ERK Inhibitor Enhances Everolimus Efficacy through the Attenuation of dNTP Pools in Renal Cell Carcinoma

Abstract

The clinical efficiency of everolimus, an mammalian target of rapamycin (mTOR) inhibitor, is palliative as sequential or second-line therapy for renal cell carcinoma (RCC). However, the limited response of everolimus in RCC remains uncertain. In the present study, everolimus-resistant RCC models were established to understand the mechanisms and to seek combination approaches. Consequently, the activation of ERK was found to contribute toward everolimus-acquired resistance and poor prognosis in patients with RCC. In addition, the efficacy and mechanism of combination treatment underlying RCC using everolimus and ERK inhibitors was investigated. The ERK inhibitor in combination with everolimus synergistically inhibited the proliferation of RCC cells by arresting the cell cycle in the G1 phase. The combination treatment markedly attenuated the deoxyribonucleoside triphosphate (dNTP) pools by downregulating the mRNA expression of RRM1 and RRM2 through E2F1. The overexpression of E2F1 or supplementation of dNTP rescued the anti-proliferation activity of the everolimus-SCH772984 combination. The antitumor efficacy of combination therapy was reiterated in RCC xenograft models. Thus, the current findings provided evidence that the everolimus-ERK inhibitor combination is a preclinical therapeutic strategy for RCC.

Keywords: ERK inhibitor; dNTP; everolimus; renal cell carcinoma; ribonucleotide reductase.

Figure 1

Activation of ERK Signal Contributes to Everolimus Resistance and Poor Prognosis of RCC (A) Caki-1 and 786-O cells were treated with chronic (>3 months) everolimus therapy to establish everolimus-resistant cell line models (Caki-1-E and 786-O-E). IC₅₀ of everolimus in parental and resistant cells was detected. (B) Immunoblotting was performed with the parental and resistant cell lysates for phospho-ERK expression. (C) Cells were treated with ERK inhibitor SCH772984 and everolimus for 72 h. Cell viability was assessed by CCK-8 assay. (D) IHC staining was performed in 90 cases of ccRCC tissues and 30 cases of normal tissues. The representative images of phospho-ERK expression localized in the nucleus are shown (×400). Scale bar, 50 μm. (E) IHC score of phospho-ERK in the above samples. **p < 0.01. (F) Survival analysis of RCC patients related to phospho-ERK expression was analyzed by Kaplan-Meier survival curves. Error bars represent mean ± SD of three independent experiments.

Figure 2

Combination of ERK Inhibitor and Everolimus Synergistically Reduces the Viability of RCC Cells (A) Caki-1 and 786-O cells were treated with everolimus alone or in combination with ERK inhibitor SCH772984 for 72 h. The cell viability was assessed by CCK-8 assay. ***p < 0.001. (B) Synergistic effect of everolimus and SCH772984 in Caki-1 and 786-O cells was analyzed using isobologram. The horizontal line indicates the fractional effect of the combination. CI values <1 indicate synergism whereas >1 indicate antagonism. (C) Caki-1 and 786-O cells were treated with everolimus alone or in combination with ERK inhibitor BVD-523 for 72 h. The cell viability was assessed by CCK-8 assay. **p < 0.01; ***p < 0.001. (D) Synergistic effect of everolimus and BVD-523 in Caki-1 and 786-O cells was analyzed using isobologram. The horizontal line indicates the fractional effect of the combination. CI values <1 indicate synergism whereas >1 indicate antagonism. Error bars represent mean ± SD of three independent experiments.

Figure 3

ERK Inhibitor and Everolimus Synergistically Reduce the Viability of RCC Cells by Inducing G1 Cell-Cycle Arrest (A and B) Caki-1 and 786-O cells were treated with everolimus alone or in combination with ERK inhibitor SCH772984 (A) or BVD-523 (B) for 24 h. The cell cycle distribution was detected by PI staining and is shown in the bar graph as the percentage of the cells. ***p < 0.001. (C and D) Caki-1 and 786-O cells were treated with everolimus alone or in combination with ERK inhibitor SCH772984 (C) or BVD-523 (D) for 48 h. The apoptotic cells were detected by PI and Annexin V dual staining and are shown in the bar graph as the percentage of cells. n.s. indicates not significant. Error bars represent mean ± SD of three independent experiments.

Figure 4

Attenuation of dNTP Pools Caused by the Downregulation of RRM1 and RRM2 Is Required for G1 Arrest Induced by the Combination of Everolimus and ERK Inhibitor (A and B) A targeted metabolomic assay was performed in Caki-1 cells exposed to 0.1 μM everolimus, 1 μM SCH772984, the combination of both, or an equivalent volume of DMSO for 72 h. After one-way ANOVA analysis, KEGG pathway enrichment analysis of the altered 35 metabolites revealed that the purine and pyrimidine metabolisms were highly affected by the combination therapy. The number at the top of the bar indicates the number of altered metabolites; the RichFactor indicates the percentage ratio of the altered metabolites to all metabolites in this pathway (A). The top 10 altered metabolites involved in purine and pyrimidine metabolism pathways were ranked by −Log10(p value) (B). (C) 100 μM dNTP was added to the everolimus-SCH772984 combination system for 72 h in Caki-1 and 786-O cells. The cell viability was measured using CCK-8 assay. ***p < 0.001. (D) Caki-1 and 786-O cells were treated with everolimus and SCH772984 for 24 h. Then, immunoblotting analysis was performed on the cell lysates to detect the essential enzymes involved in purine metabolism. (E and F) Caki-1 (E) and 786-O (F) cells were transfected with the indicated siRNAs to concurrently knock down RRM1 and RRM2 for 48 h. The knockdown efficiency was evaluated by immunoblotting analysis. The cell cycle distribution was detected in the cells by PI staining. ***p < 0.001. Error bars represent mean ± SD of three independent experiments.

Figure 5

E2F1 Downregulation Is Required for the Transcriptional Inhibition of RRM1 and RRM2 Induced by the Combination of Everolimus and ERK Inhibitor (A–C) Caki-1 and 786-O cells treated with everolimus alone or in combination with SCH772984 for 72 h were subjected to qRT-PCR analysis to elucidate the mRNA levels of RRM1 (A), RRM2 (B), and E2F1 (C). (D) Phospho-p70S6k, phospho-Erk1/2, and E2F1 protein levels were detected by immunoblotting analysis when Caki-1 and 786-O cells were treated with everolimus and SCH772984 for the indicated time points. (E) Caki-1 and 786-O cells were transfected with E2F1 overexpression plasmid or empty plasmid (mock) for 48 h, followed by everolimus-SCH772984 combination treatment for another 24 h. The overexpression efficiency and the alterations of RRM1 and RRM2 were detected by immunoblotting analysis. Also, the proliferation of cells was measured by CCK-8 assay. ***p < 0.001. (F) Caki-1 and 786-O cells were subjected to ChIP assay using E2F1 antibody, followed by qRT-PCR analysis using primers targeting the promoter regions of RRM1 and RRM2. ***p < 0.001. Error bars represent mean ± SD of three independent experiments.

Figure 6

ERK Inhibitor Sensitized the RCC Tumors to Everolimus in Xenograft Models (A and B) Caki-1 (A) and 786-O (B) xenograft models were treated with everolimus (1 mg/kg/day) and SCH772984 (50 mg/kg, twice daily) alone or in combination daily for 21 days. The inhibition rate of the tumor growth (left panel) and body weights of the mice (right panel) were assessed by two-way ANOVA analysis. The error bars represent means ± SEM (n = 6 mice/group). *p < 0.05; **p < 0.01; ***p < 0.001. (C) Tumor tissues from Caki-1 xenografts were resected after the last dose and immunostained with Ki-67, E2F1, RRM1, and RRM2 antibodies to detect the intratumoral molecular alteration. Magnification, ×200; scale bar, 100 μm.