A Metabolic Bottleneck for Stem Cell Transformation

Abstract

Although oncogenic mutations predispose tissue stem cells to tumor initiation, the rate-limiting processes for stem cell immortalization remain unknown. In this issue of Cell, Bonnay et al. identify enhanced electron transport chain activity as a critical determinant of this process, establishing metabolic reprogramming as limiting for tumor initiation.

Tissue stem cells (SCs) support tissue formation and maintenance by balancing decisions of self-renewal and differentiation. Upon acquisition of oncogenic mutations, stem cells are predisposed to initiate tumors, and tumors in many tissues preferentially arise from stem cells but not terminally differentiated cells (White and Lowry, 2015). Recent studies in humans have demonstrated that over time, stem cells in multiple tissues accumulate oncogenic mutations without succumbing to neoplastic transformation, raising the question as to what limits tumor initiation in these pre-malignant oncogenic stem cells (Blokzijl et al., 2016). In this issue of Cell, Bonnay et al. (2020) tackle this question by using a Drosophila model of neural stem cell-derived tumors. They identify a requirement for increased electron transport chain (ETC) activity as being rate limiting for stem cell transformation, establishing metabolic reprogramming as a bottleneck in stem cell commitment to tumor initiation (Figure 1).

Figure 1. Metabolic Reprogramming Is Essential for Neuroblast Immortalization.

In order to be immortalized and initiate a tumor, mutant neuroblasts must rewire their metabolism to enhance activity of the electron transport chain. This is driven by increased mitochondrial fusion and an increase in glutamine entry into the TCA cycle. The increase in electron transport chain activity sustains NAD+ regeneration, which is essential and rate limiting for transformation and tumor initiation.

Metabolic pathways are critical for established cancer cell proliferation, growth, and survival by supporting bioenergetic and anabolic demands. Moreover, metabolic pathways and particular metabolites can drive stem cells to proliferate as well as impact their fate decisions to self-renew or differentiate. In particular, in both proliferating cultured stem cells and cancer cells, glucose carbons are shunted to lactate rather than into the tricarboxylic (TCA) cycle (also known as aerobic glycolysis or the Warburg effect), while glutamine is the major substrate that fuels oxidative metabolism (Intlekofer and Finley, 2019). The TCA cycle is sustained by the ETC, which regenerates the redox cofactor NAD+ to drive oxidative TCA cycle metabolism, thereby enabling synthesis of precursors for macromolecule synthesis (Birsoy et al., 2015; Sullivan et al., 2015). While these features are well established in proliferating cells in vitro, the role of altered metabolism in stem cell immortalization and tumor initiation has remained largely unexplored.

Development and growth of the Drosophila brain is fueled by neural stem cells or neuroblasts (NBs), whose differentiation can be arrested by loss of the RNA-binding protein brat. This is associated with a transient cell cycle arrest followed by uncontrolled proliferation of un-differentiated NBs that fuel the initiation and growth of lethal brain tumors. Normal NB self-renewal is associated with aerobic glycolysis, whereas differentiation is associated with increased glucose oxidation in the TCA cycle (Homem et al., 2014). Bonnay et al. (2020) take advantage of this metabolically and temporally well-defined stem cell system to dissect the role of metabolism in tumor initiation. In striking contrast to normal NB proliferation, brat mutant oncogenic NBs are highly reliant on ETC activity and oxidative metabolism, which is predominantly fueled by glutamine, rather than glucose, entry into the TCA cycle. Using single-cell RNA sequencing, the authors identify a subset of tumorigenic NBs that are enriched for oxidative metabolism gene expression. Genetic loss of ETC genes or TCA cycle genes blunt oncogenic NB proliferation and tumor initiation. Moreover, this heightened reliance on oxidative metabolism coincides temporally with stem cell transformation, such that rewiring of intermediary metabolism becomes rate limiting for stem cell immortalization and commitment to tumor initiation.

The authors also dissect the upstream regulators and downstream consequences of this metabolic program. They discovered that mitochondrial fusion, implicated in regulating stem cell proliferation and fate in other contexts (Chen and Chan, 2017), is essential for driving increased oxidative metabolism in NBs. Downstream, the electron transport chain is essential for maintaining NAD+ regeneration. Using SoNAR, a genetic sensor of the NAD+/NADH ratio, the authors find that intriguingly, tumorigenic but not normal NBs are reliant on the ETC for sustaining proper redox homeostasis. Moreover, loss of complex V of the ETC (the ATP Synthase) was rescued by expressing yeast NADH oxidase NDI1, which regenerates NAD+ without restoring ATP synthesis. Interestingly, in order to sustain NDI1 activity in this setting, the oncogenic NBs must sustain flux through complex III and IV to enable ongoing ubiquinol oxidation without the ATP synthase to limit membrane potential, suggesting metabolicflexibility with regard to proton translocation back into the mitochondrial matrix in oncogenic NBs. What enables this adaptation in tumorigenic NBs remains to be explored but, nevertheless, clearly establishes NAD+ regeneration and not ATP synthesis as being limiting for NB immortalization.

The work by Bonnay et al. (2020) raises several important points and questions with regard to stem cell metabolism and tumor biology. First, the differential metabolic requirements of oncogenic versus normal NBs highlight that transformed SC metabolism may not simply be a reflection of the metabolism of a tumor’s cell of origin. Indeed, whereas oxidative metabolism is associated with normal NB differentiation, it sustains oncogenic NB proliferation. This is unlikely to be simply a reflection of proliferative rates, since ETC reliance arises as tumorigenic NBs transition out of quiescence and proliferate at similar rates to normal NBs. Rather, this unique metabolic dependence may be linked to oncogenic NB identity per se, raising the question as to what function the ETC plays in sustaining tumorigenic NBs. Although NBs fail to transform upon ETC or TCA cycle inhibition, the fate of these oncogenic NBs remains unknown. Given recent links between redox metabolism, TCA cycle metabolites, and mammalian stem cell fate under homeo-static and oncogenic settings (Baksh et al., 2020; Intlekofer and Finley, 2019), it will be of interest to understand whether or not ETC inhibition excludes these oncogenic NBs from the tissue by driving their terminal differentiation or whether oncogenic NB loss is driven by changes in proliferation and/or cell death.

Second, the particular metabolic role of the ETC in supporting NB transformation remains to be elucidated. Consistent with work on cultured mammalian cancer cells, the authors find that NAD+ regeneration is an essential function of the ETC in fly NBs (Birsoy et al., 2015; Sullivan et al., 2015). Sustaining NAD+ regeneration supports many metabolic reactions, and it will be of interest to learn whether any particular reactions support NB transformation. Alternatively, redox homeostasis can impact transcriptional and signaling pathways, which may then enable NB transformation.

Finally, whereas metabolism is known to play a role in tumor initiation in cancers harboring recurrent oncogenic mutations in metabolic enzymes, the current work suggests that metabolic rewiring may be a generalizable and evolutionarily conserved regulator of stem cell transformation. Given evidence that ETC inhibition in humans via the anti-diabetic drug metformin reduces the incidence of various malignancies (Quinn et al., 2013), it is tempting to speculate that the findings presented here would extend to tissue stem cells in humans. Exploring the role of oxidative metabolism in mammalian stem cell transformation may provide insight into the benefits of metformin as a putative cancer prophylaxis agent.