ER Co-repressor function of SAFB in Breast Cancer

描述（由申请人提供）：我们最初克隆了SAFB1作为转录抑制因子，并且它已涉及许多过程，包括激素反应和转化。SAFB1基因在乳腺癌中显示出极高的杂合性缺失率，其缺失与高级别肿瘤有关，并降低了乳腺癌患者的生存率。结构-功能研究发现，转录抑制域和SAFB1的过表达减弱了雌激素诱导的基因表达，增强了雌激素介导的抑制。有趣的是，我们发现SAFB1的sumo化对于其协同抑制因子的活性是必要的。我们产生了safb1缺失的小鼠，这些小鼠显示出前和新生儿的死亡率，存活的小鼠在生殖系统中有明显的缺陷。为了更深入地了解SAFB1在ER1作用中的作用，我们使用SAFB1 siRNA敲除进行了无偏筛选，然后对雌激素刺激的乳腺癌细胞进行了微阵列分析。本实验揭示了SAFB1在雌激素介导的基因表达抑制中的重要作用，并确定了在雌激素介导的下调中完全需要SAFB1的候选基因。这些候选蛋白包括已知参与迁移、侵袭和存活的蛋白，如NEDD9和骨保护素（OPG）。此外，我们确定了HDAC7在雌激素介导的一些相同候选基因抑制中的新作用。因此，我们假设SAFB1是一种重要而独特的共同调节因子，对雌激素基因的下调至关重要，并且这种抑制是通过其翻译后的sumo修饰介导的，至少部分是通过与HDAC7的相互作用介导的。最后，我们假设safb1介导的靶基因的抑制对其在乳腺肿瘤发生中的作用至关重要，因为抑制的丧失可能导致乳腺癌细胞的迁移、侵袭和存活增加。我们将通过分析SAFB1如何介导雌激素应答靶基因的转录抑制，其协同抑制功能如何被翻译后修饰来验证这些假设，我们将测试其靶基因的抑制是否有助于SAFB1在乳腺肿瘤发生中的作用。这项工作非常重要，因为它将深入了解SAFB1作用的基本机制，这可能有助于改变ER1作用的范式。虽然ER1主要被研究为转录激活因子，但同样频繁的雌激素介导的基因表达抑制，特别是像SAFB1这样的共抑制因子在这种抑制中的作用，是一个研究不足的领域。最重要的是，我们还将了解SAFB1的这种抑制活性是否以及如何对其在乳腺肿瘤发生中的作用至关重要。公共卫生相关性：SAFB1是一种转录共抑制因子，它参与了许多细胞过程，包括激素、反应、永生、凋亡和转化。我们发现SAFB1在雌激素介导的基因表达抑制中起着重要作用，这是雌激素受体作用中一个尚未被研究的关键过程。在这里，我们建议深入研究这种抑制的机制，以及它与SAFB1在乳腺肿瘤发生中的作用的关系。

DESCRIPTION (provided by applicant): We originally cloned SAFB1 as a transcriptional repressor, and it has since been implicated in a number of processes including hormone response, and transformation. The SAFB1 gene shows extremely high rates of loss of heterozygosity in breast cancer, and its loss is associated with high grade tumors, and reduced survival of breast cancer patients. Structure-function studies identified a transcriptional repression domain and overexpression of SAFB1 blunted estrogen-induction of gene expression and enhanced estrogen-mediated repression. Intriguingly, we found that sumoylation of SAFB1 was necessary for its co-repressor activity. We generated SAFB1-null mice which showed pre- and neonatal lethality, with surviving mice having dramatic defects in the reproductive system. To gain more insight into SAFB1's role in ER1 action, we performed an unbiased screen using SAFB1 siRNA knockdown followed by microarray analysis of estrogen-stimulated breast cancer cells. This experiment revealed a significant role for SAFB1 in estrogen-mediated repression of gene expression, and candidate genes were identified that showed a complete requirement for SAFB1 in estrogen-mediated downregulation. These candidates include proteins known to be involved in migration, invasion, and survival, such as NEDD9 and osteoprotegerin (OPG). Furthermore, we identified a novel role for HDAC7 in estrogen-mediated repression of some of the same candidate genes. We thus hypothesize that SAFB1 is an important and unique co-regulator that is critical for estrogen downregulation of genes, and that the repression is mediated through its posttranslational modification by sumoylation and, at least in part, through interaction with HDAC7. Finally, we hypothesize that SAFB1-mediated repression of its target genes is critical for its role in breast tumorigenesis, since loss of repression might result in increased migration, invasion, and survival of breast cancer cells. We will test these hypotheses by analyzing how SAFB1 mediates transcriptional repression of estrogen-responsive target genes, how is its co-repressor function modified by posttranslational modification, and we will test whether repression of its target genes contributes to SAFB1's role in breast tumorigenesis? This work is highly significant since it will provide insight into fundamental mechanisms of SAFB1 action which may contribute to a change in the paradigm of ER1 action. While ER1 has mainly been studied as a transcriptional activator, the equally frequent estrogen-mediated repression of gene expression, and in particular the role of co-repressors like SAFB1 in this repression, is an understudied area. Most importantly, we will also learn whether and how this repression activity of SAFB1 is critical for its role in breast tumorigenesis. PUBLIC HEALTH RELEVANCE: SAFB1 is a transcriptional co-repressor, and it has since been implicated in a number of cellular processes including hormone, response, immortalization, apoptosis, and transformation. We have discovered that SAFB1 plays a significant role in estrogen- mediated repression of gene expression, which is an understudied yet critical process in estrogen receptor action. Here we propose to study the in-depth mechanism of this repression, and how this relates to SAFB1's role in breast tumorigenesis.