BackgroundMetabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized.MethodsClassical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XFe24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.ResultsInhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.ConclusionThese findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.
Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB–YY1–miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKβ, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKβ exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKβ wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial.
In contrast to normal cells that use mitochondrial oxidative phosphorylation for ATP production in aerobic conditions, many cancer cells depend on glycolysis even in the presence of sufficient oxygen. This process is known as aerobic glycolysis or the Warburg effect. Recent reports have shown that both the classical and alternative signaling pathways of NF-κB play important roles in controlling the metabolic profile of cancer and normal cells, respectively. However, how these signaling pathways affect the metabolism of pediatric sarcomas has not yet been explored. Here, we show NF-κB activity undergoes a shift in signaling from an alternative to a classical pathway in the genesis of sarcoma subtypes, rhabdomyosarcoma and osteosarcoma. Inhibition of classical NF-κB in sarcoma cells restores alternative signaling, as well as an oxidative metabolic phenotype in vitro and in vivo. In contrast, forced expression of the alternative pathway in rhabdomyosarcoma or in osteosarcoma cells is unable to reverse NF-κB classical activity, suggesting that the activation of the classical pathway in sarcomagenesis dominates over alternative signaling. Furthermore, we observed that inhibition of NF-κB in sarcoma cells reduced the expression of the glycolytic gene, hexokinase 2 (HK2). Through chromatin immunoprecipitation (ChIP) assays, we show that HK2 is a direct NF-κB target. Moreover, deletion of HK2 by using shRNA shifts the metabolic profile in sarcoma cells away from a warburg effect. These findings demonstrate a crucial role for classical NF-κB in sarcomagenesis and suggest that NF-κB metabolically reprograms sarcoma cells by regulating HK2. Citation Format: Yuichi Ijiri, Priya Londhe, Cheryl London, Peter J. Houghton, Denis C. Guttridge. NF-kB promotes a Warburg metabolic profile in pediatric sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 52.
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