Bisphosphonates (BPs) are a class of calcium-seeking drugs used to inhibit bone destruction in cancer patients. BPs such as zoledronic acid (ZOL) act by inhibiting FPP synthase. This causes accumulation of the upstream metabolite IPP and prevents synthesis of isoprenoids necessary for prenylation of small GTPases. We have recently optimized a highly sensitive in vitro prenylation assay to detect inhibitory effects of BPs on the prenylation of Rab GTPases.
Accumulating evidence suggests that BPs have pleiotropic effects outside the skeleton, including anti-tumour activity. Using 2-photon intravital imaging of a fluorescently-tagged BP, we recently demonstrated that BPs accumulate in mouse 4T1 mammary tumours as a consequence of diffusion from leaky vessels and binding to microcalcifications. These drug-bound microcalcifications are then efficiently engulfed by tumour-associated macrophages (TAMs). However, despite rapid drug uptake, we could not detect any defects in protein prenylation in TAMs from ZOL-treated animals, while by contrast, ZOL administration inhibited the prenylation of Rab proteins in CD11b+ peritoneal macrophages. To determine whether the lack of effect of ZOL on prenylation in TAMs was due to reduced drug activity of microcalcification-bound ZOL, we treated mice weekly for 4 weeks with free ZOL or an equivalent dose of liposome-encapsulated ZOL that is efficiently internalised by macrophages. Unlike free ZOL, liposomal ZOL had a clear inhibitory effect on Rab prenylation in TAMs. Importantly, while IPP was undetectable by mass spectrometry in TAMs from control mice, treatment with free ZOL did cause IPP accumulation in these cells.
Together, our data suggest that, at least in 4T1 tumours, engulfment of ZOL-bound microcalcifications leads to cysosolic ZOL concentrations that are sufficient to inhibit FPP synthase and cause accumulation of IPP, but are insufficient to inhibit protein prenylation.