Targeting 4EBP1/HSP90β/Nrf2 Sensitizes β-Catenin-Mutant Liver Cancer to mTOR Inhibitors Through Ferroptosis

Hepatocellular carcinoma, the most common primary cancer of the liver, may become more vulnerable to treatment through a molecular pathway that links mTOR signaling, stress-response proteins and ferroptosis, a form of cell death driven by iron-dependent lipid damage. In a study published in the Journal of Clinical and Translational Hepatology, researchers report that manipulating the 4EBP1/HSP90β/Nrf2 axis can sensitize β-catenin-mutant liver cancer to mTOR inhibitors and substantially improve the effects of combination therapy.

Hepatocellular carcinoma is frequently driven by several signaling networks operating at the same time. Among the most important are the mechanistic target of rapamycin, or mTOR, pathway and the extracellular signal-regulated kinase, or ERK, pathway. These systems help cancer cells grow, divide, produce proteins and adapt to metabolic stress. Their activity can also suppress ferroptosis, allowing malignant cells to survive the oxidative damage associated with rapid growth and anticancer treatment.

The new study focused on 4EBP1, a protein that regulates the initiation of protein synthesis downstream of mTOR. When 4EBP1 is phosphorylated, its ability to restrain translation is weakened, helping cells maintain the high level of protein production required for tumor expansion. The investigators examined whether altering 4EBP1 could change the response of β-catenin-mutant hepatocellular carcinoma cells to rapamycin and other targeted drugs.

Experiments were performed in MHCC97H and SNU449 liver cancer cells engineered to express either the normal form of 4EBP1, a modified form known as 4EBP1A4, or additional HSP90β. The cells were then exposed to rapamycin, an mTOR inhibitor, and analyzed for changes in signaling, oxidative stress and ferroptosis. The researchers used Western blotting, co-immunoprecipitation and immunofluorescence to track protein interactions and determine how 4EBP1 influences the machinery that controls cellular antioxidant defenses.

The results showed that rapamycin inhibited mTOR signaling unevenly. It more effectively reduced phosphorylation of the ribosomal protein S6, a marker of mTORC1 activity, than it reduced phosphorylation of 4EBP1. At the same time, rapamycin promoted ferroptosis, suggesting that cancer cells may respond to mTOR inhibition not only through reduced growth signaling but also through increased vulnerability to iron-dependent oxidative injury. Introducing 4EBP1A4 intensified this effect and made rapamycin more effective at suppressing tumor-cell proliferation.

The researchers identified a mechanism involving HSP90β, a molecular chaperone that helps stabilize other proteins, and Keap1, a regulator of the antioxidant transcription factor Nrf2. Under normal conditions, Keap1 binds Nrf2 and directs it toward ubiquitination and degradation. When Nrf2 is stabilized, it activates genes that protect cells from oxidative stress, including genes involved in glutathione production and detoxification. This antioxidant program can make cancer cells more resistant to ferroptosis.

According to the study, 4EBP1A4 competes with Keap1 for binding to HSP90β. This interaction displaces Keap1 from HSP90β and favors the formation of Keap1–Nrf2 complexes. As a result, Nrf2 undergoes increased ubiquitination and degradation, weakening the cancer cell’s antioxidant defenses. With less Nrf2 available to control protective genes, lipid peroxides accumulate in the cell membranes, pushing tumor cells toward ferroptotic death. The findings place 4EBP1 at the center of a signaling connection between protein synthesis, molecular chaperones and redox control.

The study also tested a broader drug strategy designed to block compensatory signaling. In addition to rapamycin, the researchers examined MLN0128, an inhibitor capable of targeting both mTORC1 and mTORC2, and PD901, an inhibitor of the ERK pathway. Each treatment reduced phosphorylated 4EBP1, induced markers of ferroptosis and inhibited the growth of hepatocellular carcinoma cells. However, the combination of MLN0128 and PD901 produced the strongest effects in cell-based experiments, indicating that simultaneous inhibition of mTOR and ERK may prevent the signaling escape routes that often limit targeted therapies.

The combination was also evaluated in mouse models of liver cancer generated through hydrodynamic tail vein injection of plasmids carrying c-Met and a truncated, constitutively active form of β-catenin. A second model additionally expressed 4EBP1A4. In these animals, the treatment regimens were assessed for their ability to slow tumor progression. The dual MLN0128–PD901 treatment showed superior antitumor activity, while the presence of 4EBP1A4 further supported ferroptosis and improved treatment sensitivity. These findings suggest that disrupting both mTOR and ERK signaling could be particularly relevant for tumors driven by aberrant β-catenin activity.

The researchers conclude that mTORC1, mTORC2 and ERK signaling promote β-catenin-mutant hepatocellular carcinoma partly by increasing 4EBP1 phosphorylation and suppressing ferroptosis. Restoring the ferroptosis-promoting activity of 4EBP1, or pharmacologically targeting the pathways that regulate it, may therefore offer a new way to overcome resistance to mTOR inhibitors. Although the results remain preclinical and require validation in human studies, the 4EBP1/HSP90β/Nrf2 pathway provides a potential framework for developing combination treatments that attack tumor growth and antioxidant protection at the same time.

News Publication Date:
16-Jun-2026

Web References:
Journal of Clinical and Translational Hepatology: https://www.xiahepublishing.com/journal/jcth
DOI: https://doi.org/10.14218/JCTH.2026.00072

References:
Xu M, Shang R, et al. “Targeting the 4EBP1/HSP90β/Nrf2 Axis Sensitizes β-catenin-mutant Hepatocellular Carcinoma to mTOR Inhibitors via Ferroptosis Induction.” Journal of Clinical and Translational Hepatology. DOI: 10.14218/JCTH.2026.00072