E4BP4 is a repressor of epigenetically regulated SOSTDC1 expression in breast cancer cells

We and others show that SOSTDC1 is down-regulated in breast cancer tissues compared to matched normal tissues. Previously, we found that epigenetic mechanisms underlie the down-regulation of SOSTDC1 in gastric cancer cells. The aim of this study was to assess the putative epigenetic regulation of SOSTDC1 expression in breast cancer cells.

Microarray-based expression profiling was performed in a series of primary breast cancers and matched normal tissues. Real-time PCR was performed to assess SOSTDC1 and E4BP4 mRNA levels in MCF7, BT549, MBMDA231, T47D (breast cancer) and HEK293T (normal kidney) cell lines. Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were performed to assess the methylation level of the SOSTDC1 gene promoter, and 5-Aza 2-deoxycytidine (5'-Aza-dC) treatment was used to induce its demethylation. A luciferase assay was used to measure SOSTDC1 promoter activity in vitro. Stable shRNA-mediated knockdown of E4BP4 was carried out in MCF7 cells and confirmed by Western blotting. Finally, MCF7 cell proliferation and survival were measured by MTS assay.

We found that SOSTDC1 is frequently down-regulated in primary breast cancers (98.2 %) and in all breast cancer cell lines tested. MSP and BSP analyses revealed SOSTC1 promoter hypermethylation at CpG sites. 5'-Aza-dC treatment induced a striking down-regulation of SOSTDC1 gene expression, whereas BSP analysis showed demethylation of its promoter. Subsequent in silico SOSTDC1 promoter analysis indicated the presence of putative transcriptional repressor E4BP4 binding sites, and promoter deletion studies indeed revealed repressor binding regions encompassing these E4BP4 binding sites. Relative quantification of E4BP4 expression showed an inverse correlation to SOSTDC1 expression in the breast cancer cell lines tested. Exogenous over-expression of E4BP4 in HEK-293 and BT549 cells reduced SOSTDC1 expression and its promoter activity, respectively. Stable shRNA-mediated E4BP4 BT549 and MCF7 knock-down cells treated with 5'-Aza-dC exhibited up-regulation of SOSTDC1 expression and a concomitant inhibition of cell proliferation and survival.

From our results we conclude that the transcriptional repressor E4BP4 plays a role in repressing epigenetically regulated SOSTDC1 expression in breast cancer cells, which can be reverted by E4BP4 silencing.