Supplementary MaterialsSupplementary File 1. activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cells IGFBP6 rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance. & em 2 /em 1.06 & VE-821 pontent inhibitor 1.03Serine synthesis em PHGDH /em 7.67Tyrosine synthesis em PAH /em 3.80Urea synthesis em CPS /em 3.49Folate synthesis em MDHFD1 /em 2.49 Open in a separate window The three rate-limiting glycolytic pathway steps (HK2, PFKL, and PKM) are three of the four most increased enzymes of the pathway (the 4th being aldolase). Interestingly, PFKL is repressed by high ATP/AMP ratios [101]. Since spheroids have high ATP amounts [19], high PFK levels suggest that AMP levels are also high. The glutamine-dependent cytosolic carbamoyl-phosphate synthetase 2, is upregulated in spheroids (CAD 3.49). CAD is the rate-limiting enzyme carrying out the first three steps in pyrimidine synthesis. CAD is essential for uridine diphosphate (UDP) synthesis, which in turn, is essential for glycogenesis. This correlates with the appearance of glycogen granules in hepatocyte spheroids and with protein glycosylation and the hexosamine pathway [102]. 3.7. Signal Pathways Involved in Orchestrating Metabolic Reprogramming All of the adaptations seen in glycolysis and glutaminolysis, pentose phosphate pathway, TCA cycle, and fatty acid synthesis indicate that spheroids, grown in a wide variety of 3D culture systems, are utilising a significant degree of metabolic reprogramming to aerobic glycolysis. The typical features of 3D culturediffusion gradients resulting in hypoxia (and to a less extent glucose starvation) clearly drive metabolic reprogramming. Warburg saw this phenomenon as a hallmark of cancer. In order to investigate how metabolic reprogramming VE-821 pontent inhibitor is orchestrated, we reviewed the status of pathways that are often associated with tumour development: PI3K/Akt/mTOR, Myc, p53, nuclear factor kappa-B (NF-B), and Wnt [54]. 3.7.1. PIK3/AKT/mTOR The PI3K/AKT/mTOR pathway (Figure 6) plays a key integrating role, sensing concentrations of nutrients (including glucose, oxygen, amino acids and ATP levels) and regulating the anabolic processes of the cell for growth and maintenance [103]. Open in a separate window Figure 6 mTOR signalling in 3D spheroids. See legend to Figure 4 for nomenclature. While only two key proteins from this pathway were detected (mTOR, 1.63; and ribosomal protein S6 kinase RPS6KA3, 5.54), strong downstream effects are clearly visible showing that pathway is activated in 3D (Table 1). mTOR signalling increases translation of hypoxia-inducible factor 1 (HIF-1), glucose transporters and glycolytic enzymes, and promotes metabolic reprogramming [101,104] (Figure 6). mTOR promotes pentose phosphate pathway (PPP) enzyme expression (on average by 2.14) VE-821 pontent inhibitor and channels metabolic flux into its oxidative, NADPH-producing branch [91]. mTOR strongly stimulates pyrimidine synthesis via the RPS6KA-mediated phosphorylation of CAD (3.49), thereby increasing the pool of nucleotides available [105]. AKT can phosphorylate ACL, enhancing VE-821 pontent inhibitor its lipogenic activities and mTOR signalling promotes NADPH-requiring lipid synthesis by activating sterol regulatory element-binding proteins (SREBP1 and 2) [106]. 3.7.2. Myc Myc has the potential to play a key role in metabolic reprogramming. Myc is central to growth regulation and is one of the most frequently deregulated oncogene transcription factors seen in a wide variety of cancers [107,108]. Myc directly transactivates gene expression of GLUT1, phosphofructokinase (PFK), enolase (ENO) and LDHA and indirectly increases phosphoglucose isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK1) [109] (Figure 7). This is consistent (with the exception of GLUT1) with their increased levels in spheroids. However, as described above, PIK3/AKT/mTOR can also induce these proteins (via HIF-1) and so this effect need not be attributed to Myc. HIF-1 can inactivate Myc [110], and in doing so, induce cell VE-821 pontent inhibitor cycle arrest [111]. Open in a separate window Figure 7 Myc signalling in 3D spheroids. See legend to Figure 4 for nomenclature. Low expression levels of several proteins normally induced by Myc suggest that Myc is not particularly active in 3D spheroids. Examples include: PTBP1 [112] (?1.23); GLUT1 (?1.36); SLC1A5 [107] (?1.29); and, PRDX3s [113] (?1.04). Myc regulates serine hydroxymethyl transferases and pathway hyperactivation is a driver of oncogenesis [107]. However, the moderate increase of SHMT2 (1.47).