Breast cancer oncogenes on the 8p11 amplicon

描述(申请人提供)：在前一个项目期间，我们对人乳腺癌细胞系和组织中的8p11-p12扩增子进行了广泛的分析，以确定和验证新的乳腺癌癌基因。通过拷贝数增加和过度表达的统计分析，我们确定了21个基因的子集作为候选癌基因。接下来，我们直接测试了这些基因在人乳腺上皮细胞中的转化功能。从这些实验中，我们确定了四个在MCF-10A细胞中具有潜在转化功能的基因，以及另外三个具有较弱转化功能的基因。已鉴定的转化能力最强的基因包括DDHD2、SPFH2、LSM1和WHSC1L1，它们可以诱导Matrigel细胞不依赖生长因子的增殖、不依赖锚定的生长、侵袭能力和改变形态发生。此外，我们确定了影响转化表型表达的基因组合。在这项工作的下一阶段，我们将进行实验，了解这些癌基因转化潜力的机制基础，并将检测它们在体外对人乳腺癌细胞的转化功能，以及在体内对人和小鼠乳腺上皮细胞的转化功能。下一个项目期的工作具体目标是：1)确定导致MCF-10A细胞转化表型的七个基因是否直接转化，并确定转化在过表达癌基因的细胞中是常见还是罕见事件；2)确定与正常乳腺上皮细胞或不含扩增子的乳腺癌细胞相比，在人乳腺癌细胞系中扩增及过表达的8p11区域基因是这些乳腺癌细胞生长和存活所必需的。我们还将检验以下假设：8p11扩增子上的某些癌基因协同作用会影响人乳腺癌细胞的转化生长潜能；3)检测8p11癌基因在人乳腺癌细胞体内过度表达的影响，并确定这些癌基因在体内是否能转化小鼠乳腺上皮细胞；4)测试WHSC1L1短异构体的过度表达会改变组蛋白甲基化密码和基因表达谱，从而导致细胞具有肿瘤起始细胞的特性，包括增强的自我更新能力、肿瘤干细胞标志物的表达和在培养中形成乳房的能力。我们还将测试这样的假设，即诱导这些改变的表型需要完整的PWWP结构域，而不需要蛋白质的固定结构域。为了开发针对这些癌基因的治疗策略，有必要明确地证明新发现的8p11转化基因是骨性乳腺癌癌基因，并阐明它们诱导细胞转化的机制。 公共卫生相关性：该项目旨在发展对新发现的乳腺癌癌基因的机械性理解。新靶向抗癌药物在临床上最有效的时候，是它们攻击负责癌症发展的癌基因的产物。为了开发针对乳腺癌的新的治疗策略，我们必须明确不同患者的致癌基因，并制定治疗策略，以更有效地治疗乳腺癌患者。这笔赠款的工作与开发新的针对乳腺癌的靶向药物直接相关。

DESCRIPTION (provided by applicant): In the previous project period, we performed an extensive analysis of the 8p11-p12 amplicon in human breast cancer cell lines and tissues to identify and validate novel breast cancer oncogenes. Using statistical analysis of copy number increase and over expression, we identified a subset of 21 genes as candidate oncogenes. Next, we directly tested the transforming function of these genes in human mammary epithelial cells. From these experiments, we identified four genes that are potently transforming in MCF-10A cells and three other genes with more modest transforming function. The most potently transforming genes identified, which include DDHD2, SPFH2, LSM1 and WHSC1L1, induce growth factor independent proliferation, anchorage-independent growth, invasive capacity, and altered morphogenesis in Matrigel. In addition, we identified gene combinations that effect the expression of transformed phenotypes. In the next phase of this work, we will perform experiments to understand the mechanistic basis for the transforming potential of these oncogenes, and we will examine their transforming function in human breast cancer cells in vitro, and in human and mouse mammary epithelial cells in vivo. The specific aims of the work in the next project period are: 1) To determine if the seven genes that induce transformed phenotypes in MCF-10A cells are directly transforming, and to determine if transformation is a common or a rare event in cells over expressing the oncogene; 2) To determine if the genes from the 8p11 region that are amplified and over expressed in human breast cancer cell lines are required for growth and survival of these breast cancer cells compared with normal mammary epithelial cells or breast cancer cells without the amplicon. We will also test the hypothesis that some oncogenes on the 8p11 amplicon cooperate to influence the transformed growth potential of human breast cancer cells; 3) To determine the influence of 8p11 oncogene over expression in the in vivo growth potential of human mammary epithelial cells, and to determine if these oncogenes can transform mouse mammary epithelial cells in vivo; 4) To test the hypothesis that over expression of the short isoform of WHSC1L1 alters the histone methylation code and gene expression profile, resulting in cells that exhibit properties of tumor initiating cells, including enhanced self-renewal capacity, expression of markers of cancer stem cells, and ability to form mammospheres in culture. We will also test the hypothesis that induction of these altered phenotypes requires an intact PWWP domain and does not require the SET domain of the protein. It is essential to demonstrate unequivocally that newly discovered 8p11 transforming genes are bone fide breast cancer oncogenes, and to elucidate the mechanism by which they induce cell transformation in order to develop therapeutic strategies that target these oncogenes. PUBLIC HEALTH RELEVANCE: This project is aimed at developing a mechanistic understanding of newly discovered breast cancer oncogenes. New targeted drugs against cancer are most effective clinically when they attack the products of oncogenes that are responsible for cancer development. In order to develop new therapeutic strategies against breast cancer, it is essential that we clearly identify the oncogenes that drive the disease in different patients, and develop therapeutic strategies to more effectively treat patients with breast cancer. The work in this grant is directly relevant to the development of new targeted drugs against breast cancer.