Jin Hana, Eun So Young, Lee Jong Sil, Park Sang Won, Lee Jae Heun, Chang Ki Churl, Kim Hye Jung
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Korea.
Department of Pathology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, 660-751, Korea.
Breast Cancer Res. 2014 Aug 26;16(5):R77. doi: 10.1186/bcr3694.
Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs).
ATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells.
We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells.
This study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.
细胞外核苷酸在肿瘤微环境中释放并能被检测到高浓度存在。G蛋白偶联P2Y2核苷酸受体（P2Y2R）可被三磷酸腺苷（ATP）和5'-三磷酸尿苷（UTP）等电位激活，它们介导细胞迁移和增殖等促炎反应。然而，P2Y2R在癌症转移过程中的作用仍不清楚。本研究旨在通过与内皮细胞（ECs）的相互作用来确定P2Y2R在高转移性MDA-MB-231乳腺癌细胞增殖、迁移和侵袭中的作用。
比较高转移性乳腺癌细胞MDA-MB-231和低转移性乳腺癌细胞MCF-7之间的ATP释放和P2Y2R活性。然后，在体外使用转染了对照或P2Y2R-siRNA的MDA-MB-231细胞和ECs，以及在体内使用注射了转染对照-shRNA或P2Y2R-shRNA的MDA-MB-231细胞的动物模型，研究P2Y2R通过与ECs相互作用对肿瘤生长和侵袭的作用。
我们发现，与低转移性乳腺癌细胞系MCF-7相比，这种高转移性乳腺癌细胞系释放的ATP水平更高，且P2Y2R活性更高。在MDA-MB-231细胞中，ATP或UTP激活P2Y2R可在24或72小时增加细胞增殖，而P2Y2R敲低可消除这种增殖。此外，ATP或UTP通过MDA-MB-231或ECs中细胞间黏附分子-1（ICAM-1）和血管细胞黏附分子-1（VCAM-1）的表达，显著增加MDA-MB-231细胞与ECs的黏附及细胞迁移，但在P2Y2R敲低的细胞中则无此现象。此外，ATP或UTP介导的P2Y2R激活诱导MDA-MB-231细胞穿过ECs侵袭，增加MDA-MB-231中基质金属蛋白酶-9（MMP-9）活性和血管内皮生长因子（VEGF）的产生，并诱导ECs中血管内皮（VE）-钙黏蛋白的磷酸化。与注射转染对照shRNA的MDA-MB-231细胞的小鼠相比，注射转染P2Y2R-shRNA的MDA-MB-231细胞的小鼠肿瘤生长和向其他组织的转移显著减少，且体重增加。
本研究表明P2Y2R可能通过调节癌细胞与ECs之间的相互作用在癌症转移中发挥重要作用。
