Abstract 4024: ING4 tumor suppressor binds to estrogen receptor-complex and modulates estrogen-dependent cell growth and gene expression in breast cancer

Abstract

Abstract The tumor suppressor ING4 (Inhibitor of Growth family member 4) has been shown to play a role in diverse cancer-related cellular processes, including gene regulation, apoptosis, cell migration, contact inhibition, and angiogenesis. Deletion or reduced expression of ING4 has been shown in a number of human malignancies, including breast cancer. We found ING4 to be deleted in 15% of estrogen receptor-positive (ER+) breast cancers, implicating a role for ING4 as a tumor suppressor in estrogen-driven breast cancer. In addition, we showed that over-expression of ING4 abrogated, whereas ING4 knock-down enhanced, estrogen-dependent cell growth of MCF7 ER+ breast cancer cells, indicating a suppressive function of ING4 in estrogen-dependent cell growth. The molecular mechanisms of ING4 in ER+ breast cancers have not yet been elucidated. Using a bacterial two-hybrid screen, we identified CCAR1 (Cell-Cycle and Apoptosis Regulatory Protein 1) as an ING4 binding protein. Subsequently, we validated interactions between ING4 and CCAR1 in 293T and MCF7 cells using co-immunoprecipitation assays. CCAR1 had previously been shown to be a co-activator of the estrogen receptor (ER) complex that is required for estrogen-dependent cell growth and gene transcription in ER+ breast cancer cells (Kim JH, Yang CK, Heo K, Roeder RG, An W, and Stallcup MR, 2008). Taken together, these results suggested that ING4 may interact with the ER-complex and implicated a role for ING4 in the ER-mediated cellular processes. Here we show that ING4 co-immunoprecipitates with estrogen receptor-alpha (ERα) and the co-activator AIB1 in MCF7 and T47D ER+ breast cancer cells, demonstrating ER:ING4 interactions in cells. Treatment with 17β-estradiol (E2) reduced ING4:ER interactions. These results suggested that ING4 may not be a co-factor in the estrogen-bound active ER-complex, supporting our hypothesis that ING4 plays a role in the modulation of the ER-complex. Strikingly, tamoxifen treatment greatly enhanced ING4 association with ERα, suggesting that tamoxifen stabilizes ER:ING4 interactions and/or ING4 has a greater binding affinity to the tamoxifen-bound ER-complex. It is possible that ING4 plays a role in the recruitment of co-repressors to the tamoxifen-bound ER-complex. These results implicate a role for ING4 in tamoxifen response. We conclude that ING4 may function as a tumor suppressor in ER+ breast cancers by binding to the ER-complex and modulating estrogen-dependent cellular processes including cell proliferation and gene transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2011-4024