Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University School of Medicine, Dayton, OH, USA.
Oncogene. 2013 Dec 5;32(49):5551-62. doi: 10.1038/onc.2013.207. Epub 2013 Jun 10.
Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.
乳腺癌是全球女性最常见的恶性肿瘤之一。在体外培养的乳腺癌中，一种具有升高的酶脂酶活性的蛋白质是磷脂酶 D（PLD），它也参与细胞迁移。我们证明，直接在肿瘤中分析的 PLD2 同工型对于细胞侵袭至关重要，而细胞侵袭对于体内乳腺癌和肺转移瘤的生长和发展至关重要。我们在 SCID 小鼠模型中使用了三种互补策略，并解决了潜在的分子机制。首先，我们使用基于慢病毒的短发夹 RNA 沉默了高转移性、侵袭性乳腺癌细胞（MDA-MB-231）中的 PLD2 基因，然后将其异种移植到 SCID 小鼠中。与对照肿瘤相比，由此产生的小鼠原发性乳腺肿瘤的大小减小（65%，P<0.05），并且其发病时间延迟。其次，我们使用双顺反子 MIEG 逆转录病毒载体在低侵袭性乳腺癌细胞（MCF-7）中稳定过表达 PLD2，并观察到这些细胞转变为高度侵袭性表型，因为异种移植后形成的原发性肿瘤更大，生长更快，更容易发生肺转移。第三，我们将渗透泵植入 SCID 异种移植小鼠中，这些泵输送两种不同的 PLD 活性小分子抑制剂（5-氟-2-吲哚基去氯卤代苯并脒和 N-[2-（4-氧代-1-苯基-1,3,8-三氮杂螺[4,5]癸-8-基）乙基]-2-萘甲酰胺）。这些抑制剂导致原发性肿瘤生长、腋窝转移瘤和肺转移瘤的显著抑制（>70%，P<0.05）。为了确定潜在的机制，我们确定了由 PLD 诱导的细胞侵袭的机制，该机制是由磷酸脂酸、Wiscott-Aldrich 综合征蛋白、生长受体结合蛋白 2 和 Rac2 信号事件介导的，这些信号事件最终影响肌动蛋白聚合和细胞侵袭。总之，这项研究首次表明 PLD2 在乳腺癌的发展、转移和侵袭性水平中起核心作用，这增加了 PLD2 可能作为新的治疗靶点的可能性。
