Significant association of serum autoantibodies to TYMS, HAPLN1 and IGFBP5 with early stage canine malignant mammary tumours

Abstract

Canine mammary tumours (CMTs) are the most prevalent neoplasms in female dogs. Despite the high incidence of such tumours, a lack of easily accessible biomarkers still impedes early diagnosis of malignant CMTs. Herein we identify thymidylate synthetase (TYMS), hyaluronan and proteoglycan link protein 1 (HAPLN1) and insulin‐like growth factor‐binding protein 5 (IGFBP5) as CMT antigens eliciting corresponding autoantibodies in CMT cases. We establish enzyme‐linked immunosorbent assays (ELISAs) to detect autoantibodies to TYMS (TYMS‐AAb), HAPLN1 (HAPLN1‐AAb) and IGFBP5 (IGFBP5‐AAb) in sera from 81 dogs with malignant CMTs (41 in Stage I), 24 with benign CMTs and 35 healthy controls. Levels of all the three autoantibodies are elevated in the malignant group compared with the healthy or the benign group; notably, the elevated autoantibody levels significantly correlate with the stage‐I CMTs. For discriminating malignant CMTs from healthy control, the area under curve (AUC) of TYMS‐AAb is 0.694 with specificity of 82.9% and sensitivity of 50.6%. The AUC of utilising HAPLN1‐AAb for distinguishing the stage‐I CMTs from healthy controls is 0.711 with specificity of 77.1% and sensitivity of 58.5%. In differentiating malignant CMTs from the benign, the AUC of IGFBP5‐AAb reaches 0.696 with specificity of 70.8% and sensitivity of 67.9%, and a combination of IGFBP5‐AAb and TYMS‐AAb increases the AUC to 0.72. Finally, the AUC of combined HAPLN1‐AAb and IGFBP5‐AAb in discriminating the stage‐I CMTs from the benign achieves 0.731. Collectively, this study highlights a significant association of the three serum autoantibodies with early stage malignant CMTs.

The Implication of Serum Autoantibodies in Prognosis of Canine Mammary Tumors

“…To verify the correlation between CMT progression and serum levels of TYMS-AAb, IGFBP5-AAb, HAPLN1-AAb, and AGR2-AAb, we conducted an in-house ELISA established previously [ 25 ] to assess the AAbs in sera collected from 20 BMT, 70 MMT, and 27 healthy dogs. Signalments of 90 CMT dogs were summarized in Table 1 .…”

“…Recombinant canine proteins of TYMS, IGFBP5, HAPLN1, and AGR2 were prepared as previously described [ 8 , 25 ]. In brief, cDNA derived from CMT tissues or CMT cell lines was used as template to amplify DNA fragments encoding the full-length canine HAPLN1, canine IGFBP5 (with deletion of the signal peptide), and canine AGR2 (with deletion of the signal peptide) by polymerase chain reaction (PCR) with forward and reverse primer pairs targeting to canine HAPLN1 (NCBI Gene ID: 488921), IGFBP5 (NCBI Gene ID: 610316), and AGR2 (NCBI Gene ID:482333), respectively.…”

“…An in-house ELISA was established for serum AAb detection [ 25 ]. The recombinant proteins diluted in 1× PBS were coated onto a 96-well microplate (ExtraGene, Davis, CA, USA) at a concentration of 250 ng/100 µL per well at 4 °C for 16 h. The wells were washed with 1× PBS (200 µL/well) five times and blocked with BlockPRO TM (Visual Protein, Taipei, Taiwan; 200 μL/well) at RT for 1 h. After washing with 1× PBS five times, the wells were incubated with canine serum samples 30-fold diluted in 100 µL BlockPRO TM at RT for 1 h, followed by washing with 1× PBS-T (0.05% Tween 20; VWR International, Avantor, Radnor, PA, USA) five times.…”

“…We have verified TYMS, IGFBP5, and HAPLN1 as CMT-associated antigens and the presence of corresponding autoantibodies, namely TYMS-AAb, IGFBP5-AAb, and HAPLN1-AAb, respectively, in serum of CMT patients. These AAbs show validity in distinguishing early-stage MMT from benign CMT (BMT) or healthy dogs [ 25 ]. Whether these AAbs can be used for prediction of CMT outcomes has not yet been evaluated.…”

Ρύθμιση Βιοσύνθεσης Υαλουρονικού Οξέος Σε Κυτταρικά Μοντέλα Καρκίνου Του Μαστού

“…Η χημική αυτή ένωση (SMD1736) δεν έχει εξεταστεί σε καμία προηγούμενη βιολογική μελέτη αλλά σύμφωνα με υπολογιστικές προβλέψεις πιθανώς αλληλεπιδρά με τις POLA1 (DNA polymerase alpha catalytic subunit), TYMS (Thymidylate synthase), TMK (Thymidylate kinase), TYMP (Thymidine phosphorylase) και TK (Thymidine kinase) [286]. Η TK έχει βρεθεί να εκφράζεται υψηλότερα σε πληθυσμούς κυττάρων του παχέος εντέρου που εμφανίζουν έκφραση CD44 (CD44 + CD133 + ) [287] και η TYMS έχει εντοπιστεί μαζί με την HAPL1 (Hyaluronan And Proteoglycan Link Protein 1) στα αρχικά στάδια καρκίνου του μαστού σε σκύλους [288]. Πέρα από αυτές τις μελέτες, καμία από αυτές τις πρωτεΐνες δεν έχει συσχετιστεί με το σύστημα του υαλουρονικού ή με απευθείας ρύθμιση των HASes.…”