ER Co-repressor function of SAFB in Breast Cancer

描述(申请人提供)：我们最初克隆的SAFB1是一个转录抑制因子，自那以后，它参与了包括激素反应和转化在内的许多过程。SAFB1基因在乳腺癌中表现出极高的杂合性缺失率，它的缺失与高级别肿瘤和乳腺癌患者的生存减少有关。结构-功能研究证实了SAFB1的转录抑制结构域和过度表达可钝化雌激素诱导的基因表达并增强雌激素介导的抑制作用。有趣的是，我们发现SAFB1的苏木糖基化是其协同抑制活性所必需的。我们产生了SAFB1缺失的小鼠，显示出前期和新生儿的致死性，存活的小鼠在生殖系统中有显著的缺陷。为了更深入地了解SAFB1在ER1作用中的S作用，我们使用SAFB1siRNA敲除了SAFB1siRNA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，并确定了候选基因，表明SAFB1在雌激素介导的下调中是完全必需的。这些候选蛋白包括已知参与迁移、侵袭和生存的蛋白质，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的抑制中的一个新角色。因此，我们假设SAFB1是一个重要的和独特的辅助调节因子，对于雌激素下调基因是至关重要的，并且这种抑制是通过其SUMO基化的翻译后修饰以及至少部分通过与HDAC7的相互作用来介导的。最后，我们假设SAFB1介导的靶基因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如何介导雌激素反应靶基因的转录抑制，翻译后修饰如何改变其辅助抑制子功能来检验这些假说，并检验其靶基因的抑制是否有助于SAFB1在乳腺肿瘤发生中的S作用？这项工作意义重大，因为它将为SAFB1行动的基本机制提供洞察，这可能有助于改变ER1行动的范式。虽然ER1主要作为转录激活因子被研究，但同样频繁的雌激素介导的基因表达抑制，特别是SAFB1等辅助抑制因子在这种抑制中的作用，是一个研究较少的领域。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起关键作用。公共卫生相关性：SAFB1是一种转录共抑制因子，自那以后，它参与了许多细胞过程，包括激素、反应、永生化、细胞凋亡和转化。我们发现，SAFB1在雌激素介导的基因表达抑制中发挥着重要作用，这是雌激素受体作用中一个尚未被充分研究但却至关重要的过程。在这里，我们打算深入研究这种抑制的机制，以及这与SAFB1的S在乳腺肿瘤发生中的作用有何关系。

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.