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Obesity induces numerous physiological changes that can impact cancer risk and patient response to therapy. Obese patients with cervical cancer have been reported to have superior outcomes following chemoradiotherapy, suggesting that free fatty acids (FFA) might enhance response to radiotherapy. Here, using preclinical models, we show that monounsaturated and diunsaturated FFAs (uFFA) radiosensitize cervical cancer through a novel p53-dependent mechanism. UFFAs signaled through PPARγ and p53 to promote lipid uptake, storage, and metabolism after radiotherapy. Stable isotope labeling confirmed that cervical cancer cells increase both catabolic and anabolic oleate metabolism in response to radiotherapy, with associated increases in dependence on mitochondrial fatty acid oxidation for survival. In vivo, supplementation with exogenous oleate suppressed tumor growth in xenografts after radiotherapy, an effect that could be partially mimicked in tumors from high fat diet–induced obese mice. These results suggest that supplementation with uFFAs may improve tumor responses to radiotherapy, particularly in p53 wild-type tumors. Significance: Metabolism of monounsaturated and diunsaturated fatty acids improves the efficacy of radiotherapy in cancer through modulation of p53 activity. See related commentary by Jungles and Green, p. 4513

Purpose:Chemoradiotherapy for locally advanced cervical cancer fails in over a third of patients. Biomarkers with therapeutic implications are therefore needed. We investigated the relationship between an established prognostic marker, maximum standardized uptake value (SUVmax) on 18F-fluorodeoxyglucose positron emission tomography, and the inflammatory and immune state of cervical cancers.Experimental Design:An SUVmax most prognostic for freedom from progression (FFP) was identified and compared with known prognostic clinical variables in a cohort of 318 patients treated with definitive radiation with prospectively collected clinical data. Gene set enrichment analysis (GSEA) and CIBERSORT of whole-transcriptome data from 68 patients were used to identify biological pathways and immune cell subpopulations associated with high SUVmax. IHC using a tissue microarray (TMA, N = 82) was used to validate the CIBERSORT findings. The impact of macrophages on cervical cancer glucose metabolism was investigated in coculture experiments.Results:SUVmax P = 0.001). The GSEA showed that high SUVmax is associated with increased gene expression of inflammatory pathways, including JAK/STAT3 signaling. CIBERSORT and CD68 staining of the TMA showed high SUVmax tumors are characterized by a monocyte-predominant immune infiltrate. Coculture of cervical cancer cells with macrophages or macrophage-conditioned media altered glucose uptake, and IL6 and JAK/STAT3 signaling contribute to this effect.Conclusions:SUVmax is a prognostic marker in cervical cancer that is associated with activation of inflammatory pathways and tumor infiltration of myeloid-derived immune cells, particularly macrophages. Macrophages contribute to changes in cervical cancer glucose metabolism.See related commentary by Williamson et al., p. 4136

Individual mouse data for in vivo experiments.

Effects of TR1 and TR2 knock down on 2-DG treated CaSki cells. (A & C) CaSki and SiHa cells were transfected with control and TR1, TR2 and TR1+TR2 siRNA and after 24h, treated with 20mM 2-DG, 48h and CFA was performed. *p

Obesity induces numerous physiological changes that can impact cancer risk and patient response to therapy. Obese patients with cervical cancer have been reported to have superior outcomes following chemoradiotherapy, suggesting that free fatty acids (FFA) might enhance response to radiotherapy. Here, using preclinical models, we show that monounsaturated and diunsaturated FFAs (uFFA) radiosensitize cervical cancer through a novel p53-dependent mechanism. UFFAs signaled through PPARγ and p53 to promote lipid uptake, storage, and metabolism after radiotherapy. Stable isotope labeling confirmed that cervical cancer cells increase both catabolic and anabolic oleate metabolism in response to radiotherapy, with associated increases in dependence on mitochondrial fatty acid oxidation for survival. In vivo, supplementation with exogenous oleate suppressed tumor growth in xenografts after radiotherapy, an effect that could be partially mimicked in tumors from high fat diet–induced obese mice. These results suggest that supplementation with uFFAs may improve tumor responses to radiotherapy, particularly in p53 wild-type tumors.Significance:Metabolism of monounsaturated and diunsaturated fatty acids improves the efficacy of radiotherapy in cancer through modulation of p53 activity.See related commentary by Jungles and Green, p. 4513

Highly glycolytic cervical cancers largely resist treatment by cisplatin and coadministered pelvic irradiation as the present standard of care. In this study, we investigated the effects of inhibiting glycolysis and thiol redox metabolism to evaluate them as alternate treatment strategies in these cancers. In a panel of multiple cervical cancer cell lines, we evaluated sensitivity to inhibition of glycolysis (2-deoxyglucose, 2-DG) with or without simultaneous inhibition of glutathione and thioredoxin metabolism (BSO/AUR). Intracellular levels of total and oxidized glutathione, thioredoxin reductase activity, and indirect measures of intracellular reactive oxygen species were compared before and after treatment. Highly radioresistant cells were the most sensitive to 2-DG, whereas intermediate radioresistant cells were sensitive to 2-DG plus BSO/AUR. In response to 2-DG/BSO/AUR treatment, we observed increased levels of intracellular oxidized glutathione, redox-sensitive dye oxidation, and decreased glucose utilization via multiple metabolic pathways including the tricarboxylic acid cycle. 2-DG/BSO/AUR treatment delayed the growth of tumors composed of intermediate radioresistant cells and effectively radiosensitized these tumors at clinically relevant radiation doses both in vitro and in vivo. Overall, our results support inhibition of glycolysis and intracellular redox metabolism as an effective alternative drug strategy for the treatment of highly glycolytic and radioresistant cervical cancers.Significance: This study suggests a simple metabolic approach to strike at an apparent Achilles' heel in highly glycolytic, radioresistant forms of cervical cancers, possibly with broader applications in cancer therapy. Cancer Res; 78(6); 1392–403. ©2018 AACR.

Prognostic significance of SUVmax for local (cervix) recurrence (A) and any pelvic recurrence (B).

Oleic acid and radiation modulates PPARγ regulated gene expression

Role of anti-oxidant NAC on 2-DG, BSO and AUR induced cell viability. (A-B) Cells were seeded and treated with 2-DG and BSO for 24h followed by 5hrs of 10mM NAC with replacement of 2-DG, BSO and AUR for another 24h. Cell viability was determined by Alamar blue. Statistical Analysis: (A) BA vs NAC+BA, p

Differential effect of polyunsaturated fatty acid, Arachidonic acid (AA) in cervical cancer cells.

Number of patients in each of the patient cohorts used, and overlap between the cohorts.

Unsaturated fatty acids radio-sensitize cervical cancer cells through a mechanism that includes upregulation in pp53 and p53 and induction of apoptosis.

Supplemental Material and Methods

Tissue microarray staining for CD163, a marker for M2 polarized macrophages.

Representative image of an auto-contoured metabolic tumor volume on simulation CT scan fused with a diagnostic PET scan.

Tumor growth kinetics of TC-1 orthograft tumors

Effect of catalase over expression in the clonogenic survival of 2-DG, BSO and AUR treated cells. (A-B) Cells were seeded and transduced with Ad CAT and Ad empty viruses and CFA was done as described in the materials and methods section. (C) CaSki cells were treated with 20mM 2-DG, 1mM BSO and 100nM AUR for 24h along with or without CQ (60μM) and cell viability was determined by Alamar Blue. Statistical Analysis: (A) *p < 0.05 Ad-empty vs Ad-CAT BSO (B) p < 0.07 Ad-empty vs Ad-CAT for DG, BSO and BA. (C) ***DBA vs DBA+CQ, p

Bootstrap multivariate analysis results.

Oleic acid and linoleic acid supplementation does not affect radiation sensitivity in TP53