Phosphoprotein Secretome of Tumor Cells as a Source of Candidates for Breast Cancer Biomarkers in Plasma

Abstract

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

“…Plasma/serum phosphoproteome analyses fared even worse in this aspect, 40 -100 l of plasma (ϳ2-5 mg protein) yielded 30 -70 phosphoproteins using TiO 2 enrichment (either alone or in combination with SCX) or Fe(III)-IMAC (27)(28)(29). The largest phosphopeptide data set from plasma was reported by Zawadzka et al in parallel to the breast cancer cell line secretome analysis (24). On average, 130 phosphoproteins could be identified from 1 ml plasma (ϳ50 mg protein) of control or breast cancer patients using HILIC fractionation in combination with TiO 2 enrichment.…”

“…Immunodepletion was used in some cases for the removal of up to the 14 most abundant plasma proteins to increase the depth of the analysis (24,25,28). However, comparison of TiO 2 enrichment applied to serum with or without depletion of albumin and IgG showed considerable loss of phosphopeptides in the depleted fraction (27).…”

“…However, there are initial attempts to find biomarker candidates in plasma or to assess variation in the phosphorylation level of plasma phosphoproteins (24,63). Continuous improvements in MS detection sensitivity will facilitate detection of lower abundance phosphoproteins.…”

“…In a phosphorylation analysis of the secretome of different luminal and basal type breast cancer cell lines over 1700 phosphoproteins were identified altogether combining hydrophilic interaction liquid chromatography (HILIC) with TiO 2 phosphopeptide enrichment (24). Approximately half of these phosphoproteins were predicted to be extracellular or membrane proteins according to gene ontology (GO) annotation and bioinformatics analyses using SignalP (classical secretion), SecretomeP (unconventional secretion), and TMHMM (transmembrane domains) algorithms.…”

Recent advances in proteomically subtyping pancreatic ductal adenocarcinomas and their potential clinical impact

“…Recently, Zawadzka managed to identify and qualify six breast cancer tumor type specific phosphosites [23]. The serum-free PACO cell cultures could offer an optimal starting point for the identification of subtype-specifically secreted phosphoproteins.…”