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.

在2014财年，我们继续分析TGF-β从肿瘤抑制剂到促进因子的活动转换的肿瘤细胞自主分量，并特别着重于对癌症干细胞群体的影响。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，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。在2014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癌症干细胞（CSC）种群。我们的基于慢病毒的CSC报告基因使用合成启动子，其中荧光蛋白的表达是由干细胞主转录因子Oct4和Sox2驱动的。记者标志着富含CSC活性的肿瘤细胞，包括自我更新，不对称分裂，通过常规化学治疗剂抗拒细胞杀死的能力，并在体内启动肿瘤发生和转移。我们确认该方法在原发性人类乳腺癌细胞以及许多已建立的乳腺癌细胞系中起作用。现在，我们正在利用此记者来解决调节CSC定位，可塑性和行为的因素。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。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。 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，我们开发了一种特异性反映肿瘤抑制作用的TGF-β响应特征，因为已发表的TGF-beta特征并未设计为将肿瘤抑制反应与促进肿瘤促进反应区分开的先验。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。我们证明，TGF-BETA诱导的细胞增殖抑制和细胞分化的诱导均导致肿瘤抑制作用，并且我们确定了ephrin信号传导在介导TGF-beta肿瘤抑制作用中的新功能作用。但是，我们表明TGF-BETA调节的转录组高度依赖上下文，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。对乳腺癌模型中SMAD结合区域的详细分析已经确定了一种可毒的转录因子，该因子可能反对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该因子是否可以恢复乳腺癌模型中TGF-beta介导的肿瘤抑制，在这种模型中，我们正在分析基本机制。