Function of V-ATPases in Breast Cancer Metastasis

项目摘要 转移是乳腺癌死亡的主要原因，但抑制转移的有效治疗方法 还没有确定身份。因此，识别限制乳腺癌转移的新靶点将填补 重要的未得到满足的临床需求。转移是指肿瘤细胞从原发部位逃逸到 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 侵袭性，V-ATPase最近被牵连的一个过程。这样做的长期目标是 研究目的是确定V-ATPase在乳腺肿瘤转移中的作用。V-ATPase依赖于ATP 在多种细胞的细胞内隔室和质膜中起作用的质子泵 流程。V-ATPase对不同细胞膜的靶向是由a亚基(A1-ATPase)的亚型控制的。 A4)，其中A3和A4能够将V-ATPase靶向质膜。我们已经鉴定出V-ATPase 在高侵袭性乳腺肿瘤细胞(MDA-MB231、MCF10CA1a、4T1)的质膜上，而在 侵袭性差的品系(MCF10A、MCF7)。此外，只有高侵袭性品系的体外侵袭力是 被特定的V-ATPase抑制剂抑制。此外，高侵袭性细胞表达更高水平的A3或A4 异构体，并使用异构体特异性siRNAs敲除这些异构体既抑制侵袭又抑制血浆 V-ATPase的膜定位。重要的是，A3在非侵袭性MCF10A细胞中过表达 显著增加质膜V-ATPase和侵袭力。最近，我们已经证明了 选择性抑制质膜V-ATPase抑制MB231细胞侵袭，其中A3上调 人乳腺肿瘤标本，其在浸润性乳腺癌中的表达水平最高 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-ATPase靶向质膜，在那里它们起到增加侵袭性的作用。我们的首要目标是 是制备针对V-ATPase胞外表位的抗体，这些表位能够抑制 细胞表面V-ATPase。我们之前的研究使用了针对细胞外的抑制性抗体 表位标签为这一目标提供了强有力的原则性证明。我们预测这些抑制性抗体将是 有效降低乳腺肿瘤细胞侵袭力。我们的第二个目标是检验这样一个假设 膜和含A3或A4的V-ATPase在乳腺肿瘤体内转移中的作用这将是 与夏洛特博士合作，使用乳腺癌转移的小鼠异种移植模型完成 联合首席调查员Kuperwasser是使用这种方法分析乳腺癌转移的专家 模特们。我们将测定目标1中制备的抑制性抗体对肿瘤转移的影响 使用CRISPR/CAS9阻断高侵袭性乳腺癌细胞系中的亚基异构体。 这些目标的实现将为质膜和含有A3或A4的V- ATPase作为限制乳腺癌转移的新治疗靶点。

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.