Dataset Information

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

ABSTRACT: 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 Tks5? fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5? fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5?). 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 Tks5? 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.

SUBMITTER: Thuault S

PROVIDER: S-EPMC7176661 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Thuault Sylvie S Mamelonet Claire C Salameh Joëlle J Ostacolo Kevin K Chanez Brice B Salaün Danièle D Baudelet Emilie E Audebert Stéphane S Camoin Luc L Badache Ali A

Scientific reports 20200422 1

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 ...[more]

PMID: 32321993

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Project description:Proximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

Project description:Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.

Dataset Information

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

Publications

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

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