[P1-06-09] Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2.
Viswanadhapalli S, Mann M, Sareddy GR, Xaionan L, Vankayalapati H, Brann D, Vadlamudi RK.
UT Health Sciences Center at San Antonio, San Antonio, TX.
Oncolexis Therapeutics, Inc., Dallas, TX.
Medical College of Georgia, Augusta, GA.
BACKGROUND: Evolving evidence suggests that cancer stem cells (CSCs) evade hormonal therapy and therapy resistance occurs due to regrowth of tumor cells from cancer stem cells that escaped hormonal therapy or remained in the body after tumor resection. Recent studies suggest that estrogen stimulates breast cancer stem-cells and G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene that functions as a critical coregulator of several nuclear receptors and other transcription factors. PELP1 is commonly overexpressed in hormone-related cancers, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered PELP1 interacts with G9a/EHMT2. However, it remains unknown whether PELP1-G9a signaling plays a role in breast cancer stem cell proliferation. The objective of this study is to develop small molecular inhibitors that block G9a/EHMT2 interactions and to test their utility.
METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. To test the effect of PELP1 inhibitors on CSCs, we cultured CSCs in SFM in the presence or absence of PELP1 inhibitors for a period of 7-10 days. 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.
RESULTS: Using mapping studies, 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 (#20, #29, #34) showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in assays using three different breast cancer cell lines. Further, PELP1 targeting peptidomimetic disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen therapy 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. Accordingly, in mammosphere formation assays, PELP1 targeting peptidomimetic significantly inhibited the formation of mammospheres and the size of the mammospheres was also substantially decreased. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity.
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.
Wednesday, December 9, 2015 5:00 PM
Poster Session 1: Tumor Cell and Molecular Biology: Stem/Progenitor Cells (5:00 PM-7:00 PM)
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