Role of hsnf5/BAF47 Loss in Human Cancer Development

描述（由申请人提供）：肿瘤抑制基因的鉴定导致了对人类癌症发展机制的新见解。这些基因的正常功能通常在于控制基因表达，特别是在细胞周期控制和细胞分化领域。最近的几项研究表明，染色质重塑复合物的异常活性与人类癌症的发生有关。INI 1/SNF 5/BAF 47基因突变是SWI/SNF染色质重塑复合物的一个组成部分，几乎发生在所有恶性横纹肌样瘤（MRT）中。SWI/SNF复合物作为一种全局转录激活因子，通过能量依赖机制改变核小体在DNA上的定位。在之前的资助期间，我们已经确定SNF 5表达的缺失会影响所有MRT细胞系的细胞周期调控。我们还开发了2种用于MRT发展的基因工程小鼠（GEM）模型，其中pRb家族成员表达的丧失或p53表达的降低加速了脊髓MRT的出现。重要的是，我们的初步研究现在已经提出了4个候选基因，怀疑支持SNF 5肿瘤抑制作用。基于这些结果，我们假设SNF 5表达的缺失可以通过破坏神经分化导致MRT的发展。为了验证这一假设，我们提出了三个具体目标。在第一个具体目标中，我们将使用Taq-Man（R）分析，蛋白质印迹和ChIP来表征人类MRT细胞系中SNF 5的新靶点，使用细胞培养模型来确定它们在MRT肿瘤发生中的作用，并评估SNF 5缺失对染色质结构及其启动子区域修饰的影响。第二个具体目标将使用高通量DNA测序方法（ChIP-seq）和基因表达阵列分析的组合来确定与MRT发展相关的SNF 5下游靶点。在第三个具体目标，我们将确定Snf 5失活的作用，在神经细胞发育和恶性横纹肌样瘤的发生，使用新的基因工程小鼠模型，神经球文化和比较小鼠脊髓MRT与人类MRT的中枢神经系统。SNF 5在MRT发展中所起作用的解剖将拓宽我们对其正常生物学和生化活性的理解，为SWI/SNF复合物在细胞周期调节和正常发育中的功能提供新的见解，并确定治疗这种高度侵袭性肿瘤的新途径。公共卫生相关性：肿瘤抑制基因在许多人类癌症中被关闭，并且通常是这种疾病的家族形式的原因。我们正在研究其中一种名为INI 1/SNF 5/BAF 47的基因，它在一种名为恶性横纹肌样瘤（MRT）的侵袭性儿童肾癌、脑癌和软组织癌的发展中起着重要作用。我们以前的研究表明，出生前或出生后早期这种基因的丢失可能会干扰正常的神经系统发育，导致儿童出现MRT。目前的资助将进行进一步的研究，以测试这一假设，使用在培养中生长的MRT细胞，来自这些细胞的DNA和蛋白质，以及在神经组织中失去INI 1/SNF 5/BAF 47表达的转基因小鼠。通过这些研究，我们将扩大我们对其正常生物学和生化活性的理解，并确定治疗这种高度侵袭性肿瘤的新途径。

DESCRIPTION (provided by applicant): The identification of tumor suppressor genes has led to new insights into the mechanisms of human cancer development. The normal functions of these genes often lie in the control of gene expression, especially in the realm of cell cycle control and cellular differentiation. Several recent studies have implicated aberrant activity of chromatin remodeling complexes in the development of human cancer. Mutations in the INI1/SNF5/BAF47 gene, a component of the SWI/SNF chromatin remodeling complex, occur in virtually all malignant rhabdoid tumors (MRTs). The SWI/SNF complex acts as a global transcriptional activator that alters nucleosome positioning on DNA via an energy-dependent mechanism. During the previous funding period, we have determined that loss of SNF5 expression affects cell cycle regulation in all MRT cell lines. We also developed 2 genetically engineered mouse (GEM) models for MRT development where loss of expression of the pRb family members or decreased expression of p53 accelerates the appearance of spinal cord MRTs. Importantly, our preliminary studies have now put forward 4 candidate genes suspected of supporting SNF5 tumor suppressor action. Based on these results, we hypothesize that loss of SNF5 expression can lead to MRT development through disruption of neural differentiation. To test this hypothesis, we propose 3 specific aims. In the first specific aim, we will characterize novel targets of SNF5 in human MRT cell lines using Taq-Man(R) assays, Western blotting and ChIPs, determine their role in MRT oncogenesis using cell culture models and assess the effects of SNF5 loss on chromatin structure and modifications in their promoter regions. The second specific aim will identify downstream targets of SNF5 associated with MRT development using a combination of high-throughput DNA sequencing approach (ChIP-seq) and gene expression array analyses. In the third specific aim, we will determine the role of Snf5 inactivation in neural cell development and malignant rhabdoid tumorigenesis using novel genetically engineered mouse models, neurosphere cultures and a comparison of mouse spinal cord MRTs with human MRTs of the central nervous system. The dissection of the role SNF5 plays in MRT development will broaden our understanding of its normal biological and biochemical activities, provide new insights into the function of the SWI/SNF complex in cell cycle regulation and normal development and identify new avenues of treatment for this highly aggressive tumor. PUBLIC HEALTH RELEVANCE: Tumor suppressor genes are turned off in many human cancers and are often responsible for the familial forms of this disease. We are studying one of these genes called INI1/SNF5/BAF47 that plays an important role in the development of an aggressive childhood cancer of the kidney, brain and soft tissues called malignant rhabdoid tumor (MRT). Our previous studies have suggested that the loss of this gene before or early after birth may interfere with normal nervous system development leading to the appearance of MRT in children. This current grant will carry out further studies to test this hypothesis using MRT cells grown in culture, DNA and protein from these cells and genetically-modified mice that will lose INI1/SNF5/BAF47 expression in nervous tissues. By doing these studies, we will broaden our understanding of its normal biological and biochemical activities and identify new avenues of treatment for this highly aggressive tumor.