Epigenetic silencing of TFPI-2 in canine diffuse large B-cell lymphoma.
Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs (17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 (82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 (p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation (p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy.
Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy.
Serena
S
Ferraresso
Department of Women's and Children's Health, University of Padova, Padova, Italy.
Silvia
S
Bresolin
Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy.
Arianna
A
Aricò
Department of Animal Pathology Hygiene and Veterinary Public Health, University of Milano, Milano, Italy.
Stefano
S
Comazzi
Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy.
Maria Elena
ME
Gelain
Department Veterinary Science, University of Torino, Torino, Italy.
Fulvio
F
Riondato
Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy.
Luca
L
Bargelloni
Centro Oncologico Veterinario, Sasso Marconi, Italia.
Laura
L
Marconato
Department of Women's and Children's Health, University of Padova, Padova, Italy.
Geertruy te
Gt
Kronnie
Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy.
Luca
L
Aresu
D000818:Animals; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000378:metabolism; D004283:Dog Diseases / Q000378:metabolism / Q000473:pathology; D004285:Dogs; D005260:Female; D020868:Gene Silencing; D006023:Glycoproteins / Q000096:biosynthesis*; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism / Q000473:pathology / Q000662:veterinary; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic
D004273:DNA, Neoplasm; D006023:Glycoproteins; D009363:Neoplasm Proteins; C021240:tissue-factor-pathway inhibitor 2