Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes

Abstract

Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A (non-malignant), MCF7 (estrogen and progesterone receptor-positive, metastatic), and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO2, followed by LC-MS/MS, were performed. The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.

Lysine Acetylome of Breast Cancer-Derived Small Extracellular Vesicles Reveals Specific Acetylation Patterns for Metabolic Enzymes

“…Our previous work focused on identifying metabolic enzymes in BC-derived EVs and MVs that could be explored as diagnostic BC biomarker [ 8 , 16 ]. We chose to focus on enzymes as biomarkers since they can be easily incorporated into quick, accurate and sensitive diagnostic assays that can assess specific enzymatic activity.…”

“…Post-translational modification of proteins intersects with cancer, playing a decisive role in regulating various cellular processes and being implicated in the major regulatory mechanisms of cell signaling networks. In our previous investigation, using proteomics analysis, we demonstrated that EVs contain functional, phosphorylated, metabolic enzymes that could be potential candidates in early BC diagnostic and therapeutic applications [ 16 ]. In this study, 25 novel acetylated sites were identified in sEVs from two breast cancer cell lines (MCF7 and MDA-MB-231) and the non-metastatic (MCF10A) cell line.…”

“…Culturing of epithelial breast cancer cell line MDA-MB-231 (ATCC HTB-26), MCF7 (ATCC HTB-22) and MCF10A non-tumorigenic epithelial breast cancer cell line (ATCC CRL-10317) was performed as described previously [ 7 , 8 , 16 ]. sEVs were isolated by using differential ultracentrifugation as described in our previous investigation [ 7 , 16 ].…”

“…The concentration and size distribution of sEVs were determined using The ZetaView nanoparticle tracking microscope PMX-110 (Particle Metrix, Meerbusch, Germany) at 85 and 40 camera shutter speeds [ 16 ].…”

“…In our previous investigations, using proteomics approaches, numerous proteins have already been identified from EVs as potential biomarkers for early diagnosis or as therapeutic targets [ 7 , 8 ]. Moreover, using a phosphoproteomics approach, we previously assessed the enzymatic activity of three enzymes associated with cancer and only present in sEVs derived from the cancerous cell lines MCF7 and MDA-MB-231: ATP citrate lyase (ACLY), sirtuin-1 (SIRT1) and sirtuin-6 (SIRT6) [ 16 ]. The specific activity of these enzymes was significantly higher in MDA-MB-231 sEV fractions when compared with similar MCF10A fractions.…”