Abstract
Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μM, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.
Highlights
Stable isotope/chemical probe mass spectrometry was used to monitor cancer cell metabolism of exogenous isoprenols
Incorporation of Exogenous Isoprenols into Isoprenoid Diphosphates by MDA-MB-231 Cells—Farnesyl diphosphate (FPP), GGPP, anilinecontaining analogs, and deuterium-labeled variants of these compounds and their corresponding isoprenols shown in Fig. 2 were synthesized as described under “Experimental Procedures” and analyzed by electrospray ionization HPLC-MS/MS with quantitation accomplished by stable isotope dilution
We concurrently discovered that cells formed anilinofarnesol diphosphate (AFPP) from AGOH, which we presume is a result of condensation of Anilinogeraniol diphosphate (AGPP) with an additional isoprene unit (Fig. 3D; see Fig. 1)
Summary
Stable isotope/chemical probe mass spectrometry was used to monitor cancer cell metabolism of exogenous isoprenols. P53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. The relative efficiency and biochemical relationship of this “isoprenol-dependent,” “scavenging,” or “recycling” pathway to the much better characterized mevalonate pathway are not known This information could be of fundamental importance to understanding the efficacy and side effects associated with pharmacological inhibition of the rate-limiting enzyme of the mevalonate pathway, HMG-CoA reductase, which is a widely. Metabolism of exogenous isoprenols is up-regulated as result of p53 silencing or mevalonate pathway inhibition in breast cancer cells, indicating that this process is regulated independently of components of the mevalonate pathway that are controlled by p53 or inhibited by statins.
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