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的双基因治疗方法。在正常细胞中，p53蛋白主要位于细胞核中，在细胞核中起肿瘤抑制作用并引起细胞凋亡。在一定条件下，当它被引导到线粒体时，它也具有凋亡活性。在许多类型的癌症中，p53错误地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，并被积极追求作为最终的癌症治疗靶点。为了将p53靶向到细胞核，我们将使用我们新兴的蛋白质开关技术捕获细胞质中错误定位的p53，可以通过添加外部药物将其拖到细胞核。一旦进入细胞核，p53就会导致癌细胞死亡。为了将p53靶向到线粒体，一种改进的线粒体导向版本的p53将被创造出来。这种2基因治疗方法可能会增加p53细胞杀伤能力的效力。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袭性和致命性的乳腺癌。IBC有p53定位错误或突变，因此应该很容易对这种类型的治疗有反应。该方法适用于所有涉及p53错位、核排斥、突变或失活的癌症类型。本项目的目的如下：1)证明带有核定位信号（nuclear localization signal， NLS）的p53蛋白开关版本在配体加入后会从细胞质转移到细胞核，并在本质上导致细胞凋亡，或在结合内源性错位p53时启动细胞凋亡增强；2)证明核定位缺陷型p53经过改进的线粒体靶向信号修饰后，可通过内在凋亡途径触发细胞凋亡；3)评估来自Aim 1的核靶向蛋白开关p53和来自Aim 2的线粒体优化p53在联合治疗中诱导乳腺癌细胞凋亡的能力，与单独构建的作用相比，其效力更高。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)验证通过腺病毒载体传递的Aim 3联合治疗将在体内根除或减少人类异种移植实体瘤小鼠模型中的乳腺癌。最后，这里描述的方法比目前的策略（包括在某些情况下给予wt p53或小分子抑制剂可以恢复肿瘤抑制功能）更有利，因为将p53直接靶向细胞的活性区室将允许以特定的、同时的或协同的方式触发细胞凋亡的内在和外在途径。这种双重基因治疗也有望对目前没有有效治疗方法的其他类型的侵袭性癌症有益。

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.