Abstract
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 approximately 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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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3-Phosphoinositide-Dependent Protein Kinases
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Adaptor Proteins, Signal Transducing
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Amino Acid Transport System ASC / genetics
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Amino Acid Transport System ASC / metabolism*
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Apoptosis* / drug effects
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Carcinoma, Hepatocellular / genetics
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Carcinoma, Hepatocellular / metabolism*
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Carcinoma, Hepatocellular / pathology
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Carrier Proteins / metabolism
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Cell Line, Tumor
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Cell Proliferation*
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Cell Survival
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Eukaryotic Initiation Factor-4E / metabolism
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Gene Expression Regulation, Neoplastic
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Humans
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Liver Neoplasms / genetics
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Liver Neoplasms / metabolism*
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Liver Neoplasms / pathology
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Minor Histocompatibility Antigens
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Phosphorylation
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Protein Biosynthesis
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases / metabolism
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Proteins / metabolism
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Proto-Oncogene Proteins c-akt / metabolism
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RNA Interference*
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RNA, Antisense / genetics
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RNA, Antisense / metabolism
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RNA, Messenger / metabolism
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Rapamycin-Insensitive Companion of mTOR Protein
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Regulatory-Associated Protein of mTOR
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Ribosomal Protein S6 Kinases, 70-kDa / metabolism
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Signal Transduction*
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TOR Serine-Threonine Kinases
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Time Factors
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Transfection
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bcl-Associated Death Protein / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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Amino Acid Transport System ASC
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BAD protein, human
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Carrier Proteins
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Eukaryotic Initiation Factor-4E
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Minor Histocompatibility Antigens
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Proteins
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RICTOR protein, human
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RNA, Antisense
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RNA, Messenger
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RPTOR protein, human
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Rapamycin-Insensitive Companion of mTOR Protein
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Regulatory-Associated Protein of mTOR
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SLC1A5 protein, human
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bcl-Associated Death Protein
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Protein Kinases
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MTOR protein, human
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3-Phosphoinositide-Dependent Protein Kinases
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PDPK1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Ribosomal Protein S6 Kinases, 70-kDa
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TOR Serine-Threonine Kinases