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，可以作为肿瘤抑制因子并引起细胞凋亡。在某些条件下，当它定向到线粒体时也具有细胞凋亡活性。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，通过添加外部药物可以将其拖至细胞核。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 p53 的改进的线粒体定向版本。这种 2-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袭性和致命性的乳腺癌。 IBC 的 p53 定位错误或突变，因此应该很容易对此类治疗产生反应。该方法适用于涉及 p53 错误定位、核排斥、突变或失活的所有类型的癌症。该项目的目标如下： 1) 证明具有核定位信号（NLS）的 p53 蛋白开关版本将在添加配体后从细胞质易位到细胞核并内在地引起细胞凋亡，或在结合内源性错误定位的 p53 时启动增强的细胞凋亡; 2) 证明具有改进的线粒体靶向信号的 p53 核定位缺陷版本将通过内在凋亡途径触发细胞凋亡； 3) 评估联合疗法中来自 Aim 1 的核靶向蛋白开关-p53 和来自 Aim 2 的线粒体优化 p53 诱导乳腺癌细胞凋亡的能力，与单独使用任一构建体的作用相比，其效力增加。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传递； 4) 验证通过腺病毒载体传递的 Aim 3 联合疗法将在体内人异种移植实体瘤小鼠模型中根除或减少乳腺癌。最后，这里描述的方法比目前的策略（包括施用wt 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触发内在和外在的以特定且同时或协同的方式进行细胞凋亡途径。这种双基因疗法也有望对目前尚无有效疗法的其他类型的侵袭性癌症有益。公共健康相关性：肿瘤抑制因子 p53 在大多数类型的癌症中失活或出现故障，因此恢复 p53 成为癌症治疗的主要候选者。我们的目标是将 p53 添加回癌细胞中，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 p53 的靶向递送作为有效的癌症治疗剂。