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: 10608059
• 负责人: Jane B. Lian
• 金额: $ 41.86万
• 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/10608059
• 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 Death Induction; 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; Lung; MCF10A cells; MCF7 cell; MDA MB 231; Malignant Neoplasms; Maps; Mediating; Mediator; Metastatic breast cancer; Mitosis; Mitotic; Mus; Names; Necrosis; Neoplasm Metastasis; Non-Malignant; Normal Cell; Normal tissue morphology; Oncogenic; Organ; Organoids; Outcome; 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 Promotion; 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; genomic locus; 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.

概括 长非编码 RNA (lncRNA) 是染色质组织的表观遗传调节因子，支持 文献中存在一个空白，以确定建立染色质相互作用。 我们对浸润性乳腺癌 (BCa) 细胞的表型进行了表征，并将其命名为 lncRNA。 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 具有临床意义的 MANCR 抑制会导致细胞死亡，因此具有作为有丝分裂的潜力。 预防侵袭性肿瘤生长的治疗目标要达到这一转化目标需要一个完整的方案。 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。 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。 目的旨在获得多个层面的机械洞察力，以确定 MANCR 功能 维持侵袭性细胞表型的 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 以获得可访问的图谱 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 生存并通过 MANCR 敲低与这些域进行比较，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 促进侵袭性 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；以及 iii) 显示 MANCR 正常乳腺上皮细胞的抑制是由肿瘤抑制因子 RUNX1 介导的。 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 乳腺脂肪垫中的肿瘤会因细胞死亡而坏死；ii) 切除的 BCa 肿瘤组织； MANCR 阳性，可培养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。 表明 MANCR 作为抑制晚期 BCa 原发肿瘤生长的治疗靶点的潜力。