Genetic Interaction Between the pRB and Warts Tumor Suppressor Pathways

描述（申请人提供）：视网膜母细胞瘤蛋白（retinoblastoma protein, pRB）是非常重要的肿瘤抑制因子，在癌症形成过程中经常发生突变或失活。作为一种肿瘤抑制因子，pRB调节E2F转录因子家族的活性。E2F家族的特征是“激活因子”和“抑制因子”，两者传统上与靶基因启动子处的DP蛋白形成异源二聚体。在缺乏pRB的情况下，激活因子E2Fs可以通过激活G1周期蛋白的转录来驱动细胞进入细胞周期。因此，在pRB功能丧失的癌症中，E2F活性高，因此，肿瘤继续生长和分裂。由于在肿瘤形成过程中pRB功能丢失的发生率很高，因此有研究表明，靶向抑制E2F家族可能是一种很有前途的抗癌治疗方法。然而，由于E2F、pRB和Dp家族成员数量众多，在哺乳动物模型系统中开展这些研究具有挑战性。幸运的是，果蝇（Drosophila melanogaster）在E2F、pRB和DP家族之间的复杂性较低。有一个激活因子E2F（dE2F1）和一个抑制因子E2F(dE2F2)，当激活因子E2F因突变或靶向抑制而丢失时，会导致严重的增殖阻滞。因此，强化了E2F活性的丧失可以帮助减缓癌症生长的观点。然而，当所有E2F活性丧失时，无论体内还是体外，细胞都能相对正常地增殖。最近，在果蝇中发现了一种新的肿瘤抑制通路，称为Hippo通路。该通路的所有成员在人类中都有同源物或同源物，并且在人类癌症中被发现发生突变。当这一途径变得不活跃时，细胞获得了比邻近野生型组织更强的增殖优势，并对凋亡应激具有抵抗力。这项研究的目的是测试E2Fs的缺失，在所有组合中，对具有非活性Hippo肿瘤抑制通路的组织有什么影响。本研究将结合新的和经典的果蝇遗传学、分子生物学和细胞生物学来解决这个问题。免疫组织化学分析将用于确定在缺乏E2Fs的情况下对Hippo突变组织增殖和凋亡的体内影响。此外，对dE2F依赖性转录的任何要求将通过ChIP分析确认。由于这两种途径（pRB/E2F和SWH）在果蝇和人类中的进化守恒，本研究将深入了解这两种重要的肿瘤抑制途径之间的功能关系。因此，本研究的结果可以证明靶向抑制激活剂E2Fs可能被证明是一种有效的抗癌治疗方法。

DESCRIPTION (provided by applicant): The retinoblastoma protein (pRB) is very important tumor suppressor that is often mutated or inactivated during the formation of cancer. As a tumor suppressor pRB regulates the activity of the E2F transcription factor family. The E2F family is characterized by "activators" and "repressors", both of which traditionally form a heterodimer with the DP protein at the promoters of target genes. The activator E2Fs, in the absence of pRB, can drive the cell into the cell cycle by activating the transcription of the G1 cyclins. Thus, in cancers in which pRB function is lost, E2F activity is high, and therefore, the tumor continues to grow and divide. Due to the high incidence in which pRB function is lost during tumor formation it has been suggested that targeted inhibition of the E2F family could be a promising anti-cancer therapeutic. However, to carry out these studies in the mammalian model system has proven challenging due to the overwhelming number of E2F, pRB, and Dp, family members. Fortunately, the fruit fly, Drosophila melanogaster, has less complexity between the E2F, pRB, and DP families. There is one activator E2F(dE2F1) and one repressor E2F(dE2F2), and when the activator E2F is lost due to mutation or targeted inhibition it results in a severe proliferation block. Thus, reinforcing the idea that loss of E2F activity could help slow cancerous growths. However, when all E2F activity is lost, both in vivo and in vitro, cells can proliferate with relative normality. Recently, a new tumor suppressor pathway referred to'as the Hippo pathway has been delineated in Drosophila. All members of this pathway have orthologs or homologs in humans, and are found to be mutated in human cancers. When this pathway becomes inactive, cells gain a proliferative advantage over neighboring wildtype tissue and are resistant to apoptotic stresses. This study aims to test what effects the loss of the E2Fs, in all combinations, has in tissue that has an inactive Hippo tumor suppressor pathway. This study will use a combination of new and classical Drosophila genetics, molecular biology, and cell biology to address this issue. Immunohistochemistry analysis will be used to determine in vivo effects upon proliferation and apoptosis Hippo mutant tissue in the absence of the E2Fs. Furthermore, any requirement for dE2F dependent transcription will be confirmed via ChIP analysis. Due to the evolutionary conservation between both of these pathways (pRB/E2F and SWH) in Drosophila and humans, this study will provide insight into the functional relationship between two important tumor suppressor pathways. Thus, the outcome of this study could demonstrate that targeted inhibition of activator E2Fs could prove to be a productive anticancer therapy.