Project 3: MANCR Mediates Epigenetic Mechanisms for Survival of Advanced Breast Cancer

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SUMMARY Long non-coding RNAs (lncRNAs) are epigenetic regulators of the chromatin organization supporting cancer cell activities. There is a gap in the literature identifying the chromatin interactions that establish the phenotype of invasive breast cancer (BCa) cells. We have characterized a lncRNA, we named MANCR (Mitotically Associated Non-Coding RNA (Tracy K Mol Can Res 2018) which is highly expressed in aggressive triple negative and tamoxifen resistant (TAMR) BCa cell lines and increases further during mitosis. Of clinical significance, MANCR inhibition results in cell death, thus having potential as a therapeutic target to prevent invasive tumor growth. To reach this translational goal requires a complete understanding of mechanisms by which MANCR is suppressed in non-malignant cells and activated in activated in aggressive cancer cell and suppressed in normal mammary epithelial cells (NMEs). Our findings support the hypothesis that MANCR alters the epigenetic environment via chromatin interactions with genomic regions that support survival mechanisms in invasive breast cancer cells; and that inhibition of MANCR will contribute to cell death and regression of tumors in vivo. The specific aims are designed to obtain mechanistic insight at multiple levels to determine the MANCR functions that sustain the aggressive cell phenotype. Aim1 identifies chromatin organization mediated by MANCR’s epigenetic properties that support BCa survival by using: i) ChIRP-Seq to reveal MANCR target gene binding sites at specific genomic locations; ii) ATAC-seq to obtain a map of accessible chromatin regions that will identify genomic regulatory in aggressive MDA-231 cell supporting cell survival and comparing to those domains altered by MANCR knockdown which will reveal mechanisms resulting in cell death; iii) Capture Hi-C will discover the 3-dimensional chromatin interactions contributing to survival of invasive BCa cells. Aim 2 focuses on mechanisms contributing to MANCR suppression in NMEs and MCF7 ER+ BCa cells. We will i) examine if MANCR suppression is mediated by estrogen signaling in ER+ cells, ii) examine MANCRs effect on TAMR; and iii) show that MANCR suppression in normal mammary epithelial cells is mediated by the tumor suppressor RUNX1. Aim 3 will use two in vivo models: i) a pre-clinical xenograft mouse to demonstrate MDA-231 CRISPR inhibited tumors in the mammary fat pad will become necrotic due to cell death; ii) resected BCa tumor tissue that is MANCR positive to grow organoids for transplantation into mammary fat pad to determine if high MANCR organoids have aggressive tumor growth compared to low expressing MANCR organoids. Impact: MANCR expression correlates with poor prognosis and survival, its inhibition results in cell death and MANCR is nearly absent from all normal tissues except testis and spleen. These properties indicate MANCR’s potential as a therapeutic target to inhibit primary tumor growth of advanced BCa.