Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma

Abstract

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.

Keywords: ASCT2; CD44 variant; glutamate dehydrogenase; head and neck cancer; xCT.

Figure 1

Sulfasalazine‐induced oxidative stress requires glutamine uptake mediated by ASCT2. A, Immunoblot analysis of CD44v, xCT, involucrin and β‐actin (loading control) in HSC‐2 cells cultured under normal (Undiff) or adhesion‐restricted conditions for 96 h (Diff). B, Gene ontology (GO) analysis of genes whose expression was upregulated (blue) or downregulated (red) in HSC‐2 cells cultured under the adhesion‐restricted condition. C, Heat map for SLC family genes whose expression was upregulated (red) or downregulated (green) with an absolute fold change value of >2.5 and a P value of <0.01 as revealed by microarray analysis of HSC‐2 cells cultured under normal (Undiff) or adhesion‐restricted conditions for 72 h (Diff). The gene names of glutamine transporter are shown in red, and those of glucose transporter in blue. D, Quantitative RT‐PCR analysis of SLC1A5, SLC6A15, SLC38A5, SLC7A11, involucrin (IVL) and MYC mRNA in HSC‐2 cells cultured under normal (Undiff) or adhesion‐restricted conditions for 72 h (Diff). Data were normalized by the amount of RPS17 mRNA and are means ± SD from 3 independent experiments. **P < 0.01 (Student's t test). E, Immunoblot analysis of ASCT2, MYC and involucrin in HSC‐2 cells cultured under normal (Undiff) or adhesion‐restricted conditions for 72 h (Diff). F and G, Survival of HSC‐2 cells cultured under the normal condition with sulfasalazine (400 μM) for 48 h in the absence or presence of 4 mM glutamine (F) or of 2 mM GPNA (G). Data are expressed relative to the corresponding value for cells not treated with sulfasalazine and are means ± SD from 3 independent experiments. **P < 0.01 (Student's t test). H, HSC‐2 cells cultured under the normal condition with sulfasalazine (400 μM) or DMSO vehicle for 24 h in the absence of glutamine or in the presence of GPNA (2 mM) were stained (or not) with dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) and subjected to flow cytometric analysis for measurement of intracellular reactive oxygen species. RFI, relative fluorescence intensity

Figure 2

ASCT2⁺/CD44v^high head and neck squamous cell carcinoma (HNSCC) cells are highly sensitive to xCT inhibition. A, Flow cytometric analysis of ASCT2 and CD44v expression in HSC‐2 cells. FSC, forward scatter. B, Immunohistofluorescence staining of ASCT2 (green) and CD44v (red) in tumors formed in athymic mice injected with HSC‐2 cells. Nuclei in the merged image were stained with 4ʹ,6‐diamidino‐2‐phenylindole (DAPI, blue). Scale bar, 50 μm. C, Volume of tumors formed by HSC‐2 cells in nude mice treated with saline (control), sulfasalazine (350 mg/kg per day) or cisplatin (2 mg/kg every 3 days). Data are means ± SD for 5 mice per group. *P < 0.05 (Student's t test); NS, not significant. D, Flow cytometric analysis of CD44v expression in lineage marker‐negative cells isolated from tumors formed by HSC‐2 cells in nude mice treated with saline, sulfasalazine or cisplatin as in (C). E, Quantification of CD44v^high tumor cells as in (D). Data are means ± SD for 3 mice per group. *P < 0.05 (Student's t test). F, Immunohistofluorescence staining for ASCT2 (green) and CD44v (red) in tumors formed by HSC‐2 cells in nude mice treated as in (C). Nuclei in the merged images were stained with DAPI (blue). Scale bars, 50 μm. G, Quantification of the area occupied by ASCT2⁺ cells in the CD44v‐expressing region of tumors formed by HSC‐2 cells in nude mice as in (F). Each data point corresponds to an individual field of view, and bars indicate mean ± SD values. **P < 0.01 (Student's t test)

Figure 3

A stemlike gene signature is associated with poor patient survival and glutaminolysis‐related gene expression in head and neck squamous cell carcinoma (HNSCC). A, Kaplan‐Meier plots of overall survival for patients with HNSCC tumors characterized by CD44v9^high or CD44v9^low (left) or CD44v9^high/involucrin^low or CD44v9^low/involucrin^high (right) gene expression signatures. Data are from the TCGA cohort. P values were determined with the log‐rank test. B, Box‐whisker plot of the expression of KRT10 (differentiation marker) and glutaminolysis‐related (SLC1A5, SLC6A15, SLC38A5, GLS, GLUD1 and GLUD2) genes on the CD44v9^high/involucrin^low and CD44v9^low/involucrin^high gene signatures for head and neck squamous cell carcinoma (HNSCC) tumors in the TCGA cohort. **P < 0.01 (Student's t test); NS, not significant

Figure 4

Inhibition of xCT promotes mitochondrial metabolism in CD44v^high head and neck squamous cell carcinoma (HNSCC) cells. A, The content of metabolites related to the GSH synthesis pathway (GSSG, oxidized glutathione) or the TCA cycle was determined for HSC‐2 and OSC19 cells cultured in the presence of sulfasalazine (SSZ, 400 μM) or DMSO vehicle for 6 h. Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01 (Student's t test). N.D., not detected. B, Immunoblot analysis of xCT in HSC‐4 cells stably infected with a retrovirus encoding xCT or with the corresponding empty virus (Mock). C, Glutamate release over 24 h and glutathione (GSH) content for cells as in (B). Data are means ± SD from 3 independent experiments. **P < 0.01 (Student's t test). D, Oxidative consumption (OCR) and extracellular acidification (ECAR) for cells as in (B). Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01 (Student's t test)

Figure 5

Role of α‐KG production in oxidative stress induced by xCT inhibition. A, Quantitative RT‐PCR analysis of GLUD1 and GLUD2 mRNA abundance in OSC19 cells transfected with control or GLUD1/2 (#1 or #2) siRNAs. Data were normalized by the amount of GAPDH mRNA and are means ± SD from 3 independent experiments. **P < 0.01 (Student's t test). B and C, OSC19 cells transfected with control or GLUD1/2 #2 siRNAs were cultured with sulfasalazine (400 μM) or DMSO vehicle for 6 h and then subjected to metabolome analysis for determination of the amounts of metabolites related to glutaminolysis (B) or to the glutathione (GSH) antioxidant system (C). Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01 (Student's t test). N.D., not detected. D, OSC19 cells transfected with control or GLUD1/2 (#1 or #2) siRNA were cultured with sulfasalazine (400 μM) or DMSO vehicle for 24 h and were then stained with DCFH‐DA for determination of ROS abundance by flow cytometry. E, OSC19 cells transfected with control or GLUD1/2 (#1 or #2) siRNA were cultured with the indicated concentrations of sulfasalazine for 48 h and then assayed for cell viability. Data are means ± SD from 3 independent experiments. **P < 0.01 vs the corresponding value for cells transfected with the control siRNA (Student's t test). F, Competition between xCT‐mediated cystine uptake and GLUD‐mediated α‐KG generation for the utilization of glutamate in HNSCC cells either depleted of GLUD (left) or treated with the xCT inhibitor sulfasalazine (right)