RAB20 downregulation mediates the release of extracellular vesicles that stimulate hepatocellular carcinoma

Abstract

Accompanied with complicated genetic and histological alterations, hepatocellular carcinoma (HCC) remains as the third cancer killer worldwide, with limited effective therapeutic options available. Extracellular vesicles (EVs) have gained increasing attention on their emerging role on carcinogenesis, particularly in cancer metastasis. As critical cell-cell communicators, EVs allow the transfer of biological materials, which lead to phenotype transformation and signalling pathway change in the recipient cells. Accumulating evidences comprehensively described the importance of EVs in facilitating a wide spectrum of cancer traits. As the major regulators of intracellular membrane traffic, Rab GTPases take the key roles in controlling EV biogenesis. Understanding the interplay of Rab GTPases and EVs in HCC has become the objective of this study.

By RNA sequencing, Rab20 was found to be significantly downregulated among the detected Rab proteins in the tumorous sample isolated from a late-HCC patient, when compared with corresponding non-tumorous liver tissue. Former studies delineated the role of Rab20 in endosomal and immune regulation, but questions remain on its role in cancer. Reduced Rab20 expression was consistently detected in the in-house HCC clinical cohort, TCGA and GSE databases. Moreover, its expression was only detected in the immortalized hepatocyte cell line MIHA. Functionally, studies on Rab20 overexpression in metastatic MHCC97L cells and knockdown in MIHA collectively indicated Rab20 posed an inhibitory effect on HCC invasiveness, anchorage-independent growth and in vivo tumor growth. Analyses on the conditioned medium uncovered the inhibitory effect induced by Rab20 was conferred by the biological activity of EVs. In comparison with PBS-treated cells, EVs derived from MHCC97L cells enhanced migratory potential, invasiveness and proliferation in non- metastatic HCC recipient cells, as well as in vivo colonization of genetically modified murine p53-/-;myc transduced hepatoblasts. However, such promotion was abolished by EVs from MHCC97L Rab20-overexpressing cells. Conversely, the promoting effect was detected in motility and proliferation of HCC cells educated with EVs of MIHA Rab20-knockdown cells, when compared to EVs of non-target control cells. Taken altogether, these findings illustrated Rab20 expression coordinated the functions of EVs.

To determine how Rab20 regulates EV functions, mass spectrometry was employed and identified exosomal triosephosphate isomerase 1 (TPI1) level was positively correlated with the cellular Rab20 expression in HCC cells. TPI1 catalyses the isomerization of glyceraldehyde-3-phosphate and dihydroxy-acetone phosphate in glycolysis and gluconeogenesis. Through overexpressing TPI1 in EVs of Rab20-knockdown cells, it was found that activity of EVs derived was dependent on exosomal TPI1 expression, indicating the functional correlation between Rab20 and exosomal TPI1. Independently, exosomal TPI1 demonstrated a suppressing role in hepatocarcinogenesis. Overexpressing exosomal TPI1 in MHCC97L cells diminished the invasiveness and growth of recipient HCC cells, and in vivo colonization of p53-/-;myc hepatoblasts in lung. In contrast to TPI1-overexpressing EVs, TPI1- reduced EVs derived from PLC/PRF/5 TPI1 knockdown cells posed a reversed effect on the recipient cells.

Despite how the interaction between Rab20 and exosomal TPI1 regulates HCC remains obscure, this study reported a previously unknown role of Rab20 in HCC, and its interplay with exosomal TPI in governing in vitro motility and proliferation of HCC cells.