Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors.

ReportReport Cell Cycle 13:7, 1152–1161; April 1, 2014; © 2014 Landes Bioscience Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors Sameer H Issaq1, Beverly A Teicher2, and Anne Monks1,* Molecular Pharmacology Branch; Leidos Biomedical Research, Inc.; Frederick National Laboratory for Cancer Research; Frederick, MD USA; 2Division of Cancer Treatment and Diagnosis; National Cancer Institute; Rockville, MD USA 1 Keywords: sarcoma, rhabdomyosarcoma, osteosarcoma, bioenergetics, 2-DG, metformin Abbreviations: aRMS, alveolar rhabdomyosarcoma; eRMS, embryonal rhabdomyosarcoma; 2-DG, 2-deoxy-D-glucose; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; SKMC, normal human skeletal muscle cells; NHDF, normal human dermal fibroblasts; ATP, adenosine triphosphate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. Introduction Sarcomas represent a diverse group of mesenchymal malignancies that arise from connective and soft tissues, including bone, muscle, and cartilage. Sarcomas affect approximately 200 000 individuals worldwide each year and represent a higher percentage of overall cancer morbidity and mortality in children and adolescents than in adults.1,2 Research identifying alterations associated with specific histological subtypes of sarcoma has indicated that previous classifications based on the site of tumor (bone or soft tissue) are less important than the tumor molecular and pathological features.1 Thus, a better understanding of the genetic and molecular alterations present in specific sarcoma subtypes is critically important to improve sarcoma treatment and develop new therapeutic approaches. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence, with approximately 350 new cases diagnosed each year in the United States.3 Like other sarcomas, RMS tumors are molecularly diverse.4 There are 2 major histologic subtypes of RMS, embryonal (eRMS) and alveolar (aRMS), each having distinct underlying genetic alterations that participate in pathogenesis.3 Patients with eRMS generally have a more favorable prognosis than patients with aRMS, which has a 5-year survival rate of <50%.4 aRMS is further characterized by chromosomal translocations, resulting in fusion genes such as PAX3-FOXO1. PAX3-FOXO1-positive aRMS patients, with metastatic disease survive in <10% of cases.4 Current treatment for both types of RMS includes surgery, chemotherapy (vincristine, actinomycin-D, and cyclophosphamide), and radiation, with an overall survival rate of about 70%. Despite this aggressive multimodal treatment, patients with metastatic disease have an overall survival rate of <20%.3-5 Osteosarcoma is the most common primary tumor of bone and mainly affects adolescents and young adults.6,7 Osteosarcoma is the eighth most common malignancy of childhood, with approximately 400 new cases diagnosed in children and young adults in *Correspondence to: Anne Monks; Email: Submitted: 12/13/2013; Revised: 01/26/2014; Accepted: 01/27/2014; Published Online: 02/10/2014 http://dx.doi.org/10.4161/cc.28010 1152 Cell Cycle Volume 13 Issue 7 the United States each year.7 Like other sarcomas, osteosarcoma tumors are molecularly diverse.6 Despite treatment protocols that combine chemotherapy, surgery, and sometimes radiotherapy, the 5-year survival rate for patients diagnosed with osteosarcoma is 60–70%.6,8 The long-term survival rate for patients with metastatic disease detectable at presentation is 30–35%.7 Current treatments for osteosarcoma are associated with significant morbidity, and there have been no significant improvements in prognosis in the last 2 decades, hence there is a significant need for improved therapies for osteosarcoma.6,7 Many signaling pathways that are affected by genetic events in cancer, as well as the tumor microenvironment, significantly alter cellular bioenergetics to support growth and survival.9 Cancer cells exhibit a metabolic phenotype known as aerobic glycolysis, or the Warburg effect, which is characterized by increased glycolysis regardless of oxygen availability.9,10 Alterations in cellular metabolism are recognized as a crucial hallmark of cancer.11 Exploiting the fundamental differences between cancer and normal cell metabolism may provide an opportunity for therapeutic intervention through selective targeting of the metabolic dependencies of cancer cells. Despite a lack of drugs that target specific metabolic enzymes, several readily available compounds, including 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, and metformin, a widely used diabetes drug that can inhibit complex I of the mitochondrial respiratory chain, have been described as having effects on cancer cell growth and cellular bioenergetics.9 Additionally, several recent studies have shown that simultaneous inhibition of glycolysis and mitochondrial respiration can act synergistically to reduce tumor cell survival in vitro and in vivo.12-14 With respect to sarcomas, 2-DG induces apoptosis in huma (...truncated)