A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape.</jats:p>

CD9 mediates the uptake of extracellular vesicles from cancer-associated fibroblasts that promote pancreatic cancer cell aggressiveness

“…Briefly, protein extracts from EVs derived from either Ctr-shRNA– or sh461 (ANXA6-shRNA)–transfected CAFs were stacked on NuPAGE 4 to 12% Bis-Tris acrylamide gels (Life Technologies) in a single band, stained with Imperial Blue (Pierce, Rockford, IL), and cut from the gel. Gel pieces were subjected to in-gel trypsin digestion as previously described ( 51 ). Peptide mixtures were extracted, dried with speed vacuum, and reconstituted with 0.1% trifluoroacetic acid in 4% acetonitrile (ACN) before analysis by LC-MS/MS using an LTQ Velos Orbitrap Mass Spectrometer (Thermo Electron, Bremen, Germany) online with a an Ultimate 3000RSLCnano chromatography system (Thermo Fisher Scientific, Sunnyvale, CA).…”

Intersection of TKS5 and FGD1/CDC42 signaling cascades directs the formation of invadopodia

“…Note added in proof. During the final revision of the present article, Thuault et al (2020) identified an interaction between TKS5 and FGD1 using the BioiD proximity biotinylation technology.…”

Deciphering the involvement of the Hippo pathway co-regulators, YAP/TAZ in invadopodia formation and matrix degradation

“…The assignment of proteins belonging to Hippo signaling, cell adhesion, and ECM remodeling pathways was inferred using the GeneCards suite (33). A list of invadopodia-related proteins was compiled by data mining in the Harmonizome database and the related literature (34)(35)(36)(37)(38)(39)(40)(41)(42)(43).…”

“…To determine whether knockdown of YAP+TAZ affects the levels of invadopodia associated proteins, we compiled a list of proteins associated with invadopodia structure and functionality (Supplementary Table S7). The list was primarily based on the scientific literature (35,(40)(41)(42), the Harmonizome database (34) and selected proteomic reports (36)(37)(38)(39)43). Given that these resources are based on data obtained by different methods, diverse cell types, and that association with invadopodia (e.g., structural, functional, and regulatory) was not strictly and uniformly defined, our list is rather comprehensive albeit with limited editing.…”