Simultaneous Targeting of p53 to the Nucleus and Mitochondria for Cancer Therapy

描述（由申请人提供）：该项目的目标是将肿瘤抑制因子（p53）靶向2个不同的细胞区室，作为乳腺癌和其他类型癌症的新双基因治疗方法。在正常细胞中，p53蛋白主要位于细胞核中，在那里它可以作为肿瘤抑制因子并引起细胞凋亡。在某些条件下，它在被引导至线粒体时也具有抗肿瘤活性。在许多类型的癌症中，p53错误定位于细胞质或失活。事实上，p53已经成为癌症预防的主开关，并被积极追求为最终的癌症治疗靶点。为了将p53靶向细胞核，我们将使用我们新兴的蛋白质开关技术来捕获细胞质中错误定位的p53，通过添加外部药物可以将其拖到细胞核中。一旦进入细胞核，p53就会导致癌细胞死亡。为了使p53靶向线粒体，将产生p53的改进的神经定向版本。这种双基因治疗方法可能会增加p53细胞杀伤能力的效力。展示这项技术的最初目标是乳腺癌，未来将用于炎症性乳腺癌（IBC），这是一种非常具有侵略性和致命性的乳腺癌。IBC具有错误定位或突变的p53，因此应该容易对这种类型的治疗作出反应。这种方法适用于涉及p53错误定位、核排斥、突变或失活的所有类型的癌症。本课题的主要目的是：1）证明具有核定位信号（nuclearlocalizationsignal，NLS）的p53蛋白开关（proteinswitch）在配体的作用下从细胞质转位到细胞核，并内在地引起细胞凋亡，或与内源性错误定位的p53蛋白结合后启动细胞凋亡; 2）证明用改进的线粒体靶向信号工程化的p53的核定位缺陷形式将通过内在凋亡途径触发凋亡; 3）评估来自Aim 1的核靶向蛋白转换-p53和来自Aim 2的经细胞学优化的p53在组合疗法中诱导乳腺癌细胞凋亡的能力，导致与任一构建体单独的作用相比增加的效力。还将测试癌症特异性启动子和使用腺病毒载体在细胞中的递送; 4）证实通过腺病毒载体递送的来自Aim 3的组合疗法将在体内根除或减少人异种移植实体瘤鼠模型中的乳腺癌。最后，本文所述的方法比目前的策略（包括施用野生型p53或在某些情况下可以恢复肿瘤抑制功能的小分子抑制剂）更有利，因为将p53直接靶向细胞的活性区室将允许以特异性和同时或协同的方式触发细胞凋亡的内在和外在途径。这种双基因疗法也有望对目前没有有效疗法的其他类型的侵袭性癌症有益。 公共卫生相关性：肿瘤抑制因子p53在大多数类型的癌症中是无活性的或发生故障的，并且使p53的恢复成为癌症治疗的主要候选者。我们的目标是将p53重新添加到癌细胞中，同时将p53靶向其最活跃的细胞区室，即细胞核和线粒体。我们的长期目标是使用靶向递送p53作为有效的癌症治疗剂。

DESCRIPTION (provided by applicant): The goal of this project is to target a tumor suppressor (p53) to 2 different cellular compartments as new dual gene therapy approach for breast and other types of cancers. In normal cells, p53 protein is located mostly in the nucleus of the cell where it can act as a tumor suppressor and cause apoptosis. Under certain conditions it is also apoptotically active when directed to the mitochondria. In many types of cancers, p53 is mislocalized to the cytoplasm or inactivated. Indeed, p53 has emerged as a master switch for cancer prevention and is actively pursued as the ultimate cancer therapeutic target. To target p53 to the nucleus, we will use our emerging protein switch technology to capture mislocalized p53 in the cell cytoplasm, which can be dragged to the nucleus with addition of an external drug. Once in the nucleus, p53 will cause death of the cancer cell. To target p53 to the mitochondria, an improved mitochondrially directed version of p53 will be created. This 2- gene therapy approach is likely to increase the potency of p53 cell-killing ability. The initial target to demonstrate this technology is breast cancer, with future use in inflammatory breast carcinoma (IBC), a very aggressive and deadly form of breast cancer. IBC has mislocalized or mutated p53 and therefore should readily respond to this type of therapy. This approach is applicable to all types of cancers involving p53 mislocalization, nuclear exclusion, mutation, or inactivation. The aims of this project are as follows: 1) Demonstrate that protein switch versions of p53 with nuclear localization signal (NLS) will translocate from the cytoplasm to the nucleus upon ligand addition and intrinsically cause apoptosis, or initiate enhanced apoptosis when binding endogenous mislocalized p53; 2) Prove that a nuclear-localization deficient version of p53 engineered with an improved mitochondrial targeting signal will trigger apoptosis via the intrinsic apoptotic pathway; 3) Assess the ability of the nuclear targeted protein switch-p53 from Aim 1, and the mitochondrially optimized p53 from Aim 2 in combination therapies to induce apoptosis in breast cancer cells, resulting in increased potency compared to the action of either construct alone. A cancer-specific promoter and delivery in cells using an adenoviral vector will also be tested; 4) Validate that the combination therapy from Aim 3 delivered via adenovirus vector will eradicate or reduce breast cancer in a human xenograft solid tumor murine model in vivo. Finally, the approach described here is more advantageous that current strategies (including administering wt p53 or small molecule inhibitors that can restore tumor suppressor function in some cases) because targeting of p53 directly to active compartments of the cell will allow triggering of both intrinsic and extrinsic pathways of apoptosis in a specific and simultaneous or synergistic manner. This dual gene therapy is also expected to be beneficial for other types of aggressive cancers that currently have no effective therapies. PUBLIC HEALTH RELEVANCE: The tumor suppressor p53 is inactive or malfunctioning in most types of cancer, and making restoration of p53 a prime candidate for cancer therapy. Our goal is to add p53 back into cancer cells, and simultaneously target p53 to its most active cellular compartments, the nucleus and mitochondria. Our long term goal is to use targeted delivery of p53 as a potent cancer therapeutic.