Characterisation of ADP-ribosylation factor 5 (ARF5) as a mediator in promoting breast cancer growth

Abstract

ADP ribosylation factors (ARFs) belong to Ras-related GTP-binding proteins family. Studies have shown that ARF1, ARF3, ARF4 and ARF6 were abnormally expressed in various cancers including breast cancer, and their aberrant expressions were associated with cancer cell migration, invasion and proliferation. However, the roles of ARF5 in breast cancer remain unclear. In the present study, we aim to investigate the functions and underlying mechanisms of ARF5 in cancer cell growth and invasion, and its clinical association with breast cancer. Evidence from public database (oncomine.org) and our data showed that ARF5 expression was significantly upregulated in breast cancer tissues and cell lines. Inhibition of ARF5 by both siRNA and brefeldin A (BFA) reduced cell proliferation, colony formation, tumorsphere formation and invasion abilities. In addition, inhibition of ARF5 stimulated apoptosis and autophagy via reduced BCL- 2 and increased LC3-II conversion respectively. Moreover, intraperitoneal injection of BFA to mice tumour MDA-MB-231 xenografts greatly inhibited tumour growth in vivo. Overexpression of ARF5 promoted MDA-MB-231 cell growth in vitro and partially reversed the inhibitory effect of BFA on tumour growth in vivo. Previous studies indicated that the biological functions of ARFs were associated with ER-to-Golgi protein transport. In the present study, ARF5 expression was co-localised in Golgi apparatus. Inhibition of ARF5 disturbed the functions of Golgi apparatus and endoplasmic reticulum (ER) through the suppression of GM-130 and ER stress proteins (calnexin and IRE1α) respectively. Due to its close relationship with Golgi apparatus and ER, COPI vesicles mediating Golgi-to-ER transport were further examined by immunofluorescence. Silencing of ARF5 reduced the formation of COPI from Golgi apparatus, and thus interrupted EGFR nuclear translocation regulated by COPI. Consequently, the downstream targets of EGFR (AURKA and MYBL2) were alleviated upon ARF5 inhibition. In conclusion, ARF5 expression was upregulated in breast cancer and silencing of ARF5 reduced breast cancer cell growth and invasion by blocking EGFR nuclear translocation via COPI vesicles. Inhibition of ARF5 reduced breast cancer cell growth in vitro and in vivo, indicating the potential use of ARF5 inhibitor as a therapeutic target for breast cancer.