Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment

定义卵巢癌微环境中癌相关间充质干细胞的形成和功能

• 批准号: 10006503
• 负责人: Lan Coffman
• 金额: $ 16.61万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006503
• 关键词: Acidosis; Activities of Daily Living; Adipocytes; Bone Marrow; Bone Morphogenetic Proteins; Carcinoma; Cell Hypoxia; Cell physiology; Cells; Chemicals; Coculture Techniques; Communication; Complex; DNA Methylation; Data; Development; Diffuse; Education; Elements; Epigenetic Process; Exhibits; Expression Profiling; Fibroblasts; Foundations; Future; Genotype; Goals; Growth; HIF1A gene; Hyperactive behavior; Hypoxia; Hypoxia Inducible Factor; Hypoxia-Inducible Factor Pathway; In Vitro; Intra-abdominal; Knock-out; Knockout Mice; Knowledge; Leadership; Learning; Malignant - descriptor; Malignant Female Reproductive System Neoplasm; Malignant Neoplasms; Malignant neoplasm of ovary; Mediator of activation protein; Mentors; Mentorship; Mesenchymal Stem Cells; Modification; Molecular; Multipotent Stem Cells; Mutation; Myofibroblast; Neoplasm Metastasis; Non-Malignant; Normal Cell; Normal tissue morphology; Phenotype; Physicians; Play; Population; Property; Research; Research Training; Resistance; Role; Scientist; Signal Pathway; Signal Transduction; Site; Stromal Cells; Stromal Neoplasm; System; Testing; Training; Transgenic Mice; Treatment outcome; Tumor Promotion; Woman; Work; behavior influence; cancer cell; cancer stem cell; career; cell growth; cell type; chemotherapy; conditioning; epigenetic regulation; genome editing; genome-wide; improved outcome; in vivo; innovation; intraperitoneal; migration; molecular phenotype; mortality; mouse model; multipotent cell; neoplastic cell; novel; novel therapeutic intervention; novel therapeutics; ovarian neoplasm; protein expression; skills; small molecule; stem-like cell; stemness; success; tumor; tumor growth; tumor microenvironment; tumorigenic

ABSTRACT: Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment Ovarian cancer is the most deadly US gynecologic malignancy with a mortality rate that exceeds 50% at 5 years. Ovarian cancer is characterized by early intraperitoneal metastasis and the development of a complex microenvironment which supports tumor cell growth, survival and spread. Understanding and eventually targeting this cancer-promoting tumor microenvironment offers the potential for powerful new therapeutic approaches. My ultimate goal is to become a world-class independent physician scientist studying the ovarian cancer microenvironment in order to develop new treatments and improve outcomes for women with ovarian cancer. This proposal describes important and innovative research which will lay the foundation for my future career in addition to providing the necessary skills and mentorship vital for my success. The ovarian tumor microenvironment (TME) is a diverse system of cellular and chemical components. The cellular TME includes tumor cells and non-malignant stromal cells. The chemical TME is marked by acidosis and hypoxia. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are multi-potent stromal cells within the cellular TME that can differentiate into multiple pro-tumorigenic stromal cell types including fibroblasts, myofibroblasts, and adipocytes. CA-MSCs are genotypically normal without malignant potential but are functionally different than normal tissue or bone marrow derived MSCs. Compared to normal MSCs, CA-MSCs demonstrate a unique molecular phenotype with very high expression of bone morphogenetic proteins (BMPs). Due to this unique phenotype, these CA-MSCs strongly promote ovarian cancer growth, enhance chemotherapy resistance and enrich the cancer stem cell-like population. How CA-MSCs develop their unique phenotype remains unclear. My preliminary data indicate that tumor secreted factors induce some of the molecular changes associated with CA-MSCs. Another potential mediator of the CA-MSC phenotype is hypoxia. Hypoxia is a hallmark of the chemical TME known to impact normal MSC function. In cancer, hypoxia influences tumor:stromal interactions and hypoxia is a key regulator of BMP expression—high levels of which characterize ovarian cancer CA-MSCs. Preliminary data indicates that hypoxia enhances the ability of tumor cells to induce a CA-MSC expression profile in normal MSCs. While the mechanism of this induction is unknown, given CA-MSCs are genetically normal yet maintain their unique phenotype across multiple passages, tumor-induced epigenetic regulation may be critical to the formation of the CA-MSC phenotype. Indeed, preliminary data indicates CA-MSCs exhibit significant hypomethylation compared to normal MSCs. In addition to influencing the formation of a CA-MSC, hypoxia may also critically regulate the function of CA- MSCs already established in the ovarian TME. My preliminary data suggests that hypoxia maintains the “stemness” of CA-MSCs slowing growth and maintaining differentiation capacity. Further, my data suggests that the hypoxia inducible factor pathway, the main hypoxia signaling pathway, is hyper-active in CA-MSCs compared to normal MSCs. Thus hypoxia may be a critical modulator of CA-MSCs within the ovarian TME. My main research goal is to understand how CA-MSCs obtain their unique phenotype and subsequently interact with and influence the function of the ovarian cancer microenvironment. To achieve this goal, I propose two specific aims: 1) Determine the ability of normal MSCs to acquire a CA-MSC-like phenotype 2) Determine the impact of hypoxia on established CA-MSCs within the tumor microenvironment. In aim 1, I hypothesize that tumor cell conditioning under hypoxia induces normal MSCs to become CA-MSCs. To test this I will perform cancer cell: normal MSC co-cultures under normoxia and hypoxia to determine if cancer cells can functionally turn a normal MSC into a CA-MSC. I will also explore differential DNA methylation as a mechanism for the creation of a CA-MSC. In aim 2, I focus on already established CA-MSCs. I hypothesize that hypoxia enhances the pro-tumorigenic effects of established CA-MSCs within the tumor microenvironment. To test this, I will utilize conditional HIF pathway knockout mice and CRISPER/CAS9 genome editing to assess the impact of hypoxia and HIF signaling on established CA-MSCs. In addition to furthering our understanding of CA-MSCs in ovarian cancer, the proposed research and training will cultivate expertise necessary for an independent career studying the ovarian TME. Through the support of Dr. Laird, I will master the assessment of genome-wide epigenetic modifications and the analysis of large scale “omics” data. Dr. Schipani will facilitate my education in hypoxia and HIF signaling. Through Dr. Schipani and Dr. Cho, I will learn to generate and manipulate transgenic mouse models. Dr. Buckanovich's ongoing mentorship will further my expertise in the function of the ovarian TME and, together with my mentoring committee, will help develop my leadership, team-building and communication skills. By the end of the training period, I will have developed a novel skill set which merges the expertise of multiple scientific leaders yielding a uniquely trained physician scientist ideal for the study of the ovarian cancer microenvironment.

摘要：定义癌症相关间充质干细胞的形成和功能 卵巢癌微环境 卵巢癌是美国最致命的妇科恶性肿瘤，5岁时死亡率超过50% 年。卵巢癌的特点是早期腹膜内转移和复杂的发展 支持肿瘤细胞生长、存活和扩散的微环境。理解并最终 针对这种促癌肿瘤微环境提供了强大的新疗法的潜力 接近。我的最终目标是成为研究卵巢的世界级独立医师科学家 癌症微环境，以开发新的治疗方法并改善卵巢癌女性的预后 癌症。该提案描述了重要且创新的研究，将为我的未来奠定基础 除了提供对我的成功至关重要的必要技能和指导之外，我的职业生涯也是如此。 卵巢肿瘤微环境（TME）是一个由细胞和化学成分组成的多样化系统。这 细胞TME包括肿瘤细胞和非恶性基质细胞。化学TME的特点是酸中毒 和缺氧。癌相关间充质干细胞 (CA-MSC) 是多能基质细胞 细胞 TME 可以分化成多种促肿瘤基质细胞类型，包括成纤维细胞， 肌成纤维细胞和脂肪细胞。 CA-MSCs 基因型正常，无恶性潜能，但 其功能与正常组织或骨髓来源的 MSC 不同。与正常MSCs相比，CA-MSCs 表现出独特的分子表型，骨形态发生蛋白（BMP）的表达非常高。 由于这种独特的表型，这些 CA-MSC 强烈促进卵巢癌的生长，增强 化疗耐药性并丰富癌症干细胞样群。 CA-MSCs 如何形成其独特的表型仍不清楚。我的初步数据表明肿瘤 分泌因子诱导一些与 CA-MSC 相关的分子变化。另一个潜在的调解人 CA-MSC表型的主要特征是缺氧。缺氧是化学 TME 的一个标志，已知会影响正常 间充质干细胞功能。在癌症中，缺氧影响肿瘤：基质相互作用和缺氧是 BMP 的关键调节因子 表达——高水平表达是卵巢癌 CA-MSC 的特征。初步数据表明 缺氧增强了肿瘤细胞在正常 MSC 中诱导 CA-MSC 表达谱的能力。虽然 鉴于 CA-MSC 基因正常但仍保持其独特性，这种诱导机制尚不清楚 跨越多个传代的表型，肿瘤诱导的表观遗传调控可能对于形成 CA-MSC 表型。事实上，初步数据表明 CA-MSC 表现出显着的低甲基化 与正常MSC相比。 除了影响 CA-MSC 的形成外，缺氧还可能关键地调节 CA-MSC 的功能。 MSC 已在卵巢 TME 中建立。我的初步数据表明缺氧可以维持 CA-MSCs的“干性”减缓生长并保持分化能力。此外，我的数据表明 缺氧诱导因子途径（主要的缺氧信号传导途径）在 CA-MSC 中高度活跃 与正常MSC相比。因此，缺氧可能是卵巢 TME 内 CA-MSC 的关键调节剂。 我的主要研究目标是了解 CA-MSCs 如何获得其独特的表型，并随后 与卵巢癌微环境相互作用并影响其功能。为了实现这个目标，我建议 两个具体目标： 1) 确定正常MSC获得CA-MSC样表型的能力 2) 确定缺氧对肿瘤微环境中已建立的 CA-MSC 的影响。 在目标 1 中，我假设肿瘤细胞在缺氧条件下的调理会诱导正常 MSC 转变为 CA-MSC。 为了测试这一点，我将在常氧和缺氧下进行癌细胞：正常 MSC 共培养，以确定是否 癌细胞可以在功能上将正常 MSC 转变为 CA-MSC。我还将探索差异 DNA 甲基化 作为创建 CA-MSC 的机制。在目标 2 中，我重点关注已经建立的 CA-MSC。我 假设缺氧增强了肿瘤内已建立的 CA-MSC 的促肿瘤作用 微环境。为了测试这一点，我将利用条件 HIF 通路敲除小鼠和 CRISPER/CAS9 基因组编辑评估缺氧和 HIF 信号对已建立的 CA-MSC 的影响。 除了进一步加深我们对卵巢癌中 CA-MSC 的了解外，拟议的研究和培训 将培养研究卵巢 TME 的独立职业所需的专业知识。通过支持 Laird博士，我将掌握全基因组表观遗传修饰的评估和大规模的分析 “组学”数据。 Schipani 博士将促进我在缺氧和 HIF 信号传导方面的教育。通过 Schipani 博士和 Cho 博士，我将学习生成和操作转基因小鼠模型。巴卡诺维奇博士正在进行的 指导将进一步提升我在卵巢 TME 功能方面的专业知识，并且与我的指导一起 委员会，将帮助培养我的领导能力、团队建设和沟通能力。培训结束时 在此期间，我将开发出一套新颖的技能，融合了多个科学领导者的专业知识，从而产生 一位经过独特培训的医师科学家，非常适合研究卵巢癌微环境。