TGF-betas in breast cancer progression

在2008财年，我们主要专注于阐明tgf - β活性从肿瘤抑制因子到促进展因子转换的肿瘤细胞自主成分。tgf - β是一种有效的上皮细胞增殖抑制剂，人们普遍认为这种特性对tgf - β作为肿瘤抑制因子的能力至关重要。然而，我们已经确定了一种新的肿瘤抑制机制，它不依赖于对细胞增殖的直接影响。使用乳腺癌异种移植模型，我们已经证明内源性tgf - β可以通过减少假定的癌症干细胞群的大小和促进高度增殖的癌症祖细胞的分化来抑制肿瘤发生。在该模型系统中，实验阻断tgf - β反应可将肿瘤的基因表达谱和组织学从分化的管腔状态转变为分化程度较低的基底状态，先前的乳腺癌临床研究表明，这与较差的预后有关。我们发现转录调节因子Id1是tgf - β调节分化的关键下游靶点，我们提出阻断分化的分子变化可以选择性地阻断tgf - β的这种肿瘤抑制作用，从而促进转移开关。tgf - β信号由两个结构上高度相关的蛋白Smad2和Smad3以及其他信号级联如MAPK激酶和PI3激酶途径转导。我们假设，这些不同信号通路之间的平衡可能是决定tgf - β信号输出是抑制肿瘤还是促进肿瘤的关键。为了系统地解决这个问题，我们从不同Smad基因型的小鼠中产生了一组有条件永生化的乳腺上皮细胞。这种方法使我们能够比通过RNA干扰方法更精确地控制Smad2/3的水平。我们发现乳腺上皮对tgf - β的生长抑制和促凋亡反应只需要Smad3而不需要Smad2。相反，tgf - β的促迁移和促侵袭作用需要两个smad之间的强制性合作。数据表明Smad3可能对肿瘤抑制和促进展反应都至关重要，而Smad2对促进展反应更为重要。此外，在对Smad3的需求中存在有趣的基因剂量效应，Smad3的一个等位基因的丢失导致对tgf - β的促凋亡反应的丢失，同时仍然允许其他反应。因此，尽管Smad3在肿瘤抑制和肿瘤促进活性中都是至关重要的，但预测Smad3的减少可以选择性地促进肿瘤进展。为了支持这一预测，我们发现Smad3+/-小鼠在MMTV-PVT转基因乳腺癌小鼠模型中具有更高的转移发生率。Smad3多态性与转移性乳腺癌风险之间的潜在关联将被研究。来自我们实验室和其他实验室的数据暗示Smad3在肿瘤细胞的肿瘤抑制和促tgf - β的进展作用中都有作用。我们假设，在癌症进展过程中发生的遗传或表观遗传变化可能会改变smad3介导的tgf - β信号的读数，从而使促肿瘤活性占主导地位。为了解决这个问题，我们进行了全基因组染色质免疫沉淀，在两个密切相关的细胞系中鉴定Smad3靶点，MCF10Ca1h中tgf - β发挥肿瘤抑制作用，MCF10Ca1a中tgf - β发挥促进展因子作用。启动子占用数据与全球基因表达数据的整合产生了与两种不同结果相关的基于smad3的核心基因签名。这些特征应该对开关过程背后的机制产生重要的见解，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。

In FY08, we have focused primarily on elucidating the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor. TGF-beta is a potent inhibitor of the proliferation of epithelial cells, and it has been widely assumed that this property is critical for the ability of TGF-beta to function as a tumor suppressor. However, we have identified a novel tumor suppression mechanism that is independent of direct effects on cell proliferation. Using a breast cancer xenograft model, we have shown that endogenous TGF-betas can suppress tumorigenesis by reducing the size of the putative cancer stem cell population and by promoting differentiation of the highly proliferative cancer progenitor cells. Experimental blockade of TGF-beta response in this model system converted the gene expression profile and histology of the tumor from a differentiated luminal to a less differentiated basal state, which previous clinical breast cancer studies have shown to be associated with a poorer prognosis. We identified the transcriptional regulator Id1 as a critical downstream target of TGF-beta in regulating differentiation, and we propose that molecular changes that block differentiation can selectively block this tumor suppressor effect of TGF-beta, thereby contributing to the metastatic switch. The TGF-beta signal is transduced by two structurally highly related proteins, Smad2 and Smad3, as well as by other signaling cascades such as the MAPK kinase and PI3 kinase pathways. We have hypothesized that the balance between these different signaling pathways may be critical in determining whether the output of the TGF-beta signal is tumor suppressive or tumor promoting. To systematically address this question, we have generated a panel of conditionally immortalized mammary epithelial cells from mice of differing Smad genotypes. This approach allows us more precise control over Smad2/3 levels than can be achieved through RNA interference approaches. We have found that the growth inhibitory and pro-apoptotic responses of the mammary epithelium to TGF-beta require only Smad3 and not Smad2. In contrast, the pro-migratory and pro-invasive effects of TGF-beta require obligatory cooperation between both Smads. The data suggest that Smad3 may be critical for both tumor suppressor and pro-progression responses, while Smad2 is more important for the pro-progression responses. In addition, there are interesting gene dosage effects in the requirement for Smad3, with loss of one allele of Smad3 resulting in loss of the pro-apoptotic response to TGF-beta, while still permitting the other responses. Thus, despite the critical requirement for Smad3 in both tumor suppressive and tumor promoting activities, a reduction in Smad3 is predicted selectively to promote progression. In support of this prediction, we have found that Smad3+/- mice have a higher incidence of metastasis in the MMTV-PVT transgenic mouse model of breast cancer. The potential association between Smad3 polymorphisms and risk of metastatic breast cancer will be investigated. The data from our lab and others implicates Smad3 in both the tumor suppressor and pro-progression effects of TGF-beta on the tumor cell. We hypothesize that genetic or epigenetic changes that occur during cancer progression may alter the Smad3-mediated readout of the TGF-beta signal so that tumor promoting activities dominate. To address this question, we have performed genome-wide chromatin immunoprecipitation to identify Smad3 targets in two closely related cell lines, MCF10Ca1h in which TGF-beta functions as tumor suppressor, and MCF10Ca1a in which TGF-beta functions as a pro-progression factor. Integration of the promoter occupancy data with global gene expression data has yielded core Smad3-based gene signatures that are associated with the two different outcomes. These signatures should yield important insights into mechanisms underlying the switch process, 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.