Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.
Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.
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DOI：
10.1016/j.molonc.2015.03.012
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发表时间：
2015-08
影响因子：
6.6
通讯作者：
Bates PJ
中科院分区：
文献类型：
--
作者：
Reyes-Reyes EM;Šalipur FR;Shams M;Forsthoefel MK;Bates PJ
AS 1411是一种富含G的四链体形成寡脱氧核苷酸，可特异性结合核仁素，核仁素是一种在大多数恶性细胞的表面和细胞质中发现的蛋白质，但在大多数正常细胞的表面/细胞质中不存在。AS 1411已显示出有前途的临床活性，并被广泛用作肿瘤靶向药物，但其作用机制尚未完全了解。以前，我们表明，AS 1411是采取在癌细胞中的巨胞饮（液相内吞），随后刺激进一步的巨胞饮的核仁依赖性机制。在本研究中，我们研究了AS 1411诱导巨胞饮的意义和分子机制。我们的研究结果表明，AS 1411在各种细胞系中的抗增殖活性与其刺激巨胞饮的能力相关。在DU 145前列腺癌细胞中，AS 1411诱导EGFR、Akt、p38和Rac 1的活化。Akt和p38的激活对于AS 1411活性并不关键，因为在所有AS 1411应答细胞系中均未观察到Akt激活，并且p38的敲低对AS 1411抑制增殖的能力没有影响。另一方面，EGFR和Rac 1的激活似乎在所有检查的癌细胞系（DU 145、MDA-MB-468、A549、LNCaP）中的AS 1411活性中发挥作用，并且其抑制显著降低了AS 1411介导的巨胞饮作用和AS 1411抗增殖活性。有趣的是，通过siRNA下调核仁素表达也产生了激活的Rac 1的大幅增加，揭示了核仁素作为Rac 1激活的负调节剂的先前未知的作用。我们的研究结果是一致的模型，其中AS 1411结合核仁素导致持续激活的Rac 1，并导致methuosis，一种新型的非凋亡性细胞死亡的特点是过度刺激巨胞饮。我们推测，methuosis是一种肿瘤/转移抑制机制，反对Rac 1的恶性功能，癌细胞可能过度表达核仁素，以克服这一障碍。
AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced As1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.