Loss of Neogenin1 in human colorectal carcinoma cells causes a partial EMT and wound-healing response

Vishal Chaturvedi,Helen M Cooper,Alexandre Fournier-Level,Michael J Murray

doi:10.1038/s41598-019-40886-y

Abstract

Neogenin1 (NEO1) is a receptor of the Deleted in Colorectal Carcinoma (DCC)/Frazzled/UNC-40 family, which regulates axon guidance but can also stabilize epithelial adherens junctions. NEO1 and DCC are also tumor suppressors that can inhibit metastasis by acting as dependence receptors. Given the role of NEO1 in maintaining adherens junctions we tested whether loss of NEO1 also promoted metastasis via an epithelial mesenchymal transition (EMT). Loss of NEO1 disrupted zonula adherens but tight junctions were unaffected. Neo1-depleted epithelial cells exhibited a more migratory morphology, had reduced F-actin rich stress-fibres and more basal lamellipodia. Microtubule density was decreased while microtubule outgrowth was faster. Live imaging showed that Neo1-depleted epithelial islands had increased lateral movement. Western blots and immunostaining revealed increased expression of mesenchymal markers such as Fibronectin and MMP1. Furthermore, RNA-seq analysis showed a striking decrease in expression of genes associated with oxidative phosphorylation, and increased expression of genes associated with EMT, locomotion, and wound-healing. In summary, loss of NEO1 in intestinal epithelial cells produces a partial EMT response, based on gene expression, cellular morphology and behaviour and cytoskeletal distribution. These results suggest that loss of NEO1 in carcinomas may contribute to metastasis by promoting a partial EMT and increased motility.

Highlights

Receptors of the Neogenin/Deleted in Colorectal Carcinoma (DCC)/Frazzled/UNC-40 family were originally identified as axon guidance receptors[1] but have been increasingly linked to epithelial morphogenesis events[2]
Neo1-siRNA was transfected into 1-day old epithelia and cells were immunostained after 2 days for E-Cad and NEO1
In Neo1-siRNA treated cells actin localisation was largely intact in the most apical regions but became weaker in middle regions, and there was a clear reduction in stress fibres at the basal side of cells (Fig. 1b, arrowheads)

Summary

Introduction

Receptors of the Neogenin/Deleted in Colorectal Carcinoma (DCC)/Frazzled/UNC-40 family were originally identified as axon guidance receptors[1] but have been increasingly linked to epithelial morphogenesis events[2]. The Neo/DCC ortholog, inhibits a partial epithelial-mesenchymal transition (EMT) event that occurs during pupal metamorphosis[5] and promotes the formation of the gut epithelium in the embryo[6]. In the Caco-2 colorectal cancer cell line, NEO1 localises to AJs in an E-Cadherin (E-Cad) dependent way, where it promotes F-Actin formation and junctional tension by recruiting the WAVE Regulatory Complex (WRC) and Arp2/37. As might be expected for genes that promote epithelial stability, loss of Neo-family receptors is strongly linked to metastasis. Genes that were down-regulated are strongly enriched for those involved in oxidative phosphorylation These results confirm the importance of NEO1 in maintaining epithelial integrity and provide insight into the transcriptional response of intestinal epithelial cells when cadherin-dependent adhesion is disrupted

Methods

Results

Conclusion