Proteomic characterisation of primary breast tumour extracellular matrix

Abstract

Breast cancer is the most commonly diagnosed cancer in women and is the leading cause of female cancer mortality worldwide. High cancer mortality rates, mostly due to late-stage diagnosis and the lack of appropriate personalised therapy, highlights treatment failure that prompts the need for continued research to identify new and improved breast cancer detection methods and treatment. The purpose of this study was to use advanced mass spectrometry-based proteomics to characterise and compare the proteome but especially extracellular matrix (ECM) protein components from solid invasive ductal carcinoma tumours to matched non-tumorous breast tissue with the aim of identifying potential prognostic markers or new therapeutic drug targets for breast cancer treatment. Breast tumours are dense, complex tissue masses made up from a number of different proliferating cell types that are embedded in an intricate tumour microenvironment. Several studies have highlighted the role of the tumour microenvironment, more specifically the ECM, in tumour development and progression from localised invasion to advanced metastasis. The ECM consists of numerous protein components that provide a scaffold for both cell and growth factor binding, where ECM changes have been associated with tumour advancement. By implication, characterisation of tumour ECM components can potentially be used as prognostic or staging markers for breast cancer or to identify new targets for anticancer therapies. In this research study, cryotome cut slices of snap-frozen tumour biopsies resected from patients diagnosed with invasive ductal carcinoma (IDC) were used to characterise the primary breast tumour proteome especially for the ECM. Haematoxylin and eosin staining, the gold standard for routine histopathological diagnosis of cancer, was used to visualise tissue morphology and to confirm the clinical IDC diagnosis. An optimised protein extraction method involving high pressure cycling technology was used for tissue homogenisation and protein solubilisation of tumour biopsies. Proteomics analysis involving innovative semi-automated and cutting-edge sample preparation and liquid chromatography tandem mass spectrometry-based methods that are at the fore-front of drug target validation, drug discovery and prognostic marker identification, were used to acquire proteomic data from the tissue isolated from both tumour biopsies and equivalent non-tumorous breast tissue. A semi-automated sample preparation method using hydrophilic affinity-based protein capture, clean-up and off-bead trypsin digestion was used to produce peptides, followed by analysis using a Dionex Ultimate 3000 RSLC system coupled to an SCIEX 6600 TripleTOF mass spectrometer. Data independent acquisition using sequential window acquisition of all theoretical mass spectra (SWATH) data was collected and bioinformatic protein identification and relative quantification was performed. SWATH data provided reliable proteomic assessment, could identify low abundance proteins as well as provide relative quantitation of differentially expressed proteins in tumour samples. Tumour associated ECM changes were classified through STRING pathway analysis comparing the relative protein abundance between non-tumorous and tumour masses. Pathway analysis revealed that ribosomal, spliceosome and endoplasmic reticulum protein processing pathways with associated protein components were significantly upregulated in breast tumour samples. Proteomic data confirmed that protein homeostasis, associated with protein synthesis, protein folding and alternative splicing, is severely affected in solid tumours in order to meet the demands of uncontrolled tumour growth and promotion of tumour metastasis. SWATH-based quantification and pathway enrichment analysis did identify several ECM protein networks containing a number of differentially expressed ECM proteins in breast tumour samples. These ECM proteins within the tumour microenvironment are involved in several cancer related biological processes that include structural integrity, cancer cell proliferation, tumour growth, tumour tissue invasion, and metastasis. These differentially expressed ECM proteins could potentially be used as putative biological prognostic signatures for breast cancer or be used as new drug targets to slow or completely inhibit breast cancer advancement and progression. This exploratory study provides valuable proteomic data for breast cancer research associated with the tumour microenvironment and has laid the foundation for prognostic and pharmacological based studies for cancer therapeutics by identifying putative ECM protein candidates that can be further assessed in independent verification and validation breast cancer studies.