Epigenetic Control of Trefoil Factor Family (TFF) Peptide Expression in Human Retinoblastoma Cell Lines

Abstract

Background: Recent studies demonstrated that epigenetic mechanisms are involved in the regulation of trefoil factor family (TFF) peptide expression in cancer. In human tissues with endogenous TFF1, TFF2 or TFF3 gene expression, the corresponding promoter is unmethylated and in organs without TFF expression, the promoter of the three genes is highly methylated. Methods: Retinoblastoma (Rb) cell lines were treated with the DNA methyltransferase inhibitor 5-Aza-2`deoxycytidine (5-Aza-dC), the histone deacetylase inhibitor 4-Phenylbutyric acid (PBA) or both and analyzed for changes (i) in TFF mRNA expression by Real-time PCR and (ii) in the methylation status of the TFF promoters by genomic bisulfite sequencing. Results: The degree of promoter methylation correlates with endogenous TFF expression in the retinoblastoma cell lines analyzed. Nearly all Rb cell lines exhibiting high endogenous TFF1 expression displayed low methylation of the CpGs in the corresponding promoter region. Low expression of TFF3 in Rb cell lines is linked with high density methylation of the TFF3 promoter. 5-Aza-dC treatment induced TFF1 and TFF3 expression in nearly all cell lines investigated and combined treatment with PBA further increased this effect. The number of methylated CpG dinucleotides of the TFF promoter is clearly reduced upon treatment with 5-Aza-dC and combined treatment with PBA further extended the degree of demethylation. Conclusion: Our data clearly show that the expression of TFF3 in retinoblastoma cell lines is epigenetically regulated, whereas the level of TFF1 and TFF2 seems to be regulated by other or additional mechanisms.

Reduction of the tumorigenic potential of human retinoblastoma cell lines byTFF1overexpression involves p53/caspase signaling and miR-18a regulation

“…The loss of TFF1 in gastric cancer and its overexpression in ovarian cancer promote tumorigenesis involving the Wnt/ß‐catenin signaling pathway . Loss of TFF1 expression is epigenetically regulated by promotor methylation in gastric cancer and likewise in retinoblastoma cell lines as recently published by our group . The mechanisms by which TFF1 promotes apoptosis and changes proliferation behavior of tumor cells remain, however, largely unknown.…”

“…We performed TFF1 knockdown (KD) experiments in the RB cell line RBL30, exhibiting decently detectable endogenous TFF1 levels, to confirm TFF1 effects on growth and apoptosis. Testing different TFF1 ‐specific shRNA clones, we achieved a very efficient knockdown with an 80–95% reduction in TFF1 expression confirmed by quantitative Real‐time PCR (data not shown).…”

“…). Previous studies by our group revealed that only TFF3, but not TFF1 is expressed in the healthy human retina, whereby RB cell lines exhibit high levels of TFF1 …”

Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

“…Our group was the first to investigate retinal expression of TFF peptides. Previous studies by our group revealed that only TFF3, but not TFF1 and TFF2 are expressed in the healthy human retina [ 11 ; 12 ], whereby retinoblastoma (RB) cell lines, established from malignant eye tumors of children, exhibit high levels of TFF1 [ 11 ; 12 ], but only trace amounts of TFF3 and no detectable TFF2 . Most recently, we demonstrated that the expression of TFF3 in retinoblastoma cell lines is regulated epigenetically [ 12 ].…”

Orchestrating epigenetic roles targeting ocular tumors

“… 42 , 46 Interestingly, deletions of some of these tumor suppressor genes may drive RB. 46 Moreover, high-density methylation of numerous other genes, such as TFF3 , 47 the apoptotic effector CASP8, 48 , 49 the DNA repair gene MLH1 , 50 APC-2 , 31 and the RB2/p130 gene, is involved in RB 51 , 52 ( Table 1 ).…”