The Epigenetic Modifier 5-Aza-2-deoxycytidine Triggers the Expression of CD146 Gene in Prostate Cancer Cells

Abstract

Background/Aim: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT).Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro. Materials and Methods: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing. Results: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype. Conclusion: The demethylating agent 5-aza-2deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression

“…However, de novo hypermethylation of canonical CpG islands has been observed in cancer cells in association with inactivation of gene expression (Jones & Baylin, 2007). Although we observed the CpG island methylation of the CD146 promoter in 2 out of 3 analysed breast cancer cell lines, it does not have to be the only and unique methylated region which controls CD146 expression, since in the T47D breast cancer cell line and three prostate cancer cell lines published previously (Dudzik et al, 2019), the CD146 expression was induced by demethylating treatment, while the analysed CpG island was methylation free. Importantly, recent studies revealed that methylation in the upstream and downstream CpG island shores may inhibit gene transcription in cancer cells, whereas the CpG island itself can remain methylation free (Rao et al, 2013;Irizarry et al, 2009).…”

“…To our knowledge, this is the first study showing methylation of the CD146 promoter region in breast cancer cells, and concomitantly suggesting that an epigenetic mechanism may be important in expression control of this relevant metastasis related-oncogene. In our previous paper, using prostate cancer cell lines, we have shown that the CD146 gene is induced by a demethylating compound, 5 aza-2-deoxycytidine, however, analysis of the CpG island region in those cells did not confirm presence of promoter methylation (Dudzik et al, 2019). Of note, the link between epigenetics and expression of the CD146 gene was reported previously in prostate cancer patients but the authors of that study incorrectly localized the CpG island in the CD146 gene promoter (Liu et al, 2008), and in consequence misinterpreted the results suggesting, contrary to the dogma, that presence of the CpG island methylation correlates with increase in the CD146 gene expression in prostate cancer cells (Liu et al, 2008).…”

“…Sequences of reverse and forward primers used in this study are presented in Table 1. For CD146, HPRT1, SNAI1, TWIST, ZEB1 and SNAI2, the PCR reactions were carried out as previously described (Dudzik et al, 2019). For CAD-HERIN1 and CADHERIN2, the conditions were as following: initial denaturation at 95°C for 5 min; followed by 30 cycles: 30 s at 95°C, 30 s at 58°C and 30 s at 72°C; and final extension at 72°C for 10 min.…”

“…Concentration and quality of isolated DNA were assessed by means of NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The DNA was modified, and bisulfite sequencing procedure was carried out as previously described (Dudzik et al, 2019). Primers for bisulfite-converted DNA were as following: forward (5'-GTTGTAAATTGGTTGTAAAGAAGAGTTG-3′), reverse (5′-CAACCTTCCCAAACCAAAAAC-3′), generating a product of 502 bp, which was cut out of the gel, purified according to the manufacturer's instructions (Agarose Out DNA purification kit, EURx, Gdansk, Poland), and subsequently sequenced (Genomed, S.A, Poland).…”

“…In order to check whether CD146 expression is regulated at the transcriptional level by an epigenetic modifier, 5-aza-2-deoxycytidine, as it has been shown previously in prostate cancer cell lines (Dudzik et al, 2019), two epithelial breast cancer cell lines (MCF7 and T47D) (Dai et al, 2017) and a mesenchymal one (MDA-MB-231) (Dai et al, 2017) were cultured with and without 5-aza-2-deoxycytidine (10 μM) for 6 days, as previously described, followed by CD146 expression analysis at the mRNA and protein level. As expected, basal expression of CD146 was low in the MCF7 and T47D cancer cell lines with epithelial characteristics, and high in the MDA-MB-231 cell line with mesenchymal expression profile.…”