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 空泡H+-ATP酶（V-ATP酶）是一种重要的质子泵，被癌细胞利用以促进细胞增殖。 增殖、迁移和耐药性。在正常细胞中，该泵在亚细胞中产生确定的pH值。 细胞器是细胞器通信和功能所必需的，因此，与V-ATP酶密不可分。 to different多样fundamental基本cellular细胞processes处理.在正常的酸分泌细胞中，V-ATP酶存在于血浆中， 膜，在那里它将质子泵出细胞，这是例如尿酸化和骨形成所需的过程。 再吸收V-ATP酶功能对各种细胞过程的影响是由细胞膜上的 因此，在正常细胞中，酶存在于哪种细胞中，其丰度和定位受到严格控制。在 然而，在许多癌症中，V-ATP酶被上调和错误定位，这是癌症的一种基本适应， 生存事实上，V-ATP酶的抑制导致转移的抑制、药物敏感性的增加和 最终导致癌细胞死亡然而，V-ATP酶功能的完全丧失是胚胎致死的， 酶的~15种不同亚基以多种亚型表达，对两种酶的表达都施加了显著的障碍。 酶的研究和治疗靶向。重要的是，亚基a以四种同种型（a1-4）存在， 差异组织和（亚）细胞定位，最近，特异性同种型已被证明是 在乳腺癌（a3，a4）和卵巢癌（a2）中过表达和错误定位。此外，我们的初步数据显示， 表明A4在肾癌中高度上调。这项工作的长期目标是改善 通过揭示治疗开发的新靶点，即亚单位亚型， 人的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， 结构域抗体或纳米抗体（Nbs），用于研究特定的V-ATP酶群体。NB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是小的、高度稳定的，并且可以在细胞内使用。我们将使用生物化学和 生物物理方法、活细胞成像和基于细胞的测定，具体目的如下：1.）生成和 针对人V-ATP酶的亚基a同种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 目前通过开发新的创新工具研究V-ATP酶亚型的局限性， 对人类健康中特定V-ATP酶群体的理解具有高度的变革潜力， 疾病在拟议的研究结束时，我们希望已经建立了NBS作为一个强大的手段， 研究V-ATPase同工型和这些Nbs的实施将阐明同工型的特定作用 A4促进肾癌存活和恶性表型。作为结果而产生的信息 这些研究将为开发亚单位同种型特异性的新疗法提供坚实的基础。 靶向癌症中的V-ATP酶群体。