Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells

Spencer Alford,Heather Lorimer,Amanda Carette,Jessa Bazowski,Nadia Scott,Adam Watson-Hurthig,Perry L Howard

doi:10.1186/1475-2867-10-41

Spencer Alford, Heather Lorimer + Show 5 more

Open Access

https://doi.org/10.1186/1475-2867-10-41

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Abstract

BackgroundEphrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. In malignancy, the precise role of EFNA1 and its preferred receptor, EPHA2, is controversial. Several studies have found that EFNA1 may suppress EPHA2-mediated oncogenesis, or enhance it, depending on cell type and context. However, little is known about the conditions that influence whether EFNA1 promotes or suppresses tumorigenicity. EFNA1 exists in a soluble form as well as a glycophosphatidylinositol (GPI) membrane attached form. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2.ResultsUsing a RNAi strategy to reduce the expression of endogenous EFNA1 and EPHA2, we found that both EFNA1 and EPHA2 are required for growth of HeLa and SK-BR3 cells. The growth defects could be rescued by conditioned media from cells overexpressing soluble EFNA1. Interestingly, we found that overexpression of the membrane attached form of EFNA1 suppresses growth of HeLa cells in 3D but not 2D. Knockdown of endogenous EFNA1, or overexpression of full-length EFNA1, resulted in relocalization of EPHA2 from the cell surface to sites of cell-cell contact. Overexpression of soluble EFNA1 however resulted in more EPHA2 distributed on the cell surface, away from cell-cell contacts, and promoted the growth of HeLa cells.ConclusionsWe conclude that soluble EFNA1 is necessary for the transformation of HeLa and SK-BR3 cells and participates in the relocalization of EPHA2 away from sites of cell-cell contact during transformation.

Highlights

Ephrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family
To determine whether the endogenous EFNA1 was attached to the cell surface or released, conditioned media and lysates were prepared from cultured cells
To confirm that this band corresponded to EFNA1, we knocked down EFNA1 expression in HeLa cells using short hairpin RNA (shRNA)

Summary

Introduction

Ephrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2. The Eph receptors are the largest family of receptor tyrosine kinases. They are activated by protein ligands, known as ephrins, which are attached to the cell membrane by either a membrane-spanning protein domain (B-type) or by a glycosylphosphatidylinositol (GPI) anchor (A-type). The functions regulated by Eph receptors and their ephrin ligands are diverse and cell-type dependent.

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