (Thr308)/AKT expressions. B: p-ERK1/2/ERK1/2 expressions. C: p-p70S6K/p70S6K expressions. *P<0.05, **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01 vs PDGF-BB (Mean±SD, n=3).

图6 U-0126和LY294002对依维莫司诱导的RT-112- RS迁移与侵袭能力的影响

Fig.6 Effects of U-0126 and LY294002 on migration and invasion capabilities of RT-112-RS cells treated with everolimus ( ×40). A: RT-112-RS group. B: RT-112-RS+0. 1 μmol/L everolimus group. C: RT-112-RS+1 μmol/L everolimus group. D: RT-112-RS+0.1 μmol/L everolimus+U-0126 group. E: RT-112-RS+0.1 μmol/L everolimus+LY294002 group. **P<0.01, ***P<0.001 vs RT-112-RS; ##P<0.01 vs RT-112-RS+PDGF (Mean±SD, n=3).

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