Folate metabolism limits cancer cell proliferation

One-carbon metabolism is a cellular pathway that employs folic acid (vitamin B9) derivatives to transfer single carbon groups from one molecule to another. The sum of all folic acid derivatives is called folates. The one-carbon groups transferred by folates are the smallest molecular Lego bricks available to build up more complex molecules such as DNA. It is also the source of one-carbon groups required for controlling the activity of individual stretches of DNA via histone methylation. One- carbon metabolism is therefore essential for cellular reproduction. One group of diseases where cell division is especially important is cancer, which is defined by abnormal cell growth with the potential to invade or spread to other parts of the body. The importance of one-carbon metabolism in cancer is highlighted by the fact that the first chemotherapeutics invented in the 1950s actually target this pathway, and some of them are still in use today. The flux of one-carbon units through folate metabolism to the individual follow-up pathways is controlled by enzymes which establish an equilibrium of the individual folates, dependent on cellular demand. Much information can be gained by measuring these individual pools of folates, since one can then also conclude which follow-up pathways are active to which extent. Unfortunately the folates are rather unstable metabolites and start to degrade and interconvert to each other upon disruption of cells or tissue. So far the solution to this is to measure folates in groups irrespective of their individual precursors or measure the folates as quickly as possible after disruption and try to calculate back the original concentration by monitoring standards. The first approach sacrifices a lot of information while the latter is not feasible in many settings, e.g. for clinical samples. In this project we aim to elucidate the molecular basis of a cellular model of a cancer aggressiveness. We will do so by chemically stabilizing the individual folates directly at the cell / tissue disruption step. The stabilized folates cannot interconvert and are more resistant to degradation. This also allows sample preparation methods not feasible for unmodified folates and the setup of a high throughput direct infusion mass spectrometry method, reducing sample cost and allowing application in a more routine environment. In the long run we aim to use our method in a clinical setup to be able to distinguish between potential responders and non-responders to a drug before the actual administration. Moreover, detailed knowledge of which of the follow-up pathways of one-carbon metabolism is disturbed will allow a more precisely targeted medical intervention with fewer side effects.

The aim of this Erwin-Schrödinger Fellowship was the development of a method for analysis of individual folate species. Folates play a crucial role in many cellular synthetic pathways, as they carry the smallest potential building block, i.e. one carbon units, in various oxidation states. These building blocks are needed for the biosynthesis of nucleic acids and amino acids and also play a crucial role in the regulation of gene expression and protection against oxidative stress. Many diseases including cardiovascular disease, neurological disorders and cancer are closely linked to changes in cellular folate metabolism. Inauspiciously, many folate species are highly unstable and cannot be quantified using common analytical methods. In the course of this fellowship a new analytical approach employing a derivatisation strategy which allows stabilization of folate species at the time of sampling was developed. Stabilized folates can then be analyzed using liquid chromatography coupled mass spectrometry. This approach does not only allow for the first time to analyse every cellular folate species, it also enables the parallel measurement of all folate species in a single analysis. Precluding loss of unstable folates also improved method sensitivity and, maybe practically even more important, greatly simplifies sample handling. In contrast to other methods, stabilizing folates allow storage between sampling and analysis, a benefit which is especially important in a clinical environment where strictly time controlled processes are often unfeasible.