Publication Date

2017

Document Type

Dissertation/Thesis

First Advisor

Bode, Barrie P.

Degree Name

Ph.D. (Doctor of Philosophy)

Department

Department of Biological Sciences

LCSH

Cytology||Molecular biology||Oncology

Abstract

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.

Comments

Advisors: Barrie P. Bode.||Committee members: Sherine Elsawa; Elizabeth Gaillard; Linda Yasui; Yanbin Yin.||Includes bibliographical references.||Includes illustrations.

Extent

xii, 134 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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