Targeting glutamine-addiction and overcoming CDK4/6 inhibitor resistance in human esophageal squamous cell carcinoma

Abstract

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.

Fig. 1

Dysregulated Fbxo4-cyclin D1 axis leads to Gln-addiction. a Western analysis for cleaved PARP and cleaved caspase-3 in Fbxo4−/− mouse embryonic fibroblasts (MEFs) following Gln-depletion. b FACS for Annexin V-positive cells following 48 h Gln-depletion. c Western analysis for cleaved PARP and cleaved caspase-3 following 24 h Gln-depletion in Fbxo4−/− MEFs with ectopic expression of Fbxo4 WT, ΔN, ΔF, ΔC2, or ΔC3. Magenta stars indicate Fbxo4 bands. d FACS for Annexin V-positive cells following 48 h Gln-depletion. e Cyclin D1 knockout antagonizes apoptosis in a Fbxo4−/− background following 24 h Gln-depletion. In order to show cyclin D1 expression, cyclin D1 blot was performed in medium with Gln, because Gln-depletion reduces endogenous cyclin D1 expression. f Overexpression of cyclin D1 promotes apoptosis in NIH3T3 cells upon 24 h Gln-depletion. g One micromolar PD-0332991 (PD) suppresses apoptosis induced by 24 h Gln-depletion in NIH3T3 cells with ectopic cyclin D1 or D1T286A. SE: short exposure; LE: long exposure. Arrow: specific band; open triangle: non-specific band

Fig. 2

Dysregulated Fbxo4-cyclin D1 axis promotes Gln-addiction in esophageal squamous cell carcinoma (ESCC) cells. a Gene set enrichment analysis (GSEA) enrichment of ESCC versus normal tissues for gene set “Cell Cycle Regulation” in NCBI GEO dataset (GSE100942): NES = 1.4644697, FDR q-val = 0.45459393. b GSEA enrichment of ESCC versus normal tissues for gene set related to “Gln Metabolism” in NCBI GEO dataset (GSE100942): NES = 1.5255251, FDR q-val = 0.06451613. c Expression of Gln metabolism genes correlates with poor prognosis of ESCC patients. Dysregulated Gln Met: dysregulated Gln metabolism; HR: hazard ratio; CI: confidence interval. d Increased PARP cleavage following 24 h Gln-depletion in TE7 and TE10 versus TE15 cells. e WT Fbxo4 suppresses PARP cleavage following 24 h Gln-depletion in TE10 cells. f Overexpression of cyclin D1 or D1T286A promotes the sensitivity of TE15 cells to 24 h Gln-depletion. g Increased PARP cleavage in TE1 versus TE15 cells upon 24 h Gln-depletion. h FACS for Annexin V-positive cells with or without 48 h Gln-depletion in TE7, TE10, TE1, and TE15 cells. Arrow: band of interest; open triangle: non-specific band

Fig. 3

mTORC1 contributes to Gln-addiction in cells with dysregulated cyclin D1. a, b Gene set enrichment analysis (GSEA) enrichment plots for expression profiles of esophageal squamous cell carcinoma (ESCC) versus normal tissues for gene sets correlated to mTORC1 signaling in NCBI GEO datasets: a GSE100942—NES = 1.4599689, FDR q-val = 0.12097056 and b GSE20347—NES = 1.269795, FDR q-val = 0.3669077. c Western blot for the indicated mTORC1 effectors in NIH3T3 cells expressing cyclin D1 or D1T286A. d Western blot analysis for mTORC1 effectors in TE7, TE10, and TE15 cells. e Rapamycin compromises mTORC1 activation and apoptosis induced by ectopic cyclin D1 expression in NIH3T3 cells upon 24 h Gln-depletion. f Rad001 inhibits mTORC1 activation and apoptosis in TE7 and TE10 cells upon 24 h Gln-depletion. g Raptor knockdown suppresses apoptosis in TE7 and TE10 cells upon 24 h Gln-depletion. SE: short exposure; LE: long exposure. Arrow: specific band

Fig. 4

Cyclin D1 accumulation promotes mitochondrial dysfunction. a–c ATP/ADP ratio in Fbxo4+/+ and −/− mouse embryonic fibroblasts (MEFs) (a), NIH3T3 cells (b), and esophageal squamous cell carcinoma (ESCC) cells (c) detected by ApoSENSOR™ ADP/ATP ratio bioluminescent assay. a Data represent as mean ± s.d., two-tailed Student t-test was used to compare means (n = 3); b, c Data represent as mean ± s.d., one-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 3); p-values are listed. d Western analysis for the indicated proteins in Fbxo4+/+ versus −/− MEFs upon Gln-depletion. Arrow: band of interest; open triangle: non-specific band. e Seahorse analysis for Fbxo4+/+ and −/− MEFs; data were normalized to protein concentration. Injections: Port D: Gln; Port A: Oligomycin; Port B: Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and Port C: Antimycin A & Rotenone. f, g Mitochondrial membrane potential analysis of Fbxo4+/+ and −/− MEFs; g Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment as positive control. h, i Mitochondrial membrane potential analysis of TE7, TE10, and TE15 cells; i CCCP treatment as positive control. TMRE, tetramethylrhodamine, ethyl ester; RFU, relative fluorescence units. All data in f–i represent as mean ± s.d., one-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 3). *p < 0.05; **p < 0.01

Fig. 5

CB-839 plus Phenformin/Metformin synergistically induces apoptosis. a Western analysis of PARP and caspase-3 cleavage in Fbxo4−/− versus +/+ mouse embryonic fibroblasts (MEFs) treated with Phenformin following 24 h Gln-depletion. b Western analysis for the indicated proteins in Fbxo4+/+ versus −/− MEFs treated with CB-839 plus phenformin for 24 h. c Western analysis for PARP cleavage following 24 h treatment with CB-839 and/or phenformin in TE7, TE10, and TE15 cells. d Combined treatment for 24 h induces more apoptosis in TE15 cells with ectopic cyclin D1 or D1T286A expression. SE: short exposure; LE: long exposure. e FACS for Annexin V-positive cells following combined CB-839 (CB) and metformin (Met) treatment for 48 h. f, g Mitochondrial membrane potential analysis of Fbxo4+/+ and −/− MEFs with the indicated treatments; g CCCP treatment as positive control. h, i Mitochondrial membrane potential analysis of TE7, TE10, and TE15 cells with indicated treatments; i CCCP treatment as positive control. All data represent as mean ± s.d., one-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 3). *p < 0.05; **p < 0.01. CB-839 concentration is 10 μM and phenformin/metformin concentration is 1 mM for panels a–i. Arrow: specific band; open triangle: non-specific band

Fig. 6

Combined treatment inhibits xenograft growth and induces apoptosis in vivo. a, b Growth curve of TE7 (a) and TE15 (b) xenografts. Data represent as mean ± s.d., two-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 8). **p < 0.01. c H&E staining sections from TE7 and TE15 xenografts. d IHC staining of Ki-67 in xenograft tissues. e IHC staining of cleaved caspase-3 in xenograft tissues, magenta arrow indicates positive cells. f Comparison of Ki-67 index among different groups. g Quantification of cleaved caspase-3 positive cells per 2000 cells. All data in f and g represent as mean ± s.d., one-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 8). **p < 0.01. Scale bar, 10 μm

Fig. 7

Combined treatment overcomes CDK4/6 inhibitor resistance in esophageal squamous cell carcinoma (ESCC) cells. a Blue-Pink O’ Gram in the Space of the Analyzed Gene Set with NCBI GEO (GSE40513), the comparison of Gln metabolism genes between palbociclib and vehicle-treated mouse breast cancer V720 cells. Glutamate-ammonia ligase (GLUL), glutamate dehydrogenase (GLUD), asparagine synthetase (ASNS), glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT). Red color indicates gene upregulation; blue color indicates gene downregulation. b The expression of GLS1 and GLS2 mRNAs in parental and PDR cells. Data represent as mean ± s.d., two-tailed Student t-test was used to compare means (n = 3). *p < 0.05, **p < 0.01. c Western blot analysis of GLS1 levels in parental and PDR cells. d PDR ESCC cells exhibit increased [³H]Gln uptake relative to parental counterparts (1 × 10⁵ cells used). Data represent as mean ± s.d., one-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 3); p-values are listed. e CB-839 plus metformin treatment for 24 h induces more apoptosis in both TE7PDR and TE10PDR cells. f GLS1 knockdown increases apoptosis in both TE7PDR and TE10PDR cells. g Rb knockdown induces cell apoptosis in TE15 cells upon combined treatment for 24 h. Arrow: band of interest

Fig. 8

Combined treatment suppresses TE7PDR xenograft growth and induces cell apoptosis in vivo. a Growth curve of TE7PDR xenografts in nude mice. Data represent as mean ± s.d., two-way ANOVA was used to compare means with Bonferroni as Post Hoc test (n = 8). **p < 0.01. b IHC staining of Ki-67 in xenograft tissues. c IHC staining of cleaved caspase-3 in xenograft tissues, magenta arrow indicates positive cells. d Comparison of Ki-67 index between vehicle and treated groups. e Quantification of cleaved caspase-3 positive cells per 2000 cells between vehicle and treated groups. All data in d and e represent as mean ± s.d., two-tailed Student t-test was used to compare means (n = 8). **p < 0.01. Scale bar, 10 μm. f Schematic illustration of the working model. In normal cells, the homeostatic regulation of Fbxo4-cyclin D1 axis keeps the downstream pathway balanced; however, in tumor cells, dysregulated Fbxo4-cyclin D1 axis suppresses Rb and hyperactivates mTORC1, leading to the imbalance between energetic production and consumption, and finally, Gln-addiction. By targeting this genetic vulnerability, combined CB-839 and metformin/phenformin disrupts the metabolic balance, leading to cell apoptosis and the suppression of cell proliferation. In addition, palbociclib-resistant ESCC cells demonstrate metabolic reprogramming characterized by Gln-addiction, resulting in increased sensitivity to combined treatment. This model provides promising therapeutic targets for cancer treatment