Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer

Abstract

Abstract BACKGROUND Carboxypeptidase N (CPN) is important in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. We investigated whether circulating peptides specifically cleaved by CPN in the tumor microenvironment can be stage-specific indicators of breast cancer. METHODS CPN activity was measured using an ex vivo peptide cleavage assay by incubating synthesized C3f peptide (His6-C3f_S1304-R1320-His6) in interstitial fluids of breast tumors and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. The nature and extent of peptide cleavage by CPN was investigated by fragment profiling using nanopore fractionation and mass spectrometry. The fragment profiles in interstitial fluid correlated with concentrations of CPN-catalyzed peptides in blood samples taken from the tumor-bearing mice, healthy women, and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. RESULTS We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, CPN was clearly increased in tumor tissues compared with normal breast tissue, whereas corresponding CPN abundance in blood remained constant. Concentrations of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n = 8) at 2 weeks after orthotopic implantation. Six homologous peptides displayed significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. CONCLUSIONS Circulating CPN-catalyzed peptide concentrations reflect the CPN activity in tumors. These biomarkers show strong potential for the noninvasive and early diagnosis of breast cancer.