Breast Cancer-Derived Microvesicles Are the Source of Functional Metabolic Enzymes as Potential Targets for Cancer Therapy

Abstract

Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.

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

“…Among these cell cycle proteins, five proteins belonged to kinases: cyclin-dependent kinase 1 (CDK1) and 2 (CDK2), glycogen synthase kinase 3 beta (GSK3B), polo-like kinase 1 (PLK1), and protein kinase/DNA-activated, catalytic subunit (PRKDC) ( Table S5 ). We previously studied the presence of three functional metabolic enzymes in BC-derived MVs that might be used as potential biomarkers in BC therapy [ 7 ]. This investigation indicated that sEV enzymes can effectively be incorporated into accurate, quick, and sensitive early diagnostic assays that work by measuring their enzymatic activities.…”

“…To process samples, the Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Mississauga, ON, Canada) coupled to an UltiMate 3000 nanoRSLC (Thermo Fisher Scientific, Mississauga, ON, Canada) was utilized, as previously described with modified instrument parameters [ 6 , 7 ]. Following the reconstitution of digested peptides with 20 μL MS-grade H 2 O/1% formic acid ( v / v ), three microliters of sample was injected onto an in-house packed column and eluted for 65 min at a flow rate of 300 nL/min (0–10 min, 2–2% ACN; 10–40 min, 2–38% ACN; 40–45 min, 38–98% ACN; 45–50 min, 98–98% ACN; 50–55 min, 98–2% ACN; 55–65 min, 2–2% ACN).…”

“…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 act as transport vectors or signal transducers that can deliver specific biological information by transferring bioactive content (nucleic acids, proteins, and metabolites) from donor to nearby or distant cells [ 8 , 9 , 10 ].…”

“…Comparative analyses of body fluids and cell lines may help find more tissue specific biomarkers. In recently published work, the difference in proteomes between cancerous (MCF7 and MDA-MB-231) and noncancerous (MCF10A) cell line-derived EVs has been investigated [ 6 , 7 ]. The results revealed that 87 sEV [ 6 ] and 112 mEV [ 7 ] proteins relevant to BC in which MDA-MB-231 cell line-derived sEV proteins were proposed as potential breast cancer biomarkers for disease diagnosis and prognosis as well as for potential therapeutic targets and resistance against chemotherapy agents.…”

“…In recently published work, the difference in proteomes between cancerous (MCF7 and MDA-MB-231) and noncancerous (MCF10A) cell line-derived EVs has been investigated [ 6 , 7 ]. The results revealed that 87 sEV [ 6 ] and 112 mEV [ 7 ] proteins relevant to BC in which MDA-MB-231 cell line-derived sEV proteins were proposed as potential breast cancer biomarkers for disease diagnosis and prognosis as well as for potential therapeutic targets and resistance against chemotherapy agents. Moreover, the study also validated three enzymes—ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH)—using standard enzymatic assays.…”