A novel tool for organelle and isoform specific targeting of V-ATPase in cancer

癌症中 V-ATP 酶的细胞器和亚型特异性靶向的新工具

基本信息

批准号：
9764745
负责人：
Stephan Wilkens
金额：
$ 21.14万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9764745
关键词：
Ablation Acidosis Acids Affinity Aging Antibodies Area Binding Biochemical Biological Biological Assay Bone Resorption Breast Cancer Patient Cell Death Cell Proliferation Cell Survival Cell membrane Cell physiology Cell surface Cells Cellular Membrane Collaborations Communication Complement Complex Data Development Disease Drug resistance Embryo Enzyme Inhibition Enzymes Epidermal Growth Factor Receptor Eukaryotic Cell FRAP1 gene Foundations Funding Generations Goals Growth Health Human Institution Intercalated Cell Kidney Label Link Malignant Neoplasms Malignant neoplasm of ovary Mediating Membrane Metabolic Metastasis Suppression Neoplasm Metastasis Normal Cell Organelles Osteoclasts Pathway interactions Patient-Focused Outcomes Phosphotransferases Physiological Play Population Process Protein Isoforms Proton Pump Pump Recording of previous events Renal Cell Carcinoma Renal carcinoma Request for Proposals Research Role Signal Pathway Signal Transduction Stimulus Structure Testing Tissues Urology Work antigen binding base biophysical techniques biophysical tools cancer cell cancer survival drug sensitivity experimental study human disease improved inhibitor/antagonist innovation live cell imaging mRNA Differential Displays malignant phenotype migration nanobodies new therapeutic target novel novel therapeutics overexpression pH Homeostasis receptor response study population targeted treatment therapeutic development therapeutic target tool trafficking tumor tumor progression urinary vacuolar H+-ATPase

项目摘要

Project Summary The vacuolar H+-ATPase (V-ATPase) is an essential proton pump that is exploited by cancer cells to promote proliferation, migration and drug resistance. In normal cells, this pump creates a defined pH in subcellular organelles that is essential for organelle communication and function, and thus, inextricably ties the V-ATPase to diverse fundamental cellular processes. In normal acid secreting cells, the V-ATPase is found on the plasma membrane where it pumps protons out of the cell, a process required for e.g. urinary acidification and bone resorption. The impact of V-ATPase function on various cellular processes is determined by the membrane on which the enzyme resides, therefore, in normal cells, its abundance and localization are tightly controlled. In many cancers, however, the V-ATPase is upregulated and mislocalized, an essential adaptation for cancer survival. Indeed, inhibition of the V-ATPase leads to suppression of metastasis, increased drug sensitivity and ultimately, cancer cell death. However, total loss of V-ATPase function is embryonic lethal and most of the enzyme's ~15 different subunits are expressed as multiple isoforms, imposing significant barriers to both the study and therapeutic targeting of the enzyme. Importantly, subunit a exists as four isoforms (a1-4), with differential tissue and (sub)cellular localization and recently, specific isoforms have been shown to be overexpressed and mislocalized in breast (a3, a4) and ovarian cancers (a2). Further, our preliminary data indicates that a4 is highly upregulated in renal cancers. The long term objectives of this work are to improve cancer patient outcomes by revealing novel targets for therapeutic development, namely subunit isoforms of the human V-ATPase. The immediate goal of the here proposed work is to generate a powerful new tool, single domain antibodies or Nanobodies (Nbs), for the study of specific V-ATPase populations. Nbs are derived from the unique heavy chain antibodies found in Camelidae and have many advantages over traditional antibodies. For example, Nbs are small, highly stable, and can be used intracellularly. We will use biochemical and biophysical methods, live cell imaging and cell based assays in the following Specific Aims: 1.) Generation and characterization of Nanobodies against subunit a isoforms of human V-ATPase and 2.) Nb mediated characterization and ablation of V-ATPase isoform a4 in kidney cancer. This project is aimed at overcoming the current limitations in the study of V-ATPase isoforms by developing novel and innovative tools, which will have highly transformative potential for the understanding of specific V-ATPase populations in human health and disease. At the end of the proposed research, we expect to have established Nbs as a powerful means to study isoforms of the V-ATPase and that implementation of these Nbs will illuminate the specific role of isoform a4 in promoting kidney cancer survival and malignant phenotypes. The information generated as a result of these studies will provide a firm foundation for developing novel therapeutics for subunit isoform specific targeting of V-ATPase populations in cancer.

项目概要液泡 H+-ATP 酶（V-ATP 酶）是一种重要的质子泵，癌细胞利用它来促进增殖、迁移和耐药性。在正常细胞中，该泵在亚细胞中产生特定的 pH 值细胞器对于细胞器通讯和功能至关重要，因此与 V-ATP 酶有着千丝万缕的联系不同的基本细胞过程。在正常的酸分泌细胞中，V-ATP酶存在于血浆中膜将质子泵出细胞，这是例如细胞所需的过程。尿液酸化与骨骼吸收。 V-ATP酶功能对各种细胞过程的影响是由细胞膜决定的因此，在正常细胞中，酶的丰度和定位受到严格控制。在然而，在许多癌症中，V-ATP 酶上调且定位错误，这是癌症的重要适应生存。事实上，抑制 V-ATP 酶可抑制转移、增加药物敏感性和最终，癌细胞死亡。然而，V-ATP酶功能的完全丧失对于胚胎来说是致命的，并且大多数酶的约 15 个不同亚基被表达为多种亚型，这对两种酶都造成了显着的障碍该酶的研究和治疗靶向。重要的是，亚基 a 以四种亚型 (a1-4) 的形式存在，其中差异组织和（亚）细胞定位，最近，特定亚型已被证明是在乳腺癌 (a3、a4) 和卵巢癌 (a2) 中过度表达和错误定位。此外，我们的初步数据表明 a4 在肾癌中高度上调。这项工作的长期目标是改进通过揭示治疗开发的新靶标（即亚单位亚型）来改善癌症患者的结果人类 V-ATP 酶。这里提出的工作的直接目标是生成一个强大的新工具，单一域抗体或纳米抗体 (Nbs)，用于研究特定的 V-ATP 酶群体。 Nbs 源自骆驼科动物中发现的独特重链抗体，与传统抗体相比具有许多优势。例如，Nbs体积小，稳定性高，并且可以在细胞内使用。我们将使用生化和生物物理方法、活细胞成像和基于细胞的测定，具体目标如下：1.) 生成和针对人 V-ATP 酶亚基 a 亚型的纳米抗体的表征和 2.) Nb 介导肾癌中 V-ATP 酶亚型 a4 的表征和消融。该项目旨在克服通过开发新颖和创新的工具来克服目前 V-ATP 酶亚型研究的局限性，这将有对于了解人类健康中特定 V-ATP 酶群体的高度变革潜力疾病。在拟议的研究结束时，我们希望将 Nbs 建立为一种强有力的手段研究 V-ATP 酶的亚型，这些 Nb 的实施将阐明亚型的具体作用 a4 促进肾癌存活和恶性表型。所产生的信息这些研究将为开发亚单位亚型特异性的新疗法奠定坚实的基础靶向癌症中的 V-ATP 酶群体。