GLUT5-mediated fructose utilization drives lung cancer growth by stimulating fatty acid synthesis and AMPK/mTORC1 signaling - PubMed (original) (raw)

. 2020 Feb 13;5(3):e131596.

doi: 10.1172/jci.insight.131596.

Xing Jin 1, Mingsong Wang 2, Dan Liu 1, Qin Luo 1, Hechuan Tian 1, Lili Cai 1, Lifei Meng 2, Rui Bi 2, Lei Wang 2, Xiao Xie 2, Guanzhen Yu 1, Lihui Li 1, Changsheng Dong 1, Qiliang Cai 3, Wei Jia 4, Wenyi Wei 5, Lijun Jia 1

Affiliations

GLUT5-mediated fructose utilization drives lung cancer growth by stimulating fatty acid synthesis and AMPK/mTORC1 signaling

Wen-Lian Chen et al. JCI Insight. 2020.

Abstract

Lung cancer (LC) is a leading cause of cancer-related deaths worldwide. Its rapid growth requires hyperactive catabolism of principal metabolic fuels. It is unclear whether fructose, an abundant sugar in current diets, is essential for LC. We demonstrated that, under the condition of coexistence of metabolic fuels in the body, fructose was readily used by LC cells in vivo as a glucose alternative via upregulating GLUT5, a major fructose transporter encoded by solute carrier family 2 member 5 (SLC2A5). Metabolomic profiling coupled with isotope tracing demonstrated that incorporated fructose was catabolized to fuel fatty acid synthesis and palmitoleic acid generation in particular to expedite LC growth in vivo. Both in vitro and in vivo supplement of palmitoleic acid could restore impaired LC propagation caused by SLC2A5 deletion. Furthermore, molecular mechanism investigation revealed that GLUT5-mediated fructose utilization was required to suppress AMPK and consequently activate mTORC1 activity to promote LC growth. As such, pharmacological blockade of in vivo fructose utilization using a GLUT5 inhibitor remarkably curtailed LC growth. Together, this study underscores the importance of in vivo fructose utilization mediated by GLUT5 in governing LC growth and highlights a promising strategy to treat LC by targeting GLUT5 to eliminate those fructose-addicted neoplastic cells.

Keywords: Lung cancer; Metabolism.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Activity of fructose utilization in LC tissues of patients.

(A) Visualization of clinical parameters of enrolled patients in the study for metabolomic survey. ADC, n = 22; SCC, n = 13. TNM, extent of the primary tumor, involvement of lymph nodes, and distant metastases. (B and C) Concentrations of fructose and fructose-derived metabolites between paired adjacent normal lung tissues and tumor tissues from patients with lung ADC (n = 22) (B) or SCC (n = 13) (C). The midline represents the median of the data, with the upper and lower limits of the box being the third and first quartiles. Additionally, the whiskers of the box plot extend up to 1.5 times the interquartile range from the top or bottom of the box. P values were computed using 2-tailed Wilcoxon rank-sum test. Fructose-1-P, fructose-1-phosphate; DHAP, dihydroxyacetone phosphate. (D) Ex vivo study of fructose uptake between paired adjacent normal lung tissues (N) and tumor lung tissues (T) from patients with lung ADC or SCC. Tissues were cultured in complete medium containing 1.5 mM fructose and different levels of glucose for 48 hours. Statistical analysis was conducted using 1-way ANOVA test. After conducting a homogeneity of variance test to confirm equal variances among subgroups, P values were obtained from post hoc test using least significant difference (LSD) method. Cumulative data are shown; n = 3. Data shown as mean ± SEM; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

Figure 2

Figure 2. GLUT5 not GLUT2 is upregulated in LC tissues of patients.

(A and B) SLC2A2 expression between paired adjacent normal (N) and tumorous (T) lung tissues of patients with LC. Data were obtained from public databases, including 2 TCGA RNA-Seq data sets (n = 57 for patients with ADC and n = 50 for patients with SCC) (A) and 2 microarray data sets of GSE10072 (n = 32) and GSE75037 (n = 83) (B). (C) TCGA data sets showing SLC2A5 expression between paired adjacent normal and tumorous lung tissues of patients with lung ADC (n = 57) or SCC (n = 50). (D) SLC2A5 expression between paired adjacent normal and tumorous lung tissues from patients with lung ADC (n = 21) or SCC (n = 12) from our hospital. (E and F) Representative Western blot images and statistical analysis of GLUT5 expression between paired adjacent normal and tumorous tissues from patients with lung ADC (n = 20) (E) or SCC (n = 12) (F). Actin was used to normalize GLUT5 expression. For box plots, the midline represents the median of the data, with the upper and lower limits of the box being the third and first quartiles. Additionally, the whiskers of the box plot extend up to 1.5 times the interquartile range from the top or bottom of the box. P values were calculated using 2-tailed Wilcoxon’s rank-sum test.

Figure 3

Figure 3. Increased GLUT5 expression in LC tissues is validated in 2 independent patient cohorts.

(A and B) IHC staining of lung tissue microarrays using GLUT5 antibody for patients with lung ADC (A) or SCC (B). N, paired adjacent normal lung tissues; T, tumorous lung tissues. The midline represents the median of the data, with the upper and lower limits of the box being the third and first quartiles. Additionally, the whiskers of the box plot extend up to 1.5 times the interquartile range from the top or bottom of the box. P values were calculated using 2-tailed Wilcoxon’s rank-sum test. (C and D) Overall survival curves of patients with lung ADC (C) or lung SCC (D) stratified by low and high GLUT5. GLUT5 expression in LC tissues was measured by IHC staining. P values were computed using log-rank test.

Figure 4

Figure 4. Abrogation of SLC2A5 impairs fructose uptake and fructose-induced cell proliferation of LC cells in vitro.

(A) Western blot showing CRISPR/Cas9-mediated deletion of SLC2A5 in A549 and EKVX cells. (B) Fructose uptake by A549 and EKVX cells with or without SLC2A5 ablation in vitro. Statistical analysis was conducted using 1-way ANOVA test. After carrying out a homogeneity of variance test to confirm equal variance among subgroups, P values were acquired from post hoc test using LSD method. Cumulative data are shown; n = 3. (C) Fructose-induced cell proliferation of A549 and EKVX cells with or without SLC2A5 abrogation in vitro. Cells were cultured in fructose medium for 72 hours. Statistical analysis was conducted using 1-way ANOVA test. After performing a homogeneity of variance test to verify equal variance among subgroups, P values were gained from post hoc test using LSD method. Cumulative data are shown; n = 3. NC, nontarget control; KO, knockout. Data shown as mean ± SEM. ***P < 0.001, 2-tailed Student’s t test.

Figure 5

Figure 5. Blockade of in vivo fructose utilization via deleting SLC2A5 hinders the neoplastic growth of LC xenografts.

(A) GLUT5 expression in LC xenografts with or without SLC2A5 abrogation. (B) 13C-fructose concentration in A549 (n = 5 tumors for each group) and EKVX (n = 5 tumors for each group) xenografts with or without SLC2A5 deletion. (C) Subcutaneous tumor growth of A549 cells with or without SLC2A5 deletion in nude mice. (D) Xenograft tumor images and tumor weight of A549 cells with or without SLC2A5 ablation (n = 5 tumors for each group). Scale bar: 1 cm. (E) Subcutaneous tumor growth of EKVX cells with or without GLUT5 ablation in nude mice (n = 5 tumors for each group). (F) Tumor images and tumor weight of EKVX cells with or without SLC2A5 abrogation (n = 5 tumors for each group). Scale bar: 1 cm. (G) Representative Western blot images displaying PCNA expression in A549 and EKVX tumor xenografts with or without SLC2A5 ablation. (H) Representative IHC images and quantitative bar plot showing Ki-67 expression in A549 tumor xenografts with or without SLC2A5 deletion (n = 5 tumors for each group). Scale bar: 50 μm. (I) Representative IHC images and quantitative bar plot showing cyclinD1 expression in A549 tumor xenografts with or without SLC2A5 deletion (n = 5 tumors for each group). Scale bar: 20 μm. Data shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

Figure 6

Figure 6. Enhanced fructose utilization via enforcing GLUT5 expression exacerbates the malignancy of LC xenografts.

(A) Western blot showing GLUT5 expression in A549 tumor xenografts with or without enforced GLUT5 expression. (B) 13C-fructose concentration in A549 xenografts with or without enforced GLUT5 expression (n = 5 tumors for each group). (C) Tumor growth of A549 xenografts with or without ectopic GLUT5 expression. (D) Xenograft images and tumor weight of A549 cells with or without ectopic GLUT5 expression (n = 10 tumors for each group). Scale bar: 1 cm. (E) Representative Western blot images exhibiting PCNA expression in A549 xenografts with or without enforced GLUT5 expression. Data shown as mean ± SEM. **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

Figure 7

Figure 7. In vivo fructose utilization mediated by GLUT5 stimulates fatty acid synthesis and palmitoleic acid production.

(A) Impact of impaired fructose utilization caused by SLC2A5 deletion on fructose input, fructose-derived metabolite generation, and synthesis of fatty acid precursors and fatty acids in A549 xenografts. (B) Influence of enhanced fructose utilization caused by ectopic GLUT5 expression on fructose input, fructose-derived metabolite production, and synthesis of fatty acid precursors and fatty acids in A549 xenografts. (C) Production of 13C-metabolites derived from 13C-fructose in A549 xenografts with or without SLC2A5 ablation (n = 5 tumors for each group). (D) Generation of 13C-metabolites derived from 13C-fructose in A549 xenografts with or without enforced GLUT5 expression (n = 5 tumors for each group). (E) Tracer study in ex vivo lung tissues from LC patients with ADC. Fresh adjacent normal tissues (n = 5) and tumor tissues (n = 5) were enrolled for measurement of 13C-fructose uptake and 13C-fructose–derived metabolite generation. (F) Analysis of fatty acids between adjacent normal and tumorous lung tissues of patients with lung ADC (n = 22). Data shown as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test.

Figure 8

Figure 8. Downstream fatty acid synthesis from fructose is essential for LC growth.

(A) Construction of A549 and EKVX cells with FASN knockout. (B and C) Impact of FASN ablation on fructose-induced cell growth of A549 and EKVX cells. Statistical analysis was conducted using 1-way ANOVA test. After conducting a homogeneity of variance test to confirm equal variance among subgroups, P values were acquired from post hoc test using LSD algorism. Cumulative data are shown; n = 3. (D) Palmitoleic acid supplement (50 μM) restored the in vitro growth of A549 cells with SLC2A5 abrogation. Cumulative data are shown; n = 3. (E) Restoration of A549 tumor growth with SLC2A5 knockout by palmitoleic acid (PA) administration. Mice bearing A549 tumors were divided into 4 groups and treated with vehicle or PA. (F) Tumor images and tumor weight of A549-NC cells with vehicle feeding (n = 6), A549-NC cells with PA feeding (n = 6), A549-_SLC2A5_-KO cells with vehicle feeding (n = 6), and A549-_SLC2A5_-KO cells with PA feeding (n = 6). Scale bar: 1 cm. Data shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

Figure 9

Figure 9. Fructose utilization mediated by GLUT5 influences the activity of AMPK/mTORC1 signaling in LC.

(A) Phospho-protein profiling showing enhanced phosphorylation of AMPK and ACC1 and decreased phosphorylation of 4E-BP1, P70S6K, and S6 in A549 tumor xenograft with SLC2A5 deletion as relative to control A549 tumor xenograft. (B) Western blot approach validating the phosphorylation states of AMPK, ACC1, and 4E-BP1 between A549 tumor xenografts with SLC2A5 deletion and control A549 tumor xenografts. (C) RNA-Seq analysis showing repressed transcription of downstream target genes of mTORC1 signaling in A549 tumor xenografts with SLC2A5 abrogation. (D and E) Impact of fructose and glucose on AMPK (D) and 4E-BP1 (E) phosphorylation of A549 cells with or without SLC2A5 ablation. Control A549 cells and A549 cells with SLC2A5 deletion were starved in sugar-free medium for 2 hours and then were treated with 6 mM fructose or 6 mM glucose for 1 hour. Subsequently, cells were harvested for analysis. (F) The influence of an AMPK agonist, AICAR, on phosphorylation status of AMPK and 4E-BP1 in A549 cells cultured in fructose medium. Cells were treated with 1 mM AICAR or vehicle for 6 hours. Samples from the same batch were run at different times. The corresponding loading controls were shown for each measurement. (G) The influence of AICAR (1 mM) on proliferation of A549 cells cultured in fructose medium. Cumulative data were shown; n = 3. Data shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test.

Figure 10

Figure 10. In vivo pharmacological blockage of fructose utilization by 2,5-AM ameliorates the malignancy of LC.

(A and B) 2,5-AM treatment inhibited fructose uptake and fructose-1-phosphate generation in A549 (A) (n = 3 tumors for each group) and EKVX (B) (n = 5 tumors for each group) xenografts. (C and D) Subcutaneous tumor growth of A549 (C) and EKVX (D) cells treated with vehicle or 2,5-AM. (E and F) Heatmap showing the alteration of fatty acids in A549 (E) (n = 3 tumors for each group) and EKVX (F) (n = 5 tumors for each group) xenografts treated with 2,5-AM. (G) A model depicting that in vivo fructose utilization mediated by GLUT5 promotes LC growth. On the one hand, incorporated fructose is used to synthesize downstream fatty acids. On the other hand, fructose utilization activates the oncogenic AMPK/mTORC1 signaling pathway. This gives rise to a therapeutic opportunity by blocking fructose utilization using a GLUT5 inhibitor, 2,5-AM. Data shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.

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