Abstract Background Radiation-induced lung injury (RILI) occurs in cancer patients treated with thoracic radiation. Although 5-10% of cancer survivors develop clinically significant symptoms like dry cough or dyspnea, up to 90% of patients develop signs of RILI on CT. These fibrotic lesions can obscure tumor recurrence or impact treatment for recurrence. Our lab was the first to link RILI to metabolic dysfunction using metabolomic analysis of exhaled breath condensate, including elevated lactate production, which drives fibroblast-to-myofibroblast transdifferentiation (FMT), a key process in fibrosis. Here, we investigate whether ionizing radiation drives additional metabolic reprogramming in lung fibroblasts and if these processes can be therapeutically targeted. Methods Primary human lung fibroblasts (HLFs) were irradiated with 2-5 Gy ionizing radiation from an X-ray source to induce FMT and were harvested after 5 days. Protein expression was determined by western blot, and lactate was measured in conditioned medium by a colorimetric assay. Glycolysis rates were measured using the Seahorse Xfe system. 13C-labelled glucose or ribose was added to medium as the only sugar for 4 hours prior to harvest, and targeted metabolomics analysis was performed on cell lysates by liquid chromatography/tandem mass spectrometry. To investigate potential therapeutic targets, irradiated HLFs were treated with shikonin to inhibit pyruvate kinase M2 (PKM2), or with N3-pyridyl thiamine (N3PT) to inhibit transketolase (TKTL), and FMT was assessed by immunocytochemistry. Results Radiation markedly induces alpha-smooth muscle actin and extracellular lactate (Fig. 1A- B). Radiation significantly accelerates both basal and compensatory glycolysis (Fig. 1C). Radiation decreases glucose-derived glycolysis products but increases ribose-derived glycolysis products, as shown by analysis of labeled phosphoenolpyruvate (Fig. 1D- E). Ribose is a significant source of lactate in irradiated fibroblasts (Fig. 1F). Inhibition of PKM2, the final step in glycolysis, attenuates FMT and lactate accumulation (Fig. 1G-H and data not shown). Surprisingly, inhibition of TKTL, which catalyzes the entry of ribose products into glycolysis, also inhibits FMT and lactate secretion (Fig. 1G-H and data not shown). Conclusions We observed that radiation-induced FMT of primary HLFs is marked by glycolytic reprogramming with increased rates of glycolysis and lactate production that is fueled, in part, by the pentose phosphate pathway. This supports results from RILI patients, suggesting that reverse flux through the pentose phosphate pathway compensates for the increased energy demand of FMT and the diversion of glycolysis products to lactate. Pharmacologically targeting these pathways may be a useful therapeutic approach to treat fibrosis by depriving fibroblasts of fuel. This abstract is funded by: This work was supported in part by National Institute of Health (NIH) Grant R01HL127001. J.P.-L.O. was funded in part by a Ford Foundation Predoctoral Fellowship. Services in support of the research project were provided by the VCU Massey Cancer Center Lipidomics and Metabolomics Shared Resource, which is supported, in part, with funding from NIH-National Cancer Institute Cancer Center Support Grant P30 CA016059. P.D.J. was supported by the Pulmonary Fibrosis Foundation Scholars program. M.A.T.F was supported in part by the Pulmonary Fibrosis Foundation Scholars Program and by NIH grant K99HL169903. This content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Pierre-Louis et al. (Fri,) studied this question.
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