Arf6-driven endocytic recycling of CD147 determines HCC malignant phenotypes

Shanshan Qi,Qing Zhang,Linjia Su,Chuanshan Zhang,Sihe Zhang,Guhe Jia,Jing Li,Zhe Ma,Yongjun Piao,Guiqiu Liu

doi:10.1186/s13046-019-1464-9

Abstract

BackgroundAdhesion molecules distributed on the cell-surface depends upon their dynamic trafficking that plays an important role during cancer progression. ADP-ribosylation factor 6 (Arf6) is a master regulator of membrane trafficking. CD147, a tumor-related adhesive protein, can promote the invasion of liver cancer. However, the role of Arf6 in CD147 trafficking and its contribution to liver cancer progression remain unclear.MethodsStable liver cancer cell lines with Arf6 silencing and over-expression were established. Confocal imaging, flow cytometry, biotinylation and endomembrane isolation were used to detect CD147 uptake and recycling. GST-pull down, gelatin zymography, immunofluorescence, cell adhesion, aggregation and tight junction formation, Transwell migration, and invasion assays were used to examine the cellular phenotypes. GEPIA bioinformatics, patient’s specimens and electronic records collection, and immunohistochemistry were performed to obtain the clinical relevance for Arf6-CD147 signaling.ResultsWe found that the endocytic recycling of CD147 in liver cancer cells was controlled by Arf6 through concurrent Rab5 and Rab22 activation. Disruption of Arf6-mediated CD147 trafficking reduced the cell-matrix and cell-cell adhesion, weakened cell aggregation and junction stability, attenuated MMPs secretion and cytoskeleton reorganization, impaired HGF-stimulated Rac1 activation, and markedly decreased the migration and invasion of liver cancer cells. Moreover, high-expression of the Arf6-CD147 signaling components in HCC (hepatocellular carcinoma) was closely correlated with poor clinical outcome of patients.ConclusionsOur results revealed that Arf6-mediated CD147 endocytic recycling is required for the malignant phenotypes of liver cancer. The Arf6-driven signaling machinery provides excellent biomarkers or therapeutic targets for the prevention of liver cancer.

Highlights

Adhesion molecules distributed on the cell-surface depends upon their dynamic trafficking that plays an important role during cancer progression
ADP-ribosylation factor 6 (Arf6) up-regulates the endocytic recycling of CD147 by activating Rab5 and Rab22 We made three kinds of shRNA-lentivirus for human Arf6 gene silencing, and tested their silencing efficiency in commonly used liver cancer cell lines
As Arf6(Q67L) over-expression excessively reversed CD147 uptake, we explored this phenomenon by checking the endomembrane-associated CD147 level

Summary

Introduction

Adhesion molecules distributed on the cell-surface depends upon their dynamic trafficking that plays an important role during cancer progression. CD147, a tumor-related adhesive protein, can promote the invasion of liver cancer. The role of Arf in CD147 trafficking and its contribution to liver cancer progression remain unclear. Membrane proteins containing the clathrinadapter-bound sequence are internalized via clathrinmediated endocytosis (CME). When they lack specific sorting sequence they enter the cell through clathrinindependent endocytosis (CIE). Two types of CIE cargo proteins join different trafficking itineraries within the CD147 (Basigin/EMMPRIN) is an adhesion molecule overexpressed in multiple tumors [3, 4]. Depending on the glycosylation level, CD147 exists in two forms: highglycosylated CD147 (HG-CD147) and low-glycosylated.

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