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：
8467689
负责人：
Carol S. Lim
金额：
$ 28.29万
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/8467689
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 abstracting 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 response restoration small molecule survivin targeted delivery therapeutic target tumor

项目摘要

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.

项目摘要/摘要该项目的目的是将肿瘤抑制因子（p53）靶向2个不同的蜂窝室乳腺癌和其他类型的癌症的基因治疗方法。在正常细胞中，p53蛋白主要位于该细胞的细胞核可以充当肿瘤抑制并引起凋亡。在某些条件下当针对线粒体时，也是凋亡的活动。在许多类型的癌症中，p53被错误定位到细胞质或灭活。确实，p53已成为预防癌症的主开关，是积极追求最终的癌症治疗靶点。要将p53靶向核，我们将使用我们的新兴的蛋白质开关技术以捕获细胞质中置换错误的p53，可以将其拖放到核与添加外部药物的核。一旦进入细胞核，p53将导致癌细胞死亡。到将创建目标p53到线粒体，将创建改进的线粒体定向版本的p53。这个2- 基因治疗方法可能会增加p53细胞杀伤能力的效力。最初的目标证明这项技术是乳腺癌，未来在炎症性乳腺癌（IBC）中使用，非常乳腺癌的侵略性和致命形式。 IBC的定位错误或突变p53，因此应很容易回应这种类型的疗法。这种方法适用于涉及p53的所有类型的癌症错误定位，核排斥，突变或灭活。该项目的目的如下：1）证明p53具有核定位信号（NLS）的蛋白质转换版本将从添加配体时细胞质到细胞核，并本质上引起凋亡，或引发凋亡增强结合内源性错误定位的p53时； 2）证明p53的核位置不足版本用改进的线粒体靶向信号设计的将通过内在凋亡触发凋亡途径； 3）评估AIM 1的核靶向蛋白p53-p53的能力，线粒体的能力在组合疗法中优化了AIM 2的p53，以诱导乳腺癌细胞凋亡，导致与仅两个构造的作用相比，效力提高。特定于癌症的启动子和交付使用腺病毒载体的细胞也将进行测试； 4）验证AIM 3的联合疗法通过腺病毒载体传递将消除或减少人异种移植物实体瘤鼠的乳腺癌体内模型。最后，这里描述的方法比当前的策略（包括在某些情况下，使用WT p53或小分子抑制剂，可以恢复肿瘤抑制剂的功能）因为将p53直接靶向细胞的活动室将允许触发内在和凋亡的外在途径以特定和同时或协同的方式。这种双重基因疗法预计还将对当前没有有效的其他类型的侵略性癌症有益疗法。