Amino acid transporter ASCT2 and mTOR signaling in human liver cancer cells

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 insights into the reliance of hepatocellular carcinoma (HCC) lines on ASCT2, the aim of this study is to silence ASCT2 or inhibit ASCT2 function to determine the response on growth and survival signaling.;Induced antisense ASCT2 RNA in SK-Hep cells led to reduced ASCT2 mRNA and mammalian target-of-rapamycin complex-1 (mTORC1) signaling by 8 h. mTORC1 reduction coincides with ASCT2 protein reduction, but precedes measureable reduction in ASCT2 transporter activity. Diminished glutamine delivery could not alone be sufficient to account for the suppression of mTORC1 signaling with loss of ASCT2 protein. Glutamine deprivation alone causes cell death at 72 h, compared to cell death at 48 h with ASCT2 silencing via antisense. These results suggest that ASCT2 silencing inhibits mTORC1 signaling followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.;ASCT2 silencing, via an inducible plasmid that produced shRNAs silenced ASCT2 mRNA expression to 80% of control in SK-Hep. ASCT2 protein decrease was not observed. siRNAs were implemented to silence ASCT2, and moderate reduction in mRNA and protein levels were measured in SK-Hep, but not reliably. Therefore, mTORC1 signaling among HCC cell lines with ASCT2 silenced remained undetermined.;ASCT2 transport function was impaired in 6 HCC lines of either epithelial/oncofetal Group I or aggressive/metastatic Group II to assess whether it was necessary for glutamine repletion via ASCT2 for mTORC1 growth signaling. An earlier model proposed that glutamine antiported for leucine was necessary for mTORC1 signaling. Our results show SK-Hep most reliant on glutamine repletion for mTORC1 signaling. In addition, glutamine repletion alone was sufficient to cause mTORC1 signaling without leucine repletion. The other Group II cell lines showed moderate mTORC1 signaling with glutamine repletion as well. These results suggest that the more aggressive HCC lines have a greater reliance on glutamine repletion for mTORC1 signaling.