Tracing the interplay between cellular metabolism and cell cycle

Sammanfattning: Cancer cells are more vulnerable to targeting during S-G2-M cell cycle phases due to common deregulations in cell cycle checkpoint mechanisms. Most common cancer drugs target processes in the S-G2-M phases, often causing negative effects on proliferating normal cells. On the other hand, several metabolic processes are deregulated in cancer, generating metabolic dependencies and exposing vulnerabilities specific to cancer cells. Therefore, deregulated metabolic processes in the S-G2-M phases potentially offer effective targeting strategy without harming normal cells. There are some studies reporting cyclic metabolic processes in synchronized cells, but a more systematic study of metabolism across cell cycle is lacking. The focus of this thesis is to trace metabolic events in G1 and S-G2-M phases, and identify metabolic enzymes that affect cancer cell proliferation using an approach that does not interfere with cell metabolism. Initially we developed a method for performing metabolomics in sorted cells, then applied this method on G1 and S-G2-M sorted normal and transformed cells. We mapped hundreds of metabolites and metabolic activities in G1 and S-G2-M phases, and identified arginase 2 (ARG2) enzyme as a potential target which reduced cancer cell proliferation. This study can be used as a resource of metabolism across the cell cycle. This project presents a large scale investigation of metabolism in G1 and S-G2-M phases, contributing to the understanding of the biology of metabolism across the cell cycle and reveals metabolic activities in the S-G2-M phases of cancer cells. This thesis is comprised of the following papers. In Paper I (Roci et al, 2016) we developed a method for performing metabolomics in sorted subpopulations combining Fluorescence Activated Cell Sorting (FACS) and Liquid Chromatography Mass Spectrometry (LC-MS). We evaluated relative metabolite abundance using peak areas and metabolic activities using isotope tracing. From benchmarking of sorted sampled with extraction from culture dish we found that isotope tracing is more robust and reflects the metabolism of cells in culture dish. In Paper II (manuscript) and Paper III (Roci et al, 2019) we mapped metabolism in G1 and S-G2-M phases and presented relative metabolite abundances and metabolic activities in these phases. Most metabolite abundances and 13C enrichment were constant showing that they are required throughout cell cycle. Some metabolic processes like ornithine synthesis, arginine uptake showed higher activities in the S-G2-M, while synthesis of some phosphorylated sugar metabolites was upregulated in G1 phase. In a follow-up study of the ornithine synthesizing enzyme, ARG2 presented in (Paper III), we found that silencing of ARG2 reduced cancer cell proliferation. Cancer cells synthesized ornithine only via ARG2, but normal cells used both ARG2 and the alternative pathway via Ornithine transferase (OAT). High ARG2 expression was correlated with ER negative and Basal breast cancer subtypes, and poor survival in these cancer types. In Paper IV (manuscript) we present a survey of choline metabolism by culturing several normal and transformed cell lines in 13C3-choline. Betaine was synthesized only in cancer cells and was not a methyl donor in any of the cell lines. The choline dehydrogenase (CHDH) enzyme, which synthesizes the precursor of betaine, was also expressed only in cancer cell lines, and caused an increase of G2-M phase cells when silenced. Besides, from untargeted analysis we found 122 peaks that were labeled from choline, including phosphatidylcholine synthesized in all cell lines, and other previously unknown peaks.