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)是一种重要的质子泵，被癌细胞用来促进 增殖、迁移和抗药性。在正常细胞中，这个泵在亚细胞中产生一个确定的pH 细胞器对于细胞器的通讯和功能是必不可少的，因此，V-ATPase不可避免地联系在一起 不同的基本细胞过程。在正常的酸性分泌细胞中，V-ATPase存在于血浆中 将质子泵出细胞的膜，这是尿酸和骨骼等过程所必需的 再吸收。V-ATPase功能对各种细胞过程的影响是由细胞膜决定的 因此，酶驻留在正常细胞中，其丰度和定位受到严格控制。在……里面 然而，许多癌症的V-ATPase被上调和错误定位，这是癌症的一种基本适应 生死存亡。事实上，抑制V-ATPase会抑制转移，增加药物敏感性，并 最终，癌细胞死亡。然而，V-ATPase功能的完全丧失是胚胎致命的，而且大多数 酶的~15个不同亚基以多种异构体的形式表达，对两种 该酶的研究和治疗靶向性。重要的是，a亚基以四种异构体(A1-4)存在， 不同的组织和(亚)细胞定位，最近，特定的异构体已经被证明是 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 通过揭示治疗发展的新靶点，即亚单位亚型，癌症患者的预后 人类的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，Single 结构域抗体或纳米抗体(NBS)，用于研究特定的V-ATPase群体。国家广播公司源自 在茶花科中发现的独特的重链抗体，与传统抗体相比具有许多优势。 例如，NBS很小，非常稳定，可以在细胞内使用。我们将使用生化和 生物物理学方法、活细胞成像和基于细胞的分析，具体目的如下：1.世代和 抗人V-ATPase亚基a亚型和2亚基的纳米抗体的特性。)NB中介 肾癌组织中V-ATPase亚型A4的特征及其消融这个项目的目的是克服 目前通过开发新的和创新的工具来研究V-ATPase亚型的局限性，这将具有 对人类健康和健康中特定的V-ATPase群体的理解具有极大的变革潜力 疾病。在拟议的研究结束时，我们希望建立国家统计局作为一种强有力的手段来 研究V-ATPase异构体和这些NBS的实现将阐明异构体的具体作用 A4在促进肾癌生存率和恶性表型中的作用。由于以下原因而生成的信息 这些研究将为开发亚基异构体特异性新的治疗方法提供坚实的基础 在癌症中靶向V-ATPase群体。