The Subcellular Localisation of HAX1 Isoforms and Their Roles in Cancer Cell Migration, Autophagy and Apoptosis.

Abstract

It has been reported that HAX1 is a multi-functional protein which protects cells from apoptosis, modulates autophagy, regulates membrane protein trafficking and promotes cell migration. Many studies have shown it has many different intra-cellular binding partners (including integrin β6 subunit) and exists in multiple cellular locations, including the nucleus, mitochondria and cytoplasm. This behaviour seemed unlikely for a single protein. My lab discovered that there are at least eight different HAX1 isoforms in humans and this might explain why multiple roles and sub-cellular locations are described for HAX1. In this study, I sought not only to confirm the role of HAX1 in cell behaviour, but also to examine specifically the role of HAX1 isoforms in different biological functions. I screened a panel of cancer cell lines for αvβ6-dependent migration, including breast (MCF10.CA1a), pancreatic (CFPac1 and Panc04.03), and αvβ1-dependent migration in cervical cancer (HeLa); siRNA designed to knockdown all HAX1 isoforms significantly decreased cell migration indicating HAX1 is required for cell migration in cells from multiple tissues, regulating both αvβ6 and αvβ1-dependent cell migration in various cancer types. In order to establish the functions attributable to individual isoforms, I re-introduced individual HAX1 isoforms into HAX1-null (siRNA knockdown) cells and then studied their subcellular localisation and whether they modulate migration, apoptosis and autophagy. The immunofluorescent staining revealed the predominantly mitochondrial localisation of HAX1.1, HAX1.4, HAX1.5 whereas HAX1.2 showed a very unique pattern of location in the cytoplasm. Overexpression of HAX1.2 decreased cell motility on αv-dependent ligand latency associated peptide (LAP) and knockdown of HAX1.2 increased cell migration. Furthermore, 6 HAX1.2 protected cells from cell death induced by various cytotoxic drugs. Moreover, HAX1.1, HAX1.2 and HAX1.5 increased autophagy by elevating the levels of Beclin-1 and LC3b-II. However, it also is clear that much further study is needed to thoroughly understand the biology of each isoform. These are the first experimental data that demonstrate different HAX1 isoforms have different biological activities in human cancer cells, helping to explain the conflicting literature describing HAX1.