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

p53 同时靶向细胞核和线粒体用于癌症治疗

基本信息

批准号：
8100507
负责人：
Carol S. Lim
金额：
$ 30.11万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8100507
关键词：
Adenovirus Vector Apoptosis Apoptotic Back Binding Biological Assay Breast Breast Cancer Cell Breast Carcinoma Caspase Cause of Death Cell Death Cell Fractionation Cell Nucleus Cells Cellular Morphology Combined Modality Therapy Cytochromes Cytoplasm DNA Dexamethasone Dimerization Dose DsRed Emerging Technologies Engineering Excision Exclusion Exhibits Female Fluorescence Fluorescence Microscopy Future Genes Goals Human Imagery Immunoblotting Immunocompromised Host Immunoprecipitation Inflammatory Kinetics Label Ligand Binding Domain Ligands MCF7 cell Malignant Neoplasms Measurement Measures Mediating Mitochondria Monitor Multi-Drug Resistance Mus Mutate Mutation Neoplasm Metastasis Normal Cell Noxae Nuclear Nuclear Export Nuclear Localization Signal Nude Mice Pathway interactions Pharmaceutical Preparations Play Protein p53 Proteins Reporter Genes Role Signal Transduction Signaling Protein Site-Directed Mutagenesis Solid Neoplasm Staining method Stains Subcutaneous Injections System T47D Technology Testing Therapeutic Therapeutic Effect Time Transcriptional Activation Transfection Treatment Efficacy Tumor Suppressor Proteins Two-Hybrid System Techniques Western Blotting Xenograft Model Xenograft procedure annexin A5 cancer cell cancer prevention cancer therapy cancer type cell killing effective therapy enhanced green fluorescent protein gene therapy improved in vivo in vivo Model inhibitor/antagonist malignant breast neoplasm mutant novel prevent promoter public health relevance response restoration small molecule survivin targeted delivery therapeutic target tumor

项目摘要

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.

描述（由申请人提供）：该项目的目的是将肿瘤抑制因子（p53）靶向2个不同的细胞室，作为用于乳腺癌和其他类型的癌症的新的双基因治疗方法。在正常细胞中，p53蛋白主要位于细胞的细胞核中，它可以充当肿瘤抑制剂并引起凋亡。在某些条件下，当针对线粒体时，它也凋亡。在许多类型的癌症中，p53被错误地定位于细胞质或灭活。实际上，p53已成为预防癌症的主开关，并被积极作为最终的癌症治疗靶标。为了将p53靶向核，我们将使用新兴的蛋白质开关技术在细胞细胞质中捕获错误定位的p53，可以通过添加外部药物将其拖到细胞核中。一旦进入细胞核，p53将导致癌细胞死亡。为了将p53靶向线粒体，将创建改进的线粒体指向版本的p53。这种2-基因治疗方法可能会增加p53细胞杀伤能力的效力。最初证明这项技术的目标是乳腺癌，未来在炎症性乳腺癌（IBC）中使用了一种非常侵略性和致命的乳腺癌形式。 IBC的p53错误定位或突变，因此应轻易对这种类型的治疗做出反应。这种方法适用于涉及p53错误定位，核排除，突变或失活的所有类型的癌症。该项目的目的如下：1）证明，具有核定位信号（NLS）p53的蛋白质转换版本将在添加配体时从细胞质转移到细胞核，并本质上引起凋亡，或者在结合内源性错误的偏置p53时启动增强的凋亡； 2）证明，用改进的线粒体靶向信号设计的p53的核位置不足版本将通过内在凋亡途径触发凋亡； 3）评估AIM 1的核靶向蛋白转换p53的能力，以及从AIM 2中优化的线粒体优化的p53在组合疗法中诱导乳腺癌细胞中凋亡的能力，与单独的任何一种结构的作用相比，乳腺癌细胞中的凋亡。也将测试使用腺病毒载体在细胞中的癌症特异性启动子和传递。 4）验证通过腺病毒载体传递的AIM 3的组合疗法将在体内人类异种移植实体瘤鼠模型中消除或减少乳腺癌。最后，此处描述的方法更有利，因为在某些情况下，当前策略（包括给予可以恢复肿瘤抑制剂功能的小分子抑制剂），因为将p53直接靶向细胞的主动隔室将允许在特定和同时或同步或同时或同步或同时或同时启发凋亡的固有性和额外途径。预计这种双重基因疗法对目前没有有效疗法的其他类型的侵略性癌症有益。公共卫生相关性：肿瘤抑制p53在大多数类型的癌症中都是无活性或故障，使p53恢复是癌症治疗的主要候选者。我们的目标是将p53添加到癌细胞中，同时将p53靶向其最活跃的细胞室，即细胞核和线粒体。我们的长期目标是将目标递送作为有效的癌症治疗。