Adenovirus E1B55K Functions Related to Oncolytic Replication

描述(申请人提供)：所有癌细胞的两个主要“特征”包括细胞周期失调和细胞凋亡抑制，这两个特征也与腺病毒(Ad)感染有关。这两个过程主要由病毒癌蛋白E1a和E1B进行。由于病毒E1蛋白的功能类似于促进增殖和抑制凋亡的癌细胞因子，因此癌蛋白E1a和E1B突变的病毒可以在癌细胞中选择性复制。其中一个缺失E1B55K的突变体被称为dl1520，已被用于记录溶瘤效果的临床试验。然而，病毒溶瘤复制的机制尚未得到很好的研究，治疗效果有待提高。一般认为E1B55K的主要作用是结合和抑制P53的激活，但许多研究证明E1B55K介导的P53失活并不是病毒复制所必需的。我们的实验室已经证明：(1)突变病毒在大型肿瘤中的有限传播是降低疗效的一个关键因素；(2)AdE1A表达增加增强了病毒的溶瘤复制；(3)E1B缺失引起的细胞凋亡可以部分减少病毒的复制，但不会改变病毒介导的癌症杀伤的最终结果。我们最近的研究表明，E1B55K在诱导细胞周期蛋白E和其他细胞周期相关基因方面具有新的功能。最重要的是，我们还观察到细胞周期蛋白E的表达增加与病毒复制效率相关。E1B55K诱导的细胞周期蛋白E的表达是病毒在正常细胞中复制所必需的，但在癌细胞中不是必需的。我们推测E1B55K可能靶向细胞因子(S)增加细胞周期蛋白E的表达，而该因子(S)可能已经在癌细胞中被激活。因此，癌细胞中细胞周期蛋白E的失调可能是E1B55K缺失病毒溶瘤复制的分子基础。我们的研究团队将(1)确定E1B55K诱导周期蛋白E的靶向细胞因子，(2)确定E1B55K激活周期蛋白E表达的机制，以及(3)确定周期蛋白E过表达与E1B缺失病毒溶瘤复制之间的关系。如果我们证实溶瘤复制依赖于细胞周期蛋白E的表达和细胞增殖，侵袭性生长的肿瘤和周期蛋白E异常的患者应该会从这种腺病毒疗法中受益匪浅。这项工作的长期目标是提高溶瘤基因治疗的疗效。公共卫生相关性：缺少重要调节蛋白E1B55K的腺病毒仍然可以在一些癌细胞中扩增。因此，E1B55K突变体dl1520已用于临床试验。了解E1B55K的功能和E1B55K缺失的dl1520在癌细胞中的选择性复制是很重要的。我们最近发表的研究表明，E1B55K在诱导细胞周期蛋白E的表达方面具有新的功能，而细胞周期蛋白E是DNA复制的关键。癌细胞通常表达高水平的细胞周期蛋白E或存在该基因的失调。我们推测病毒E1B55K可能激活了一些细胞因子，这些细胞因子增加了细胞周期蛋白E的表达，从而促进了病毒DNA的复制。癌细胞可能已经激活了这些因子，因此癌细胞不需要E1B55K功能。对这种可能性的研究非常重要。如果我们证实突变的病毒复制依赖于Cyclin E的表达，那么患有侵袭性生长的肿瘤和Cyclin E异常的患者应该会从这种腺病毒疗法中受益匪浅。这项工作的长期目标是提高溶瘤基因治疗的疗效。

DESCRIPTION (provided by applicant): Two major "hallmarks" of all cancer cells include dysregulated cell cycle and inhibited apoptosis, both of which are also involved in adenovirus (Ad) infection. These two processes are primarily conducted by viral oncoproteins E1A and E1B. Since the function of viral E1 protein is similar to that of cancer cellular factors that promote proliferation and inhibit apoptosis, viruses with mutations in oncoprotein E1A and E1B can selectively replicate in cancer cells. One such mutant with deletion of E1B55K, known as dl1520, has been used in clinical trials documenting oncolytic effects. However, the mechanism of viral oncolytic replication has not been well characterized, and the therapeutic efficacy needs improvement. It is generally believed that the major role of E1B55K is to bind to and inhibit p53 activation, but many studies have documented that E1B55K-mediated p53 inactivation is not required for virus replication. Our laboratory has shown: (1) that the limited spread of mutant viruses in large tumors is a key factor in decreased therapeutic efficacy; (2) increased Ad E1A expression enhances virus-oncolytic replication; and (3) apoptosis caused by E1B deletion can partially decrease virus replication but does not change the final outcome of virus-mediated cancer killing. Our recent studies have revealed that E1B55K has a novel function in the induction of cyclin E and other cell cycle-related genes. Most importantly, we also observed that increased cyclin E expression is correlated with virus replication efficiency. E1B55K-induced cyclin E expression is required for virus replication in normal cells, but is not necessary in cancer cells. We hypothesize that E1B55K may target cellular factor(s) to increase cyclin E expression, and this factor(s) may already be activated in cancer cells. Thus, cyclin E dysregulation in cancer cells may be the molecular basis for oncolytic replication of E1B55K-deleted viruses. Our research team will (1) identify cellular factors targeted by E1B55K for cyclin E induction, (2) define the mechanism by which E1B55K activates cyclin E expression, and (3) determine the relationship between cyclin E overexpression and oncolytic replication of E1B-deleted viriuses. If we confirm that oncolytic replication relies on cyclin E expression and cell proliferation, patients with aggressively growing tumors and dysregulated cyclin E should greatly benefit from this adenoviral therapy. The long-term goal of this work is to increase the efficacy of oncolytic cancer gene therapy. PUBLIC HEALTH RELEVANCE: Adenoviruses lacking an important regulative protein-E1B55K-still can amplify in some cancer cells. Therefore, the E1B55K mutant dl1520 has been used in clinical trials. It is important to understand the E1B55K function and the selective replication of E1B55K-deleted dl1520 in cancer cells. Our recently published studies have revealed that E1B55K has a novel function in the induction of cyclin E expression, which is crucial for DNA replication. Cancer cells generally express high levels of cyclin E or have a dysregulation of the gene. We reason that viral E1B55K may activate some cellular factors that increase cyclin E expression for viral DNA replication. Cancer cells may already have the factors activated; therefore cancer cells do not require the E1B55K function. The study of this possibility is very important. If we confirm that mutated virus replication relies on cyclin E expression, patients with aggressively growing tumors and dysregulated cyclin E should greatly benefit from this adenoviral therapy. The long-term goal of this work is to increase the efficacy of oncolytic cancer gene therapy.