RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells (HUVECs) are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the EGFR signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of METTL3, the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3'UTR accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, TSP-1, an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

EGF-induced nuclear translocation of SHCBP1 promotes bladder cancer progression through inhibiting RACGAP1-mediated RAC1 inactivation

Bladder cancer is a highly heterogeneous and aggressive malignancy with a poor prognosis. EGF/EGFR activation causes the detachment of SHC-binding protein 1 (SHCBP1) from SHC adapter protein 1 (SHC1), which subsequently translocates into the nucleus and promotes cancer development via multiple signaling pathways. However, the role of the EGF-SHCBP1 axis in bladder cancer progression remains unexplored. Herein, we report that SHCBP1 is upregulated in bladder cancer tissues and cells, with cytoplasmic or nuclear localization. Released SHCBP1 responds to EGF stimulation by translocating into the nucleus following Ser273 phosphorylation. Depletion of SHCBP1 reduces EGF-induced cell migration and invasiveness of bladder cancer cells. Mechanistically, SHCBP1 binds to RACGAP1 via its N-terminal domain of amino acids 1 ~ 428, and this interaction is enhanced following EGF treatment. Furthermore, SHCBP1 facilitates cell migration by inhibiting RACGAP-mediated GTP-RAC1 inactivation, whose activity is indispensable for cell movement. Collectively, we demonstrate that the EGF-SHCBP1-RACGAP1-RAC1 axis acts as a novel regulatory mechanism of bladder cancer progression, which offers a new clinical therapeutic strategy to combat bladder cancer.

Mutual Regulation between PFKP and VEGF Promotes Glioblastoma Tumor Growth

Glioblastoma (GBM) is highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, it remains unknown whether PFKP and VEGF are reciprocally regulated during GBM tumor growth. Here, we show that PFKP promotes EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in the AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated β-catenin S552 phosphorylation, to fully enhance VEGF transcription and subsequent blood vessel formation and brain tumor growth. The levels of PFKP Y64 phosphorylation in human GBM specimens positively correlate with HIF-1α expression, β-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth.

EGFR inhibition reverses resistance to lenvatinib in hepatocellular carcinoma cells

Background Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Lenvatinib is approved as a first-line treatment for unresectable HCC. The therapeutic duration of lenvatinib is limited by resistance, but the underlying mechanism is unclear. Methods To establish lenvatinib-resistant cells, Hep3B cells were initially treated with 3 µM lenvatinib. The concentration was gradually increased by 1µM or 0.5µM per week and it reached to 7.5µM 2 months after the initial exposure to lenvatinib. The biological characteristics of these cells were analyzed by ERK activation in the MAPK signaling pathway and a human phospho‐receptor tyrosine kinase (RTK) antibody array. Factors possibly related to lenvatinib resistance were analyzed using inhibitors, and cell proliferation was analyzed. Results We established lenvatinib-resistant HCC cells (LR cells) by long-term exposure to lenvatinib. Lenvatinib reduced ERK activation in the parent cells, but not in the LR cells. RTK array analysis showed that the activities of EGFR and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) were significantly increased in LR cells, whereas the activities of other RTKs were unchanged. Erlotinib, a widely used EGFR inhibitor, downregulated ERK activation in LR cells. The proliferation of LR cells will also be affected when lenvatinib is combined with erlotinib to treat LR cells. In contrast, inhibition of IGFR/INSR did not affect ERK activation or cell proliferation. Scavenging of reactive oxygen species (ROS) ameliorated the enhanced EGFR activation in LR cells. Conclusions Lenvatinib resistance was induced by enhanced EGFR activation, possibly via ROS accumulation, in lenvatinib-resistant cells.These findings may enable the development of lenvatinib combination therapies for HCC.