Bode, Barrie P.
Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
Cytology; Molecular biology; Oncology
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 epithelial and mesenchymal HCC cell lines. In addition, direct genomic editing of the ASCT2 and LAT1 genes (SLC1A5 and SLC7A5, respectively) was performed using six unique CRISPR-Cas9 vectors. Both targeting systems were successful, and glutamine and leucine uptake rates were reduced proportionally to diminished transporter protein expression in each cell line. A greater degree of knockout was achieved by the CRISPR-Cas9 gene editing system versus the stable shRNA expression system (75-90% versus 20-80% respectively). Contrary to expectations, diminished glutamine and leucine influx failed to substantially affect cellular proliferation. Analysis of mTOR complex 1 (mTORC1) signaling output showed no compromise to basal or amino acid-stimulated signaling to growth and autophagy, as assessed by the phosphorylation of three targets: eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase beta-1 (p70S6K), and Unc-51 like autophagy activating kinase 1 (ULK1). In addition, quantitative RT-PCR indicated no significant upregulation of other amino acid transporters or glutamine synthetase (GLUL) to supplement the intracellular availability of glutamine. Western blot analysis revealed an increased expression of glucose transporter 1 (GLUT1) expression in response to ASCT2 knockout, a response that was unique to mesenchymal type HCC. Subsequent evaluation of prolonged uptake of radiolabeled glutamine and leucine demonstrated that diminished transport rates do not affect the total intracellular accumulation of either amino acid in ASCT2 and LAT1 transporter knockout cell lines. Further in vivo analysis will be essential to understanding the notable upregulation of ASCT2 and LAT1 across cancers, and these findings do not exclude the possibility that these transporters play a role in tumorigenesis and metastasis. The results presented herein establish that, in an in vitro context, HCC cell lines are able to adapt to decreased rates of amino acid transport.
Bothwell, Paige Jay, "Human hepatocellular carcinoma cells adapt to ASCT2 and LAT1 amino acid transporter knockdown mediated by shRNA and CRISPR-Cas9" (2017). Graduate Research Theses & Dissertations. 3419.
xii, 134 pages
Northern Illinois University
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