Provost J J, Rastedt D, Canine J, Ngyuen T, Haak A, Kutz C, Berthelsen N, Slusser A, Anderson K, Dorsam G, Wallert M A
Department of Chemistry, Minnesota State University Moorhead, 407 Hagen Hall, Moorhead, MN, 56563, USA,
Cell Oncol (Dordr). 2012 Apr;35(2):95-110. doi: 10.1007/s13402-011-0068-y. Epub 2012 Jan 31.
Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na(+)/H(+) exchanger isoform 1 (NHE1) expression and activity. METHODS AND RESULTS: The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 μM EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm(3). This was compared to 20% of H460 uPAR K/D injected mice forming tumors with an average volume of 15 mm(3) and 10% of H460 NHE1 K/D injected mice forming tumors with an average volume of 5 mm(3). CONCLUSION: Taken together, these data demonstrate that uPA/uPAR-mediated tumor progression and metastasis requires NHE1 in NSCLC cells and suggests a potential therapeutic approach to blocking cancer progression.
非小细胞肺癌（NSLC）是侵袭性癌症，对化疗不敏感，在美国占所有癌症死亡人数的近33%。癌症的两个允许细胞侵袭和转移的标志是持续增殖和增强的运动能力。在本研究中，我们调查尿激酶型纤溶酶原激活剂（uPA）/uPA受体（uPAR）信号传导与钠/氢交换体1型（NHE1）表达和活性之间的关系。
添加10nM uPA增加了三种NSCLC细胞系NCI-H358、NCI-H460和NCI-H1299的致癌潜力。这包括细胞增殖速率增加1.6至1.9倍；显示应力纤维的细胞百分比增加3.05至3.17倍；以及锚定非依赖性生长增加1.64至2.0倍。在每种情况下，当用10μM EIPA（乙基异丙基氨氯地平）抑制NHE1进行实验时，这种增加被阻断。为了进一步评估uPA/uPAR和NHE1在肿瘤进展中的作用，我们使用全长uPA与uPA氨基末端片段（ATF）比较评估信号事件。比较H460细胞中的uPA和ATF信号传导，我们发现uPA和ATF均使应力纤维形成增加约2倍，而与ATF的2.81倍相比，uPA使基质金属蛋白酶9（MMP9）活性增加5.44倍。为了扩展这项信号研究，生成了两个新的细胞系，一个NHE1表达降低（H460 NHE1 K/D），一个uPAR表达降低（H460 uPAR K/D）。使用K/D细胞系，我们发现在NHE1或uPAR表达显著降低的细胞中，uPA和ATF均不能刺激应力纤维形成或MMP9活性。最后，在无胸腺小鼠中进行体内肿瘤形成研究，我们发现当给小鼠注射H460细胞时，80%的小鼠形成肿瘤，平均体积为390立方毫米。相比之下，注射H460 uPAR K/D的小鼠中有20%形成肿瘤，平均体积为15立方毫米，注射H460 NHE1 K/D的小鼠中有10%形成肿瘤，平均体积为5立方毫米。
综上所述，这些数据表明uPA/uPAR介导的肿瘤进展和转移需要NSCLC细胞中的NHE1，并提示了一种阻断癌症进展的潜在治疗方法。
