ARHI is a maternally imprinted tumor suppressor gene that maps to chromosome 1p31. ARHI is expressed in normal breast and ovarian epithelial cells, but its expression is lost in majority of breast and ovarian cancers. Our previous study has revealed that the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) can activate ARHI expression. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation. Our recent data have further confirmed that transcriptional factors E2F1 and E2F4 and their complexes with HDACs play an important role in downregulating ARHI expression in breast cancer cells. TSA treatment can reverse the repression of E2F1 and E2F4. Human HDACs are divided into three classes: class I (HDAC1, -2, -3, -8, -11), class II (HDAC4, -5, -6, -7, -9, -10), and class III (SIRT1 to -7). To determine which HDAC is responsible for the repression of the ARHI, we co-transfected SKBr3 breast cancer cells with vectors expressing HDAC1 to 11 and with an ARHI/luciferase reporter. Expression of HDAC1, -3, and 11 significantly reduced ARHI promoter activity 3 to 4 fold (p<0.01). Expression of HDAC2, -6, -7, -9, -10 also significantly reduced ARHI promoter activity (p<0.05). Whereas HDAC4 and 5 did not affect ARHI promoter activity. Expression of HDAC8 slightly increased ARHI promoter activity, but this effect did not achieve statistical significance. When SKBr3 cells were co-transfected with serial dilutions of HDAC1, -3, -8, -11 expression vectors, the changes of ARHI promoter activity were correlated with HDAC expression levels. To confirm that different HDACs play different roles in the repression of the ARHI promoter, small interfering RNAs (siRNA) were used to decrease expression of HDAC3 or HDAC8 in SKBr3 cells. SiRNA targeting HDAC3 or HDAC8 knocked down the expression of HDAC3 and HDAC8 respectively, but not the expression of a β-actin control, as monitored by immunoblotting. Depletion of HDAC3, but not HDAC8, significantly increased the activity of the ARHI promoter. Taken together, our results suggest that several different HDACs lead to the repression of ARHI tumor suppressor gene. HDAC inhibitors may serve as new reagents for treatment that activate ARHI and reverse epigenetic changes.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]