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  • By measuring the accumulation of

    2021-11-30

    By measuring the accumulation of unprenylated Rap1a as a surrogate marker for the inhibition of FPPS [8], [9], [10], [11], we found that upregulation of FPPS expression in Hela cells reduced the accumulation of unprenylated Rap1a in response to 24h' treatment with 0–50μM ZOL. As expected, cells cultured in 10% FCS showed robust accumulation of unprenylated Rap1a following treatment with 2μM ZOL (Fig. 3A), whereas in cells cultured in LDS (in which endogenous FPPS levels were increased; Fig. 3B), unprenylated Rap1a was not detectable in cells treated with 5μM ZOL and was barely detectable in cells treated with 10μM ZOL (Fig. 3D). Hence, the upregulation of endogenous FPPS conferred partial resistance to the pharmacological effect of ZOL. Intracellular cholesterol and fatty resazurin metabolism are regulated by SREBP-2 and SREBP-1a respectively, although in cultured cells the effect of SREBP-1 predominates [33]. Overexpression of SREBP-1a in HeLa cells caused upregulation of endogenous FPPS (Figs. 4A,B), similar to the effect of culturing cells in LDS. Furthermore, the inhibitory effect of ZOL on Rap1a prenylation was reduced in HeLa cells overexpressing SREBP-1a compared to untransfected cells (Figs. 4A,C). Together, these studies demonstrate that increasing the level of endogenous FPPS (either by removal of exogenous sterols, or by overexpression of SREBP-1a) can confer at least partial resistance to the pharmacologic effects of N-BP drug. A single nucleotide polymorphism (rs2297480) within the promoter region of FPPS was recently reported to be associated with low BMD [34] and reduced response to BP treatment in post-menopausal women [34], [35], with carriers of the rare allele demonstrating significantly less improvement in BMD with long term BP treatment compared to homozygous carriers of the common allele [35]. It was suggested that the variant allele creates a Runx1 binding site, likely leading to reduced FPPS transcription [34] although this was not studied. Our observations indicate that reduced expression of FPPS would actually lead to increased sensitivity to BP therapy rather than decreased responsiveness. Consequently the relevance of this polymorphism to BP treatment remains to be determined. Despite the punctate appearance of FPPS following immunofluorescence staining, the LDS-induced, endogenous FPPS did not colocalise to peroxisomes (Fig. 2C), which were labelled in cells transfected with the pDsRed2-Peroxi vector (the product of which colocalised with the peroxisomal marker PMP-70; Fig. 2A). A myc-tagged isoform of FPPS that includes exon 2 (containing an apparent mitochondrial targeting sequence; [26]) colocalised with MitoTracker (Fig. 2D). However, by comparison with the distribution of MitoTracker, the LDS-induced endogenous FPPS did not localise to mitochondria (Fig. 2E). This is consistent with a previous study in Trypanosoma species [25], in which the presence of FPPS was not detected in mitochondria. Taken together, these results suggest that endogenous FPPS is predominantly cytosolic, consistent with a proposed model of the mevalonate pathway in which FPPS, together with other enzymes of the mevalonate pathway, is predominantly cytosolic rather than peroxisomal [23].
    Acknowledgements This study was funded by a PhD studentship for S.D. supported by Roemex Ltd, Portlethen, UK. We thank Dr Fraser Coxon for helpful discussions. P.A.E. was supported by NIH grant HL30568.
    Farnesyl diphosphate synthase (FPPS) catalyzes the formation of the isoprenoid, farnesyl pyrophosphate (FPP), from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) during mevalonate metabolism . FPP is incorporated into diverse classes of proteins that participate in a multitude of cellular processes relating to cellular protein–protein interactions and membrane-associated protein trafficking . The isoprenylation of proteins seems to be tightly regulated within the mevalonate pathway and some of these proteins are known to be involved in the pathogenesis and progression of some cancers . Increased FPPS activity and expression in human colon cancer and rat prostate tumor cell lines have been well documented , , and an increase in FPPS activity in tumor tissues has been implicated in the pathology of certain neoplastic disorders .