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Human Hepatocellular Carcinoma Cells Adapt to ASCT2 and LAT1 Amino Acid Transporter Silencing by shRNA and CRISPR‐Cas9

Amino acid transporters ASCT2 and LAT1 are coordinately enhanced in human cancers where, among other roles, they are thought to drive mammalian target‐of‐rapamycin (mTOR) growth signaling. To assess the value of ASCT2 and LAT1 as therapeutic targets in hepatocellular carcinoma (HCC), nine unique shRNA sequences in lentiviral vectors were used to produce stable transporter suppression in mesenchymal and epithelial HCC cell lines (SK Hep and Hep 3B, respectively). In addition, direct editing of the ASCT2 and LAT1 genes was performed using six CRISPR‐Cas9 vectors in SK Hep and HUH7 HCC cell lines. Glutamine and leucine transport rates were reduced proportionally to diminished transporter protein expression in each cell line, and a greater degree of knockout was achieved by the CRISPR‐Cas9 gene editing system versus the stable shRNA expression system (75–85% vs. 20–80% respectively). Contrary to expectations, diminished glutamine and leucine influx failed to affect cellular proliferation. Analysis of mTOR complex 1 (mTORC1) signaling output assessed by 4E‐BP1 showed no compromise to basal or amino acid‐stimulated mTORC1 signaling. Quantitative RT‐PCR indicated no significant upregulation of other transporters or glutamine synthetase (GLUL) to supplement intracellular availability of glutamine. Prolonged uptake of radiolabeled glutamine and leucine revealed that diminished transport rates did not affect the total intracellular accumulation of either glutamine or leucine. Further in vivo analysis will be essential to understanding upregulation of ASCT2 and LAT1 across cancers, and these findings suggest that, in an in vitro context, HCC cell lines are able to adapt to decreased rates of amino acid transport.