Glucose metabolism and hexosamine pathway regulate oncogene-induced senescence

Abstract

Oncogenic stress-induced senescence (OIS) prevents the ability of oncogenic signals to induce tumorigenesis. It is now largely admitted that the mitogenic effect of oncogenes requires metabolic adaptations to respond to new energetic and bio constituent needs. Yet, whether glucose metabolism affects OIS response is largely unknown. This is largely because of the fact that most of the OIS cellular models are cultivated in glucose excess. In this study, we used human epithelial cells, cultivated without glucose excess, to study alteration and functional role of glucose metabolism during OIS. We report a slowdown of glucose uptake and metabolism during OIS. Increasing glucose metabolism by expressing hexokinase2 (HK2), which converts glucose to glucose-6-phosphate (G6P), favors escape from OIS. Inversely, expressing a glucose-6-phosphatase, [corrected] pharmacological inhibition of HK2, or adding nonmetabolizable glucose induced a premature senescence. Manipulations of various metabolites covering G6P downstream pathways (hexosamine, glycolysis, and pentose phosphate pathways) suggest an unexpected role of the hexosamine pathway in controlling OIS. Altogether, our results show that decreased glucose metabolism occurs during and participates to OIS.

Figure 1

Glucose metabolism decreases during oncogenic stress-induced senescence. Immortalized human epithelial cells expressing the inducible MEK/ER oncogene were treated or not with 4-OHT. (a) Cell extracts were prepared after 0, 3, or 4 days of 4-OHT treatment and analyzed by immunoblotting with the indicated antibodies. (b) Cells were seeded at the same density and treated or not for 3 days with 4-OHT. After 5 days, they were PFA fixed and crystal violet stained. (c) After 3 days with or without 4-OHT treatment, cells were fixed and stained for detection of SA-β-Gal activity. Percentages of stained cells were calculated and representative pictures are shown. (d) After 3 days with or without 4-OHT treatment, RNA was prepared and the expression of the indicated senescence markers was analyzed by RT-qPCR and normalized with respect to actin expression. (e and f) Cells were treated or not for 2 days with 4-OHT, counted, seeded back, and subjected or not to 4-OHT treatment. After 24 h, glucose uptake (e) and lactate production (f) were determined. (g) Cells were treated with or without 4-OHT for 3 days. ATP concentration was determined and normalized with respect to the protein content

Figure 2

Glucose-6-phosphatase blocks glucose metabolism and causes premature senescence. (a) Schematic representation of actors involved in the first glucose metabolism step. (b–i) Immortalized human epithelial cells were infected with a control or G6PC3-encoding vector and puromycin selected. (b) RNAs were extracted and analyzed by RT-qPCR to check for constitutive G6PC3 expression. (c–e) Equal numbers of cells were seeded and glucose uptake (c) or lactate production (d) was analyzed after 24 h. (e) ATP concentrations were determined and normalized with respect to protein content. (f) Cells were seeded at the same density. At 6 days after seeding, they were fixed with PFA and stained with crystal violet. (g) Cell lysates were prepared and protein expression analyzed by immunoblotting with the indicated antibodies. (h) Cells were PFA fixed and tested for SA-β-Gal activity. (i) RNAs were extracted and analyzed by RT-qPCR for expression of the indicated senescence markers

Figure 3

HK2 expression restores glucose metabolism and allows escape from OIS. (a–h) Immortalized human epithelial cells expressing the inducible RAF/ER oncogene were infected with a control or HK2-encoding retroviral vector and neomycin selected. (a) Immunofluorescence against the Flag tag was performed. Nuclei were counterstained with Hoechst. (b–d) Cells were treated or not for 2 days with 4-OHT, counted, and seeded back with or without 4-OHT. After 24 h, glucose uptake (b) and lactate production (c) were determined. (d) Cells were treated with or without 4-OHT for 3 days as indicated. ATP concentrations were determined and normalized with respect to the protein content. (e) Cells were seeded and treated or not for 3 days with 4-OHT. After 5 days, they were PFA fixed and crystal violet stained. (f) Cells were seeded and treated or not for 3 days with 4-OHT. After 2 days, cell extracts were prepared and analyzed by immunoblotting with the indicated antibodies. (g) Cells were PFA fixed and stained for SA-β-Gal detection. Percentages of stained cells were calculated and representative photographs are displayed. (h) RNAs corresponding to the indicated genes were prepared and analyzed by RT-qPCR. Expression levels were normalized with respect to actin expression

Figure 4

The hexosamine pathway favors OIS escape. (a) Schematic representation of key pathways and metabolites involved in glucose metabolism. (b) Human epithelial cells expressing MEK/ER were seeded at the same density and treated or not for 3 days with 4-OHT, and every 2 days by G6P at 5 mM. After 5 days, they were PFA fixed and crystal violet stained. (c) Human epithelial cells expressing MEK/ER were seeded at the same density and treated every day as indicated with or without NAcGluc at 40 mM, pyruvate at 5 mM, and NADPH at 100 μM, and with 4-OHT for the first 3 days. (d) After treatment as indicated in (b) and (c), cells were PFA fixed and stained for SA-β-Gal detection. Percentages of stained cells were calculated and representative photographs are displayed