Background and aims

The aberrant activation of the mTOR pathway and its crosstalk with other signaling cascades represent key drivers of hepatocellular carcinoma (HCC) progression. mTOR-mediated ferroptosis suppression has been implicated in HCC resistance to chemotherapy. This study aimed to elucidate the mechanisms underlying mTOR inhibitor resistance and to evaluate the therapeutic potential of multidrug combinations in β-catenin-mutant HCC.

Methods

MHCC97H and SNU449 cells were transfected with 4EBP1WT, 4EBP1A4, or HSP90β expression plasmids and then treated with rapamycin to assess their effects on ferroptosis and rapamycin sensitivity. The role of 4EBP1 in regulating ferroptosis was further explored by Western blotting, co-immunoprecipitation, and immunofluorescence. The inhibitory effects of mTOR inhibitors (rapamycin, MLN0128), ERK inhibitors (PD901), and their combination (MLN0128 + PD901) on tumor cells were evaluated. HCC mouse models were generated via hydrodynamic tail vein injection of c-Met/β-cateninΔN90 or c-Met/β-cateninΔN90/4EBP1A4 plasmids to evaluate the therapeutic effects of the four treatment regimens.

Results

Rapamycin more potently inhibited mTOR/RPS6 than mTOR/4EBP1 and concurrently induced ferroptosis. 4EBP1A4 promoted ferroptosis and potentiated rapamycin efficacy. Mechanistically, 4EBP1A4 competitively bound HSP90β, displacing Keap1, thereby increasing Keap1–Nrf2 complex formation and promoting Nrf2 degradation. Furthermore, rapamycin, MLN0128, PD901, and their combination reduced p-4EBP1 levels, induced ferroptosis, and inhibited HCC cell proliferation, thereby suppressing tumor growth, with the combination exhibiting the strongest effect.

Conclusions

Activation of mTOR (mTORC1 and mTORC2) and ERK signaling inhibits ferroptosis by increasing 4EBP1 phosphorylation, thereby promoting β-catenin-mutant HCC progression. This study demonstrates that 4EBP1A4 and Keap1 competitively bind to HSP90β, increasing Keap1–Nrf2 complexes to accelerate Nrf2 degradation, thereby relieving mTORC1 activation-mediated ferroptosis inhibition (as illustrated in the graphical abstract). Additionally, 4EBP1 serves as a critical downstream effector common to both ERK and mTOR pathways. The combination therapy of MLN0128 and PD901 not only effectively suppresses mTOR compensatory activation but also synergistically induces ferroptosis, leading to significantly improved therapeutic efficacy against β-catenin-mutant HCC. This study provides novel insights for advancing therapeutic strategies and targeted drug development in HCC.

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