Inhibition of AXL enhances chemosensitivity of human ovarian cancer cells to cisplatin via decreasing glycolysis

Abstract

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases (RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

Keywords: AXL; PKM2; R428; Warburg effect; cisplatin resistance; glycolysis; ovarian neoplasms; tumor metabolism.

Fig. 1. AXL is a potential therapeutic target for the treatment of ovarian cancer.

a GEO2R analysis comparing differences in AXL mRNA expression levels between normal ovarian epithelial tissue and OvCa tissue in GSE14407. b The levels of AXL mRNA between OvCa tissues and normal ovarian tissues were determined by qRT-PCR. Actin served as a loading control. c The effect of AXL mRNA levels on the overall survival of OvCa patients was analyzed in the GSE9891 dataset. d The expression of AXL protein in the normal ovarian epithelial cell line and OvCa cell lines was measured by Western blot analysis. Actin served as a loading control. e The cell proliferation of HO-8910 and SKOV3.ip cells stably transfected with shAXL or treated with 1 μM R428 was measured by the CCK-8 assay. f Cell proliferation was measured by a colony formation assay in SKOV3.ip cells transfected with AXL shRNA. g SKOV3.ip-shNT and SKOV3.ip-shAXL cells were plated in 0.7% agar. After 14 days, colonies were photographed and quantified. **P < 0.01.

Fig. 2. AXL enhances aerobic glycolysis in ovarian cancer cells.

a Enrichment plots from gene set enrichment analysis (GSEA) of AXL in glycolysis. b HO-8910 and SKOV3.ip cells were transfected with the nontargeting RNA or AXL siRNA and then tested for glucose uptake, lactate production, and ATP production. c A2780 cells transfected with the empty vector or His-AXL plasmid were tested for glucose uptake, lactate production, and ATP production. d HO-8910 and SKOV3.ip cells were transfected with nontargeting RNA or AXL siRNA, and the expression of AXL, PKM2, and p-PKM2 (Y105) was measured by WB. β-Actin served as a loading control. e HEK293T cells were transfected with His and GST-PKM2 plasmids, GST and His-AXL plasmids, or His-AXL and GST-PKM2 plasmids, followed by immunoprecipitation with anti-His and anti-GST antibodies. f Electrostatic surface view of AXL compounded with the PKM2 peptide. The close-up view shows that the PKM2 (100–110) peptide is suitable for the catalytic domain of AXL. The Y105 residue of PKM2 is highlighted in green, and the γ-phosphate of ATP is highlighted in red. g A2780 cells were transfected with either the nontargeting RNA or PKM2 siRNA, followed by transfection with a His-AXL plasmid, and then the expression of p-PKM2 and glycolysis level were measured. h HO-8910 or SKOV3.ip cells were transfected with nontargeting RNA or AXL siRNA, followed by transfection with the PKM2 plasmid. Then, the expression of p-PKM2 and glycolysis level were measured. **P < 0.01.

Fig. 3. AXL is involved in cisplatin-regulated aerobic glycolysis in ovarian cancer cells.

a HO-8910, SKOV3.ip, and A2780 cells were treated with cisplatin, and glucose uptake, lactate production, and ATP production were measured. b HO-8910 and SKOV3.ip cells were treated with cisplatin in the presence or absence of 0.5 mM 2-DG. Cell viability was measured by the CCK-8 assay after 48 h. c SKOV3.ip cells were treated with 1 μM cisplatin with or without 0.5 mM 2-DG, and cell proliferation was measured by the colony formation assay. d HO-8910, SKOV3.ip, and A2780 cells were treated with cisplatin; AXL, PKM2, and p-PKM2 protein expression was measured by WB; β-actin served as a loading control. e HO-8910 and SKOV3.ip cells were transfected with nontargeting RNA or AXL siRNA, followed by 1 μM cisplatin treatment for 48 h, and then the glycolysis levels were measured. **P < 0.01.

Fig. 4. AXL affects the chemotherapy sensitivity of ovarian cancer to cisplatin.

a Kaplan–Meier curves for the PFS of 93 OvCa patients who received platinum as the chemotherapy drug (GSE26193). The patients were divided into two groups according to the level of AXL mRNA. b The mRNA expression of AXL in parent cells and cisplatin-resistant cells was compared in the GSE58470 dataset. c HO-8910, SKOV3.ip, and A2780 cells were transfected with AXL siRNA and then treated with cisplatin for 48 h, and cell viability was measured via the CCK-8 assay. d SKOV3.ip cells were transfected with AXL shRNA and treated with 1 μM cisplatin, and cell proliferation was measured by the colony formation assay. e HO-8910 and SKOV3.ip cells were transfected with AXL siRNA and then treated with 1 μM cisplatin for 48 h, and cell proliferation was assessed via the EdU assay. f HO-8910 and SKOV3.ip cells were pretreated with 1 μM R428 for 4 h and then treated with various concentrations of cisplatin. Cell viability was measured by the CCK-8 assay. g SKOV3.ip cells were pretreated with 1 μM R428 and then treated with 1 μM cisplatin, and cell proliferation was detected by the colony formation assay. **P < 0.01.

Fig. 5. Inhibition of AXL increases the cisplatin sensitivity of ovarian cancer in vivo.

Four-week-old female nude mice were inoculated subcutaneously with SKOV3.ip cells. The tumor-bearing mice were grouped and received the indicated treatment. a Tumors were harvested and photographed at the termination of the experiment. b Tumor weights were measured after excision. c The average tumor volumes were measured every two days from the first day of administration. d Each mouse was weighed every 2 days. **P < 0.01.

Fig. 6. Inhibition of AXL enhances the efficacy of cisplatin in chemoresistant ovarian cancer cells.

a The ROC Plotter was used to analyze the transcriptome data of 961 patients, and the differences in AXL expression levels between patients with and without significant pathological response after platinum treatment were compared. b The mRNA expression levels of AXL in the parental cisplatin-sensitive IGROV-1 cell line and acquired cisplatin-resistant IGROV-1/Pt1 cell line were compared in GSE58470. c Western blotting detection of AXL, PKM2, and p-PKM2 expression in A2780 and A2780/DDP cells; β-actin was used as a loading control. d The glycolysis levels of A2780 and A2780/DDP cells were measured. e A2780/DDP cells were transfected with either the nontargeting RNA or AXL siRNA, and the expression of AXL, PKM2, and p-PKM2 was detected by Western blotting; β-actin was used as a loading control. f A2780/DDP cells were transfected with AXL siRNA, and the glycolysis level was measured. g A2780/DDP cells were pretreated with 1 μM R428, and the glycolysis level was measured. h A2780/DDP cells were transfected with either the nontargeting RNA or PKM2 siRNA, followed by treatment with cisplatin, and then cell viability was measured by the CCK-8 assay. i A2780/DDP cells were transfected with siNT or siAXL, followed by treatment with cisplatin, and cell viability was measured by the CCK-8 assay. j A2780/DDP cells were pretreated with 1 μM R428 for 4 h and then treated with cisplatin for 48 h. Cell viability was measured by the CCK-8 assay. **P < 0.01.