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

项目 3：MANCR 介导晚期乳腺癌生存的表观遗传机制

• 批准号: 10380073
• 负责人: Jane B. Lian
• 金额: $ 42.01万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380073
• 关键词: 3-Dimensional; ATAC-seq; Accounting; Address; Binding Sites; Biological Markers; Biopsy; Biopsy Specimen; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer Prevention; Breast Cancer Treatment; Breast Cancer cell line; Breast Epithelial Cells; CRISPR interference; CRISPR/Cas technology; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Chromatin; Chromosomal Rearrangement; Chromosome Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; DNA Repair; Data; Development; Diagnosis; Distal; ESR1 gene; Effectiveness; Environment; Epigenetic Process; Estradiol; Estrogen receptor positive; Estrogens; Fatty acid glycerol esters; Gene Expression; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Segment; Genomics; Goals; Hi-C; Intervention; Investigation; Literature; Location; Lung; MCF10A cells; MCF7 cell; MDA MB 231; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Metastatic breast cancer; Metastatic to; Mitosis; Mitotic; Mus; Names; Necrosis; Neoplasm Metastasis; Non-Malignant; Normal Cell; Normal tissue morphology; Oncogenic; Organ; Organoids; Outcome; Outcome Study; Patients; Phenotype; Pre-Clinical Model; Primary Neoplasm; Prognosis; Property; Publishing; RNA Binding; RNA purification; RUNX1 gene; Regulation; Regulatory Element; Resected; Resistance; Resolution; Signal Transduction; Site; Spleen; Supporting Cell; Tamoxifen; Testing; Testis; Therapeutic Intervention; Tissue Microarray; Transplantation; Tumor Suppressor Proteins; Tumor Tissue; Untranslated RNA; Xenograft Model; Xenograft procedure; advanced breast cancer; aggressive breast cancer; bone; breast cancer survival; cancer cell; cancer invasiveness; chromatin isolation by RNA purification sequencing; clinical candidate; clinically significant; cofactor; design; hormone therapy; in vivo; in vivo Model; innovation; insight; knock-down; malignant breast neoplasm; mammary; neoplastic cell; organoid transplantation; overexpression; pre-clinical; prevent; protein complex; therapeutic target; translational goal; triple-negative invasive breast carcinoma; tumor; tumor growth

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.

总结 长链非编码RNA（lncRNA）是染色质组织的表观遗传调节因子， 癌细胞活动。在鉴定建立免疫反应的染色质相互作用的文献中存在空白。 侵袭性乳腺癌（BCa）细胞的表型。我们描述了一种lncRNA，我们命名为 MANCR（有丝分裂相关非编码RNA）（Tracy K Mol Can Res 2018），在哺乳动物中高度表达， 侵袭性三阴性和他莫昔芬耐药（TAMR）BCa细胞系，并在 分裂。具有临床意义的是，MANCR抑制导致细胞死亡，因此具有作为抗肿瘤药物的潜力。 治疗靶点，以防止侵袭性肿瘤生长。要实现这一转化目标， 了解MANCR在非恶性细胞中被抑制和在恶性细胞中被激活的机制。 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 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中存活机制的基因组区域的相互作用; 并且MANCR的抑制将有助于体内细胞死亡和肿瘤消退。具体 目标是在多个层次上获得机械的洞察力，以确定MANCR的功能 维持细胞的攻击性表型。aim 1识别染色质组织介导的 MANCR的表观遗传特性通过使用以下来支持BCa存活：i）ChIRP-Seq揭示MANCR ii）ATAC-seq，以获得可访问的基因的图谱; 在侵袭性MDA-231细胞支持细胞中识别基因组调控的染色质区域 存活并与MANCR敲低改变的那些结构域进行比较，这将揭示机制 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侧重于有助于MANCR的机制 抑制NME和MCF 7 ER+ BCa细胞。我们将i）检查MANCR抑制是否介导 通过ER+细胞中的雌激素信号传导，ii）检查MANCR对TAMR的作用;和iii）显示MANCR 正常乳腺上皮细胞中的抑制由肿瘤抑制因子RUNX 1介导。目标3 i）临床前异种移植小鼠，以证明MDA-231 CRISPR抑制 乳房脂肪垫中的肿瘤将由于细胞死亡而坏死; ii）切除的BCa肿瘤组织 其对于生长用于移植到乳房脂肪垫中类器官是MANCR阳性的， 与低表达MANCR类器官相比，MANCR类器官具有侵袭性肿瘤生长。 影响：MANCR表达与不良预后和生存相关，其抑制导致细胞凋亡。 死亡和MANCR在除睾丸和脾外的所有正常组织中几乎不存在。这些属性 表明MANCR作为抑制晚期BCa原发性肿瘤生长的治疗靶点的潜力。