Abstract 1123: The role of proto-oncogene PELP1 in breast cancer stem cell maintenance and therapy resistance

BACKGROUND: Cancer stem cells (CSCs) are known to evade hormonal therapy and regrowth of tumor cells from cancer stem cells is a major clinical problem. Histone methyltransferase G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene, functions as a coregulator of nuclear receptors, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered that PELP1 interacts with G9a/EHMT2. The objective of this study is to develop small molecular inhibitors that block PELP1 interactions with G9a/EHMT2. METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. Effect of inhibitors on the cell viability of breast cancer cells was determined using cell titer glow assays. Xenograft studies were used to determine the in vivo efficacy of the inhibitors. RESULTS: Using yeast based genetic screen, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in MTT assays and in biochemical assays disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen and letrozole resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. In mammosphere assays, PELP1 targeting peptidomimetics significantly inhibited the formation of mammospheres. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. In xenograft assays using ZR-75 cells, PELP1 inhibitor significantly reduced the tumor growth. CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1-G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance. Citation Format: Suryavathi Viswanadhapalli, Monica Mann, Gangadhara Reddy Sareddy, Xaionan Lix, Hariprasad Vankayalapati, Ratna K. Vadlamudi. The role of proto-oncogene PELP1 in breast cancer stem cell maintenance and therapy resistance. [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 1123.