Cancer cells are characterized by uncontrolled proliferation, a process that comes along with high demands of building blocks to give rise to daughter cells. Although the necessity of glycolysis in supporting tumor growth i.e. the Warburg effect had been well established, the contribution of mitochondrial metabolism in tumor growth was not fully understood. A decade ago, our group using genetic mouse models of lung adenocarcinoma demonstrated that the mitochondrial electron transport chain (ETC) function is required for oncogenic Kras-mediated tumorigenicity1. Since then, multiple laboratories have confirmed that mitochondrial metabolism is essential for tumor growth in a wide variety of cancer models. Human data have also corroborated that lung and brain tumors display a robust oxidative TCA cycle compared to adjacent normal tissues and pathogenic mutations in mitochondrial DNA are selected against in a wide variety of tumors. Moreover, some drugs targeting mitochondrial ETC like metformin have shown promising results in preclinical models and are currently being tested in clinical trials.
As we recognized that the field was appreciating the essential role of the mitochondrial ETC for tumor growth, we began to examine the underlying mechanism for the dependency on ETC function. Mitochondrial ETC is necessary for ATP generation i.e. oxidative phosphorylation and regenerating NAD+ and FAD to allow oxidative TCA cycle to function, which generates metabolites that serve as building blocks for macromolecule synthesis. How to discern the contribution of ATP versus TCA cycle function for tumor growth? This required the use of a new set of genetic tools. In the past two decades, there have been multiple genetic tools from lower organisms including yeast and sea squirt that can complement the loss of distinct mitochondrial ETC complex functions2,3. Previously, we used some of these techniques in vitro to demonstrate that the TCA cycle is necessary for histone acetylation, a post-translational modification with a direct impact in controlling gene expression4. We also demonstrated that the maintenance of the mitochondrial membrane potential is essential for the production of mitochondrial reactive oxygen species to control cell proliferation and the cellular hypoxic response4. These are all essential biological responses for tumor growth. Thus, we asked what are the essential mitochondrial ETC functions that support tumor growth in vivo?
We manipulated different mitochondrial functions in cancer cells by CRISPR–Cas9-mediated deletion of subunits of mitochondrial complexes and dihydroorotate dehydrogenase (DHODH), a mitochondrial enzyme involved in de novo pyrimidine synthesis that requires mitochondrial complex III activity, and analyzed the ability of cells to develop tumors. Following our previous in vitro approach, we used the non-mammalian proteins alternative oxidase (AOX) from C. intestinalis (sea squirt) or the NADH oxidase (NDI1) from S. cerevisiae (yeast) to complement defects in the ETC complexes III or I, respectively. Our findings indicate that individual loss of complex I, II, or III as well as DHODH impairs tumor growth in vivo by diminishing the oxidative TCA cycle and de novo pyrimidine synthesis functions but not due to lack of ATP generation5. The water-forming NADH oxidase (LbNOX) from L. brevis (bacteria) that allows maintaining NAD+ levels in cells with mitochondrial dysfunction6, allowed us to uncover that NAD+ regeneration by ETC is necessary but not sufficient to efficiently support tumor growth. The use of these genetic tools in vivo could help discern the importance of the bioenergetic versus biosynthetic functions of mitochondria in a variety of biological contexts moving forward.
1 Weinberg, F. et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci U S A 107, 8788-8793, doi:10.1073/pnas.1003428107 (2010).
2 Perales-Clemente, E. et al. Restoration of electron transport without proton pumping in mammalian mitochondria. Proc Natl Acad Sci U S A 105, 18735-18739, doi:10.1073/pnas.0810518105 (2008).
3 Fernandez-Ayala, D. J. et al. Expression of the Ciona intestinalis alternative oxidase (AOX) in Drosophila complements defects in mitochondrial oxidative phosphorylation. Cell Metab 9, 449-460, doi:10.1016/j.cmet.2009.03.004 (2009).
4 Martinez-Reyes, I. et al. TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions. Mol Cell 61, 199-209, doi:10.1016/j.molcel.2015.12.002 (2016).
5 Martínez-Reyes, I. et al. Mitochondrial ubiquinol oxidation is necessary for tumour growth. Nature, doi:10.1038/s41586-020-2475-6 (2020).
6 Titov, D. V. et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio. Science 352, 231-235, doi:10.1126/science.aad4017 (2016).