TGF-betas in breast cancer progression

在2013财年，我们继续专注于阐明tgf - β从肿瘤抑制因子到促进展因子活性转换的肿瘤细胞自主成分。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。我们之前已经证明，在该模型中，tgf - β从肿瘤抑制因子转换为促进展因子，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一个异常高的信号-噪声系统，在该系统中可以解决tgf - β转换的潜在机制。然而，今年我们也从刚切除的患者肿瘤中建立了一些新的乳腺癌细胞株，将我们的发现扩展到原代细胞。我们在2013财年的工作主要集中在两个方面：1。tgf - β在调节癌症干细胞动力学中的作用。在2013财年，我们继续验证和采用一种新的功能成像方法来识别假定的癌症干细胞（CSC）群体。我们的CSC报告因子响应干细胞主转录因子Oct4和Sox2的存在，并标记富含CSC活性的细胞，包括不对称分裂的能力，抵抗常规化疗的细胞杀伤，以及在体内启动肿瘤发生和转移。我们证实，这种方法在原发性人类乳腺癌细胞和已建立的细胞系中都有效，并且我们在慢病毒载体设计方面取得了一些进展，以提高灵敏度。我们现在正在利用各种方法，包括体外延时荧光显微镜和体外肿瘤共聚焦显微镜来研究调节CSC定位、可塑性和行为的因素，并筛选针对CSC群体的治疗方法。一个主要的焦点是tgf - β超家族成员在CSC生物学中的作用。通过使用tgf - β作为肿瘤抑制因子的异种移植乳腺癌模型，我们将我们的干细胞报告因子与tgf - β途径报告因子结合起来，发现内源性tgf - β信号在CSCs中被激活的程度高于普通人群，这表明tgf - β在干细胞区室中起着特别重要的作用。内源性tgf - β信号激活的CSCs本质上比未激活该途径的CSCs的增殖能力更低。此外，我们发现tgf - β选择性地抑制了CSC室中不对称的自我更新有丝分裂，并且特异性地抑制了CSC通过基底膜的侵袭，而对大部分肿瘤细胞群的侵袭几乎没有影响。这些观察结果表明，tgf - β是一种重要的CSC生物学调节剂，并确定了tgf - β抑制肿瘤作用的新机制。为了将我们的成像方法从移植肿瘤扩展到自发产生的肿瘤，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CSCs是如何被调节的对于开发更有效的癌症治疗方法至关重要，因为这些细胞对现有的治疗方法有很大的抵抗力，最终导致复发。2. 从基因组方法了解tgf - β介导的肿瘤发生作用。tgf - β拮抗剂正被开发作为癌症治疗药物。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仍有完整肿瘤抑制反应的患者。目前尚不清楚在任何乳腺癌的诊断和手术中是否仍然保留对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反应特征，因为已发表的tgf - β特征并没有先验地设计来区分肿瘤抑制反应与中性或肿瘤促进反应。利用基于mcf10的人类乳腺癌进展模型，我们在体外和体内应用整合全基因组染色质免疫沉淀和转录组学方法特异性地解剖出与tgf - β / smad3介导的肿瘤抑制相关的核心基因特征。在一项针对1300多例人类乳腺癌的荟萃分析中，该特征的高表达与雌激素受体阳性（ER+）乳腺癌患者良好的无远处转移生存率相关，这表明tgf - β的肿瘤抑制作用仍然活跃，并影响一部分患者的疾病结局。我们继续探索潜在的生物学和分子机制，并证明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。我们进一步确定了Ephrin信号在介导tgf - β的肿瘤抑制作用中的功能作用。然而，我们已经证明，tgf - β调节的转录组是高度依赖环境的，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量身定制的tgf - β信号，并且不会有单一的信号可以作为所有癌症类型中tgf - β肿瘤抑制的生物标志物。这一发现对基于特征的生物标志物的开发具有重要意义，可用于tgf - β拮抗剂临床肿瘤试验中的患者纳入/排除。我们也在同样的基于mcf -10的模型系统中研究tgf - β对miRome的影响，以构建tgf - β在乳腺癌进展中改变作用的综合分子机制图。为此，我们对四种mcf10衍生细胞系的基础和tgf - β调节的miRomes进行了Next-Gen测序，这些细胞系分别代表乳腺癌进展中的正常、癌前、低度恶性和高度恶性阶段。对这些数据集的分析正在进行中。

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.