The functional role of tumor-derived small extracellular vesicles and particles in promoting hepatocellular carcinoma carcinogenesis and reshaping the surrounding tumor microenvironment

Abstract

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, which has a high mortality rate but only limited treatment options. With an expanding understanding of the heterogenous nature of cancer biology, the active role of the surrounding tumor microenvironment (TME) in substantiating tumor progression has come to the spotlight. The TME is comprised of multiple players, ranging from immune cells, stromal cells and non-cellular components; together, they interact dynamically to create a favorable carcinogenic niche. Therapies that target components of the TME, ranging from anti-angiogenetic monoclonal antibodies to immune checkpoint inhibitors (ICIs), have recently proven clinical success. Therefore, by studying components of the TME, we explored new approaches and druggable targets to enhance treatment efficacy against liver cancer. Small extracellular vesicles (sEV) are lipid membrane-enclosed nanoparticles that are secreted by almost all cell types, including cancer cells. In fact, tumor-derived sEV was shown to support tumor progression by delivering different oncogenic biomolecules to prime both their local TME and distant pre-metastatic sites. Given the contributing role of tumor-derived sEV in cancer development, we studied their functions and mechanisms in HCC. Using mass spectrometry (MS) proteomic analysis, we identified that the proteins upregulated in metastatic HCC cell-derived sEV were significantly related to the glycolytic pathway, and among all, nicotinamide phosphoribosyltransferaese (NAMPT) was top-ranked. NAMPT is an enzyme involved in the adenine dinucleotide (NAD+) salvage pathway, and we demonstrated that sEV-NAMPT promoted HCC oncogenesis and glycolysis. The detailed mechanism of sEV-NAMPT in mediating HCC glycolysis has not been elucidated. We revealed that the proteomic and transcriptional levels of solute carrier family 27 member 4 (SLC27A4) were elevated upon sEV-NAMPT treatment. We further demonstrated that SLC27A4 expression was induced by NFB transcription factor, whose activation was dependent on Toll-like receptor 4 (TLR4) expression. The lipidomic and metabolomic analysis then unveiled SLC27A4 to be positively correlated with triglyceride (TG) and dihydroxyacetone phosphate (DHAP) levels. Elevated TG deposition favoured a metabolic shift from Glycerol-3-Phosphate (G3P) to DHAP, which promoted the transition from lipid metabolism to glycolysis. Therapeutically, we explored the potential of targeting sEV-NAMPT using inhibitor FK866. We showed that FK866 successfully inhibited sEV-NAMPT oncogenic and glycolytic promotion using various HCC animal models.

Due to technological restrictions, sEV was often collected as a heterogenous population. With better isolation techniques, a new subtype of extracellular vesicles and particles (EVP), termed exomeres (EM), was discovered. Hence, we continued our study on EM by profiling the proteomic composition of a number of normal and cancer cell lines. Here, we ascertained that sEV and EM contained distinctive biophysical characteristics. We further identified and validated 2 proteins, galactosamine (N-acetyl)-6-sulfatase (GALNS) and mannosidase alpha class 2B member 1 (MAN2B1), as potential EM markers. Finally, we explored the carcinogenic role of HCC-derived EM and demonstrated their ability to activate the PI3K/AKT/mTOR pathway.

In conclusion, our study uncovered the oncogenic role of HCC-derived EVP in oncogenesis. It also provided new insights about the diverse nature of EVP. Targeting these HCC-derived EVP brings new therapeutic opportunities for liver cancer patients.