CDK6 increases glycolysis and suppresses autophagy by mTORC1-HK2 pathway activation in cervical cancer cells

Abstract

Cervical carcinoma is a leading malignant tumor among women worldwide, characterized by the dysregulation of cell cycle. Cyclin-dependent kinase 6 (CDK6) plays important roles in the cell cycle progression, cell differentiation, and tumorigenesis. However, the role of CDK6 in cervical cancer remains controversial. Here, we found that loss of CDK6 in cervical adenocarcinoma HeLa cell line inhibited cell proliferation but induced apoptosis as well as autophagy, accompanied by attenuated expression of mammalian target of rapamycin complex 1 (mTORC1) and hexokinase 2 (HK2), reduced glycolysis, and production of protein, nucleotide, and lipid. Similarly, we showed that CDK6 knockout inhibited the survival of CDK6-high CaSki but not CDK6-low SiHa cervical cancer cells by regulation of glycolysis and autophagy process. Collectively, our studies indicate that CDK6 is a critical regulator of human cervical cancer cells, especially with high CDK6 level, through its ability to regulate cellular apoptosis and metabolism. Thus, inhibition of CDK6 kinase activity could be a powerful therapeutic avenue used to treat cervical cancers.

Keywords: CDK6; Cervical carcinoma; apoptosis; autophagy; glycolysis; mTOR.

Figure 1.

CRISPR/Cas9-mediated knockout of CDK6 leads to suppression of HeLa cell growth and increase of apoptosis. (a) Western blotting analysis of CDK6 protein in CDK6 knockout (KO) and wide type (WT) HeLa cell line. β-Tubulin is used as internal loading control. (b) Cell viability was measured by the CCK8 assay. (c) Colony formation assay for WT and KO cells. Quantification was shown in the diagram. The numbers represented the average colony number of four areas per plate. (d) cell cycle profiles of WT and KO cells. Column diagram showed the percentage of cells in each phase of the cell cycle (G0/G1, S, G2/M). (n = 10 for each group). (e) Western blotting analysis of a group of cell-cycle related proteins in WT and KO cells. (f) Apoptosis of WT and KO cells. Percentage of apoptotic cells with early, late and total apoptosis was shown in diagram. (g) Western blotting analysis of apoptosis associated proteins (caspase 3, cleaved caspase 3, Bax, Bcl-2). Data are presented as the mean ± SEM (n = 3 for each group), *p < 0.05, **p < 0.01, t-test, vs WT. ns indicates no significance.

Figure 2.

CDK6 knockout restricts glycolysis in HeLa cells. (a) Western blotting analysis of mTOR and its downstream target p70 S6K, Phospho-p70 S6K (Thr389) and HIF-1α protein in WT and KO cells. (b) Glucose uptake and lactate production in WT and KO cells. (c) ECAR measured with Seahorse XFe96 bioanalyzer. The calculated glycolysis and glycolytic capacity were presented as bar graph. (d) Western blotting analysis of glycolytic enzymes (HK2, PFKP, PKM2) in WT and KO cells. Data shown are mean ± SEM of three replicates. *p < 0.05, **p < 0.01, t-test, vs WT.

Figure 3.

CDK6 knockout induces autophagy in HeLa cells. (a) The ultrastructure of cells observed by TEM. (a and b): WT cells, (c and d): KO cells. Arrows indicate representative autophagic vacuoles. (b) Western blotting analysis of autophagy protein (mTOR, ULK1, Phospho-ULK1 (Ser757), p62, LC3) of WT and KO cells. (c) Cells were transfected with mRFP-GFP-LC3 lentivirus and autophagic flux was monitored by a confocal microscope. The autophagosome in Cells appears as yellow puncta, while autolysosome as red puncta. (a-c): WT cells, (d-f): KO cells. (d) The ultrastructure of mRFP-GFP-LC3 lentivirus transfected cells observed by TEM. (a and b): WT cells, (c and d): KO cells. Arrows indicate representative autophagic vacuoles. (e) Western blotting analysis of glycolytic enzymes (HK2, PFKP, PKM2) and autophagy protein (p62, LC3) in WT cells treated by Baf A1 100 nM for 24 h. (f) Western blotting analysis of glycolysis and autophagy related proteins in WT and KO cells treated by Baf A1 100 nM for 24 h. (g) Cell viability of WT and KO supplemented with Baf A1 or vehicle for 4 h. Data are presented as the mean ± SEM (n = 4 for each group), *p < 0.05, **p < 0.01, t-test, vs WT. ##p < 0.01, t-test, Baf A1-treated KO vs vehicle-treated KO. ns indicates no significance between the corresponding groups.

Figure 4.

Raman spectra and map of HeLa WT and KO cells. (a) Raman mean spectra of cells measured. (b) Raman mean difference spectrum (KO−WT) of HeLa cells. (c-l) Different images of WT (c-g) and KO (h-l) cells under 60× magnification. (c, h) Normal light microscope. The pseudocolor Raman intensities of cells at wavenumber of 676 cm⁻¹ (d, i), 1001 cm⁻¹ (e, j), 2854 cm⁻¹ (f, k). (g, l) Combined chemical images of cells at 676, 1001, 2854 cm⁻¹. Thirty cells were measured in each group.

Figure 5.

CDK6 knockout inhibits growth of HeLa xenograft in vivo. (a) The presentative mice tumors from HeLa cells xenografts. The average of tumor weights was shown in diagram. Tumor volumes were calculated on the basis of tumor diameters examined. (b) Body weights of mice. (c) Representative TUNEL staining results from tumor xenografts in nude mice. (d) Western blotting analysis of glycolysis, autophagy and cell cycle related proteins in tumor xenografts. Data are presented as the mean ± SEM (n = 6 for each group), *p < 0.05, **p < 0.01, t-test, vs WT.

Figure 6.

The effect of CDK6 on HPV integrated CaSki or SiHa cells. (a) CDK6 protein level in WT and KO CaSki cells. (b) Western blotting analysis of CDK6 protein in HeLa, CaSki, WT and KO SiHa cells. (c) Colony formation assay for WT and KO CaSki cells. (d) Colony formation assay for WT and KO SiHa cells. In c and d, quantification was shown in the diagram. The numbers represented the average colony number of each well. Data are presented as the mean ± SEM (n = 3 for each group), *p < 0.05, **p < 0.01, t-test, vs CaSki. ns indicates no significance between WT and KO SiHa group. (e) Western blotting analysis of glycolysis and autophagy related proteins in WT and KO CaSki cells. (f) A model depicting how CDK6 couple tumor cell growth with metabolism. CDK6 increases the expression of mTOR, thereby leading to the activation of mTORC1 and consequent upregulation of S6K and HIF-1α, which increases the cellular biosynthesis and glycolysis to promote cell growth and proliferation, and inhibits autophagy and apoptosis.