TGF-betas in breast cancer progression

TGF-β 在乳腺癌进展中的作用

• 批准号: 8763004
• 负责人: Lalage Wakefield
• 金额: $ 81.75万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763004
• 关键词: Address; Affect; Area; Basement membrane; Behavior; Binding; Biological; Biological Markers; Biological Models; Breast; Breast Cancer Cell; Breast Cancer Model; Cell Line; Cells; Chromatin; Clinical; Clinical Oncology; Complex; Confocal Microscopy; Coupled; Data Set; Development; Diagnosis; Disease Outcome; Distant Metastasis; Ephrins; Epithelial; Epithelial Cells; Estrogen receptor positive; Exclusion; Fluorescence Microscopy; Functional Imaging; Gene Expression Profile; Genes; Genetic; Genomics; Goals; Homeostasis; Human; Image; In Vitro; Inhibition of Cell Proliferation; Lentivirus Vector; Ligands; Maintenance; Malignant - descriptor; Malignant Neoplasms; Mediating; Mediator of activation protein; Meta-Analysis; Mitosis; Modeling; Molecular; Molecular Profiling; Neoplasm Metastasis; Noise; Normal tissue morphology; Operative Surgical Procedures; Pathway interactions; Patients; Play; Population; Pre-Clinical Model; Premalignant; Process; Production; Publishing; Relapse; Reporter; Resistance; Role; Sampling; Series; Signal Transduction; Staging; Stem cells; System; Testing; Therapeutic; Time; Transforming Growth Factor beta; Transgenic Mice; Transgenic Model; Transplantation; Tumor Promotion; Tumor Suppression; Tumor Suppressor Proteins; Woman; Work; Xenograft Model; Xenograft procedure; base; cancer stem cell; cancer therapy; cancer type; carcinogenesis; cell killing; cell type; chromatin immunoprecipitation; design; established cell line; functional genomics; genetic regulatory protein; genome-wide; in vivo; insight; malignant breast neoplasm; member; mouse model; neoplastic cell; novel; novel strategies; prevent; response; self-renewal; stem cell biology; stem cell population; therapeutic target; transcription factor; transcriptomics; tumor; tumor progression; tumorigenesis

In FY13, we have continued to focus primarily on elucidating the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor. Our main experimental platform is a xenograft model of breast cancer progression based on the MCF10A human breast epithelial cell line. We have previously demonstrated that TGF-beta switches from tumor suppressor to pro-progression factor in this model, and the high degree of genetic relatedness between the different cell lines of the progression series gives us an exceptionally high signal-to-noise system in which to address mechanisms underlying the TGF-beta switch. However, this year we have also established a number of new breast cancer cell strains from freshly excised patient tumors to extend our findings to primary cells. Our work in FY13 has focused in two main areas: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY13 we have continued to validate and employ a novel functional imaging approach for the identification of the putative cancer stem cell (CSC) population. Our CSC reporter responds to the presence of the stem cell master transcription factors Oct4 and Sox2, and marks cells that are enriched for CSC activities, including the ability to divide asymmetrically, to resist cell killing by conventional chemotherapeutics, and to initiate tumorigenesis and metastasis in vivo. We confirmed that the approach works in primary human breast cancer cells as well as in established cell lines, and we have made a number of advances in the lentiviral vector design to increase sensitivity. We are now exploiting this reporter using a variety of approaches including timelapse fluorescence microscopy in vitro and confocal microscopy on tumors ex vivo to address factors that regulate CSC localization, plasticity and behavior, and to screen for therapeutics that target the CSC population. A major focus is the role of TGF-beta superfamily members in CSC biology. Using a xenograft breast cancer model in which TGF-beta functions as a tumor suppressor, we have combined our stem cell reporter with a TGF-beta pathway reporter and shown that endogenous TGF-beta signaling is activated more highly in the CSCs compared with the bulk population, suggesting a particularly important role for TGF-betas in the stem cell compartment. CSCs with endogenous TGF-beta signaling active were intrinsically less proliferative than CSCs that had not activated the pathway. Furthermore, we showed that TGF-beta selectively inhibited asymmetric self-renewing mitoses in the CSC compartment, and that it specifically inhibited the invasion of CSCs through basement membrane, while having little effect on the invasion of the bulk tumor cell population. These observations show that TGF-beta is an important modulator of CSC biology, and identify new mechanisms that could underlie the tumor suppressive effects of TGF-beta. To extend our imaging approach from transplanted to spontaneously arising tumors, we have successfully generated transgenic mice expressing the stem cell reporter. Understanding how CSCs are regulated will be critical to development of more effective cancer therapies as these cells are largely resistant to existing therapeutic approaches, leading ultimately to relapse. 2. INSIGHTS INTO TGF-BETA-MEDIATED EFFECTS ON TUMORIGENESIS FROM GENOMIC APPROACHES. TGF-beta antagonists are being developed as cancer therapeutics. However, the complex role of TGF-beta in cancer progression makes it imperative to avoid treating patients whose tumors still have intact tumor suppressive responses to TGF-beta. Currently it is not clear whether the tumor suppressive responses to TGF-beta are still retained by any breast cancers at the time of diagnosis and surgery. To address this question, we chose to develop a TGF-beta response signature that specifically reflects the tumor suppressive effects, since published TGF-beta signatures were not designed a priori to distinguish the tumor suppressive responses from the neutral or tumor promoting responses. Using the MCF10-based model of human breast cancer progression, we applied integrated genome-wide chromatin immunoprecipitation and transcriptomic approaches in vitro and in vivo to specifically dissect out a core gene signature that is associated with TGF-beta/Smad3-mediated tumor suppression. In a meta-analysis of more than 1300 human breast cancers, high expression of this signature associated with good distant metastasis-free survival in women with estrogen receptor positive (ER+) breast cancer, suggesting that the tumor suppressor effects of TGF-beta are still active and affecting disease outcome in a subset of patients. We have continued to probe the underlying biological and molecular mechanisms, and we demonstrated that TGF-beta-induced inhibition of cell proliferation and induction of cellular differentiation both contribute to tumor suppression. We have further identified a functional role for Ephrin signaling in mediating the tumor suppressive effects of TGF-beta. However, we have shown that the TGF-beta-regulated transcriptome is highly context-dependent, since the downstream transcriptional mediator, Smad3, appears only to bind into regions of chromatin that are already transcriptionally active. As a result, we believe that it will be necessary to build tailored TGF-beta signatures for different tumor types, and that there will be no single signature that will serve as a biomarker of TGF-beta tumor suppression in all cancer types. This finding has important implications for the development of signature-based biomarkers to use for patient inclusion/exclusion in clinical oncology trials with TGF-beta antagonists. We are also addressing effects of TGF-beta on the miRome in the same MCF-10-based model system to build an integrated molecular mechanistic picture of the changing role of TGF-beta in breast cancer progression. To this end, we have performed Next-Gen sequencing of the basal and TGF-beta-regulated miRomes of four of the MCF10-derived cell lines representing normal, premalignant, low-grade malignant and high-grade malignant stages in breast cancer progression. Analysis of these datasets is ongoing.

在2013财年，我们继续专注于阐明TGF-β从肿瘤抑制剂到促进因子的肿瘤细胞自治成分。我们的主要实验平台是基于MCF10A人乳腺上皮细胞系的乳腺癌进展的异种移植模型。我们先前已经证明，在该模型中，TGF-beta从肿瘤抑制剂转变为促进因子，并且进展系列的不同细胞系之间的高度遗传相关性为我们提供了一个异常高的信号到噪声系统，在该系统中，该系统可以解决TGF-beta开关的基础机制。但是，今年，我们还从新鲜切除的患者肿瘤中建立了许多新的乳腺癌细胞菌株，以将我们的发现扩展到原代细胞。我们在2013财年的工作集中在两个主要领域：1。TGF-beta在调节癌症干细胞动力学中的作用。在2013财年期间，我们继续验证并采用一种新型的功能成像方法来鉴定假定的癌症干细胞（CSC）种群。我们的CSC报告基因对干细胞主转录因子Oct4和Sox2的存在做出了反应，并标记了富含CSC活性的细胞，包括不对称分裂的能力，通过传统的化学治疗剂抗拒细胞杀死细胞，并启动肿瘤发生和体内的转移。我们确认该方法在原发性人类乳腺癌细胞以及已建立的细胞系中起作用，并且我们在慢病毒载体设计中取得了许多进步，以提高敏感性。现在，我们正在使用多种方法来利用此记者，包括在体外进行及时荧光显微镜和肿瘤的共聚焦显微镜，以解决调节CSC定位，可塑性和行为的因素，并筛选针对CSC种群的疗法。主要重点是TGF-Beta超家族在CSC生物学中的作用。使用异种移植乳腺癌模型，其中TGF-beta充当肿瘤抑制剂，我们将干细胞报告基因与TGF-BETA途径记者相结合，并表明内源性TGF-BETA信号在CSC中激活了更高的激活，与大量的人群相比，TGF-Betas在TGF-Betas中的作用非常重要。具有内源性TGF-β信号传导活性的CSC本质上比未激活该途径的CSC的CSC具有较小的增殖。此外，我们表明TGF-beta在CSC室中有选择地抑制了不对称的自我更新有丝分裂，并且它特别抑制了通过基底膜侵袭CSC的侵袭，同时对散发肿瘤细胞种群的侵袭几乎没有影响。这些观察结果表明，TGF-beta是CSC生物学的重要调节剂，并确定可以构成TGF-β抑制肿瘤抑制作用的新机制。为了将成像方法从移植到自发产生的肿瘤扩展，我们成功地产生了表达干细胞报告基因的转基因小鼠。了解如何调节CSC对于开发更有效的癌症疗法至关重要，因为这些细胞在很大程度上对现有的治疗方法具有抵抗力，最终导致复发。 2。对TGF-β介导的对基因组方法肿瘤发生的影响的见解。 TGF-β拮抗剂正在作为癌症疗法发展。然而，TGF-β在癌症进展中的复杂作用使得避免治疗肿瘤仍然对TGF-β的肿瘤完全抑制反应的患者。目前，尚不清楚在诊断和手术时，任何乳腺癌仍保留了对TGF-β的肿瘤抑制反应。为了解决这个问题，我们选择开发一个特异性反映肿瘤抑制作用的TGF-β反应签名，因为发表的TGF-beta特征并未设计为将肿瘤抑制反应与中性或肿瘤促进反应区分开的先验性。使用基于MCF10的人类乳腺癌进展模型，我们在体外和体内应用了整合的全基因组染色质免疫沉淀和转录组方法，以特别剖析与TGF-β/SMAD33-介导的肿瘤抑制的核心基因签名。在对1300多种人类乳腺癌的荟萃分析中，这种特征的高表达与雌激素受体阳性（ER+）乳腺癌女性良好远处转移生存有关，这表明TGF-β的肿瘤抑制剂抑制作用仍然是活跃的，并且在患者子群中仍具有活性并影响疾病。我们继续探测潜在的生物学和分子机制，并证明TGF-beta诱导的细胞增殖抑制和细胞分化的诱导都会导致肿瘤抑制。我们进一步确定了源自源自TGF-β的肿瘤抑制作用在埃弗林信号传导中的功能作用。但是，我们已经表明，TGF-BETA调节的转录组高度取决于上下文，因为下游转录介质SMAD3似乎仅结合已经转录活性的染色质区域。结果，我们认为有必要为不同的肿瘤类型建立量身定制的TGF-β签名，并且在所有癌症类型中都不会有单一的签名作为TGF-β抑制TGF-β肿瘤的生物标志物。这一发现对开发基于签名的生物标志物的开发具有重要意义，以便在TGF-beta拮抗剂的临床肿瘤学试验中使用患者包容/排除。我们还在基于MCF-10的模型系统中解决TGF-BETA对Mirome的影响，以构建TGF-β在乳腺癌进展中作用不断变化的综合分子机械图。为此，我们对MCF10衍生的四种细胞系中的四种基础和TGF-Beta调节的miomes进行了下一代测序，这些细胞系代表乳腺癌进展中正常，预抗剂，低度恶性肿瘤和高级恶性阶段。这些数据集的分析正在进行中。