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

• 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
• 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

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.

项目摘要 液泡H+-ATPase（V-ATPase）是一个必需的质子泵，由癌细胞利用以促进 增殖，迁移和耐药性。在正常细胞中，该泵在亚细胞中产生定义的pH 对于细胞器的通信和功能至关重要的细胞器，因此与V-ATPase密不可分 到各种基本的细胞过程。在正常的酸分泌细胞中，在血浆上发现了V-ATPase 膜将质子从细胞中抽出，例如尿酸化和骨骼 吸收。 V-ATPase功能对各种细胞过程的影响由膜对 因此，酶驻留在正常细胞中，其丰度和定位受到严格控制。在 但是，许多癌症的V-ATPase被上调和定位错误，这是对癌症的重要适应 生存。实际上，抑制V-ATPase会导致转移的抑制，药物敏感性提高和 最终，癌细胞死亡。但是，V-ATPase功能的总损失是胚胎致死的，大多数是 酶的〜15个不同亚基表示为多种同工型，对这两种亚型施加了明显的障碍 酶的研究和治疗靶向。重要的是，亚基A作为四种同工型（A1-4），有 差分组织和（子）细胞定位以及最近的特定同工型已被证明为 在乳房（A3，A4）和卵巢癌（A2）中过表达和误置。此外，我们的初步数据 表明A4在肾脏癌中高度上调。这项工作的长期目标是改善 癌症患者的结局是通过揭示用于治疗发育的新靶标的，即 人类V-ATPase。这里提出的工作的直接目标是生成一个强大的新工具 用于研究特定V-ATPase种群的域抗体或纳米型（NBS）。 NB是从 在Camelidae中发现的独特的重链抗体，比传统抗体具有许多优势。 例如，NBS很小，高度稳定，可在细胞内使用。我们将使用生化和 以下特定目的中的生物物理方法，活细胞成像和基于细胞的测定：1。）生成和 针对人V-ATPase的亚基A同工型的纳米体的表征和2。）NB介导 V-ATPase同工型A4在肾癌中的表征和消融。该项目旨在克服 通过开发新颖和创新的工具对V-ATPase同工型的研究中的当前局限性，这将具有 在人类健康中了解特定V-ATPase人群的高度变革潜力和 疾病。在拟议的研究结束时，我们希望将NBS建立为有力的手段 V-ATPase的研究同工型和这些NBS的实施将阐明同工型的特定作用 A4在促进肾癌生存和恶性表型中。由于 这些研究将为开发针对亚基特定亚基的新型治疗剂提供坚定的基础 癌症中V-ATPase种群的靶向。