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Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.

ABSTRACT: Amino acid transporters alanine-serine-cysteine transporter 2 (ASCT2) and L-Type Amino Acid Transporter 1 (LAT1) are coordinately enhanced in human cancers where among other roles, they are thought to drive mechanistic target-of-rapamycin (mTOR) growth signaling. To assess ASCT2 and LAT1 as therapeutic targets, nine unique short hairpin RNA (shRNA) vectors were used to stably suppress transporter expression in human epithelial (Hep3B) and mesenchymal (SK-Hep1) hepatocellular carcinoma (HCC) cell lines. In addition, six unique CRISPR-Cas9 vectors were used to edit the ASCT2 (SLC1A5) and LAT1 (SLC7A5) genes in epithelial (HUH7) and mesenchymal (SK-Hep1) HCC cells. Both approaches successfully diminished glutamine (ASCT2) and leucine (LAT1) initial-rate transport proportional to transporter protein suppression. In spite of profoundly reduced glutamine or leucine transport (up to 90%), transporter suppression or knockout failed to substantially affect cellular proliferation or basal and amino acid-stimulated mTORC1 growth signaling in either HCC cell type. Only LAT1 knockout in HUH7 slightly reduced growth rate. However, intracellular accumulation of radiolabeled glutamine and leucine over longer time periods largely recovered to control levels in ASCT2 and LAT1 knockout cells, respectively, which partially explains the lack of an impaired growth phenotype. These data collectively establish that in an in vitro context, human epithelial and mesenchymal HCC cell lines adapt to ASCT2 or LAT1 knockout. These results comport with an emerging model of amino acid exchangers like ASCT2 and LAT1 as "harmonizers", not drivers, of amino acid accumulation and signaling, despite their long-established dominant role in initial-rate amino acid transport.

SUBMITTER: Bothwell PJ

PROVIDER: S-EPMC6073291 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.

Bothwell Paige J PJ Kron Clare D CD Wittke Evan F EF Czerniak Bradley N BN Bode Barrie P BP

International journal of molecular sciences 20180719 7

Amino acid transporters alanine-serine-cysteine transporter 2 (ASCT2) and L-Type Amino Acid Transporter 1 (LAT1) are coordinately enhanced in human cancers where among other roles, they are thought to drive mechanistic target-of-rapamycin (mTOR) growth signaling. To assess ASCT2 and LAT1 as therapeutic targets, nine unique short hairpin RNA (shRNA) vectors were used to stably suppress transporter expression in human epithelial (Hep3B) and mesenchymal (SK-Hep1) hepatocellular carcinoma (HCC) cell ...[more]

PMID: 30029480

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Project description:Introduction:Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-? signalling pathway, which is involved in corneal epithelial wound healing. Methods:HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-? secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-?2 treatment. Results:KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-? canonical signalling pathway, TGF-?2 secretion by HCECs increased up to 5 folds, and several TGF-?-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-? signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-?2 treatment. Conclusions:These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-?2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-? signalling.

Dataset Information

Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.

Publications

Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.

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