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FIBROBLAST GROWTH FACTOR RECEPTOR-4 PROMOTES ANTIOXIDANT RESPONSE VIA ACTIVATION OF NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR-2 IN GASTRIC CANCER
Date
May 21, 2024
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Background: Helicobacter pylori infection is the main risk factor for gastric cancer. Fibroblast growth factor receptor 4 is a member of transmembrane tyrosine kinase receptors that are activated in cancer. Nuclear Factor Erythroid 2-Related Factor 2, a transcription factor that plays a critical role in the antioxidant response and has a cytoprotective role against oxidative and cellular stress. In this study, we aimed to identify whether FGFR4 regulates the antioxidant response in gastric cancer.
Methods: We used public databases sets, mouse and human gastric tissues, and gastric cancer cell lines. In vitro and in vivo H. pylori infections were performed. Western blot, RT-qPCR, Immunofluorescence, flow cytometry, Immunohistochemistry, immunoprecipitation, proximity ligation assay and luciferase reporter were utilized.
Results: Public datasets analysis demonstrated that FGFR4high expression is associated with oxidative stress signature and poor survival. Gene set enrichment analysis demonstrated enrichment of NRF2 signature genes in FGFR4high tissue samples. Using in vitro cell models, we detected an increase in reactive oxygen species, following H. pylori infection. Knocking-down FGFR4 significantly reduced NRF2 protein and transcription activity levels, with subsequent increases in ROS and DNA damage levels. Immunofluorescence analysis performed in the in-vivo, ex-vivo and in-vitro models confirmed the increased nuclear NRF2 accumulation, reversed by the knockdown of FGFR4. Using H. pylori-infected mice, we confirmed induction of FGFR4 and NRF2 levels. Dysplastic and neoplastic gastric lesions from the TFF1-KO mouse model showed high levels of FGFR4, NRF2. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, decreased NRF2 with a remarkable reduction in the size and numbers of gastric cancer spheroids. Mechanistically, FGFR4 levels were associated with elevated P62 protein expression. Using immunoprecipitation and proximity ligation assay, we detected binding between FGFR4 and P62 proteins that competed with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. Immunohistochemistry analysis on tissue arrays demonstrated an increase of FGFR4 immunostaining in neoplastic tissues as compared to adjacent normal samples; this increase correlated with an increase in the levels of NRF2 and P62.
Conclusion: Our findings suggest that the activation of NRF2 antioxidant response prevents accumulation of lethal high levels of ROS in response to H. pylori infection. The interaction between FGFR4 and P62 interfered with NRF2 binding to KEAP1, allowing it to escape KEAP1-dependent degradation mechanisms, leading to NRF2 accumulation and activation. The FGFR4-NRF2 signaling network may serve as a molecular vulnerability that can be targeted by FGFR4 inhibitors.
Methods: We used public databases sets, mouse and human gastric tissues, and gastric cancer cell lines. In vitro and in vivo H. pylori infections were performed. Western blot, RT-qPCR, Immunofluorescence, flow cytometry, Immunohistochemistry, immunoprecipitation, proximity ligation assay and luciferase reporter were utilized.
Results: Public datasets analysis demonstrated that FGFR4high expression is associated with oxidative stress signature and poor survival. Gene set enrichment analysis demonstrated enrichment of NRF2 signature genes in FGFR4high tissue samples. Using in vitro cell models, we detected an increase in reactive oxygen species, following H. pylori infection. Knocking-down FGFR4 significantly reduced NRF2 protein and transcription activity levels, with subsequent increases in ROS and DNA damage levels. Immunofluorescence analysis performed in the in-vivo, ex-vivo and in-vitro models confirmed the increased nuclear NRF2 accumulation, reversed by the knockdown of FGFR4. Using H. pylori-infected mice, we confirmed induction of FGFR4 and NRF2 levels. Dysplastic and neoplastic gastric lesions from the TFF1-KO mouse model showed high levels of FGFR4, NRF2. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, decreased NRF2 with a remarkable reduction in the size and numbers of gastric cancer spheroids. Mechanistically, FGFR4 levels were associated with elevated P62 protein expression. Using immunoprecipitation and proximity ligation assay, we detected binding between FGFR4 and P62 proteins that competed with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. Immunohistochemistry analysis on tissue arrays demonstrated an increase of FGFR4 immunostaining in neoplastic tissues as compared to adjacent normal samples; this increase correlated with an increase in the levels of NRF2 and P62.
Conclusion: Our findings suggest that the activation of NRF2 antioxidant response prevents accumulation of lethal high levels of ROS in response to H. pylori infection. The interaction between FGFR4 and P62 interfered with NRF2 binding to KEAP1, allowing it to escape KEAP1-dependent degradation mechanisms, leading to NRF2 accumulation and activation. The FGFR4-NRF2 signaling network may serve as a molecular vulnerability that can be targeted by FGFR4 inhibitors.
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