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

项目摘要/摘要 这个项目的目标是将肿瘤抑制基因(P53)定位于两个不同的细胞隔间，作为新的双重基因 乳腺癌和其他类型癌症的基因治疗方法。在正常细胞中，P53蛋白主要位于 细胞的核，在那里它可以起到肿瘤抑制因子的作用并引起细胞凋亡。在某些情况下，它 当被定向到线粒体时，也是凋亡活跃的。在许多类型的癌症中，P53被错误定位为 细胞质或失活。事实上，p53已经成为癌症预防的总开关，并正在 积极追求成为癌症治疗的终极目标。为了将P53靶向细胞核，我们将使用我们的 新兴的蛋白质开关技术可以捕获细胞质中错误定位的P53，它可以被拖动到 加了外源性药物的细胞核。一旦进入细胞核，p53就会导致癌细胞死亡。至 将P53靶向线粒体，将产生一个改进的线粒体导向的P53版本。本条例草案2- 基因治疗方法有可能提高P53细胞的杀伤能力。最初的目标是 证明这项技术是乳腺癌，未来将用于炎症性乳腺癌(IBC)，一种非常 侵袭性和致命性乳腺癌。IBC已经错位或突变了P53，因此应该 对这种疗法很容易有反应。这种方法适用于所有涉及p53基因的癌症。 定位错误、核排斥、突变或失活。本项目的目标如下：1) 证明带有核定位信号(NLS)的P53蛋白开关版本将从 当配基增加时，细胞质进入细胞核，并本质上导致细胞凋亡或启动增强的细胞凋亡 当结合内源性错位P53时；2)证明核定位缺陷的P53版本 带有改进的线粒体靶向信号的工程将通过内在的凋亡来触发细胞凋亡 3)评估Aim 1核靶蛋白Switch-P53的能力，以及线粒体 在联合治疗中优化Aim 2中的P53，以诱导乳腺癌细胞凋亡，导致 与任何一种构造单独作用相比，增强了效力。一种癌症特异性启动子并在 使用腺病毒载体的细胞也将进行测试；4)验证Aim 3的联合疗法 腺病毒载体可根除或减少人移植瘤实体瘤小鼠的乳腺癌 活体模型。最后，这里描述的方法比当前的策略(包括 在某些情况下，给予可恢复肿瘤抑制功能的wt p53或小分子抑制剂) 因为将P53直接靶向细胞的活性隔间将允许触发固有和 以一种特定的、同时的或协同的方式实现外在的细胞凋亡。这种双基因疗法 也有望有益于其他类型的侵袭性癌症，目前还没有有效的 治疗。

Project Summary/Abstract 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.