Reprogramming of Glutamine Amino Acid Transporters Expression and Prognostic Significance in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes (p < 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p < 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p < 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.

Keywords: glutamine; hepatocellular carcinoma; liver; metabolic reprogramming; transporters.

Figure 1

Glutamine is required for the optimal survival and growth of liver cancer cells. Viability relative to 0 mM glutamine of murine (A) and human (B) liver cancer cells after 24 h of culture with increasing exogenous glutamine up to 4 mM. Cell doubling time of murine (C) and human (D) liver cancer cells cultured with increasing exogenous glutamine up to 4 mM. All experiments were performed a minimum of n = 3 times, each in quadruplicate. Dt81: Dt81Hepa1-6 cells.

Figure 2

Highly tumorigenic murine HCC cells exhibit increased uptake of exogenous glutamine. (A) ³H-glutamine (20 nM) uptake kinetics over 24 h in Dt81Hepa1-6 (Dt81) HCC cells and normal primary hepatocytes (PH). (B) Linear phase of ³H-glutamine uptake in Dt81 cells and PH (0–6 h). Linear regressions of ³H-glutamine uptake were compared by two-tailed ANCOVA. Intracellular quantities of ³H-glutamine were compared between Dt81 and PH at each timepoint. Experiments were performed a minimum of n = 4 times, each in duplicate. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 3

Murine HCC is characterized by the reprogramming of glutamine transporter mRNA levels. mRNA expression of glutamine transporters SLC6A14 (A), SLC38A1 (B), SLC38A5 (C), SLC38A2 (D), SLC38A3 (E), SLC7A6 (F), and SLC1A5 (G) relative to housekeeping genes (PPIA, HPRT1, and H2AFZ), measured using qPCR analyses. For cell cultures, mRNA was extracted after 24 h. NH: normal primary murine hepatocytes, H1-6: Hepa1-6 murine HCC cells, Dt81: Dt81Hepa1-6 murine HCC cells. Nor: normal mouse liver controls, Peri: peritumoral tissue, Tum: murine HCC tumor. AU: arbitrary units. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 4

Glutamine transporter reprogramming in human HCC cells. (A) mRNA expression levels of the glutamine transporters SLC1A5, SLC6A14, SLC7A6, SLC38A1, SLC38A2, SLC38A3, and SLC38A5 relative to housekeeping genes (S9 and HMBS) in normal human liver tissue (NL), normal human hepatocyte isolates (NH), HepG2 and SK-HEP-1 non-HCC liver cancer cells, and a panel of 15 HCC cell lines, measured using qPCR analysis. (B) Representative Western blot image of ASCT2 protein expression in NL, NH, as well as HepG2, Huh7, and Hep3B cells after 24 h of cell culture. mASCT2: glycosylated surface membrane-bound ASCT2 (75 kDa), cASCT2: non-glycosylated cytosolic ASCT2 (49 kDa). (C) Quantification of mASCT2 (75 kDa) relative to actin. (D) Quantification of cASCT2 (49 kDa) relative to actin. AU: arbitrary units. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5

Glutamine transporter expression profiles are associated with differential overall survival of HCC patients. Analysis of overall survival within TCGA-LIHC HCC patient cohort between low and high intratumoral mRNA expression (RNAseq data) of glutamine transporters SLC6A14 (A), SLC38A3 (B), SLC7A6 (C), SLC38A1 (D), SLC1A5 (E), SLC38A2 (F), and SLC38A5 (G). (H) Summary table of cohort data, including RNAseq expression data, median survival, and log-rank analysis between low and high expression cohorts for each TCGA-LIHC gene dataset. Appropriate cutoff mRNA values were determined between lower and upper quartiles. (I) Forest plot depiction of log-rank hazard ratios comparing risk of overall mortality in high-expressing patients to low-expressing patients.

Figure 6

SLC1A5 and SLC38A2 are significant contributors to glutamine uptake in human HCC cells. siRNA transfection was performed to target SLC1A5 (A–C), SLC38A2 (D–F), or in combination (G–I). Validation of siRNA targeting was performed by measuring relative mRNA levels of targets SLC1A5/ASCT2 (A), SLC38A2/SNAT2 (D), or both (G). Intracellular uptake of radiolabeled glutamine (gln), ³H-glutamine (20 nM), over one hour was measured after cell conditioning with no additional gln (0 mM), low gln (0.25 mM), high gln (4 mM), DMEMc (gln-rich, glucose-rich medium), and gamma-p-nitroanilide (GPNA) treatment in DMEMc [0.5 mM], for SLC1A5 inhibition (B), SLC38A2 inhibition (E), or dual inhibition (H). Glutamine-dependent cell viability was compared between non-targeting siRNA control-treated Huh7 and Hep3B cells and those subject to SLC1A5 (C), SLC38A2 (F), or combined (I) inhibition. Intracellular uptake of radiolabeled glutamine (gln), ³H-glutamine (20 nM), over one hour was also measured under gamma-p-nitroanilide (GPNA) treatment [0.5 mM] compared to control (DMEMc) (J). Glutamine-dependent cell viability was assessed under GPNA treatment [0.5 mM] in Huh7 and Hep3B cells (K). AU: arbitrary units, CPM: counts per minute. All experiments were performed a minimum of n = 3 times, each in duplicate. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.