This study established an erdafitinib-resistant bladder cancer cell model (RT-112-RS) by continuous exposure of parental RT-112 cells to 5 μmol/L erdafitinib for 16 weeks. The investigators evaluated the regulatory effect of everolimus across a concentration range (0.001–1 μmol/L) on the biological behavior of these resistant cells.
Cell viability was assessed using the CCK-8 assay to determine cytotoxicity and calculate the IC50 for everolimus. Within the 0.001–0.1 μmol/L range, everolimus produced a dose-dependent inhibition of RT-112-RS proliferation (statistical significance reported as P < 0.05). The abstract indicates an IC50 was computed but does not provide the numeric IC50 value.
RT-112-RS cells demonstrated significantly enhanced migration and invasion compared with parental RT-112 cells. These changes were quantified by scratch wound healing assay and Transwell invasion assays; example values cited in the abstract indicate a marked increase in migratory/invasive capacity (reported means with standard deviations in the abstract), with P < 0.05 for the difference versus parental cells.
The resistant cell line was generated by gradient induction, using 5 μmol/L erdafitinib as the terminal concentration and continuous induction over 16 weeks. Experimental groups included control RT-112-RS, PDGF-BB stimulation, and PDGF-BB combined with everolimus at 0.01, 0.1, or 1 μmol/L. Assays performed were:
Everolimus inhibited RT-112-RS proliferation in a dose-dependent manner at concentrations up to 0.1 μmol/L. Although 1 μmol/L everolimus significantly suppressed phosphorylation of p70S6K, that higher concentration did not yield a correspondingly greater antiproliferative effect relative to lower concentrations. The abstract reports statistical significance for the inhibitory trends (P < 0.05) but does not list the absolute IC50 value or full concentration–response curve data in the abstract.
The study measured apoptosis by flow cytometry and monitored changes in gene expression by RT-qPCR across everolimus concentrations from 0.001 to 1 μmol/L. The abstract does not provide detailed numeric apoptosis rates or specific gene expression fold changes; those data are not reported in the abstract and would require consulting the full article.
Western blot analysis showed that 1 μmol/L everolimus produced a significant reduction in p70S6K phosphorylation (P < 0.05). In contrast, phosphorylation of PI3K/Akt at Thr308 and mTORC2/Akt at Ser473 was not notably affected by everolimus treatment in this model, according to the abstract.
Crucially, high-dose everolimus (1 μmol/L) selectively activated the Raf1/MEK/ERK arm of the MAPK pathway (increased ERK1/2 phosphorylation). This activation occurred despite suppression of p70S6K and without detectable changes in the tested Akt phosphorylation sites, suggesting a context-dependent feedback response.
To dissect the mechanism of ERK activation, the authors applied pathway inhibitors. The MEK inhibitor U-0126 reversed the everolimus-induced ERK1/2 activation (reported effect sizes with statistical significance). The PI3K inhibitor LY294002 partially reduced ERK1/2 activation in everolimus-treated cells but did not fully reverse it. These pharmacologic data implicate a PI3K–Raf1/MEK/ERK feedback loop: everolimus-mediated suppression of mTORC1/p70S6K may relieve negative feedback restraints and allow activation of Ras/Raf1–MEK–ERK signaling, with a partial contribution from PI3K activity.
From the experiments summarized in the abstract, the principal conclusions are:
Everolimus inhibits proliferation of the erdafitinib-resistant RT-112-RS bladder cancer cells primarily via suppression of p70S6K activity within the mTORC1 pathway.
At higher concentrations (1 μmol/L), everolimus can activate the Raf1/MEK/ERK (MAPK) pathway, likely by relieving negative feedback inhibition, and this activation may partially offset the drug’s antiproliferative effects.
Pharmacologic blockade with MEK inhibitor U-0126 reverses ERK activation induced by everolimus, while PI3K inhibition with LY294002 only partially attenuates it, supporting a model in which PI3K contributes but is not solely responsible for the feedback-driven ERK activation.
Clinical and experimental notes
All data presented here are derived from in vitro experiments in a single erdafitinib-resistant bladder cancer cell line (RT-112-RS). The abstract reports statistical significance for key findings but omits some numeric details (for example, the exact IC50 value, full RT-qPCR results, and detailed apoptosis metrics). The full article (Chinese) and supplemental data would be required to extract complete quantitative results and experimental replicates. The findings point to pathway cross-talk between mTORC1/p70S6K, PI3K, and MAPK signaling as a determinant of everolimus response in this resistant model, and they suggest that combined pathway modulation could be explored to enhance antiproliferative effects in erdafitinib-resistant bladder cancer cells.