TGF-betas in breast cancer progression

在2011财年，我们继续将重点放在阐明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上。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。我们在2011财年的工作主要集中在三个方面：1。tgf - β在调节癌症干细胞动力学中的作用。在2011财年，我们继续开发和使用我们的新型功能成像方法来识别假定的癌症干细胞（CSC）群体，我们已经研究了tgf - β如何调节CSC动态。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，包括体内不对称分裂和启动肿瘤发生和转移的能力。我们之前已经证明tgf - β在MCF10Ca1h人乳腺癌模型中具有肿瘤抑制活性。现在我们已经证明，在这个模型中，CSC群体的很大一部分具有内源性激活的tgf - β信号。通过延时荧光显微镜，我们发现这些细胞大多数是非增殖的。此外，我们发现csc本质上比整体肿瘤细胞群更具迁移性和侵袭性，tgf - β可以抑制csc中的这些活动，同时对整体细胞群具有轻度刺激作用。因此，tgf - β似乎在维持CSCs亚群的静止和保持该群体的静止中起着重要作用。为了将这种方法从移植到自发产生的肿瘤，我们产生了表达相同干细胞报告结构的转基因小鼠。已经获得了多种方正线，其中一些看起来很有前途。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. 从核心tgf - β / smad3基因表达特征的产生深入了解tgf - β介导的肿瘤抑制。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。已发表的tgf - β反应特征并没有将肿瘤抑制反应与中性反应或肿瘤促进反应区分开来。我们的目标是开发一种特异性反映tgf - β肿瘤抑制活性的特征，希望它能提供对潜在生物学的见解，并对tgf - β拮抗剂临床试验中的患者分层有用。Smad2和Smad3是高度同源的转导tgf - β信号的蛋白。利用不同Smad基因型的有条件永生化乳腺上皮细胞，我们发现Smad3在大多数针对tgf - β的生物反应中起关键作用，而Smad2起较小甚至相反的作用。通过shRNA敲低方法，我们进一步证明Smad3在MCF10Ca1h乳腺肿瘤异种移植物中介导tgf - β的肿瘤抑制活性，而这种活性在密切相关的MCF10Ca1a肿瘤中缺失。因此，我们专注于Smad3来解决潜在的机制。首先，我们进行了全基因组染色质免疫沉淀，以鉴定MCF10进展系列中的tgf - β /Smad3靶基因。将启动子占用数据与体外和体内的全局基因表达数据整合，得出了一个基于smad3的核心基因特征，该特征与tgf - β介导的肿瘤抑制有关。在一项针对1300多例人类乳腺癌的荟萃分析中，这一特征的高表达与良好的无远处转移生存率相关，这表明tgf - β的肿瘤抑制活性确实在人类乳腺癌中发挥了重要作用。与大多数预后特征不同，我们的特征是丰富的分化相关基因而不是增殖相关基因，这表明它反映了乳腺癌生物学的一个新方面。研究潜在的生物学和分子机制的实验正在进行中。这一特征将在临床前模型中用于预测对tgf - β拮抗剂反应的能力，并将在基于基因表达的筛选中被利用，以发现可能逆转开关并恢复tgf - β肿瘤抑制活性的新化合物。3. 在人类临床样本中鉴定和定量非典型tgf - β信号的工具的开发。最近来自其他实验室的数据表明，通过非规范机制的tgf - β信号传导与tgf - β的促肿瘤作用有关，而规范信号传导对肿瘤抑制作用至关重要。因此，非典型tgf - β信号的激活可能导致tgf - β从肿瘤抑制因子转换为肿瘤启动因子。非规范信号机制包括tgf - β Smads （Smad2/3）和BMP Smads （Smad1/5/8）混合Smad信号复合物的形成，以及由于tgf - β Smads在中间连接区域的磷酸化而导致的信号改变。原位接近结扎（Duolink）是一种利用配对抗体检测和定量冷冻或固定组织切片中蛋白质/蛋白质相互作用或复杂的翻译后修饰的新技术。在2011财年，我们在开发用于评估非典型tgf - β信号的技术方面取得了实质性进展。我们将应用这种方法来观察临床前和临床乳腺癌材料进展过程中不同阶段非典型信号的开始，并评估非典型tgf - β信号是否可以作为tgf - β拮抗剂试验的有用预测生物标志物（见相关项目Z1A BC 010881）。

In FY11, 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. Our work in FY11 has focused in three main areas: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY11 we have continued to develop and employ our novel functional imaging approach for the identification of the putative cancer stem cell (CSC) population, and we have examined how TGF-beta regulates CSC dynamics. 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 and to initiate tumorigenesis and metastasis in vivo. We have previously shown that TGF-beta has tumor suppressor activity in the MCF10Ca1h human breast cancer model. Now we have demonstrated that a significant fraction of the CSC population in this model has endogenously activated TGF-beta signaling. By timelapse fluorescence microscopy, we have shown that the majority of these cells are non-proliferative. Furthermore, we find that CSCs are intrinsically more migratory and invasive than the bulk tumor cell population, and that TGF-beta can inhibit these activities in CSCs while having a mildly stimulatory effect on the bulk cell population. Thus TGF-beta appears to play an important role in maintaining quiescence in a subpopulation of CSCs and in keeping this population stationary. In order to extend this approach from transplanted to spontaneously arising tumors, we have generated transgenic mice expressing the same stem cell reporter construct. Multiple founder lines have been obtained and several look promising. 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 TUMOR SUPPRESSION FROM THE GENERATION OF A CORE TGF-BETA/SMAD3 GENE EXPRESSION SIGNATURE. 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. Published TGF-beta response signatures have not distinguished the tumor suppressive responses from the neutral or tumor promoting responses. Our goal here was to develop a signature that specifically reflects the tumor suppressor activity of TGF-beta with the hope that it provide insights into the underlying biology and be useful for patient stratification in TGF-beta antagonist clinical trials. Smad2 and Smad3 are highly homologous proteins that transduce the TGF-beta signal. Using conditionally immortalized mammary epithelial cells of different Smad genotypes, we have shown that Smad3 is critical for the majority of the biological responses to TGF-beta, with Smad2 playing a lesser or even opposing role. Using shRNA knockdown approaches, we further demonstrated that Smad3 mediates the tumor suppressor activity of TGF-beta in MCF10Ca1h breast tumor xenografts and that this activity is lost in the closely related MCF10Ca1a tumors. We therefore focused on Smad3 to address underlying mechanisms. First we performed genome-wide chromatin immunoprecipitation to identify TGF-beta/Smad3 target genes in the MCF10 progression series. Integration of the promoter occupancy data with global gene expression data both in vitro and in vivo yielded a core Smad3-based gene signature that is associated with TGF-beta-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, suggesting that the tumor suppressor activity of TGF-beta does indeed play an important role in human breast cancer. Unlike the majority of prognostic signatures, our signature was enriched for differentiation-associated rather than proliferation-associated genes suggesting that it is reflecting a novel aspect of breast cancer biology. Experiments addressing the underlying biological and molecular mechanisms are ongoing. This signature will be tested for ability to predict response to TGF-beta antagonists in pre-clinical models, and will be exploited in gene expression based screens to find novel compounds that might reverse the switch and restore the tumor suppressor activities of TGF-beta. 3. DEVELOPMENT OF TOOLS TO IDENTIFY AND QUANTITATE NON-CANONICAL TGF-BETA SIGNALING IN HUMAN CLINICAL SAMPLES. Recent data from other labs has suggested that TGF-beta signaling via non-canonical mechanisms is associated with tumor promoting effects of TGF-beta, while canonical signaling is critical for tumor suppressor effects. Thus activation of non-canonical TGF-beta signaling may cause TGF-beta to switch from tumor suppressor to tumor promoter. Non-canonical signaling mechanisms include the formation of mixed Smad signaling complexes involving both TGF-beta Smads (Smad2/3) and BMP Smads (Smad1/5/8), and altered signaling due to phosphorylation of TGF-beta Smads in the middle linker region. In situ proximity ligation (Duolink) is a new technique that uses paired antibodies to detect and quantitate protein/protein interactions or complex post-translational modifications in frozen or fixed tissue sections. In FY11 we have made substantial progress towards developing this technique for the assessment of non-canonical TGF-beta signaling. We will apply this approach to look at the onset of non-canonical signaling at different stages in the progression process in pre-clinical and clinical breast cancer material and to assess whether non-canonical TGF-beta signaling might serve as a useful predictive biomarker for TGF-beta antagonist trials (see related project Z1A BC 010881).