Energy Metabolism during Anchorage-Independence. Induction by Osteopontin-c

Weber GF (2014) Energy Metabolism during Anchorage-Independence. Induction by Osteopontin-c. PLoS ONE 9(8): e105675. doi:10.1371/journal.pone.0105675 Energy Metabolism during Anchorage-Independence. Induction by Osteopontin-c Zhanquan Shi 0 Bo Wang 0 Tafadzwa Chihanga 0 Michael A. Kennedy 0 Georg F. Weber 0 Wing-Kin Syn, Institute of Hepatology, Foundation for Liver Research, United Kingdom 0 1 University of Cincinnati Academic Health Center , Cincinnati , Ohio, United States of America, 2 Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio , United States of America The detachment of epithelial cells, but not cancer cells, causes anoikis due to reduced energy production. Invasive tumor cells generate three splice variants of the metastasis gene osteopontin, the shortest of which (osteopontin-c) supports anchorage-independence. Osteopontin-c signaling upregulates three interdependent pathways of the energy metabolism. Glutathione, glutamine and glutamate support the hexose monophosphate shunt and glycolysis and can feed into the tricarboxylic acid cycle, leading to mitochondrial ATP production. Activation of the glycerol phosphate shuttle also supports the mitochondrial respiratory chain. Drawing substrates from glutamine and glycolysis, the elevated creatine may be synthesized from serine via glycine and supports the energy metabolism by increasing the formation of ATP. Metabolic probing with N-acetyl-L-cysteine, L-glutamate, or glycerol identified differential regulation of the pathway components, with mitochondrial activity being redox dependent and the creatine pathway depending on glutamine. The multiple skewed components in the cellular metabolism synergize in a flow toward two mechanisms of ATP generation, via creatine and the respiratory chain. It is consistent with a stimulation of the energy metabolism that supports anti-anoikis. Our findings imply a coalescence in cancer cells between osteopontin-a, which increases the cellular glucose levels, and osteopontin-c, which utilizes this glucose to generate energy. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The RNASeq data are available in GEO (accession number GSE55193). Funding: This research was supported by Department of Defense grants PR094070 and BC095225 to GFW. MAK acknowledges support by a grant from the National Institutes of Health/National Cancer Institute (1R15CA152985). The instrumentation used in this work was obtained with the support of Miami University and the Ohio Board of Regents with funds used to establish the Ohio Eminent Scholar Laboratory where the work was performed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Anchorage independence is an essential characteristic of metastasizing cells. While untransformed non-hematopoietic cells undergo programmed cell death (anoikis) consecutive to losing contact with their substratum, cancer cells can survive in the circulation for extended periods of time. In fact, the major limiting factor in the process of metastasis formation is the death of the tumor cells before their implantation in the target organs [13]. Therefore, anchorage independent survival may be more critical to the process of cancer metastasis than organ-specific homing. The molecular mechanisms underlying anchorage independence are poorly understood. The cytokine osteopontin may act as a metastasis gene, particularly through its splice variant osteopontinc, which has a deletion of exon 4. Osteopontin-c is uniquely expressed in breast cancers, but not in normal breasts [4]. It very effectively supports anchorage independent survival and expansion [5]. Osteopontin-c, but not osteopontin-a, signals through the activation of oxidoreductase gene expression [5] associated with the mitochondrial respiratory chain (NDUFV1, NDUFS4, NDUFS7, NDUFS8, NDUFA9, NDUFB9, Cytochrome c Oxidase), the hexose monophosphate shunt (PGDH) or the regulation of the hexose monophosphate shunt (GPX-4) [57]. The oxidoreductase induction may lead to the generation of reactive oxygen intermediates in the tumor cells. In fact, it has been reported that escape from anoikis can be mediated through the production of reactive oxygen species, which cause the oxidation and activation of the tyrosine kinase SRC [8], resulting in the transduction of a survival signal. The detachment of mammary epithelial cells leads to ATP deficiency, owing in part to the loss of glucose transport. Hence, reduced energy production is a feature of anoikis that needs to be overcome in cancer progression [9]. Consistently, increased cancer invasiveness under detached conditions is associated with higher mitochondrial activity, elevated ATP production, pyruvate uptake, and oxygen consumption [10]. We have found that osteopontin-a increases the glucose levels in deadherent breast tumor cells [11], which may provide the biochemical fuel for ATP generation. In conjunction with the observation (referenced above) that osteopontin-c induces oxidoreductases associated with the energy metabolism [5], we investigate the hypothesis that osteopontin-c supports the anchorage independence of cancer cells by upregulating their energy metabolism via redox signaling. Materials and Methods Reagents, cell lines, DNA constructs and transfection Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), N-acetyl-Lcysteine (NAC), glutathione (GSH), H2O2, and mannitol came from Sigma-Aldrich. MCF-7 cells and their transfectants were grown in alpha-MEM with insulin and 10% fetal bovine serum. MCF-7 cells stably transfected with osteopontin-a, osteopontin-c, or vector control have been previously described [5]. ZR-75 cells were grown in RPMI-1640 medium with 10% fetal bovine serum. They were stably transfected with osteopontin-c or vector control in pCR3.1 (selected in G418). The construct for expression of catalase targeted to the mitochondria was obtained from DR. J.A. Melendez [12]. MCF-7 cells were transfected with the FuGENE reagent (Roche) and stable clones were selected in zeocin. Immunoblot assay For the analysis of secreted osteopontin, serum-free cell culture supernatant was collected from each transfectant. 40 ml of supernatant per sample were electrophoresed on 10% SDSpolyacrylamide mini-gels with non-reducing sample buffer. For the analysis of intracellular osteopontin, the cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate). Cell lysates at equal amounts of protein (20 mg/lane) were electrophoresed on reducing 10% SDS-polyacrylamide gels. The separated proteins were transferred to PVDF membranes and probed with antibody O-17 (Assay Designs Inc.) to osteopontin. The expression levels of all transfected genes were (...truncated)