Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly...

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Published inThe Journal of pathology Vol. 236; no. 3; pp. 278 - 289
Main Authors Wang, Qian, Hardie, Rae-Anne, Hoy, Andrew J, van Geldermalsen, Michelle, Gao, Dadi, Fazli, Ladan, Sadowski, Martin C, Balaban, Seher, Schreuder, Mark, Nagarajah, Rajini, Wong, Justin J-L, Metierre, Cynthia, Pinello, Natalia, Otte, Nicholas J, Lehman, Melanie L, Gleave, Martin, Nelson, Colleen C, Bailey, Charles G, Ritchie, William, Rasko, John EJ, Holst, Jeff
Format Journal Article
LanguageEnglish
Published Chichester, UK John Wiley & Sons, Ltd 01.07.2015
Wiley Subscription Services, Inc
Subjects
Amino Acid Transport System ASC - antagonists & inhibitors
Amino Acid Transport System ASC - genetics
Amino Acid Transport System ASC - metabolism
Animals
ASCT2
Biological Transport
Cell Cycle
Cell Line, Tumor
Cell Proliferation
Down-Regulation
Fatty Acids - metabolism
Gene Expression Regulation, Neoplastic
Gene Knockdown Techniques
glutamine
Glutamine - metabolism
Heterografts
Humans
Male
Mechanistic Target of Rapamycin Complex 1
metabolism
Mice
Mice, Nude
Minor Histocompatibility Antigens
Multiprotein Complexes - genetics
Multiprotein Complexes - metabolism
Neoplasm Metastasis
Original Paper
Original Papers
Oxygen - metabolism
prostate cancer
Prostatic Neoplasms - genetics
Prostatic Neoplasms - metabolism
Prostatic Neoplasms - pathology
Prostatic Neoplasms - prevention & control
RNA, Small Interfering
SLC1A5
TOR Serine-Threonine Kinases - genetics
TOR Serine-Threonine Kinases - metabolism
prostate cancer
metabolism
SLC1A5
glutamine
ASCT2
cell cycle
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Summary:Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC‐3 prostate cancer cell lines, we showed that chemical or shRNA‐mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2‐mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC‐3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down‐regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2‐mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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ArticleID:PATH4518
AppendixS1. Supplementary InformationFigureS1. A, ASCT2 protein expression was detected on the cell membrane in patient samples. Representative images of each staining score are shown. Scale bar is 50 urn. B, scoring of ASCT2 protein expression in patient cohort with different Gleason grades. C, ASCT2 mRNA expression in the TCGA patient cohort with different Gleason scores. B, BPH, n = 36; Gleason 3,n = 59; Gleason 4, n = 44; Gleason 5, n = 7. Data are the mean ± SEM. Mann-Whitney U-test was used to analyze data. C, Gleason score 6, n = 11; Gleason score 7, n = 113; Gleason score 8, n = 12; Gleason score 9, n = 19. Data are the mean ± SEM. Mann-Whitney U-test was used to analyze data.A, glutamine uptake was assessed at a range of BenSer concentrations. Band C, glutamine uptake was assessed in the presence of bicalutamide +/- BenSer (B) or DHT +/BenSer (C) in LNCaP and PC-3. 0, glutamine uptake was assessed in the presence of GPNA (1 mM) in PC-3 cells.E and F, oxygen consumption rate (OCR) was assessed on a SeaHorse XF Analyzer in LNCaP (E) and PC-3 (F) cells pre-treated with BenSer (10 mM) or GPNA (1 mM), followed by addition of oligomycin, FCCP, or rotenone and antimycin. G and H, glutamine uptake was decreased in LNCaP and PC-3 cells in the presence of BenSer or GPNA. I, oxidative stress was measured in PC-3 cells using CellRox reagent in the presence of BenSer (10 mM), GPNA (1 mM) or positive control TBHP (250 ˜ M). A-H, data are the mean ± SEM (n = 3). 0, Mann-Whitney U-test was used to analyze data. B, C, E, F , one-way ANOVA test was used to analyze data. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s, no significance.A, ATF4 mRNA exprssion was detected by qRT-PCR in PC-3 cells in the presence of BenSer or GPNA. B, PCR products are examined in a agarose gel electrophoresis. C, gene set enrichment analysis (GSEA) plot Gene Ontology categories Amino Acid Transport in control versus GPNA group. 0, glutamine uptake was assessed in shControl and shASCT2#1 expressing PC-3 cells. E, annexin V staining was used to detect apoptosis in PC-3 cells expressing shControl and shASCT2#2. F, cell cycle phase was determined using BrdU incorporation assay in PC-3 cells expressing shControl and shASCT2#1. G, annexin V staining was used to detect apoptosis in PC-3 cells expressing shControl and shASCT2#1. A, one-way ANOVA test was performed. O-G, data represent mean ± SEM, n = 3. Mann-Whitney U test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.FigureS4. A, PC-3 cells expressing shControl or shASCT2 were transduced with GFP-2A-luciferase expressing construct and sorted for high GFP expression by FACS. B, cleaved caspase 3 expression in shControl and shASCT2. C, spontaneous metastatic PC-3-luc cells expressing shControl or shASCT2 were detected in the liver and lungs of mice bearing subcutaneous tumors.TableS1 Genes upregulated and downregulated by GPNATableS2 Genes upregulated and downregulated by BenSerTableS3 GSEA gene ontology upregulated gene sets in control vs GPNA treated PC-3 cells (Top 50).TableS4 GSEA gene ontology upregulated gene sets in control vs benzylserine treated PC-3 cells (Top 50).TableS5 GSEA motif upregulated gene sets in control vs GPNA treated PC-3 cells (Top 50).TableS6 GSEA motif upregulated gene sets in control vs benzylserine treated PC-3 cells (Top 50).
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No conflicts of interest were declared.
ISSN:0022-3417
1096-9896
1096-9896
DOI:10.1002/path.4518