Wnt/β-catenin signalling activates IMPDH2-mediated purine metabolism to facilitate oxaliplatin resistance by inhibiting caspase-dependent apoptosis in colorectal cancer

Abstract

Background: Oxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC.

Methods: An oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5'-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro.

Results: Metabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP.

Conclusions: IMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.

Keywords: Apoptosis; Colorectal cancer; IMPDH2; Oxaliplatin resistance; Purine metabolism; β‑catenin.

Fig. 1

Establishment and characterization of the acquired oxaliplatin-resistant CRC cell line HCT8/L-OHP. A CCK8 assay to assess the IC50 of oxaliplatin-resistant CRC cells HCT8/L-OHP and parental HCT8 cells after treated with increasing concentrations of oxaliplatin treatment for 48 h. B The IC50 of oxaliplatin to HCT8 and HCT8/L-OHP cells were calculated from the inhibition curves. C P-gp protein expression in HCT8 and HCT8/L-OHP cells was determined by western blotting analysis. D Flow cytometry analysis measures the apoptosis in HCT8 and HCT8/L-OHP cells at 24 h after oxaliplatin treatment. Bar charts show the percentage of apoptosis E and surviving F cells, respectively, in both untreated and oxaliplatin-treated groups. Data are presented as the mean ± SD of three independent experiments. ***p < 0.001

Fig. 2

Purine metabolism was upregulated in oxaliplatin-resistant HCT8/L-OHP cells. A PCA 3D score plot between HCT8 and HCT8/L-OHP groups. B Volcano plot of differential metabolites. C Bubble plots for KEGG metabolic pathway enrichment analysis of differential metabolites. D Hierarchical clustering analysis of differential metabolites involved in purine metabolism. E QPCR analysis of purine metabolic key enzyme genes in HCT8/L-OHP comparing to those in HCT8 cells. ACTB was used as the internal reference. F Western blotting analysis of IMPDH2 protein expression in HCT8 and HCT8/L-OHP cells. G Schematic diagram of purine metabolism. The red indicates that the metabolite is up-regulated, the blue indicates that the metabolite is down-regulated, while the gray indicates metabolite with no significant change. G-6-P Glucose-6-phosphate, R-5-P Ribose-5-phosphate, IMP Inosinic acid, XMP, Xanthylic acid; GMP, Guanosine monophosphate; dGMP, Deoxyguanosine monophosphate; AMP Adenosine monophosphate. Significance is indicated as ns  not significant, *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

IMPDH2 expression is upregulated in CRC patients and is associated with multiple drug resistance. A IMPDH2 expression in various types of cancer in the TIMER database. B The mRNA expression of IMPDH2 in 24 matched CRC and adjacent noncancerous samples from the GSE10950. The protein expression of IMPDH2 in CRC based on sample types (C), individual cancer stages D and histological subtypes E from CPTAC. F The IMPDH2 protein expression from HPA database. GSEA was performed to showing a significant association between the IMPDH2 expression and the PURINE_METABOLISM G and MULTIPLE-DRUG-RESISTANCE H. **p < 0.01, and ***p < 0.001

Fig. 4

IMPDH2 expression is associated with CRC cells sensitivity to oxaliplatin. A and B The endogenous expression of IMPDH2 in four CRC cell lines, SW620, RKO, HCT116 and HCT8, was determined by qPCR and western blotting analysis. The mRNA and protein expression of IMPDH2 in HCT8 C and D and SW620 E and F in response to oxaliplatin treatment were determined by qPCR and western blotting analysis. G Flow cytometric analysis was performed to analyse the apoptotic rate in HCT8 and SW620 cells treated with increasing concentrations of oxaliplatin at 24 h. The histograms show the percentage of apoptosis H and surviving I cells, respectively, in both untreated and oxaliplatin-treated groups. Data are presented as the mean ± SD of three independent experiments. ns  not significant, ***p < 0.001

Fig. 5

IMPDH2 regulates cell apoptosis of CRC in response to oxaliplatin. A and B Overexpression of IMPDH2 was confirmed at the mRNA and protein level in HCT8 cells by qPCR and western blotting. C Comparison of apoptosis induction between vector and IMPDH2-overexpression cells in HCT8 after oxaliplatin treatment was carried out by flow cytometry analysis. Bar charts show the percentage of apoptotic and surviving cells, respectively. D and E The mRNA and protein expression levels of stable knockdown IMPDH2 in HCT8/L-OHP cells were quantified by qPCR and western blotting analysis. F Flow cytometry analysis was performed to analyse the apoptotic rate in shNC and shIMPDH2 HCT8/L-OHP cells after oxaliplatin treatment. G Western blotting analysis of apoptotic proteins expression was carried out between vector and IMPDH2-overexpression HCT8 cells after oxaliplatin treatment. H Western blotting analysis was performed to detect the expression of apoptotic proteins in shNC or IMPDH2-knockdown HCT8/L-OHP cells after oxaliplatin treatment. I The apoptotic rate was analysed after GMP and/or oxaliplatin treatment compared with that of untreated cells for 24 h by flow cytometry analysis in HCT8 cells. J The apoptotic rate was analysed after MPA and/or oxaliplatin treatment compared with that of untreated cells for 24 h by flow cytosmetry analysis in HCT8/L-OHP cells. Data are presented as the mean ± SD of three independent experiments. **p < 0.01, and ***p < 0.001

Fig. 6

Wnt/β-catenin pathway is hyperactivated in oxaliplatin-resistant cells. A Coomassie staining of total proteins in HCT8 and HCT8/L-OHP cells transfecting with Flag-IMPDH2 plasmids separated by SDS–PAGE. B Venn diagrams displaying the number of proteins identified in HCT8 and HCT8/L-OHP cells. C KEGG pathway enrichment analysis of proteins identified in HCT8/L-OHP cells. D and E QPCR and western blotting analysis of the activation of the Wnt/β-catenin pathways in HCT8/L-OHP cells compared to that in HCT8 cells. F and G QPCR and western blotting analysis were conducted to evaluate the suppressive effect of XAV939 on Wnt/β-catenin pathways and IMPDH2 in HCT8/L-OHP cells. H and I The cell apoptosis was analysed after XAV939 and/or oxaliplatin treatment with or without GMP compared with that of untreated cells for 24 h by flow cytometry analysis in HCT8/L-OHP cells. J Western blotting analysis of apoptotic proteins expression was performed in HCT8/L-OHP cells after XAV939 and/or oxaliplatin treatment with or without GMP. Data are presented as the mean ± SD of three independent experiments. **p < 0.01, and ***p < 0.001

Fig. 7

Inhibition of IMPDH2 reversed oxaliplatin resistance in vivo. A Tumour images. B Tumour growth curve. C Tumour weight. D Body weight. E The H&E and IHC of Ki-67 of tumour tissue with indicated treatment. F A schematic diagram of the Wnt/β‑catenin/IMPDH2 mediated purine metabolism regulating oxaliplatin-induced apoptosis in CRC. ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001