For the last decade tumor metabolism has been chiefly characterized by aerobic glycolysis, which is also known as the Warburg Effect. The Warburg Effect is a phenomenon by which tumor cells exhibit an enhanced glycolytic state and secrete large amounts of lactate under aerobic conditions that are sufficient to support OXPHOS (Warburg, 1956). Even though OXPHOS is a much more energy-rewarding pathway, elevated glycolytic rates have been shown to be beneficial to rapidly proliferating cells by providing sufficient quantities of ATP and also through the maintenance of pools of biosynthetic intermediates (nucleotides, amino acids, fatty acids), which support growth (Christofk et al., 2008; DeBerardinis et al., 2008; Levine and Puzio-Kuter, 2010; Moreno-Sanchez et al., 2007; Vander Heiden et al., 2009). This is achieved through the incomplete catabolism of glucose, which allows the shunting of glycolysis-derived pyruvate towards anabolic processes via the TCA cycle and pentose phosphate pathway (PPP) (Christofk et al., 2008; DeBerardinis et al., 2008; Levine and Puzio-Kuter, 2010; Moreno-Sanchez et al., 2007; Vander Heiden et al., 2009).
Interestingly in 2011, the synonymous relationship between tumor metabolism and the Warburg Effect was challenged. Recent work now suggests that some tumor cells undergo an alternate form of metabolic reprogramming and enhance their OXPHOS capacity rather than become increasingly glycolytic. This phenomenon is referred to as the “Reverse Warburg Effect” (Pavlides et al., 2009). The underlying concept of the Reverse Warburg Effect is that the human body undergoes metabolic decline during ageing and that ageing cells acquire OXPHOS defects and become increasingly glycolytic (Ertel et al., 2012). Subsequently, glycolytic cells secrete energy rich lactate into the extracellular matrix. Tumor cells with enhanced OXPHOS capacity are then able to consume and metabolize the extracellular lactate to support their cellular function and proliferation (Ertel et al., 2012). In an in vitro co-culture system, tumor cells have been shown to induce oxidative stress in fibroblasts cells, which also resulted in premature ageing in the same cells (Lisanti et al., 2011a; Lisanti et al., 2011b). The aged fibroblasts secreted lactate into the culture media that was consumed and utilized by the tumor cells and this process was mediated through an enhanced OXPHOS state in the tumor cells (Lisanti et al., 2011a; Lisanti et al., 2011b). In another study, immunohistochemical analysis of primary breast tumor samples revealed enhanced expression of cytochrome-c-oxidase (COX; Complex III) relative to healthy neighboring cells (Whitaker-Menezes et al., 2011). These studies provide strong evidence that OXPHOS may play an important role in tumor cell metabolism and maintenance of their cellular function.
The above studies demonstrate that tumor cell metabolism is more complex than previously thought. GBM CSCs have been shown to exhibit characteristics of the Warburg Effect (DeBerardinis et al., 2008; DeBerardinis et al., 2007; Wolf et al., 2011) however, much less is known regarding the role of OXPHOS in these cell types. Furthermore, very little is known regarding how mtDNA is regulated GBM CSCs and its implications on metabolism and OXPHOS.
GBM CSCs exhibit stem cell-like properties such as self-renewal and multipotency (Galli et al., 2004; Singh et al., 2003); however, it is unknown whether GBM CSCs regulate their mtDNA in a similar manner to normal stem cells. Furthermore, it is unknown whether GBM CSCs are able to undergo the metabolic transition that occurs during the differentiation of normal stem cells. As a stem cell undergoes differentiation, there is a metabolic transition from glycolysis to OXPHOS metabolism that serves to fulfill the future energy requirements of the terminally differentiated cell type (Cho et al., 2006; Facucho-Oliveira et al., 2007; Prigione et al., 2010; Varum et al., 2011). The differentiation of embryonic stem cells (ESCs) is associated with an expansion in mtDNA copy number that enhances OXPHOS potential by increasing the number of mtDNA copies available for translation into functional subunits of the ETC (Facucho-Oliveira et al., 2007; St John et al., 2005). Since the ETC is encoded by both nuclear and mtDNA both genomes must work synergistically in order to generate a functional respiratory chain (Woodson and Chory, 2008). Human neural stem cells (hNSCs) are the closest non-transformed counterpart of GBM CSCs and it is likely that hNSCs undergo a similar mtDNA copy number
expansion and metabolic transition during differentiation that has previously been observed in ESCs, however this requires confirmation.
The direct comparison of undifferentiated and differentiated hNSCs and GBM CSCs provides an excellent opportunity to analyze how mtDNA is regulated in both normal and transformed multipotent cell populations. Furthermore metabolic profiling of undifferentiated and differentiated GBM CSCs will also further explore the role of OXPHOS in GBM CSCs.
3.2 Hypothesis
GBM CSCs and hNSCs share multiple characteristics that include self-renewal, multi-potency and gene expression profiles. Normal stem cell populations have been shown to contain low numbers of mtDNA copies and the onset of differentiation is associated with an expansion of mtDNA copy number and a transition from glycolytic to OXPHOS metabolism. Tumors cells, including GBM CSCs, show abnormal expression of factors that regulate cell metabolism, such
as c-MYC (Gordan et al., 2007), and it is likely that this will impact upon the
ability of GBM CSCs to undergo differentiation and modulate their mtDNA copy number accordingly and will therefore differ to that of normal hNSCs.
3.3 Aims
1. To determine how mtDNA copy number is modulated during the differentiation of hNSCs and GBM CSCs
2. To determine how the expression of NSC and lineage specific markers are regulated during the differentiation of hNSCs and GBM CSCs
3. To characterize the energy metabolism profiles of undifferentiated and differentiated hNSCs and GBM CSCs through functional experiments.