Function of V-ATPases in Breast Cancer Metastasis

V-ATP酶在乳腺癌转移中的功能

• 批准号: 10308465
• 负责人: MICHAEL D FORGAC
• 金额: $ 18.54万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308465
• 关键词: 4T1; Ablation; Antibodies; Biophotonics; Blood Circulation; Bone Resorption; Breast; Breast Cancer Cell; Breast Cancer cell line; Breast Carcinoma; Breast cancer metastasis; CRISPR/Cas technology; Cause of Death; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Membrane; Clinical; Collaborations; Data; Development; Epitopes; Grant; Health; Human; Image; In Vitro; Knowledge; Luciferases; Lymphatic System; MCF10A cells; MCF7 cell; MDA MB 231; Mammary Neoplasms; Membrane Protein Traffic; Membrane Proteins; Metastatic breast cancer; Mission; Modeling; Mus; Neoplasm Metastasis; Normal tissue morphology; Phage Display; Plasma; Principal Investigator; Process; Protein Isoforms; Proton Pump; Research; Role; Sampling; Site; Small Interfering RNA; Solid Neoplasm; Surface; Testing; Therapeutic Agents; Tumor Escape; Tumor Tissue; Up-Regulation; Work; Xenograft Model; cell growth; effective therapy; experimental study; extracellular; in vitro Assay; in vivo; inhibitor; knock-down; lymphatic Invasion; malignant breast neoplasm; migration; mortality; mouse model; neoplastic cell; new therapeutic target; novel; overexpression; protein degradation; therapeutic target; tumor; tumor growth; urinary; vacuolar H+-ATPase

Project Summary Metastasis is the leading cause of mortality from breast cancer, but effective therapies to inhibit metastasis have not been identified. Identifying novel targets to limit breast cancer metastasis would therefore fill an important unmet clinical need. Metastasis involves the escape of tumor cells from the primary site into the circulation and invasion of these cells into secondary sites. Both of these processes depend upon tumor cell invasiveness, a process in which V-ATPases have recently been implicated. The long-term objective of this research is to determine the role of V-ATPases in breast tumor metastasis. V-ATPases are ATP-dependent proton pumps that function in both intracellular compartments and the plasma membrane in a variety of cellular processes. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a (a1- a4), with a3 and a4 capable of targeting V-ATPases to the plasma membrane. We have identified V-ATPases in the plasma membrane of highly invasive breast tumor cells (MDA-MB231, MCF10CA1a, 4T1) but not in poorly invasive lines (MCF10a, MCF7). Moreover, the in vitro invasiveness of only the highly invasive lines is inhibited by specific V-ATPase inhibitors. In addition, highly invasive cells express higher levels of the a3 or a4 isoforms, and knock-down of these isoforms using isoform-specific siRNAs inhibits both invasion and plasma membrane localization of the V-ATPase. Importantly, overexpression of a3 in non-invasive MCF10a cells significantly increases plasma membrane V-ATPases and invasiveness. Recently, we have shown that selective inhibition of plasma membrane V-ATPases inhibits invasion of MB231 cells, that a3 is up-regulated in human breast tumor samples and is expressed at the highest levels in invasive breast carcinoma relative to solid tumors and normal tissue. These results suggest that up-regulation of a3 or a4 in breast tumor cells targets V-ATPases to the plasma membrane, where they function to increase invasiveness. Our first objective is to prepare antibodies directed against extracellular epitopes of the V-ATPase that are capable of inhibiting cell surface V-ATPases. Our previous studies employing inhibitory antibodies directed against extracellular epitope tags provide a strong proof of principal for this aim. We predict that these inhibitory antibodies will be effective at reducing breast tumor cell invasiveness. Our second objective is to test the hypothesis that plasma membrane and a3 or a4-containing V-ATPases function in breast tumor metastasis in vivo. This will be accomplished using a mouse xenograft model of breast cancer metastasis in collaboration with Dr. Charlotte Kuperwasser, co-Principal Investigator, who is an expert in the analysis of breast cancer metastasis using such models. We will determine the effect on metastasis of the inhibitory antibodies prepared in Aim 1 as well as disruption of a subunit isoforms in highly invasive breast cancer cell lines using CRISPR/Cas9. Accomplishment of these aims will provide a critical test of plasma membrane and a3 or a4-containing V- ATPases as novel therapeutic targets to limit breast cancer metastasis.

项目概要 转移是乳腺癌死亡的主要原因，但抑制转移的有效疗法 尚未被识别。因此，确定限制乳腺癌转移的新靶点将填补 重要的未满足的临床需求。转移涉及肿瘤细胞从原发部位逃逸到其他部位 这些细胞的循环和侵入次级部位。这两个过程都依赖于肿瘤细胞 最近发现 V-ATP 酶参与了侵袭性过程。本次活动的长远目标 研究目的是确定 V-ATP 酶在乳腺肿瘤转移中的作用。 V-ATP酶是ATP依赖性的 质子泵在多种细胞的细胞内区室和质膜中发挥作用 流程。 V-ATP酶针对不同细胞膜的靶向是由亚基a（a1- a4)，其中a3和a4能够将V-ATP酶靶向质膜。我们已经鉴定出 V-ATP 酶 存在于高度侵袭性乳腺肿瘤细胞（MDA-MB231、MCF10CA1a、4T1）的质膜中，但不在 侵袭性差的细胞系（MCF10a、MCF7）。此外，只有高侵袭性细胞系的体外侵袭性才是 被特定的V-ATP酶抑制剂抑制。此外，高度侵袭性细胞表达更高水平的 a3 或 a4 同种型，并且使用同种型特异性 siRNA 敲低这些同种型可抑制侵袭和血浆 V-ATP酶的膜定位。重要的是，a3 在非侵袭性 MCF10a 细胞中过度表达 显着增加质膜 V-ATP 酶和侵袭力。最近，我们已经证明 选择性抑制质膜 V-ATP 酶可抑制 MB231 细胞的侵袭，a3 在 人类乳腺肿瘤样本中，相对于浸润性乳腺癌，其表达水平最高 实体瘤和正常组织。这些结果表明乳腺肿瘤细胞中 a3 或 a4 的上调 将 V-ATP 酶靶向质膜，在那里它们的作用是增加侵袭性。我们的第一个目标 是制备针对 V-ATP 酶胞外表位的抗体，该抗体能够抑制 细胞表面 V-ATP 酶。我们之前的研究采用针对细胞外的抑制性抗体 表位标签为此目的提供了强有力的原理证明。我们预测这些抑制性抗体将 有效减少乳腺肿瘤细胞的侵袭。我们的第二个目标是检验等离子体的假设 膜和含α3或α4的V-ATP酶在体内乳腺肿瘤转移中发挥作用。这将是 与 Charlotte 博士合作，使用乳腺癌转移的小鼠异种移植模型完成 Kuperwasser，联合首席研究员，是利用此类技术分析乳腺癌转移的专家 模型。我们将确定目标 1 中制备的抑制性抗体对转移的影响以及 使用 CRISPR/Cas9 破坏高侵袭性乳腺癌细胞系中的亚基亚型。 这些目标的实现将为质膜和含 a3 或 a4 的 V-提供关键测试。 ATP酶作为限制乳腺癌转移的新治疗靶点。