Fibroblast-derived HGF drives acinar lung cancer cell polarization through integrin-dependent RhoA-ROCK1 inhibition

Highlights

• Lung fibroblast-derived HGF induces acinar morphogenesis of Calu-3 cells.

• HGF activates β1-integrin- and c-Src-dependent adhesion.

• C-Src regulates p190A RhoGAP phosphorylation, inhibiting RhoA-ROCK1 signalling and inducing polarity orientation.

Abstract

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate β1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.

Introduction

A simple epithelial monolayer consists of apical-basal polarized epithelial cells, which are the basic building block of acini in organs [1]. Epithelial cells possess functionally specialized basolateral and apical cell surfaces. The basolateral domain, although contiguous, is often described as two independent domains due to their distinct functional properties: the basal surface contacts the underlying Extracellular Matrix (ECM) and the lateral surface allows adherence to neighbouring cells, while also providing a paracellular diffusion barrier [2]. In contrast, the apical surface provides the lining of the lumen of epithelial tissues, possessing the distinct characteristic of non-adherence to neighbours or the ECM. Such polarity on an individual level must be collectively coordinated between neighbouring cells to form the lumen and thus a biological tube.

While the mechanisms of how apical-basal polarity is formed have been extensively investigated [1], [2] how such polarity is oriented has until recently been neglected [3], [4]. Traditional culture methods for epithelial cells involve growth of cells to become apical-basal polarized monolayers on rigid substrata such as glass, plastic, or semi-permeable membrane filters. In these systems, the stiff culture vessel provides an immediate and strong cue as to where basal membranes should form [5]. In contrast, in 3-Dimensional (3D) culture where single epithelial cells are embedded in ECM gels, such as Matrigel or collagen, cells are surrounded by ECM. In such isotropic conditions, mechanisms must exist to orient apical-basal polarity formation such that the basal surface faces the ECM and the apical surface lines the cavity of ‘free space’.

We have previously described that temporospatial control of the RhoA signalling pathway is essential for the correct orientation of apical-basal polarity [6], [7]. In the MDCK cyst model, upon embedding isolated MDCK cells into Matrigel gels, single cells divide to form a cell doublet that has initially inverted polarity, i.e. apical surfaces erroneously facing the ECM. Detection of the ECM by β1-integrin-dependent adhesion results in phosphorylation of the RhoA-inhibitory GAP p190A RhoGAP [6]. P190A leads to inactivation of RhoA-Rho Kinase 1 (ROCK1) signalling, at the ECM abutting surface, allowing endocytosis of apical proteins from the ECM-abutting periphery and transcytosis to the centre of the cell doublet to form a lumen. This results in the correct orientation of apical-basal polarity between cells in 3D culture.

An open question from our and others' previous studies is how generalizable signalling pathways identified to regulate polarity orientation in one cell type are to other epithelia? In addition, are these pathways defective in epithelial cancer cells? In the current work, we show that the previously identified pathway for correct polarity orientation is inactive in Calu-3 human lung adenocarcinoma cells grown in 3D at steady state. Fibroblast-derived Hepatocyte Growth Factor (HGF) can correct aberrant polarity orientation in 3D by controlling the localization of polarity pathways to the ECM-abutting surface. This suggests that future studies into modulation of this pathway may be important for understanding polarity changes in cancer.