IDENTIFYING GERMLINE GENES AS TARGETS FOR THERAPEUTIC INTERVENTION IN CANCER

说明(申请人提供)：癌症研究的最新进展突出了抗癌药物靶标的一个新类别：在生殖系中表达的基因产物(Janic等人，2010年；Georlette等人，2007年；Wu和Ruvkun等人，2010年；Liu等人，2011年；Strumane等人，2006年；卡巴列罗等人，2009年)。对果蝇的开创性研究表明，肿瘤抑制基因的缺失会导致脑部恶性肿瘤，表现为体细胞到生殖系的转化，其中25%上调的基因具有胚系相关功能(Janic等人，2010年)。这些肿瘤上调的基因中有许多是母体起源的。重要的是，阻断这些基因的表达会抑制肿瘤的生长。生殖细胞至少有三个癌症的重要特征，即维持增殖状态、抵抗细胞死亡和经历上皮-间充质转化的侵袭和转移能力。这些发现有力地支持了筛选生殖细胞成分的价值，因为它们能够促进这些行为，作为确定癌症治疗的有效治疗靶点的一步。然而，以前识别生殖系成分的工作几乎完全依赖于RNA微阵列数据，而蛋白质在很大程度上仍然未知，大多数RNA没有特征，这减缓了这一领域的进展(Yatsu等人，2008年；Molyneaux等人，2004年；Ewen和Koopman，2010年)。生殖质是生殖细胞前体所特有的亚细胞域，它包含了确定不同生物体中生殖细胞谱系所需的所有决定因素。我们的中心假设是，种质将为抗癌药物提供丰富的新靶点来源。不幸的是，在技术上很难分离出足够数量的种质来进行蛋白质分析，因此，脊椎动物的遗传信息一直受到限制。非洲爪哇提供了一个独特的机会来分离生化数量的种质，使其有可能，利用目前的技术，创建一个完整的“部件清单”的这台细胞“机器”，指定全身性生殖细胞谱系。此外，非洲爪哇是高度服从表达克隆，允许高通量的方法来评估种质组分的基因功能。为了验证我们的中心假设，我们将完成以下两个特定目标：目标1.确定种质的蛋白质和RNA成分，并使用这些信息预测在种系中运行的基因网络和在癌细胞系中上调的基因网络。目的2.在生物检测中测试这些生殖细胞成分是否有能力促进癌症的生物学“特征”：转移、不朽和增殖。在初步研究中，我们已经分离出生化数量的种质，并通过液相色谱-串联质谱仪分析鉴定了400多种蛋白质。我们现在准备开展更广泛的研究，从功能上筛选与癌症生物学可能相关的种质成分。 与公共健康相关：癌症研究的最新进展突出了抗癌药物的一种新的靶点：在生殖系中表达的基因产品。我们提议的研究将识别并从功能上筛选这些高价值的生殖系靶标，以确定它们是否有能力促进癌症的“特征”：不受控制的细胞分裂、对细胞死亡的免疫力和转移。我们的研究将为新的抗癌药物的开发找到新的候选者。

DESCRIPTION (provided by applicant): Recent progress in cancer research has highlighted a new category of targets for anti-cancer drugs: gene products expressed in the germline (Janic et al., 2010; Georlette et al., 2007; Wu and Ruvkun, 2010; Liu et al., 2011; Strumane et al., 2006; Caballero et al., 2009). Seminal work in Drosophila has shown that loss of a tumor suppressor gene causes brain malignancies that display a soma-to-germline transformation with 25% of up-regulated genes having a germline-associated function (Janic et al., 2010). Many of these tumor up-regulated genes are of maternal origin. Importantly, blocking the expression of these genes suppressed tumor growth. Germ cells share at least three important characteristics of cancer, the ability to: sustain a proliferative state, resist cell death, and undergo an epithelial-mesenchymal transition characteristic of invasion and metastasis. These findings strongly support the value of screening germ cell components for their ability to promote these behaviors as a step towards identifying potent therapeutic targets for cancer treatments. However, previous work on identifying germline components has relied almost entirely on RNA microarray data while the proteins remain largely unknown and the majority of RNAs uncharacterized, slowing progress in this area (Yatsu et al., 2008; Molyneaux et al., 2004; Ewen and Koopman, 2010). Germ plasm is the subcellular domain unique to germ cell precursors that contains all the determinants required to specify the germ cell lineage in diverse organisms. Our central hypothesis is that germ plasm will provide a rich source of new targets for anti-cancer drugs. Unfortunately, it is technically difficult to isolate germ plasm in sufficient quantities for proten analysis and thus, genetic information in vertebrates has been limited. Xenopus offers a unique opportunity to isolate biochemical quantities of germ plasm, making it possible, with current technology, to create a complete "parts list" of this cellular "machine" that specifies the totipotnt germ cell lineage. Moreover, Xenopus is highly amenable to expression cloning, allowing a high-throughput approach to assess gene function of germ plasm components. To test our central hypothesis, we will complete the following two specific aims: Aim 1. Identify the protein and RNA components of germ plasm and use this information to predict gene networks operating in the germline and up-regulated in cancer cell lines. Aim 2. Test these germ cell components for their ability to promote the biological "hallmarks" of cancer: metastasis, immortality, and proliferation in bioassays. In preliminary studies, we have isolated biochemical amounts of germ plasm and have identified over 400 proteins by Liquid Chromatography-tandem Mass Spectrometry analyses. We are now ready to launch into broader studies functionally screening germ plasm components for their possible relevance to cancer biology. PUBLIC HEALTH RELEVANCE: Recent progress in cancer research has highlighted a new category of targets for anti-cancer drugs: gene products expressed in the germline. Our proposed research will identify and functionally screen such high value germline targets for their ability to promote the "hallmarks" of cancer: uncontrolled cell division, immunity to cell death, and metastasis. Our research will discover novel candidates for the development of new anti-cancer drugs.