The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression in Breast Cancer Cells

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RAR beta is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RAR beta has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RAR beta expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RAR beta induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RAR alpha and RAR beta from Sin3A in a coimmunoprecipitation assay. This was associated with increased RAR alpha and RAR beta expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RAR alpha agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RAR beta promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RAR beta activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RAR beta expression. These data suggest that SIN3A/MAD1 acts as a second RAR beta repressor and may be involved in fine-tuning retinoid sensitivity.