Upregulation of the mevalonate pathway by cholesterol depletion abolishes tolerance to N-bisphosphonate induced Vγ9Vδ2 T cell cytotoxicity in PC-3 prostate cancer cells

Highlights

• PC-3 prostate cancer cells treated with zoledronate were shown to be resistant to Vγ9Vδ2 T cell cytotoxicity.

• Resistance was attributed to the low activity of the mevalonate pathway in these cells.

• PC-3 cells could be rendered susceptible to Vγ9Vδ2 T cell mediated cytotoxicity by activating the mevalonate pathway.

• Cholesterol depletion together with ZOL treatment led to high accumulation of IPP, which was recognized by Vγ9Vδ2 T cells.

Abstract

Zoledronate (ZOL) inhibits farnesyl pyrophosphate synthase leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI). Cytotoxic Vγ9Vδ2 T cells have been shown to recognize IPP/ApppI in breast cancer cells. Further, human breast cancer cells have been shown to differ remarkably in their ZOL treatment induced IPP/ApppI production and responses to that. In this communication we analysed the responsiveness of prostate cancer cells PC-3 and DU-145, Caki-2 renal carcinoma cells and U87MG glioblastoma cells to ZOL treatment, and the subsequent activation of Vγ9Vδ2 T-cell cytotoxicity.

Of the cell lines tested, PC-3 cells were not susceptible to Vγ9Vδ2 T-cell cytotoxicity due to low activity of the mevalonate pathway and low amount of IPP formed. However, the resistance of PC-3 cells to Vγ9Vδ2 T-cell cytotoxicity could be abrogated by upregulation of the mevalonate pathway through cholesterol depletion.

Introduction

Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of osteoclast-mediated bone resorption [1], [2] and they have demonstrated clinical utility in the treatment of bone diseases with high bone turnover, such as postmenopausal osteoporosis [3] and tumour-induced bone lysis [4]. Bisphosphonates are also effective in preventing cancer treatment-induced bone loss [5] and N-BPs have been demonstrated to show direct and indirect antitumour properties in preclinical models [6]. N-BPs can make the bone marrow a less favourable environment for cancer cell colonization by inhibiting the release of bone-derived growth factors such as TGF-β during bone resorption [6], [7]. Moreover, they may interfere with the functions of bone marrow derived cells such as endothelial progenitor cells, mesenchymal cells, monocytes and macrophages, which have an important role in priming distant tissues for tumour metastasis [8]. They can also exhibit direct antitumour effects and improve immune surveillance against neoplasia [6]. Emerging data suggest that N-BPs, such as zoledronic acid (ZOL), have immunomodulating properties, paving the way to new possibilities for the therapeutic use of these drugs.

The molecular mechanism of action of N-BPs, including ZOL, is to inhibit farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway [9], [10], [11]. This leads to accumulation of isopentenyl pyrophosphate (IPP), the metabolite immediately upstream of FPPS. IPP, acting as a phosphoantigen, activates the expansion of human Vγ9Vδ2 T cells, which then exhibit strong cytotoxic activity against N-BP treated cancer cells [11]. Vγ9Vδ2 T cells recognize phosphoantigens, naturally derived e.g. from bacterial cell wall, presented on the context of CD277 (butyrophilin 3A1) [12], [13]. There is also evidence that the ATP-analog ApppI, which results from the covalent binding of IPP to AMP [14], acts as a phosphoantigen to activate proliferation of Vγ9Vδ2 T cells [15]. Furthermore, ApppI inhibits the mitochondrial adenine nucleotide translocase (ANT) causing deregulation of the mitochondrial membrane potential which ultimately leads to apoptosis [14].

Inhibition of the FPPS halts downstream prenylation of membrane anchored small GTPases such as Ras, Rho, Rab and Rap [16], [17], [18], [19], [20]. Disturbances in intracellular signalling mediated by e.g. Ras proteins and/or intracellular trafficking mediated by Rab proteins can ultimately induce cellular apoptosis [18], [19], [21], [22].

Recently, it has been shown that ZOL-induced IPP/ApppI accumulation in breast cancer cells correlates with Vγ9Vδ2 T cell-mediated cancer cell death in vitro and in vivo [23]. In this study, we evaluated the ZOL-induced IPP/ApppI accumulation and Vγ9Vδ2 T cell-mediated cytotoxicity in human prostate cancer cell lines as well as in kidney carcinoma and glioblastoma cell lines. Moreover, we analysed the effect of ZOL treatment on abundance of various enzymes of the mevalonate pathway and prenylation of a model GTPase Rap1A. We demonstrate that the Vγ9Vδ2 T cell-mediated cytotoxicity is dependent on IPP/ApppI formation, whereas low expression of HMGR results in low IPP/ApppI formation and, consequently, resistance to ZOL induced Vγ9Vδ2 T cell-mediated cytotoxicity. By upregulating the activity of the mevalonate pathway using cholesterol depletion we were able to increase IPP/ApppI formation in PC-3 cells and abrogate their resistance to Vγ9Vδ2 T cell-mediated cytotoxicity.