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The overexpression of ATP binding cassette (ABC) transporters makes tumor cells simultaneously resistant to several cytotoxic drugs. Impairing the energy metabolism of multidrug resistant (MDR) cells is a promising chemosensitizing strategy, but many metabolic modifiers are too toxic in vivo. We previously observed that the aminobisphosphonate zoledronic acid inhibits the activity of hypoxia inducible factor-1a (HIF-1a), a master regulator of cancer cell metabolism. Free zoledronic acid, however, reaches low intratumor concentration. We synthesized nanoparticle formulations of the aminobisphosphonate that allow a higher intratumor delivery of the drug.

We investigated whether they are effective metabolic modifiers and chemosensitizing agents against human MDR cancer cells in vitro and in vivo. At not toxic dosage, nanoparticles carrying zoledronic acid chemosensitized MDR cells to a broad spectrum of cytotoxic drugs, independently of the type of ABC transporters expressed. The nanoparticles inhibited the isoprenoid synthesis and the Ras/ERK1/2-driven activation of HIF-1α, decreased the transcription and activity of glycolytic enzymes, the glucose flux through the glycolysis and tricarboxylic acid cycle, the electron flux through the mitochondrial respiratory chain, the synthesis of ATP. So doing, they lowered the ATP-dependent activity of ABC transporters, increasing the chemotherapy efficacy in vitro and in vivo.

These effects were more pronounced in MDR cells than in chemosensitive ones and were due to the inhibition of farnesyl pyrophosphate synthase (FPPS), as demonstrated in FPPS-silenced tumors. Our work proposes nanoparticle formulations of zoledronic acid as the first not toxic metabolic modifiers, effective against MDR tumors. The overexpression of ATP binding cassette (ABC) transporters makes tumor cells simultaneously resistant to several cytotoxic drugs. Impairing the energy metabolism of multidrug resistant (MDR) cells is a promising chemosensitizing strategy, but many metabolic modifiers are too toxic in vivo.

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We previously observed that the aminobisphosphonate zoledronic acid inhibits the activity of hypoxia inducible factor-1α (HIF-1α), a master regulator of cancer cell metabolism. Free zoledronic acid, however, reaches low intratumor concentration. We synthesized nanoparticle formulations of the aminobisphosphonate that allow a higher intratumor delivery of the drug. We investigated whether they are effective metabolic modifiers and chemosensitizing agents against human MDR cancer cells in vitro and in vivo. At not toxic dosage, nanoparticles carrying zoledronic acid chemosensitized MDR cells to a broad spectrum of cytotoxic drugs, independently of the type of ABC transporters expressed. The nanoparticles inhibited the isoprenoid synthesis and the Ras/ERK1/2-driven activation of HIF-1α, decreased the transcription and activity of glycolytic enzymes, the glucose flux through the glycolysis and tricarboxylic acid cycle, the electron flux through the mitochondrial respiratory chain, the synthesis of ATP. So doing, they lowered the ATP-dependent activity of ABC transporters, increasing the chemotherapy efficacy in vitro and in vivo.

These effects were more pronounced in MDR cells than in chemosensitive ones and were due to the inhibition of farnesyl pyrophosphate synthase (FPPS), as demonstrated in FPPS-silenced tumors. Our work proposes nanoparticle formulations of zoledronic acid as the first not toxic metabolic modifiers, effective against MDR tumors. INTRODUCTION The mevalonate pathway produces cholesterol and isoprenoids - such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate - which activate small G-proteins like Ras and Rho.

The high activity of the mevalonate pathway induces tumor proliferation, invasion and aggressiveness, and is correlated with poor clinical outcome of oncological patients ,. Hence, inhibitors of the pathway are attractive adjuvant anti-tumor drugs –.

The overexpression of ATP binding cassette (ABC) transporters - such as P-glycoprotein (Pgp/ABCB1), multidrug resistance related proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) - limits the intracellular retention and activity of several cytotoxic drugs, producing a multidrug resistant (MDR) phenotype in tumor cells. MDR cells have a higher mevalonate pathway than chemosensitive ones ,. Since the activity of ABC transporters is increased by the high content of cholesterol in the plasma membrane –, the inhibition of mevalonate pathway has efficiently overcome the MDR phenotype in vitro –. To achieve the maximal efficacy, ABC transporters have also a huge need of ATP.

ATP depleting agents exert higher cytotoxicity against MDR cells than against chemosensitive ones, inducing a phenomenon known as “collateral sensitivity” ,. Although ATP depleting agents are very effective in vitro, they are too toxic in vivo. Zoledronic acid (ZA), a clinically used aminobisphosphonate that inhibits the FPP synthase (FPPS) step in the mevalonate pathway , reduces the activity and expression of Pgp in MDR cells by decreasing the amount of cholesterol in plasma membrane and inhibiting the Pgp transcription mediated by the hypoxia inducible factor-1α (HIF-1α). Of note, HIF-1α also increases the energy metabolism and ATP synthesis in cancer cells. The major drawback of using ZA at clinically achievable concentrations is its fast uptake by bone tissue that limits the amount of the drug reaching the tumor. In previous studies we demonstrated that ZA has a negligible effect on different tumors in vivo, in consequence of its low intratumor accumulation. The use of nanocarriers such as nanoparticles (NPs) or liposomes made ZA a powerful anticancer agent by improving its intratumor delivery –.

The use of nanovectors have enhanced the anti-proliferative activity of ZA in a wide spectrum of chemosensitive tumors –, but it is not known whether NPs carrying ZA (here termed NZ) are also effective against MDR tumors. In this work we demonstrated that NZ is a strong chemosensitizing agent, owing to its peculiar effects on the energy metabolism of MDR tumors. NZ inhibits the mevalonate pathway/Ras/ERK1/2/HIF-1α/Pgp axis and sensitizes MDR cells to a broad spectrum of chemotherapeutic agents We investigated the effects of ZA and NZ in non-small cell lung cancer A549 cells and in the chemoresistant counterpart A549/MDR cells, which had higher IC 50 values towards different cytotoxic drugs (Table ) and higher expression of different ABC transporters. The NZ particles used in this study had a mean diameter of about 150 nm with a narrow size distribution (polydispersity index – P.I. – lower than 0.2).

A deep characterization of these particles have been described in. The IC 50 of ZA, NZ and self-assembling nanoparticles without ZA (blank NPs) in A549 and A549/MDR cells are provided in the Table: on the basis of these values, in all the experiments we used ZA, NZ and blank NPs at the not toxic concentration of 1 μM. IC 50 (μM) of ZA, NZ and blank NPs in A549 and A549/MDR cells In A549/MDR cells NZ lowered the IC 50 of different cytotoxic drugs, unrelated for structure, mechanism of action and efflux through specific ABC transporters, more than ZA (Table ). Similar results were obtained in chemosensitive HT29 cells and in their resistant counterpart HT29/MDR cells. NZ and – at a lesser extent ZA – reduced the expression of Pgp, but did not change the levels of the other ABC transporters. We next analyzed if NZ reduced the mevalonate pathway activity, which favors the MDR phenotype and is inhibited by ZA. NZ decreased the synthesis of cholesterol and FPP more than ZA, after 24 and 48 h; its effect was stronger in A549/MDR cells, which had a basally higher activity than A549 cells (Figure –).

In parallel, NZ lowered the activity of Ras and Ras-downstream effectors ERK1/2 (Figure ). HIF-1α, which was constitutively phosphorylated (Figure ) and bound to its DNA target sequence (Figure ) in A549/MDR cells, is a substrate of ERK. NZ reduced the HIF-1α amount, phosphorylation and DNA binding (Figure –), and lowered the transcription of the HIF-1α-target gene Pgp (Figure ) in MDR cells. By reducing HIF-1α activity, NZ decreases the glycolytic flux and the ATP levels in MDR cells Compared to A549 cells, A549/MDR cells had higher expression of the HIF-1α-target genes glucose transporter 1 ( GLUT1), hexokinase ( HK), phosphofructokinase-1 ( PFK1), aldolase-A ( ALDO-A), glyceraldehyde 3-phosphate dehydrogenase ( GAPDH), phosphoglycerate kinase ( PGK), enolase-A ( ENO-A), pyruvate kinase ( PK), lactate dehydrogenase ( LDH; Figure ), which are involved in glucose uptake and metabolism. NZ down-regulated all these genes (Figure ), as well as other canonical HIF-1α-target genes, such as vascular endothelial growth factor, erythropoietin, carbonic anhydrase IX and XII , in MDR cells. NZ reduces the expression of glycolytic genes in MDR cancer cells In keeping with the higher expression of the glycolytic genes, A549/MDR cells showed higher uptake of glucose (Figure ), higher activity of PFK-1 (Figure ), GAPDH (Figure ), enolase (Figure ), PK (Figure ) and LDH (Figure ), higher flux of glucose into the tricarboxylic acid (TCA) cycle (Figure ), higher levels of ATP (Figure ). NZ significantly reduced all these parameters more efficiently than ZA.

Again NZ was more effective in A549/MDR cells than in A549 cells (Figure –). NZ inhibits the mitochondrial metabolism and increases the reactive oxygen species production in MDR cells The synthesis of ubiquinone, whose isoprenoid tail is a side product of the mevalonate pathway, was higher in A549/MDR cells than in A549 cells (Figure ). The higher amount of ubiquinone was paralleled by the higher activity of the respiratory chain (Figure ) and by the higher level of mitochondrial ATP (Figure ). NZ and ZA reduced the electron flux and the ATP levels proportionally to their ability to decrease ubiquinone (Figure –). NZ reduces the efflux activity of ABC transporters and increases the intracellular drug retention in MDR cells Since doxorubicin is a substrate of Pgp, MRP1, MRP2, MRP3 and BCRP , we measured its efflux kinetics as a sensitive index of the activity of these transporters. As expected, A549/MDR cells had a higher Vmax of doxorubicin efflux than A549 cells (Figure ).

Neither ZA nor NZ changed the Vmax in A549 cells (Figure ). By contrast, NZ and – at lesser extent – ZA decreased the Vmax in A549/MDR cells (Figure ).

Moreover NZ increased the doxorubicin Km, suggesting that it reduced the affinity of the drug for the transporters. The intracellular accumulation of doxorubicin, carboplatin (a substrate of MRP1, MRP2, MRP4), gemcitabine (a substrate of MRP5) and mitoxantrone (a substrate of BCRP, Pgp, MRP1) were all significantly increased by NZ in A549/MDR cells (Figure –). The chemosensitizing effect of NZ is due to the inhibition of FPPS The chemosensitizing properties of NZ were not due to the use of the NPs scaffold: indeed blank NPs did not reduce the activity of mevalonate pathway, the activation of HIF-1α, the levels of ATP, the transcription of Pgp and the efflux of doxorubicin in A549/MDR cells (–). To investigate whether the properties of NZ were due to the inhibition of the ZA-target enzyme FPPS, we produced a A549/MDR subclone inducibly knocked-down for FPPS (Figure ). As expected, FPPS-silenced A549/MDR cells had extremely low levels of cholesterol, FPP and ubiquinone (–). Moreover, they had lower activity of Ras and ERK1/2 (Figure ), lower phosphorylation (Figure ) and DNA binding of HIF-1α (Figure ), lower glucose flux into glycolysis and TCA cycle (Figure ), lower mitochondrial respiratory activity (Figure ), lower levels of total (Figure ) and mitochondrial (Figure ) ATP, reproducing the same metabolic effects of NZ. NZ reverses drug resistance in human lung cancer xenografts In keeping with the in vitro results, doxorubicin and carboplatin reduced the growth of A549 xenografts, but not of A549/MDR ones.

NZ rescued the antitumor efficacy of these chemotherapeutic drugs in MDR tumors (Figure ). Of note, NZ did not increase liver, heart and kidney toxicity, as suggested by the hematochemical parameters of the animals. Similarly to what observed in NZ-treated mice, the FPPS-silencing rescued the efficacy of doxorubicin and carboplatin in A549/MDR xenografts (Figure ). DISCUSSION In this work we investigated the potential use of self-assembling NPs encapsulating ZA, here named NZ, as not toxic metabolic modifiers and inducers of collateral sensitivity against human MDR cells. NZ decreased the expression of Pgp, without changing the expression of other ABC transporters, but it chemosensitized MDR cells also to cytotoxic agents that are not Pgp substrates. Such mechanism is extremely surprising in the field of chemosensitizing agents, because up-to-day most chemosensitizer compounds inhibit one or few specific ABC transporters ,. By decreasing the synthesis of cholesterol, which is critical for the activity of Pgp , , and the activity of Ras/ERK1/2/HIF-1α-axis, which mediates the transcription of Pgp , NZ reversed the resistance towards Pgp substrates.

The chemosensitization towards cytotoxic agents that are not substrates of Pgp was due to the effects on the energy metabolism of MDR cells. Many HIF-1α-target genes involved in the glycolytic flux were up-regulated in MDR cells if compared with chemosensitive ones. This condition, which is compatible with the Warburg effect observed in many solid tumors, increased the glucose flux through the glycolysis and TCA cycle, and the intracellular levels of ATP. Also the electron flux through the mitochondrial respiratory chain and the mitochondrial synthesis of ATP were increased in MDR cells. Such increase can be explained by the higher supply of reducing equivalents through the accelerated TCA cycle and/or by the higher levels of the electron shuttle ubiquinone, which is a side product of the mevalonate pathway.

Chemoresistant cells often activate both glycolysis and oxidative phosphorylation to ensure an adequate supply of ATP , which is constantly hydrolyzed by ABC transporters. This observation is in keeping with the metabolic profile of our MDR cells, which had higher activity of both anaerobic and aerobic energy pathways and higher expression of ABC transporters than chemosensitive cells. On the other hand, MDR cells show a paradoxical hypersensitivity - the so called “collateral sensitivity” - to agents lowering ATP or inducing oxidative stress ,. Our work suggests that NZ is a strong inducer of collateral sensitivity: it reduced glucose anaerobic and aerobic metabolism, increased ROS production and lowered intracellular ATP, by decreasing the mevalonate pathway/Ras/ERKs/HIF-1α axis and the supply of ubiquinone to the mitochondrial respiratory chain. As a consequence, NZ reduced the ATP-dependent activity of ABC transporters in MDR cells, increased the intracellular retention and cytotoxicity of multiple chemotherapeutic agents in vitro and in vivo. These results are in line with previous data showing that agents depleting cellular ATP or lowering glucose uptake and oxidative phosphorylation overcome chemoresistance. Differently from other ATP depleting agents, which are highly toxic , NZ chemosensitized MDR cells at a concentration (1 μM) not toxic in our animal models and compatible with the concentration of ZA found in patients ,.

Interestingly, NZ was significantly more effective in MDR cells than in chemosensitive ones. By targeting the mevalonate pathway and the activity of HIF-1α, which are basally more active in chemoresistant cells, NZ exploited two metabolic features that are crucial to maintain the MDR phenotype. The linkage between the inhibition of the mevalonate pathway and the resulting chemosensitizing effects was demonstrated by FPPS-silenced cells, which reproduced the same phenotype of NZ-treated MDR cells. Chemoresistant cells often activate multiple survival pathways in response to stress conditions, such as JAK/STAT3 axis, Akt/mTOR axis, peroxisome proliferator activated receptor gamma-dependent pathways and cyclooxygenase 2-dependent pathways: these redundant pro-survival pathways promote cell proliferation and inhibit apoptosis, contributing to drug resistance –.

We recently observed that JAK/STAT3 axis is constitutively activated in MDR cells and that ZA inhibits it by reducing the Ras/ERK1/2 activity. Moreover, it has been reported that ZA effects can be mediated by peroxisome proliferator activated receptor gamma and cyclooxygenase 2 activity.

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We cannot exclude that ZA and NZ chemosensitized A549/MDR cells also by targeting some of these pathways. The use of drugs encapsulated within liposomes has been proposed as an effective strategy to overcome the drug resistance mediated by ABC transporters , for the different kinetics of drug release , , the different drug intracellular distribution , , the reduction of Pgp expression and activity –, the changes in the lipid environment where Pgp works ,. We excluded that the chemosensitizing effect of NZ was due to the liposomal envelope, because self-assembling NPs without ZA did not reverse the MDR phenotype. On the other hand, it is known that NZ induces greater anti-proliferative effects than free ZA on tumor cells, because the use of NPs produced a higher intratumor uptake of the aminobisphosphonate –. Also in this study, the greater efficacy of NZ over free ZA can be explained by the higher uptake of ZA when administered as NZ. Our work unveils that NPs encapsulating ZA reverse the MDR phenotype by inhibiting the mevalonate pathway and the HIF-1α-dependent signaling, two events that impair the energy metabolism and the activity of ABC transporters (Figure ).

These observations may pave the way to the pre-clinical use of NZ, in combination with other cytotoxic drugs, as the first not toxic metabolic modifier, effective against MDR tumors. Chemicals Fetal bovine serum and culture medium were from Invitrogen Life Technologies (Carlsbad, CA). Plasticware for cell cultures was from Falcon (Becton Dickinson, Franklin Lakes, NJ).

Dihydroethidium (DHE), N-acetylcysteine (NAC) and H 2O 2 were purchased from Sigma-Aldrich (Milan, Italy). ZA was a gift from Novartis (Basel, Switzerland). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Hercules, CA). The protein content of cell monolayers and lysates was assessed with the BCA kit from Sigma-Aldrich. Unless otherwise specified, all the other reagents were purchased from Sigma-Aldrich. Preparation and characterization of NZ Self-assembling NPs encapsulating ZA were prepared as previously reported.

Briefly, an aqueous solution of 18 mM CaCl 2 was added, dropwise and under magnetic stirring, to an aqueous solution of 10.8 mM Na 2HPO 4. The resulting suspension (termed CaPNPs) was filtered through a 0.22 μm polycarbonate filter (MF-Millipore, Microglass Heim, Italy) and stored at 4°C before use. ZA was then complexed with CaPNPs (to obtain CaPZNPs), at a volume ratio of 50:1, with a final ZA concentration of 50 mg/ml.

Cationic liposomes (N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride/cholesterol/1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 at a ratio of 1:1:0.5) were prepared by hydration of a thin lipid film followed by extrusion. The lipid mixture dissolved in chloroform/methanol (2:1 v/v) was added to a 50 ml round-bottom flask and the solvent was removed under reduced pressure by a rotary evaporator (Laborota 4010 digital, Heidolph, Schwabach, Germany) in nitrogen atmosphere. The resulting lipid film was hydrated with 1 ml of 0.22 μm-filtered distilled water and the resulting suspension was gently mixed in the presence of glass beads followed by incubation at room temperature for 2 h. The liposome suspension was then extruded using a thermobarrel extruder system (Northern Lipids Inc., Vancouver, BC, Canada) passing repeatedly the suspension under nitrogen atmosphere through polycarbonate membranes with decreasing pore sizes from 400 to 100 nm (Nucleopore Track Membrane 25 mm, Whatman, Brentford, UK). The liposomes were stored at 4°C.

Each formulation was prepared in triplicate. Finally, equal volumes of suspensions of the liposomes and CaPZNPs, respectively, were mixed in a glass tube and the resulting dispersion was maintained at room temperature for 10 min. NPs without ZA (blank NPs) were also prepared similarly, starting from CaPNPs and cationic liposomes. Each formulation was prepared in triplicate. The mean diameter of stealth liposomes and CaPZNPs were determined at 20°C by photon correlation spectroscopy (N5, Beckman Coulter, Miami, FL). Each sample was diluted in deionizer/filtered water and analyzed with detector at 90° angle.

Was used as measure of the particle size distribution. For each batch, the mean diameter and size distribution were the mean of three measures. For each formulation, the mean diameter and P.I. Were calculated as the mean of three different batches. The zeta potential (ζ) of the NPs surface was measured in water by means of a Zetasizer Nano Z (Malvern, UK). Data of ζ were collected as the average of 20 measurements. Encapsulation efficiency of ZA ZA dosage was carried out as previously reported.

1 ml of NPs dispersion was ultra-centrifuged (Optima Max E, Beckman Coulter) at 80,000 x g at 4°C for 40 min. Supernatants were carefully removed and ZA concentration was determined by high pressure liquid chromatography. The ZA encapsulation efficiency into CaPZNPs was calculated as (TS ZA – AS ZA)/TS ZA × 100, where TS ZA is the theoretical ZA in the supernatant and AS ZA is the actual ZA concentration in the supernatant. For each formulation, the results are the mean of measures on three different batches.

Cells Human chemosensitive non-small cell lung cancer A549 cells were cultured in HAM's F12 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine, and were maintained in a humidified atmosphere at 37°C and 5% CO 2. A549/MDR cells were generated by culturing parental A549 cells in medium containing increasing concentrations of doxorubicin for 30 passages and then maintaining cells at a final concentration of 100 nM doxorubicin. HT29 and HT29/MDR cells have been already described ,. Western blot Cells were rinsed in lysis buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl 2, 5 mM EDTA, 25 mM NaF, 1 mM NaVO 4, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, pH 7.5), sonicated and centrifuged at 13,000 x g for 10 min at 4°C. Glucose uptake, enzymatic assays and TCA cycle activity The uptake of glucose was measured as described earlier and expressed as pmol 2-deoxy-D- 3H-glucose/mg cell proteins. PFK1 assay was performed according to.

The activities of GAPDH, enolase, LDH were measured as reported in. The activity of PK was detected with the Enzymatic Assay of Pyruvate Kinase kit (Sigma-Aldrich). Results were expressed as nmol NAD +/min/mg cell proteins (for PFK1, enolase, PK, LDH) or nmol NADH/min/mg cell proteins (for GAPDH). The glucose flux through glycolysis and TCA cycle was measured as described in and expressed as pmol CO 2/h/mg cell proteins.

. Corrected online 13 October 2017 In the version of this article initially published, Lodato, M.A. Science 350, 94–98 (2015) (reference 2) was cited as an example of a single-cell sequencing study with high CG-to-TA transitions that applies heat lysis. However, that work used alkaline lysis on ice (Walsh, C.A.

And Lodato, M.A., personal communication); therefore, we have changed the third sentence of the paper from 'This pathway may explain the observed excess of such mutations in single neurons 2 compared with unamplified neuronal clones 3' to 'Amplification artifacts could, in general, explain the observed excess of such mutations in single neurons 2 compared with unamplified DNA from neuronal clones 3.' The error has been corrected in the HTML and PDF versions of the article. Fryxell, K.J. & Zuckerkandl, E. 17, 1371–1383 (2000). Science 350, 94–98 (2015).

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& Brandon Milholland These authors contributed equally to this work. Affiliations. Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, USA.

Xiao Dong., Lei Zhang., Brandon Milholland., Moonsook Lee., Alexander Y Maslov. & Jan Vijg. Department of Epidemiology & Population Health, Albert Einstein College of Medicine, Bronx, New York, USA. Tao Wang. Department of Ophthalmology & Visual Sciences, Albert Einstein College of Medicine, Bronx, New York, USA. Authors.

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Contributions J.V., L.Z. Conceived this study and designed the experiments. Performed the experiments. Developed the software.

Analyzed the data. J.V., X.D., L.Z., B.M. Wrote the manuscript. Competing interests X.D., L.Z., M.L., A.M.

Are cofounders of SingulOmics Corp. Corresponding author Correspondence to.