Unravelling the biogenesis mechanisms and functional significance of tumour-derived small extracellular vesicles in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly vascular cancer whose growth and propagation are significantly influenced by tumour-derived small extracellular vesicles (sEVs) in the tumour microenvironment. The delayed diagnosis of HCC leads to limited curative treatment, thereby offering minimal survival benefit. This study comprises two parts: first, to explore the functional roles and mechanisms of tumour-derived sEV in angiogenesis, and second, to delineate the sEV biogenesis pathway with the aim to disrupt intercellular communications mediated by tumour-derived sEV through the blockade of sEV secretion. Proteomic profiling of sEVs from healthy controls and HCC patients revealed a progressive upregulation of von Willebrand factor (vWF) across different stages of HCC. Functionally, anti-vWF antibodies effectively abrogated the pro-angiogenic and vascular permeability inducing function of late-stage patient sEVs. Moreover, vWF-enriched sEVs from vWF-overexpressing HCC cells significantly promoted angiogenesis by inducing the release of VEGFA and FGF2 from endothelial cells via the STAT3 pathway. The enhanced tumour-endothelial adhesion and vascular permeability, with FGF2 facilitating a positive feedback signalling to HCC cells, thus promoting their growth and motility through the FGF2-FGFR4-ERK pathway. Treatment with anti-vWF antibody in combination with the FGFR inhibitor Erdafitinib enhanced the efficacy of Sorafenib treatment in a patient-derived xenograft (PDX) mouse model.

Metastatic HCC cells were found to exhibit increased secretion of sEVs compared to non-metastatic and normal liver cells, and high levels of circulating sEVs were detected in HCC patients compared to healthy individuals. In light of the frequent dysregulation of kinases in HCC, we aimed to identify kinases involved in regulating sEV secretion in HCC. Screening of a kinase inhibitor library utilizing Amplified Luminescent Proximity Homogeneous Assay (ALPHA) identified MRT68921 as a potent inhibitor of Unc-51-like kinase 1 (ULK1) that inhibited secretion of sEVs by metastatic HCC cells. ULK1 knockdown decreased the levels of synaptosomal-associated protein 23 (SNAP23), a key regulator of sEV biogenesis by compromising multivesicular bodies (MVBs) and intraluminal vesicles (ILVs). Immunoblotting of immunoprecipitated SNAP23 revealed that inhibition of ULK1 resulted in reduced serine phosphorylation of SNAP23. Phospho-mutagenesis of SNAP23 at serine 5 highlighted the critical role of this site in protein stabilization of SNAP23 and modulation of sEV biogenesis. The ULK1/SNAP23 serine 5 pathway was demonstrated to increase the size of MVBs and number of ILVs. Mass spectrometry data revealed a significant alteration in the protein composition of sEVs from ULK1-knockdown cells, suggesting the involvement of ULK1 in sEV cargo sorting. Anion exchange protein 2 (AE2), downregulated in these sEVs, was shown to promote proliferation and metastasis both in vitro and in vivo, underscoring its functional significance in sEV.

This study identifies a reciprocal interaction between HCC cells and endothelial cells mediated by the sEV-vWF axis, highlighting the therapeutic potential of targeting sEV-induced cancer-endothelial communication in HCC treatment. Furthermore, our work sheds light on a novel role of ULK1, regulated through SNAP23 phosphorylation, in facilitating sEV biogenesis and cargo sorting. Collectively, the disruption of sEV-vWF-mediated tumour-endothelial intercellular communication and the blockade of sEV biogenesis via ULK1 inhibition present a promising avenue for therapeutic intervention in HCC management.