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.
Highlights
Introduction iationsExtracellular vesicles (EVs) derived from human cancer cell lines are involved in multiple biological processes in tumor biology, such as modulation of the microenvironment, angiogenesis, sustained growth, and tissue invasion [1,2,3,4,5,6,7]
We carried out a systematic analysis of the phosphoproteome of small extracellular vesicles (sEVs) derived from MCF10A, MCF7, and MDA-MB-231 cell lines to provide insight into the molecular mechanism of breast cancer
The results revealed the presence of markers in all free sEV fractions derived from three cell lines: MCF10A, MCF7, and MDA-MB-231 (Figures 1B and S1)
Summary
Extracellular vesicles (EVs) derived from human cancer cell lines are involved in multiple biological processes in tumor biology, such as modulation of the microenvironment, angiogenesis, sustained growth, and tissue invasion [1,2,3,4,5,6,7]. EVs include several subtypes of membranebound vesicles, including exosomes and microvesicles, distinguished by their biogenesis pathway. These subtypes may be roughly categorized by their measured diameter, with small extracellular vesicles (sEVs) or exosomes in the range of 30 to 150 nm [11,12] and medium extracellular vesicles (mEVs) in the range of 50 nm to 1000 nm in diameter [13], Licensee MDPI, Basel, Switzerland.
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