Abstract
E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.
Highlights
Human prostate cancer (PC), the second cause of male cancerrelated death in North America, has a variable behavior (Siegel et al, 2019)
Dogs have been reported as a model for human PC and the knowledge regarding molecular aspects of canine PC has increased in recent years (Fonseca-Alves et al, 2018a; Costa et al, 2019; Laufer-Amorim et al, 2019; Rivera-Calderón et al, 2019)
Eleven of 20 dogs with PC had metastasis (55%); eight of them (8/11) presented bone and lung metastasis while pelvic bones, intestine and liver were observed in one patient each
Summary
Human prostate cancer (PC), the second cause of male cancerrelated death in North America, has a variable behavior (Siegel et al, 2019). Dogs have been reported as a model for human PC and the knowledge regarding molecular aspects of canine PC has increased in recent years (Fonseca-Alves et al, 2018a; Costa et al, 2019; Laufer-Amorim et al, 2019; Rivera-Calderón et al, 2019). CDH1 promoter methylation is widely studied as a cause of E-cadherin down-regulation in human PC (Graff et al, 1995; Yoshiura et al, 1995; Li et al, 2001; Mostafavi-Pour et al, 2015). Loss of E-cadherin during the lymphatic invasion by neoplastic epithelial cells and E-cadherin re-expression in metastatic foci were previously reported in canine PC (Fonseca-Alves et al, 2015a). We investigated E-cadherin gene and protein expression in canine proliferative inflammatory atrophy (PIA), PC and its metastasis as well the methylation status of CDH1 as a silencing mechanism responsible for the dynamic E-cadherin expression
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