Flavokawain A Targets CXCR4 ‐Mediated Vasculogenic Mimicry to Reverse Hepatocellular Carcinoma Resistance to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors (TKIs), such as sorafenib, lenvatinib, and regorafenib, serve as the mainstay targeted therapies for advanced hepatocellular carcinoma (HCC), yet resistance severely limits their clinical benefits. This study aimed to elucidate the role of CXCR4 in promoting vasculogenic mimicry (VM) to drive TKI resistance and to explore the natural chalcone flavokawain A (FKA) as a novel CXCR4-targeting agent to overcome this challenge. Utilizing comprehensive experimental approaches-including transcriptome sequencing, MTT, transwell and 3D-tubule formation assays, western blotting, molecular docking, cellular thermal shift assay (CETSA), and a subcutaneous xenograft model-we confirmed that sorafenib (0-8 μM) elevated CXCR4, consistent with our prior reports, and first revealed a similar upregulation occurred following exposure to lenvatinib (0-10 μM) and regorafenib (0-2 μM), with CXCR4 further enriched in TKI-resistant cells. CXCR4 overexpression promoted VM formation and reduced TKI sensitivity, while its knockdown suppressed VM and restored TKI responsiveness. Mechanistically, CXCR4 facilitated VM through the Snail/E-cadherin/Vimentin and VE-cadherin/EphA2/MMP-14/MMP-2/Laminin5γ2 pathways. TKI-resistant cells with upregulated CXCR4 exhibited a pronounced VM phenotype, including enhanced proliferation, migration, invasion, and VM. CXCR4 depletion attenuated these VM hallmarks and resensitized resistant cells to TKIs. FKA disrupted the VM phenotype by directly targeting CXCR4, and synergized with TKI to inhibit HCC growth in vitro and in vivo, with its efficacy being CXCR4-dependent. Collectively, our findings suggest that CXCR4 promotes VM to drive TKI resistance and reveal that FKA, as a potential CXCR4 inhibitor, represents a promising therapeutic strategy in combination with TKIs to overcome resistance in HCC.