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

摘要 长非编码 RNA (lncRNA) 是染色质组织的表观遗传调节因子，支持 癌细胞的活动。在确定建立染色质相互作用的文献中存在空白 浸润性乳腺癌 (BCa) 细胞的表型。我们已经表征了一个 lncRNA，我们将其命名为 MANCR（有丝分裂相关非编码 RNA (Tracy K Mol Can Res 2018)）在 侵袭性三阴性和他莫昔芬耐药 (TAMR) BCa 细胞系，并在 有丝分裂。具有临床意义的 MANCR 抑制会导致细胞死亡，因此具有作为治疗药物的潜力 预防侵袭性肿瘤生长的治疗目标。要实现这一转化目标需要一个完整的 了解 MANCR 在非恶性细胞中被抑制并在 在侵袭性癌细胞中被激活，在正常乳腺上皮细胞（NME）中被抑制。我们的 研究结果支持 MANCR 通过染色质改变表观遗传环境的假设 与支持侵袭性乳腺癌细胞生存机制的基因组区域的相互作用； MANCR 的抑制将有助于体内细胞死亡和肿瘤消退。具体的 目的旨在获得多个层面的机械洞察力，以确定 MANCR 功能 维持攻击性细胞表型。 Aim1 识别染色质组织介导的 MANCR 的表观遗传特性通过使用以下方式支持 BCa 存活： i) ChIRP-Seq 揭示 MANCR 特定基因组位置的靶基因结合位点； ii) ATAC-seq 以获得可访问的地图 染色质区域将识别侵袭性 MDA-231 细胞支持细胞中的基因组调控 存活率并与 MANCR 敲低改变的那些域进行比较，这将揭示机制 导致细胞死亡； iii) Capture Hi-C 将发现 3 维染色质相互作用 有助于侵袭性 BCa 细胞的存活。目标 2 重点关注有助于 MANCR 的机制 NME 和 MCF7 ER BCa 细胞中的抑制。我们将 i) 检查 MANCR 抑制是否是介导的 通过 ER 细胞中的雌激素信号传导，ii) 检查 MANCR 对 TAMR 的影响； iii) 证明 MANCR 正常乳腺上皮细胞的抑制是由肿瘤抑制因子 RUNX1 介导的。目标 3 将使用两种体内模型：i) 临床前异种移植小鼠来证明 MDA-231 CRISPR 抑制 乳腺脂肪垫内的肿瘤会因细胞死亡而坏死； ii) 切除的BCa肿瘤组织 MANCR 呈阳性，可以生长类器官移植到乳腺脂肪垫以确定是否高 与低表达的 MANCR 类器官相比，MANCR 类器官具有侵袭性的肿瘤生长。 影响：MANCR 表达与不良预后和生存相关，其抑制会导致细胞 除睾丸和脾脏外，所有正常组织中几乎不存在 MANCR。这些属性 表明 MANCR 作为抑制晚期 BCa 原发肿瘤生长的治疗靶点的潜力。

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.