The role of Annexin A6 in cancer cell signalling and cholesterol homeostasis

Abstract

Annexin A6 (AnxA6) is a Ca2+ and phospholipid binding protein belonging to the well conserved annexin family. The phospholipid binding capacity of AnxA6, in addition to protein-protein interactions is thought to allow AnxA6 to affect membrane organisation and receptor/protein kinase function. Several cancer models showed a correlation between loss of AnxA6 and elevated epidermal growth factor receptor (EGFR)/Ras activity. Enhanced growth signalling and alterations in lipid biosynthesis, uptake and transport are implicated in the transformation events leading to cancer initiation and tumour progression. Thus, the identification of potential mediators of these cellular processes could provide better understanding of cancer cell biology and perhaps the development of new, rationalised drug targets. The central aim of this study was to examine AnxA6, as a scaffold protein that links cell signalling and cholesterol homeostasis in an oncogenic setting. To examine the role of AnxA6 in an oncogenic context, A431 epidermoid carcinoma cells, overexpressing AnxA6, were used in cancer growth assays and cell viability assays. AnxA6 overexpression correlated with increased membrane recruitment of protein kinase Cα (PKCα), a negative regulator of the EGFR, and PKCα-mediated inactivation of EGFR via phosphorylation of the EGFR inhibitory residue threonine 654 (T654-EGFR). Increased T654 phosphorylation translated to reduced cell growth in cell culture and in vivo xenograft models, in a PKCα-dependent manner. AnxA6 overexpression also correlated with reduced migration and invasion in 2D and 3D-in vivo-like settings. Furthermore, AnxA6 increased the potency of EGFR tyrosine kinase inhibitors, gefitinib and erlotinib to decrease cancer cell growth and migration, indicating its potential as biomarker for drug efficacy. AnxA6 is known to regulate cholesterol transport along endo- and exocytic pathways. This is exemplified in AnxA6 overexpressing cells, which represent a Niemann Pick Type C 1 (NPC1)-like phenotype, which is characterised by late endosomal (LE)-cholesterol accumulation and depletion of cholesterol at other cellular locations, such as the plasma membrane and the Golgi. To investigate the role of AnxA6 in intracellular cholesterol homeostasis, we used Chinese Hamster Ovary (CHO) cell lines lacking functional NPC1. Loss of NPC1 activity, akin to AnxA6 overexpression, correlated with reduced cell migration, reduced activation of migration signalling cascades and mislocalisation of receptors/kinases known to promote cell adhesion and spreading. Furthermore, alterations in cholesterol homeostasis were associated with increased potency of anti-cancer agents to decrease cell migration. Thus, AnxA6 alters both EGFR signal output and cholesterol-dependent membrane trafficking events, reflecting its cooperative involvement as a scaffold in signal transduction and its ability to interact with cholesterol-dependent partners. This study further exemplifies dual functions of AnxA6 in cancer cell signalling and cholesterol homeostasis.