Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

Abstract

Background: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression.

Results: Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity.

Conclusion: The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

Figure 1

Viability of Ras-LP and Ras-HP cells in standard culture conditions. Adherent and non-adherent cells were harvested after 72 hours in culture, stained using tryphan-blue, and counted. A: Data presented as percentages of total amount of cells. ***: p < 0.001 (n = 5). Ras-LP cultures contain relatively more detached cells in the medium, of which the majority is dead. B: Data presented in (A) in absolute counts (n = 5). ***: p < 0.001 (n = 5). Ras-HP cell cultures contain relatively and absolutely more viable cells.

Figure 2

Anchorage-independent growth in soft agar at different oxygen conditions. A: Growth of Ras-LP cells in soft agarat 2% and 21% environmental oxygen. Lowering external oxygen levelsresults in a 3-fold increase in the number of colonies per cm². *:p < 0.05 (Student's t-test). B: Growth of Ras-HP cells in soft agar at2% and 21% environmental oxygen. Lowering external oxygen levels inRas-HP soft agar cultures results in only a marginal 1.3-fold andnon-significant increase in colony number, showing that a glycolyticshift in these relatively glycolytic cells does not result in a largeincrease in viability and colony formation of cells in the soft agar.

Figure 3

Lactic acid production, oxygen consumption and proton production rate. A: Lactic acid production in μ moles/mg protein (mean ± SEM). Lactic acid production was measured in media samples taken from the cell cultures used to obtain the cell doubling time data in Table 1. Six independent experiments, each in duplicate, were conducted for all cell populations, except Ras-HP (n = 8) and Ras-TUM (n = 7). Lactic acid production increases with passage number in H-RasV12/E1A-transformed cells. ***:p < 0.001 compared to Prim-MEF. #:p < 0.05; ##:p < 0.01; ###:p < 0.001 for Ras-LP – Ras-HP – Ras-TUM intercomparisons (one-way ANOVA/Bonferroni). B: Oxygen consumption in NRFU/min/mg protein measured in BD oxygen biosensor plates (mean ± SEM). Each experimental value represents n = 3 (Prim-MEF, Imm-MEF, TBX2-MEF) or n = 6 (other cell populations) independent assays, each carried out in triplicate. Cells were seeded at 200,000 cells per well of a 384-well plate. Oxygen consumption peaks immediately after H-RasV12/E1A transformation, and then decreases with passage number. ***:p < 0.001 compared to Prim-MEF. ###:p < 0.001 for Ras-LP – Ras-HP – Ras-TUM intercomparisons (one-way ANOVA/Bonferroni). C: Proton production rate (PPR) and oxygen consumption rate (OCR) per cell measured in Seahorse XF24 analysis 48 hours post seeding (mean ± SEM; n = 6, except Ras-HP, n = 4). Prim-MEF, Imm-MEF and TBX2-MEF cells were seeded at 25,000/well. Ras-LP, Ras-HP, Ras-TUM cells were seeded at 30,000/well. PPR: **:p < 0.01 compared to Prim-MEF. OCR: ##:p < 0.01 compared to Prim-MEF (one-way ANOVA/Bonferroni). These data confirm the gradual increase in cellular acidification and decrease in oxygen consumption described in (A) and (B) in real time analysis.

Figure 4

Analysis of NADH autofluorescence. A: Mitochondrial and nuclear NADH autofluorescence levels (mean ± SEM of a minimum of 3 independent experiments) expressed in absolute grey levels. Note that the signals were obtained from cells with clearly distinct morphology, which makes intercomparison of Prim-MEF, Imm-MEF and TBX2-MEF on the one hand, and Ras-transformed populations on the other, difficult. Absolute NADH autofluorescence increases gradually with passage number in H-RasV12/E1A-transformed cells. B: Representative recordings of NADH autofluorescence in Ras-LP and Ras-TUM cells before and after rotenone application. Rotenone inhibition of Complex I results in a differential increase in NADH autofluorescence. The percentage NADH autofluorescence is indicative of different rates of mitochondrial respiration. These data confirm the relatively higher respiration rates in Ras-LP cells. C: Cumulative data from n = 3 NADH autofluorescence/rotenone analyses carried out on separate days, with a minimum of 3 coverslips per day. Note that this parameter is cell morphology-independent. **:p < 0.01 compared to Prim-MEF ##:p < 0.01 for Ras-LP – Ras-TUM intercomparison (one-way ANOVA/Bonferroni). D: Superoxide levels expressed as fluorescence signal of HEt oxidation products per cell. Ras-LP levels were arbitrarily set at 100%. Oxygen consumption data per cell are available in Additional file 1, Fig. S4B and Fig. S4D. HEt fluorescence in Ras-LP, HP and TUM cells was determined in three independent experiments, each in duplicate, with at least 20,000 cells per assay. Prim-MEF, Imm-MEF and TBX2-MEF HEt fluorescence was determined in one experiment, in duplicate. Superoxide levels correlate with respiration rates measured in (B) and (C). ##:p < 0.01 compared to Ras-LP in a Student's t-test.