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

摘要 长非编码RNA(LncRNAs)是染色质组织支持的表观遗传调节因子 癌细胞的活动。在确定染色质相互作用建立的文献中有一个空白 浸润性乳腺癌(BCA)细胞表型。我们已经描述了一种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 侵袭性三阴性和三苯氧胺耐药(TamR)BCA细胞株，并在 有丝分裂。具有临床意义的是，MANCR抑制导致细胞死亡，因此具有潜在的 治疗的目标是防止侵袭性肿瘤的生长。要实现这一翻译目标，需要一个完整的 对MANCR在非恶性细胞中被抑制和在非恶性细胞中被激活的机制的理解 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 与支持浸润性乳腺癌细胞生存机制的基因组区域的相互作用； 抑制MANCR将有助于细胞死亡和体内肿瘤的消退。具体的 AIMS旨在获得多个层次的机械性洞察力，以确定MANCR功能 维持着侵略性的细胞表型。Aim1鉴定染色质组织由 MANCR支持BCA存活的表观遗传学特性：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 染色质区域将识别侵袭性MDA-231细胞支持细胞的基因组调控 存活并与MANCR基因敲除改变的结构域进行比较将揭示机制 导致细胞死亡；III)捕获Hi-C将发现三维染色质相互作用 有助于侵袭性BCA细胞的存活。目标2侧重于促成MANCR的机制 对NME和MCF7 ER BCA细胞的抑制作用。我们将i)检查MANCR抑制是否通过中介 通过雌激素在ER细胞中的信号转导，II)检测MANCR对TAMR的影响；以及III)显示MANCR 正常乳腺上皮细胞的抑制是由肿瘤抑制基因RUNX1介导的。目标3 将使用两个体内模型：i)临床前异种移植小鼠显示MDA-231 CRISPR被抑制 乳腺脂肪垫肿瘤会因细胞死亡而坏死；ii)切除BCA肿瘤组织 即MANCR阳性种植有机化合物用于移植到乳房脂肪垫中以确定是否高 与低表达的MANCR有机化合物相比，MANCR有机化合物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，抑制其在细胞中的结果 死亡和MANCR在除睾丸和脾以外的所有正常组织中几乎都不存在。这些属性 提示MANCR有可能成为抑制晚期BCA原发肿瘤生长的治疗靶点。

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.