Everolimus inhibits malignant phenotype of erdafitinib-resistant bladder cancer cells through the p70S6K/PI3K/MAPK signaling pathway

doi:10.12122/j.issn.1673-4254.2026.08.14

Abstract

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

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.

Figures/Tables 6

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).

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).

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).

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).

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).

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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