Bile and urine peptide marker profiles: access keys to molecular pathways and biological processes in cholangiocarcinoma

Background Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. Methods Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. Results For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. Conclusions The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

. Observed and in silico predicted protease associations to the N-and C-terminal amino acid sequence motifs of the bile and urine peptides included in the bile and urinary proteomic models for CCA diagnosis. Confirmed cleavage site associations are based on the MEROPS peptide database, whereas in silico protease prediction was done by the software tool Proteasix. RHPY.FYAPELLFFAK

97-DGVSGGEGKGGSDGGGSHRKEGEEADAPGVIPGIVG-132
CD99 antigen MEP1B (M12.004) [7] ADLA.DGVSGGEGKGGSDGGGSHRKEGEEADAPGVIPGIVG  Table S3. Transcriptomics data table summarizing the results from differential gene expression analysis for all parental proteins and predicted proteases associated with the bile and urine CCA peptide markers. All tissue transcriptomics data sets were retrieved from the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and analyzed with GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/). The analysis was based on the following gene arrays: GSE76297 for CCA tumour and paired non-malignant tissue, GSE26566 for CCA tumour, matched non-malignant surrounding liver and normal bile duct tissue, GSE32879 for intrahepatic CCA tumour, matched non-malignant surrounding liver and benign liver lesions tissue derived from focal nodular hyperplasia patients, and GSE45001 for laser capture microdissected fibrous tissue from intrahepatic CCA tumour and non-malignant tissue. Differential tissue expression is reported as fold-change in CCA tissue versus control, supplemented by the adjusted p-value for false discovery rate and the identification number per gene array.  Figures S1-S24. Box-and-whisker plot representations of RNA-seq mRNA levels given as "transcripts per million" (TPM) for all parental proteins and predicted proteases associated with the bile and urine CCA peptide markers in 18 CCA, 18 CCA-adjacent, 100 HCC, 53 HCC-adjacent and 35 normal liver tissue replicates. Transcriptomics data sets were retrieved from the "Pan-Cancer Analysis of Whole Genomes" project RNA-Seq mRNA repository available at https://www.ebi.ac.uk/. No RNA-seq data was available for UMOD, COL6A6, COL2A1, IL1RAPL1, KLK4, CMA1 and KLK6. Table S4. Physiological and cancer-specific pathological implications of the parental proteins from which the bile peptide markers for CCA diagnosis by bile proteome analysis are derived.

Protein
Characteristic physiological functions* Link to cancer pathology Relation to CCA pathology Peptide marker Peptide marker specific information
Overexpression in tissue specimens of epithelioid sarcoma and association with cell growth and motility [20].
Upregulated in the membrane and cytosole of the human CCA cell line huCCA-1 [21].

Hemoglobin subunit α and β (HBA1 and HBB)
Oxygen transport, heme binding, control of acid-base equilibrium Peroxidase activity during inflammation and oxidative stress, and induction of cytotoxicity to macrophages by hemoglobin aggregate formation [22], hemoglobin release by cancer induced hemolysis [23].
Tumor invasion and metastasis suppressor with loss of expression in invasive tumors [25,28].
Reduced serum levels in CCA compared to benign biliary tract diseased patients [29].
Immune modulation due to binding of pathogen and danger associated molecules and inflammatory mediators [49], evidence for intracellular blocking of inflammatory signaling pathways after endocytic internalization [50].
Reduced levels in intrahepatic CCA tumor tissue samples [51], levels > 3 g/dL are an independent predictor of overall survival in hilar CCA [52].
* According to the Uniprot knowledge database unless no other reference is given Abbreviations: CCA, cholangiocarcinoma; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma. Table S5. Physiological and cancer-specific pathological implications of the parental proteins from which the urine peptide markers for CCA diagnosis by urine proteome analysis are derived.

Uromodulin (UMOD)
Salt and water retention, urinary host defense and protection against stone formation in the kidney [53], renal clearance of circulating cytokines [54], regulation of granulopoiesis via proximal epithelial activation of the IL-23/IL-17 axis [55].
Serum biomarker for advanced colorectal adenoma and colorectal cancer [65].

Protein
Characteristic physiological functions* Link to cancer pathology Relation to CCA pathology Peptide marker Peptide marker specific information Structural component of the extracellular matrix generated mainly from fibroblasts, binding of platelet-derived growth factor (PDGF) which is a potent mitogen for mesenchymal cells [74].
Overexpression in various cancer types promoting metastasis [75] and apoptosis inhibition in tumor cells [76].
Increased expression indicates tumor recurrence in high-grade serous ovarian cancer [82].
Among the genes that are associated with lymph node metastasis and perineural invasion of CCA [83].
Increased expression in bladder cancer with association to poor prognosis and focal adhesion [86].
Indicative for the presence of cancer-associated fibroblasts in the CCA tumor stroma [77].
Increased expression and prognostic marker in gastric cancer [87].
Altered lysine hydroxylation in a Pten -/mouse model representing a mixed phenotype of HCC and CCA with impact on matrix remodeling and stiffening [88].
Key determinant in the assembly of tissue matrices with binding affinity to DNA, heparan sulfate, thrombospondin, heparin, and insulin.
Increased expression in muscle-invasive bladder cancer associated with poor prognosis and tumor invasion [90].
Collagen α-6(VI) Component of the basal lamina of epithelial cells potentially involved in the regulation of epithelial cellfibronectin interactions [91].
Correlation of high tissue expression levels with early tumor stages in breast cancer [92].
Upregulation in a Pten -/mouse model representing a mixed phenotype of HCC and CCA [88].
Overexpression in epithelial cancers due to aberrant epigenetic control promoting tumor invasion [94].

Protein
Characteristic physiological functions* Link to cancer pathology Relation to CCA pathology Peptide marker Peptide marker specific information Na + /K + -ATPase γ subunit (FXYD2) Modulation of sodium ATPase activity [96].
Increased expression in ovarian clear cell carcinoma under control of the transcriptional factor HNF1B [97].
Increased serum levels as marker for early CCA diagnosis [114], increased oxidation due to oxidative stress in CCA [115].

25-EDPQGDAAQKTDTSHHDQDHP-45
Reduced urinary expression during preclinical cardiac dysfunction before heart failure [68], increase in urinary expression by inhibition of the sodium/glucose cotransporter 2 with empagliflozin in patients with diabetic kidney disease [116].
Cancer type specific marker for neuroectodermal and non-small cell lung carcinomas [126,127], increased expression in inflammatory diseases [128].
Decreased expression in gastric and gallbladder carcinomas due to promoter methylation, loss of heterozygosity and transcription factor down-regulation associated with unfavorable prognosis [129,130].
Elevated serum levels in patients with CCA compared to healthy controls and patients with PSC [139], association of elevated serum levels with shorter overall survival and high probability of tumor relapse after curative resection in patients with intrahepatic CCA [140].