Elucidating the metabolic flexibility and plasticity of one-carbon cycle in cancer cells under stressful metabolic environments

Metabolism is an interlinked series of biochemical reactions that govern biomass and energy generation at a cellular and systemic level. Due to its critical role in generating biomass, rewiring metabolism is considered an enabling hallmark of cancer progression. A key biosynthetic pathway that is often upregulated in cancer cells is the one-carbon (1C) metabolism. 1C metabolism has been successfully targeted for its biosynthetic potential since the 1950s with chemotherapeutic agents like methotrexate. However, recent advances in the cancer metabolism field have hinted at possibilities that the 1C cycle can support energy generation and antioxidant defense. Recent work by our group has shown that the 1C cycle is running in excess of the anabolic need of the cell and that excess is excreted out of the cell as formate. Furthermore, we have shown that cancer cells often increase formate release rates under growth inhibitory perturbations. Such observations suggest that the 1C cycle serves additional metabolic functions in cancer cells. However, these functions are currently not well understood and the growth-independent potential of the 1C cycle remain unexplored.
In my dissertation, we studied cancer cells under glycolytic and growth stress conditions to identify growth- independent functions of the 1C cycle. We utilized galactose as a tool compound to mimic glucose limitation, a physiological relevant feature of the tumor microenvironment. Firstly, we corroborated previous findings regarding the rewiring of glutamine metabolism under glucose limitation towards serine synthesis pathway (SSP) and TCA cycle. Moreover, we were able to separate and stratify the two rewiring pathways with the identification of a glycolytic block. Using isotope tracers, we identified pyruvate kinase M2 isozyme (PKM2) as the rewiring point under glucose limitation as it is completely blocked under these conditions. This identified PKM2 block is novel and independent of serine levels. It rewired the limited glycolytic carbons towards SSP and the 1C cycle to support nucleotide synthesis and mitochondrial ATP and NADPH generation. Furthermore, using genetic ablation of 1C cycle enzymes we showed that the PKM2 block relied on active formate overflow.
To specifically identify growth-independent functions of the 1C cycle, we utilized methotrexate as a tool compound to fully inhibit the cytoplasmic 1C cycle, halting cancer cell proliferation. We identified that cancer cells are able to sustain their mitochondrial 1C flux independently of the cytoplasmic part. In addition, the independent mitochondrial 1C cycle was significantly important to support cancer cell motility and promote metastasis.
In conclusion, cancer cells are notorious to have a highly adaptable metabolic network that allow them to be metabolically resilient and to escape treatment. By utilizing nutrient stress tools in vitro, we are able to identify rewiring strategies that can uncover vulnerabilities of the most resilient cancer cell populations.