Lenvatinib-activated NDUFA4L2/IL33/PADI4 pathway induces neutrophil extracellular traps that inhibit cuproptosis in hepatocellular carcinoma

Lenvatinib is a potent first-line therapy for patients with hepatocellular carcinoma (HCC), but it also increased the number of neutrophils in HCC tumor microenvironment. CitH3, MPO-DNA, elastase and MPO activity were measured for assessing neutrophil extracellular traps (NETs) in vivo and in vitro. Cell cuproptosis was assessed by measurement of copper content, FDX1, and pyruvate. The functions of lenvatinib, DNase I, interleukin 33 (IL33) neutralizing antibody and GPX4 in tumor growth were explored in mice. Lenvatinib induced NETs in the HCC tumor microenvironment via HCC cells, but not through the direct stimulation of neutrophils. In addition, NET clearance by DNase I improves the efficacy of lenvatinib therapy in HCC mouse models. Mechanistically, lenvatinib promoted the expression and secretion of IL33 by HCC cells that triggered NET formation. Moreover, IL33 knockdown in Hepa1-6 cells improved lenvatinib efficacy in Hepa1-6-bearing HCC model mice and reduced NET formation in the tumor microenvironment. Subsequently, lenvatinib increased IL33 production by increasing the NDUFA4L2 expression in HCC cells. Furthermore, we found that IL33 triggered NET formation in neutrophils by increasing the protein expression of PADI4 via the Akt/mTOR signaling pathway. Rapamycin inhibition of mTOR reduced PADI4 expression and NET formation. Consistently, PADI4 inhibition by the selective PAD4 inhibitor GSK484 hydrochloride (GSK484) improved lenvatinib response to HCC therapy. Importantly, NETs contribute to lenvatinib resistance by inhibiting cuproptosis, but not apoptosis, pyroptosis, or ferroptosis in HCC cells. Treatment with GSK484 reversed the inhibitory effects of NETs on cuproptosis and sensitized the HCC cells to lenvatinib. Our study revealed that lenvatinib-induced NETs inhibited the cuproptosis of HCC cells, suggesting that targeting the IL33/PADI4/NET axis represents a promising therapeutic strategy for ameliorating lenvatinib resistance in HCC.