ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells
ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells
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DOI：
10.1152/ajpcell.00330.2006
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发表时间：
2007-07-01
影响因子：
5.5
通讯作者：
Bode, Barrie P.
中科院分区：
文献类型：
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作者：
Fuchs, Bryan C.;Finger, Richard E.;Bode, Barrie P.
系统ASC氨基酸转运蛋白-2（ASCT 2）先前被证明是人类肝癌细胞生长和存活所必需的，因为其通过诱导性反义RNA表达的沉默导致在48小时内通过超越其在氨基酸递送中的作用的机制完全凋亡。为了获得对癌性肝细胞对ASCT 2依赖性的机制见解，本研究的目的是确定其沉默对预示凋亡的生长和存活信号传导的早期后果。在SK-Hep 1细胞中诱导的反义ASCT 2 RNA导致ASCT 2 mRNA在6小时内抑制> 90%，并抑制哺乳动物雷帕霉素靶蛋白（mTOR）/raptor（mTOR复合物-1; mTORC 1）信号转导，如通过减少的p70核糖体蛋白S6激酶-1和eIF 4 E结合蛋白-1磷酸化所表现的，而蛋白质合成速率下降了近50%，尽管帽结合蛋白eIF 4G或细胞核糖体蛋白含量没有可测量的降低。在ASCT 2活性可检测到的降低之前，mTORC 1信号传导被抑制，但与总细胞ASCT 2蛋白下降30%相一致。在ASCT 2沉默后12小时，观察到蛋白质合成速率和ASCT 2蛋白质和活性进一步降低，各自降低约50%，而来自mTOR/rictor（mTOR复合物-2; mTORC 2）的信号传导受到刺激，如通过丝氨酸-473上Akt/PKB激酶及其促凋亡底物Bad在丝氨酸-136上的磷酸化增强所指示的。这些结果表明，ASCT 2沉默抑制mTORC 1信号传导到翻译机器，随后是mTORC 2启动的生存反应，建立氨基酸转运蛋白表达和mTOR功能之间的联系。
System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to > 90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by similar to 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.