TGF-betas in breast cancer progression

在2014财年，我们继续分析了转化生长因子-β活性从肿瘤抑制因子转换为促进进展因子的肿瘤细胞自主成分，特别强调了对癌症干细胞种群的影响。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的异种移植模型。我们之前已经证明，在这个模型中，转化生长因子-β从肿瘤抑制因子转换为促进进展因子，而进展系列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噪比系统，在这个系统中，我们可以解决转化生长因子-β转换的潜在机制。然而，我们也从新切除的患者肿瘤中建立了一些新的乳腺癌细胞株，以将我们的发现扩展到原代细胞。我们在2014财年的工作主要集中在两个方面：1.转化生长因子-β在调节肿瘤干细胞动力学中的作用。在2014财年，我们继续验证和使用一种新的功能成像方法来识别癌症干细胞(CSC)群体。我们的慢病毒CSC报道使用了一种合成启动子，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、抵抗常规化疗药物的细胞杀伤以及在体内启动肿瘤形成和转移的能力。我们证实，这种方法在原代人类乳腺癌细胞以及许多已建立的乳腺癌细胞系中都有效。我们现在利用这位记者来探讨影响CSC本地化、可塑性和行为的因素。利用转化生长因子-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与转化生长因子-β途径报告相结合，发现与普通人群相比，内源性转化生长因子-β信号在CSCs中被激活得更高，这表明转化生长因子-β在干细胞室中起着特别重要的作用。与未激活该通路的CSCs相比，具有内源性转化生长因子-β信号活性的CSCs本质上具有较低的增殖能力。此外，我们发现，转化生长因子-β选择性地抑制CSC间隔室中的不对称自我更新有丝分裂，并特异性地抑制CSCs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。这些观察表明，转化生长因子-β是肿瘤干细胞生物学的重要调节因子，并确定了转化生长因子-β的肿瘤抑制作用的新机制。了解CSCs是如何被调控的对于开发更有效的癌症治疗至关重要，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.从基因组角度洞察转化生长因子-β在肿瘤发生中的作用。转化生长因子-β拮抗剂正被开发为癌症治疗药物。然而，转化生长因子-β在癌症进展中的复杂作用使得避免治疗那些肿瘤对转化生长因子-β仍有完整的肿瘤抑制反应的患者是必要的。目前尚不清楚是否有乳腺癌患者在诊断和手术时仍保留对转化生长因子-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效应的转化生长因子-β反应信号，因为已发表的转化生长因子-β反应信号并不是事先设计来区分肿瘤抑制反应和肿瘤促进反应的。利用基于MCF10的乳腺癌进展模型，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切割方法，专门剖析了与转化生长因子-β/Smad3介导的肿瘤抑制相关的核心基因信号。在一项对1300多例人类乳腺癌的荟萃分析中，这一信号的高表达与雌激素受体阳性(ER+)乳腺癌女性患者良好的无远处转移生存率有关，这表明转化生长因子-β的肿瘤抑制作用仍然活跃，并影响部分患者的疾病预后。我们证明了转化生长因子-β对细胞增殖的抑制和对细胞分化的诱导都有助于抑制肿瘤生长，并且我们确定了在介导转化生长因子-β的肿瘤抑制效应中的一种新的功能。然而，我们证明了转化生长因子-β调节的转录组是高度依赖于上下文的，因为下游的转录调节因子SMAD3似乎只结合到已经转录活跃的染色质区域。因此，我们认为有必要为不同的肿瘤类型建立量身定制的转化生长因子-β标记，而且不会有单一的标记作为所有癌症类型的转化生长因子-β肿瘤抑制的生物标志物。这一发现对于开发基于签名的生物标记物，用于在使用转化生长因子-β拮抗剂的临床肿瘤学试验中纳入/排除患者具有重要意义。对乳腺癌模型中Smad结合区的详细分析已经确定了一种可药物转录因子，它可能对抗转化生长因子-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在乳腺癌模型中恢复转化生长因子-β介导的肿瘤抑制，而这一点已经丢失，我们正在分析潜在的机制。

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.