Identification and Validation of Novel Subtype-Specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. METHODS: A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics. RESULTS: Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells. CONCLUSIONS: The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.

The pivotal role of reactivity in the design of novel biotinylation reagents for the chemical-proteomics-based identification of vascular accessible biomarkers.

A promising approach for the development of novel therapeutics with fewer side effects in healthy tissues is the targeted delivery of bioactive molecules directly to the site of disease. Thus, one prerequisite is the identification of a robust, disease-specific, vascular accessible biomarker localized on the surface of diseased cells, in the surrounding extracellular matrix or on newly formed blood vessels. One avenue towards the identification of such biomarkers consists in the enrichment of the vascular accessible surface proteome fraction prior to analysis. This can be achieved by covalent modification of the target proteins with membrane-impermeable ester derivatives of biotin, followed by streptavidin-based affinity capturing. The properties of the respective reagents are determined by the linker between the biotin moiety and the reactive group for protein coupling. In the frame of this study, novel, reactivity-improved peptide-based biotinylation reagents as well as reagents based on highly hydrophilic heparin linkers were synthesized and validated. The comprehensive evaluation of different biotinylation reagent classes with aliphatic, PEGylated, peptide-based and heparin-based linkers on single model protein BSA, HeLa cells as well as perfused kidney tissue revealed that the linker-dependent chemical reactivity is the crucial factor for the design of novel biotinylation reagents for vascular targeting approaches. Significance To obtain a reliable identification and stable quantification of vascular accessible protein targets by means of mass spectrometry, covalent modification with a membrane-impermeable ester derivative of biotin, followed by streptavidin-based affinity capturing, is frequently applied for in vivo or ex vivo biomarker identification studies. Nevertheless, no comprehensive evaluation of different biotinylation reagent classes has been performed so far. Within this study, we systematically evaluated novel peptide- and heparin-based biotinylation reagents as well as established compounds based on aliphatic and PEGylated linkers. We identified the linker-dependant chemical reactivity of biotinylation reagents to be the critical factor for the design of novel reagents with high efficiency. The novel, site-specifically activated peptide-based reagents were found to be efficient compounds for application in mass spectrometry-based discovery of novel vascular-accessible biomarkers.

Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.

Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.