Abstract 1189: Combination treatments with retinoic acid and the synthetic retinoid ST1926 in 2D and 3D breast cancer models overcome retinoic acid resistance and eradicate breast cancer stem/progenitor cells
Authors: Aouad Patrick , Saikali Melody , Abdel-Samad Rana , El–Houjeiri Leeanna , Pisano Claudio , Talhouk Rabih , Darwiche Nadine 
Published: July 2016

Abstract
Despite recent advances in breast cancer therapy, achieving complete remission in metastatic breast cancer patients remains a challenge. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is acquired resistance. Therefore, synthetic retinoids such as ST1926 emerged as potent anti-cancer agents. Here, we investigated the anti-tumor activities of ATRA, ST1926, and their combination treatments in 2D and 3D human breast cancer models and their targeting of breast cancer stem cells (CSCs)/progenitor cells. We have shown that in 2D culture models, MCF-7 and MDA-MB-231 cells are resistant to ATRA while being sensitive to ST1926 at sub-micromolar (μM) concentrations in an irreversible manner. Importantly, ST1926 had no effect on the ‘normal-like’ MCF-10A breast epithelial cells. ST1926 induced massive apoptosis in MCF-7 cells and it resulted in S-phase arrest and necrosis in the triple negative and metastatic MDA-MB-231 cells. Furthermore, ST1926 caused early DNA damage, increased the expression of the tumor suppressors p53 and p21, downregulated the Wnt/β-catenin pathway, and modulated the expression levels of the different retinoid receptors. Interestingly, combination treatments as low as 0.1 μM ST1926 and 0.5 μM ATRA synergistically inhibited the proliferation in 2D models of MCF-7 and MDA-MB-231 cells, independently of retinoid receptor signaling, while sparing the normal breast epithelial cells. Anchorage-independent growth of MCF-7 and MDA-MB-231 cells was examined using the soft agar colony formation assay where sub-μM concentrations of ST1926 or μM concentrations of ATRA were shown to reduce the size and the number of breast cancer colonies. ST1926 drastically induced cell death in 3D Matrigel ‘on-top’ assay cultures of breast cancer cells while the lumen of the normal-like breast epithelial cell line S1 was maintained. Finally, treatment with 0.01 μM ST1926 alone or 0.001 μM ST1926 in combination with 0.1 μM ATRA abrogated sphere formation and the self-renewal ability of breast CSCs in the 3D sphere formation assay. In summary, ST1926, ATRA, and their combination treatments were shown to display more potent anti-tumor properties in 3D versus 2D human breast cancer models. Our results also demonstrate the therapeutic potential of ST1926 in sensitizing breast cancer cells to ATRA and in targeting the population of breast CSCs. As 3D culture models are more representative of the tumor microenvironment and serve as valid tools in drug discovery, our results highlight the promising use of ATRA/ST1926 combinations in metastatic and triple negative breast cancers.
Citation Format: Patrick Aouad, Melody Saikali, Rana Abdel-Samad, Leeanna El–Houjeiri, Claudio Pisano, Rabih Talhouk, Nadine Darwiche. Combination treatments with retinoic acid and the synthetic retinoid ST1926 in 2D and 3D breast cancer models overcome retinoic acid resistance and eradicate breast cancer stem/progenitor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1189.