CDK2 positively regulates aerobic glycolysis by suppressing SIRT5 in gastric cancer

Abstract

Although significant progress has been made in the diagnosis and treatment of gastric cancer, the overall survival rate of the disease remains unchanged at approximately 20%-25%. Thus, there is an urgent need for a better understanding of the molecular biology aspects of the disease in the hope of discovering novel diagnosis and treatment strategies. Recent years have witnessed decisive roles of aberrant cancer cell metabolism in the maintenance of malignant hallmarks of cancers, and cancer cell metabolism has been regarded as a novel target for the treatment of cancer. CDK2, a cell cycle-dependent kinase that usually regulates cell cycle progression and the DNA damage response, is reported to be upregulated in many cancers. However, little is known about its role in cancer cell metabolism. In the present study, we showed that silencing CDK2 inhibited the aerobic glycolytic capacity of gastric cancer cell lines. Mechanism explorations showed that silencing CDK2 increased expression of the SIRT5 tumor suppressor. In addition, the physiological roles of SIRT5 in the regulation of proliferation and glycolysis were studied in gastric cancer cells. Taken together, the present study uncovered novel roles of the CDK2/SIRT5 axis in gastric cancer and suggests future studies concerning gastric cancer cell metabolism.

Keywords: CDK2; CDK2/SIRT5; SIRT5; gastric cancer; glycolysis.

Figure 1

CDK2 positively regulates aerobic glycolysis in gastric cancer. A,B, CDK2 expression effectively silenced in SGC‐7901 and MGC‐803 cells as validated by immunoblotting analysis with CDK2 antibody. C,D, Impact of CDK2 on aerobic glycolysis was confirmed by extracellular acidification rate (ECAR) measurement using Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioxcience), and silencing CDK2 expression inhibited ECAR values in SGC‐7901 and MGC‐803 cells. E,F, Oxygen consumption rate (OCR) values increased in CDK2 silenced gastric cancer cell lines, indicating that CDK2 played negative roles in mitochondrial respiration. Aerobic glycolysis is a multistep process, and glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1) are key glycolytic genes. G,H, In CDK2 silenced SGC‐7901 and MGC‐803 cells, the expression status of these genes decreased. *P < 0.05; **P < 0.01

Figure 2

CDK2 negatively regulated SIRT5 expression in gastric cancer cells. A,B, To explore the molecular mechanism underlying CDK2 in glycolysis regulation, we examined the impact of CDK2 on expression status of mitochondrial sirtuin family members, and our results indicated that CDK2 silencing increased SIRT5 expression, but had only slight impact on SIRT3 and SIRT4 in SGC‐7901 and MGC‐803 cells, suggesting that SIRT5 might be a CDK2 target. C,D, Western blot analysis confirmed the impact of CDK2 silencing on SIRT5 expression, and SIRT5 protein levels increased in CDK2 knockdown of SGC‐7901 and MGC‐803 cells. *P < 0.05; **P < 0.01

Figure 3

SIRT5 inhibited gastric cancer cell proliferation. To confirm the impact of SIRT5 on gastric cancer cell proliferation, we overexpressed SIRT5 in SGC‐7901 and MGC‐803 cells. A‐C, 5(6)‐Carboxyfluorescein diacetate N‐succinimidyl ester assay results demonstrated that overexpression of SIRT5 in gastric cancer cells inhibited cancer cell proliferation. E,G, SIRT5 introduction into SGC‐7901 and MGC‐803 cells inhibited colony formation capacity of these 2 cell lines. D,F, SIRT5 overexpression resulted in increased cell apoptosis in SGC‐7901 and MGC‐803 cells. *P < 0.05; **P < 0.01

Figure 4

SIRT5 inhibited aerobic glycolysis in gastric cancer cells. A,B, Overexpression of SIRT5 in SGC‐7901 and MGC‐803 cells inhibited extracellular acidification rate (ECAR) values, suggesting that SIRT5 is a negative regulator of glycolysis. C,D, SIRT5 overexpression increased oxygen consumption rate (OCR) values, indicating that SIRT5 has a positive role in mitochondrial respiration. E,F, SIRT5 introduction decreased the expressional status of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1), which further supports the negative role of SIRT5 on glycolysis. *P < 0.05; **P < 0.01

Figure 5

A‐C, SIRT5 inhibited tumor‐formation capacity of gastric cancer cells. To further confirm the negative roles of SIRT5 on gastric cancer cell proliferation in vivo, we carried out s.c. injection of SIRT5 overexpressing SGC‐7901 cells into nude mice. As shown, SIRT5 overexpression decreased tumor formation capacity of SGC‐7901 cells in vivo. P < 0.05

Figure 6

Working model of the present study. In gastric cancer cells, CDK2 positively regulated aerobic glycolysis by suppressing expression of SIRT5, a tumor suppressor and negative regulator of aerobic glycolysis