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. 2020 Oct 16;16(16):3100-3115.
doi: 10.7150/ijbs.48066. eCollection 2020.

NDRG2 ablation reprograms metastatic cancer cells towards glutamine dependence via the induction of ASCT2

Mingchao Ding  1   2 , Xin Bu  1 , Zhehao Li  1   3 , Haokun Xu  4 , Lin Feng  5 , Junbi Hu  6 , Xinxin Wei  1   3 , Jiwei Gao  7 , Yanyan Tao  8 , Bolei Cai  2 , Yanpu Liu  2 , Xuan Qu  5 , Liangliang Shen  1
Affiliations

NDRG2 ablation reprograms metastatic cancer cells towards glutamine dependence via the induction of ASCT2

Mingchao Ding et al. Int J Biol Sci. .

Abstract

Background: Metastasis is the most common cause of lethal outcome in various types of cancers. Although the cell proliferation related metabolism rewiring has been well characterized, less is known about the association of metabolic changes with tumor metastasis. Herein, we demonstrate that metastatic tumor obtained a mesenchymal phenotype, which is obtained by the loss of tumor suppressor NDRG2 triggered metabolic switch to glutamine metabolism. Methods: mRNA-seq and gene expression profile analysis were performed to define the differential gene expressions in primary MEC1 and metastatic MC3 cells and the downstream pathways of NDRG2. NDRG2 regulation of Fbw7-dependent c-Myc stability were determined by immunoprecipitation and protein half-life assay. Luciferase reporter and ChIP assays were used to determine the roles of Akt and c-Myc in mediating NDRG2-dependent regulation of ASCT2 in in both tumor and NDRG2-knockout MEF cells. Finally, the effect of the NDRG2/Akt/c-Myc/ASCT2 signaling on glutaminolysis and tumor metastasis were evaluated by functional experiments and clinical samples. Results: Based on the gene expression profile analysis, we identified metastatic tumor cells acquired the mesenchymal-like characteristics and displayed the increased dependency on glutamine utilization. Further, the gain of NDRG2 function blocked epithelial-mesenchymal transition (EMT) and glutaminolysis, potentially through suppression of glutamine transporter ASCT2 expression. The ASCT2 restoration reversed NDRG2 inhibitory effect on EMT program and tumor metastasis. Mechanistic study indicates that NDRG2 promoted Fbw7-dependent c-Myc degradation by inhibiting Akt activation, and subsequently decreased c-Myc-mediated ASCT2 transcription, in both tumor and NDRG2-knockout MEF cells. Supporting the biological significance, the reciprocal relationship between NDRG2 and ASCT2 were observed in multiple types of tumor tissues, and associated with tumor malignancy. Conclusions: NDRG2-dependent repression of ASCT2 presumably is the predominant route by which NDRG2 rewires glutaminolysis and blocks metastatic tumor survival. Targeting glutaminolytic pathway may provide a new strategy for the treatment of metastatic tumors.

Keywords: ASCT2; EMT; NDRG2; c-Myc; glutaminolysis; mucoepidermoid carcinoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
The aggressive derivatives of MEC cells exhibit the loss of epithelial phenotype. (A,B) Heatmap (A) and volcano plot (B) representing the genes significantly differentially expressed in MEC1 and MC3 cells (P<0.05). (C, D) GSEA analysis predicts the gene enrichment in epithelial cell morphogenesis (C) and CDH1 targets (D). (E, F) The indicated protein or mRNA levels were determined by western blotting (E) or qPCR assay (F) in MEC1 and MC3 cells. (G, H) The migratory and invasive behavior of MEC1 and MC3 cells were determined (G) and quantified (H). (I-J) Representative images (I) and statistical analysis (J) of the lung metastasis upon tail vein injection of MEC1 or MC3 cells. (K) Quantification of metastatic area in lungs (area of metastatic lesions, %of total lung area). Data are expressed as means ± SD (n = 3). **, P <0.001.
Figure 2
Figure 2
Glutamine addiction occurs in mesenchymal MC3 cells. (A, B) The glucose (A) or glutamine (B) uptake rate was determined in MEC1 and MC3 cells. (C) The normalized cell viability of MEC1 and MC3 cells grown in indicated conditions for 72 h. (D, E) Cells were grown with or without glutamine treatment for the indicated number of days (A) or with different dose of glutamine treatment for 72 h (E). Cell viability was normalized to its growth in complete medium containing glutamine. (F) The internal ATP levels were determined in medium lacking glutamine for 24 h and normalized to the levels in medium containing glutamine. (G) The cell viability was determined in MEC1 or MC3 cells following 0-100 µM CB-839 or V-9302 treatment for 72 h. (H) The internal ATP levels were determined in cells with 0.1 µM CB-839 or V-9302 treatment for 24 h, and normalized to the DMSO treatment group. (I, J) Quantification of the invasion (I) or migration (J) behavior of MEC1 and MC3 cells with or without 0.1 µM CB-839 or V-9302 treatment for 24 h. “G”, glucose; “Q”, glutamine. Data are expressed as means ± SD (n = 3). * P <0.01. **, P <0.001
Figure 3
Figure 3
NDRG2 suppresses MC3 cell survival through the blockage of glutaminolysis. (A) Western blotting analysis of indicated protein expressions in MC3 cells with or without NDRG2 expression. (B) Representative bioluminescence imaging photographs of tumor burden 4 weeks after the subcutaneous injection of indicated cells. (C) The Tumor size was measured over a 4-week period and tumor volume was calculated by the formula (width2× length × 0.5). (D-F) The glutamine uptake rate (D), internal glutamate levels and α-KG levels were determined in MC3 cells with or without NDRG2 expression. (G, H) The cell viability (G) or internal ATP levels (H) were determined in indicated cells with or without α-KG treatment. (I-J) Representative images (I) and statistical analysis (J) of the lung metastasis upon tail vein injection of MC3 cells with or without NDRG2 expression. (K) Quantification of metastatic area in lungs (area of metastatic lesions, %of total lung area). (L, M) The migratory and invasive behavior of indicated cells were determined (G) and quantified (H). Data are expressed as means ± SD (n = 3). * P <0.01. ** P <0.001.
Figure 4
Figure 4
NDRG2 represses ASCT2 transcription. (A) The differentially expressed genes were clustered and calculated in MEC cells, MC3 cells, and MC3 cells with NDRG2 expression (MC3N). Venn diagram was made with Jvenn (http://jvenn.toulouse.inra.fr/app/index.html). (B) The heatmap represents the expression of glutaminolytic-related genes in indicated cells. (C-E) The indicated protein levels (C), and ASCT2 (D) and GLS1 (E) mRNA levels were determined by western blotting and qPCR, respectively in MEC1 and MC3 cells. (F-H) The indicated protein levels (F), and ASCT2 (G) and GLS1 (H) mRNA levels were determined by western blotting and qPCR, respectively in MC3 cells with or without NDRG2 overexpression. (I-K) The indicated protein levels (I), ASCT2 mRNA levels (J) and ASCT2 promoter activity (K) were determined respectively. Data are expressed as means ± SD (n = 3). **, P <0.001.
Figure 5
Figure 5
NDRG2 represses ASCT2 through the induction of Fbw7-dependent c-Myc degradation. (A-D) We gained c-Myc function through overexpression in NDRG2-overexpressing MC3 cells. The indicated proteins levels (A), ASCT2 mRNA levels (B), c-Myc enrichment on ASCT2 promoter (C), and the activities of wild-type and E-box-mutant ASCT2 promoter (D) were determined respectively. (E) c-Myc mRNA levels were determined in MC3 cells with or without NDRG2 expression. (F) Levels of indicated protein expressions were determined in MC3 cells with or without NDRG2 expression, or NDRG2-overexpressed MC3 cells with Fbw7 knockdown. (G) MC3 cells with or without NDRG2 expression were treated with 20 µM MG132 for 2 h. Levels of indicated proteins were determined by western blot. (H-K) Levels of indicated proteins in indicated MC3 cells or MEF cells following 10 µg/ml CHX treatment for indicated time (H and J). c-Myc expression levels were further quantified by ImageJ and normalized to β-actin to determine the protein degradation rate (I and K). **, P <0.001.
Figure 6
Figure 6
NDRG2 suppression of c-Myc stabilization is mediated by AKT/GSK3β pathway. (A) GSEA analysis predicts the gene enrichment in PI3K-Akt pathway in MC3 cells with or without NDRG2 expression. (B) Levels of indicated proteins in MC3 cells with or without NDRG2 expression. (C-F) We overexpressed the wild type or activating form of Akt1 (myr-Akt1) in NDRG2-overexpressing MC3 cells. (C) The indicated proteins levels were determined by western blotting. (D) Immunoprecipitation was performed to determine the interaction between c-Myc and Fbw7 in indicated MC3 cells. After the Fbw7 protein were immunoprecipitated with an anti-Fbw7 antibody, indicated proteins were detected by western blotting. (E-F) c-Myc enrichment on ASCT2 promoter (E) and the activities of wild-type and E-box-mutant ASCT2 promoter (F) were determined respectively. (G, H) c-Myc enrichment on ASCT2 promoter (G), and the activities of wild-type and E-box-mutant ASCT2 promoter (H) were determined in wild-type or NDRG2-/- MEF cells, respectively. Data are expressed as means ± SD (n = 3). (I) The proposed mechanism of NDRG2 action in regulation of Akt/c-Myc axis and ASCT2 expression. **, P <0.001.
Figure 7
Figure 7
NDRG2 repression of ASCT2 controls EMT progression and cell survival in metastatic tumor. We gained ASCT2 expression in NDRG2-ovexpressing MC3 cells and determined the cell biological behaviors. (A) Representative bioluminescence imaging photographs of tumor burden 4 weeks after the subcutaneous injection of indicated cells. (B) The Tumor size was measured and tumor volume was calculated. (C, D) Levels of indicated protein (C) or mRNA (D) expressions were determined by western blotting or qPCR respectively. (E, F) The migratory and invasive behavior of indicated cells were determined (E) and quantified (F). (G, H) Representative images (G) and statistical analysis (H) of the lung metastasis upon tail vein injection of indicated cells. (I) Quantification of metastatic area in lungs (area of metastatic lesions, %of total lung area). Data are expressed as means ± SD (n = 3). *, P <0.01. **, P <0.001,
Figure 8
Figure 8
The interplay between NDRG2 and ASCT2 dictates tumor malignancy. (A) Immunohistochemistry staining of NDRG2 and ASCT2 in human adjacent normal and MEC tissues. (B) Negative correlation between NDRG2 and ASCT2 expressions with linear regression and Pearson's correlation significance (P <0.0001, ANOVA test). (C, D) NDRG2 (C) or ASCT2 (D) expression by immunohistochemistry staining in MEC tissues at different grade. (E-F) Survival analysis of NDRG2 (E) or ASCT2 (F) association with prognosis in all cancer types by GEPIA. (G-H) Negative correlation between NDRG2 and ASCT2 mRNA expression patterns in all cancer types from TCGA (G) or GTEx (H) data set by GEPIA with linear regression and Pearson's correlation significance (P <0.001, ANOVA test).
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