TGF-betas in breast cancer progression

TGF-β 在乳腺癌进展中的作用

• 批准号: 8937647
• 负责人: Lalage Wakefield
• 金额: $ 83.91万
• 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/8937647
• 关键词: Address; Affect; Area; Basement membrane; Behavior; Binding; Biological; Biological Markers; Breast Cancer Cell; Breast Cancer Model; Breast Epithelial Cells; Cancer cell line; Cell Line; Cells; Characteristics; Chromatin; Clinical; Clinical Oncology; Complex; Coupled; Development; Diagnosis; Disease Outcome; Distant Metastasis; Ephrins; Epithelial; Estrogen receptor positive; Exclusion; Functional Imaging; Gene Expression Profile; Genes; Genetic; Genomics; Goals; Homeostasis; Human; Image; In Vitro; Inhibition of Cell Proliferation; Ligands; MCF10A cells; Maintenance; 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; Process; Production; Proteins; Publishing; Reporter; Resistance; Role; Sampling; Series; Signal Transduction; Stem cells; System; Testing; Therapeutic; Time; Transforming Growth Factor beta; Transgenic Model; 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; functional genomics; genetic regulatory protein; genome-wide; in vivo; insight; malignant breast neoplasm; mouse model; neoplastic cell; novel; novel strategies; prevent; promoter; response; self-renewal; stem; stem cell biology; stem cell population; transcription factor; transcriptomics; tumor; tumor progression; tumorigenesis

In FY14, we have continued to analyze the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor, with a particular emphasis on effects on the cancer stem cell population. 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, 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 FY14 has focused in two main areas: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY14 we have continued to validate and employ a novel functional imaging approach for the identification of the cancer stem cell (CSC) population. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. The reporter marks tumor cells that are enriched for CSC activities, including the ability to self-renew, divide asymmetrically, resist cell killing by conventional chemotherapeutics, and initiate tumorigenesis and metastasis in vivo. We confirmed that the approach works in primary human breast cancer cells as well as in many established breast cancer cell lines. We are now exploiting this reporter to address factors that regulate CSC localization, plasticity and behavior. 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. 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. 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 developed 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 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 demonstrated that TGF-beta-induced inhibition of cell proliferation and induction of cellular differentiation both contribute to tumor suppression, and we identified a novel functional role for Ephrin signaling in mediating the tumor suppressive effects of TGF-beta. However, we showed 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. Detailed analysis of Smad binding regions in the breast cancer models has identified a druggable transcription factor that may oppose the tumor suppressive effects of TGF-beta activity on the cancer stem cell. We are currently testing whether targeting this factor can restore TGF-beta-mediated tumor suppression in breast cancer models where this has been lost and we are analyzing underlying mechanisms.

2014 财年，我们继续分析 TGF-β 活性从肿瘤抑制因子转变为促进展因子的肿瘤细胞自主成分，特别强调对癌症干细胞群的影响。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异种移植模型。我们之前已经证明，在该模型中，TGF-β从肿瘤抑制因子转变为促进展因子，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异常高的信噪比系统，其中解决 TGF-β 开关的潜在机制。然而，我们还从新切除的患者肿瘤中建立了许多新的乳腺癌细胞株，以将我们的发现扩展到原代细胞。我们 2014 财年的工作集中在两个主要领域： 1. TGF-β 在调节癌症干细胞动力学中的作用。 2014 财年，我们继续验证并采用一种新颖的功能成像方法来识别癌症干细胞 (CSC) 群体。我们基于慢病毒的 CSC 报告基因使用合成启动子，其中荧光蛋白的表达由干细胞主转录因子 Oct4 和 Sox2 驱动。记者标记了富含 CSC 活性的肿瘤细胞，包括自我更新、不对称分裂、抵抗传统化疗药物杀死细胞以及启动体内肿瘤发生和转移的能力。我们证实该方法适用于原代人类乳腺癌细胞以及许多已建立的乳腺癌细胞系。我们现在正在利用该报告器来解决调节 CSC 本地化、可塑性和行为的因素。使用 TGF-β 作为肿瘤抑制因子的异种移植乳腺癌模型，我们将干细胞报告基因与 TGF-β 通路报告基因结合起来，结果表明，与体细胞相比，内源性 TGF-β 信号在 CSC 中被激活得更高。群体，表明 TGF-β 在干细胞区室中发挥着特别重要的作用。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 增殖能力较差。此外，我们发现TGF-β选择性抑制CSC区室中的不对称自我更新有丝分裂，并且特异性抑制CSC穿过基底膜的侵袭，而对大量肿瘤细胞群的侵袭几乎没有影响。这些观察结果表明，TGF-β 是 CSC 生物学的重要调节剂，并确定了可能构成 TGF-β 肿瘤抑制作用的新机制。了解 CSC 的调控方式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有抵抗力。 2. 从基因组学方法深入了解 TGF-β 介导的肿瘤发生作用。 TGF-β 拮抗剂正在被开发为癌症治疗药物。然而，TGF-β 在癌症进展中的复杂作用使得必须避免治疗肿瘤对 TGF-β 仍具有完整肿瘤抑制反应的患者。目前尚不清楚乳腺癌在诊断和手术时是否仍保留对 TGF-β 的肿瘤抑制反应。为了解决这个问题，我们开发了一种专门反映肿瘤抑制作用的 TGF-β 反应特征，因为已发表的 TGF-β 特征并不是预先设计来区分肿瘤抑制反应与肿瘤促进反应的。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集成的全基因组染色质免疫沉淀和转录组学方法，专门剖析出与 TGF-β/Smad3 介导的肿瘤抑制相关的核心基因特征。在对 1300 多个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+) 乳腺癌女性良好的无远处转移生存相关，这表明 TGF-β 的肿瘤抑制作用仍然存在。活跃并影响一部分患者的疾病结果。我们证明，TGF-β 诱导的细胞增殖抑制和细胞分化诱导都有助于肿瘤抑制，并且我们确定了 Ephrin 信号在介导 TGF-β 肿瘤抑制作用中的新功能作用。然而，我们发现 TGF-β 调节的转录组是高度上下文依赖性的，因为下游转录介质 Smad3 似乎只结合到已经具有转录活性的染色质区域。因此，我们认为有必要为不同的肿瘤类型构建定制的 TGF-β 特征，并且不会有单一的特征可以作为所有癌症类型中 TGF-β 肿瘤抑制的生物标志物。这一发现对于开发基于特征的生物标志物具有重要意义，该生物标志物用于在使用 TGF-β 拮抗剂进行的临床肿瘤学试验中纳入/排除患者。对乳腺癌模型中 Smad 结合区域的详细分析发现了一种可药物转录因子，它可能会对抗 TGF-β 活性对癌症干细胞的肿瘤抑制作用。我们目前正在测试靶向这一因子是否可以在乳腺癌模型中恢复已丧失的 TGF-β 介导的肿瘤抑制作用，并且我们正在分析潜在的机制。