A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.

Jesica Diaz-Vera,Jim C Norman,Sarah Palmer,Joanne Edwards,Juan Ramon Hernandez-Fernaud,Iain Macpherson,Sara Zanivan,Louise E Mitchell,Emmanuel Dornier

doi:10.1242/jcs.190835

Abstract

ABSTRACTWe have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.

Highlights

The membrane trafficking events controlled by Rab GTPases influence cellular processes that accompany cancer initiation and progression, including loss of cell polarity, and the drive to invasion and metastasis (De Franceschi et al, 2015; Goldenring, 2013)
Low levels of Rab17 mRNA correlate with poor breast cancer survival We have previously shown that reduced Rab17 mRNA levels are associated with invasive migration of breast cancer cells
To determine whether this observation is pertinent to aggressiveness of the human disease, we interrogated the cancer Biomedical Informatics Grid, Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) repositories using the ‘kmplot’ analysis tool (Gyorffy et al, 2013)

Summary

Introduction

The membrane trafficking events controlled by Rab GTPases influence cellular processes that accompany cancer initiation and progression, including loss of cell polarity, and the drive to invasion and metastasis (De Franceschi et al, 2015; Goldenring, 2013). It is already known that Rab, a Rab GTPase whose expression is largely restricted to epithelia, is often lost in breast cancer (Cheng et al, 2010), and deletion of Rab accelerates tumorigenesis in a mouse model of colon cancer (Nam et al, 2010) One interpretation of this is that loss of Rab leads to disruption of trafficking pathways that maintain aspects of normal epithelial polarity.

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Results