title: A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.
abstract: We 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 carcinomain 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.
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
authors.forename: Jesica
authors.initials: J
authors.lastname: Diaz-Vera
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Palmer
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
authors.forename: Juan Ramon
authors.initials: JR
authors.lastname: Hernandez-Fernaud
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Dornier
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
authors.forename: Louise E
authors.initials: LE
authors.lastname: Mitchell
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.|Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
authors.forename: Iain
authors.initials: I
authors.lastname: Macpherson
authors.affiliation: Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Edwards
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK s.zanivan@beatson.gla.ac.uk j.norman@beatson.gla.ac.uk.|Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Zanivan
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK s.zanivan@beatson.gla.ac.uk j.norman@beatson.gla.ac.uk.|Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
authors.forename: Jim C
authors.identifier: 0000-0002-0098-3014
authors.initials: JC
authors.lastname: Norman
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007553:Isotope Labeling; D008954:Models, Biological; D060787:Neoplasm Grading; D009361:Neoplasm Invasiveness; D039943:Neuropilin-2 / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D050683:R-SNARE Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D016019:Survival Analysis; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D039943:Neuropilin-2; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D050600:SNARE Proteins; C112938:VAMP8 protein, human; C581308:neuropilin-2, human; D020691:rab GTP-Binding Proteins
keywords: Breast cancer; Cell migration; Ductal carcinoma in situ; Invasion; Invasive ductal carcinoma; Late endosome; Neuropilin-2; Rab17; SILAC; Vamp8