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

Abstract

Background Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer. Methods The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens 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, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients’ plasma as early as the first pathologic stage of breast cancer. Conclusions The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme’s activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer.

Ambient ionization and miniature mass spectrometry system for chemical and biological analysis

“…Effective real-time extraction from biological samples and subsequent ionization at relatively high efficiency have been well demonstrated with a variety of ambient ionization methods [45,66]. The circulating proteolytic products in biofluids [82] might also become suitable biomarkers for screening by the small MS systems. The spray-based methods, in comparison with plasma-based ones, might be of an advantage for the ease of extraction and ionization of biomolecules.…”

Clinical Predictive Circulating Peptides in Rectal Cancer Patients Treated with Neoadjuvant Chemoradiotherapy

“…Interesting, the C3f fragment (m/z 1098.537) posses an opposite trend with respect its precursor peptide at m/z 2021.106 and this could correspond to differences in cancer exoproteases activity. Cancer proteases have been recently recognised as possible biomarker source, since they can generate specific fragmentation patterns (Villanueva et al, ; Gianazza et al, ; Li et al, ) from common precursor proteins. Moreover, a potential implication of higher levels of C3 circulating in pCRT resistant CRC patients should be considered, since similar results have been obtained in esophageal cancer patients underwent neo‐adjuvant chemoradiation (Maher et al, ).…”

Decoding the Blood Peptidome as a New Biomarker Resource for Cancer Detection

“…Carboxypeptidase N (CPN)-associated peptide profiles in mouse and human blood samples correspond with those found in the interstitial fluid of mouse breast tumors or with CPN expression in normal breast tissue or breast tumor biopsies of women with various stage breast tumors. 3 We also found that in mice carrying ovarian tumors, serum levels of two complement C3fderived peptides were primarily generated by tumor-resident matrix metalloproteases 9 (MMP-9) activity, which decreased in response to positive responses to therapeutic siRNA intervention.…”