Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway

Abstract

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB-2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour-free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour-associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin-10 was drastically down-regulated in favour of interferon-γ production. Peritoneal macrophages and tumour-associated macrophages of ZA-treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour-associated macrophages and their reverted polarization from M2 to M1 phenotype.

Fig 1

ZA administration hampers ErbB-2 driven mammary carcinogenesis. Seven-week-old BALB-neuT female mice started to receive weekly i.v. courses of ZA 100 μg/kg (black line, n= 7) or saline (dotted grey line, n= 5). (A) Mice receiving i.v. ZA displayed a significant extension in the median tumour-free survival (P= 0.0004, log-rank, Mantel-Cox test) (24.5 weeks versus 19 weeks, P < 0.0004, Student’s t-test), as compared to control mice. (B) ZA antitumour effect on tumour multiplicity resulted in a statistically significant reduction of the mean number of tumours from week 21 to week 30 (P= 0.002, Student’s t-test). (C) ZA antitumour effect on carcinoma growth rate. The mean tumour diameter of each control and ZA-treated mice was evaluated in function of weeks. A statistically significant prolongation of time required by ZA-treated tumours to grow from 1 to 6 mm was observed (30.4 ± 1.8 days versus 41.1 ± 2.0 days; P= 0.001, Student’s t-test). (D) ZA-treated mice displayed a significant extension in the median overall survival (P= 0.0009, Log-rank, Mantel-Cox test) as compared to control mice. (E, F) whole mount images of representative 18-week-old BALB-neuT mammary glands from control (e) and ZA-treated (f) mice. Several large tumour masses were evident in control mice, whereas fewer and smaller early-stage lesions, mostly confined to the nipple area, were observed in ZA-treated mice. Black arrows indicate foci of carcinomas. N = mammary lymph node. Scale bar, 2 mm.

Fig 2

Clinically compatible doses of ZA inhibits the activity of the Mev pathway, but not the proliferative rate of TUBO cells in vitro. (A) ZA induces a dose-dependent inhibition of FPP synthase in TUBO cells, as shown by the significant intracellular reduction of FPP, ubiquinone and cholesterol, and the significant increase of IPP (P-values from <0.02 to <0.001 for all ZA concentrations, Student’s t-test). Results are expressed as the mean ± S.E.M. of three independent experiments. (B) Western blot analysis of prenylated (P) and unprenylated (U) Ras and pull-down assay for Ras-GTP in TUBO cells cultured in the absence (CTRL), or in the presence of 1, 10 and 100 μM ZA. The specific farnesyl transferase inhibitor (FTI) 277 (10 μM) was used as a positive control of Ras inhibition. The expression of GAPDH was used as a control of equal protein loading. Results are from one representative out of three experiments. (C) ZA inhibition of TUBO cells proliferation was assessed by measuring BrdU incorporation. TUBO cells were incubated in quadruplicates without ZA (♦) or with ZA at 1 μM (▪), 10 μM (▴) or 100 μM (▾). BrdU incorporation was measured after 8, 48, 96 and 102 hrs as the mean optical density measured for each quadruplicate. As shown, ZA 1 μM never affected tumour cell proliferation, whereas 10 μM ZA induced a time-dependent inhibition and 100 μM ZA a complete abrogation of BrdU incorporation. Results are expressed as mean ± S.E.M. of three experiments.

Fig 3

ZA impairs TAM recruitment, angiogenesis and VEGF release at the tumour site. Immunohistochemical analysis of the tumour site showed a strong reduction in interstitial CD11b⁺ TAMs in ZA-treated (B) as compared to control mice (A). CD31 immunostaining showed substantial neovascularisation in 18-week-old control mice (C), which was clearly inhibited by ZA treatment (D). Reduced VEGF expression was detected in TAMs of ZA-treated mice (F) as compared to control mice (E), as revealed by colocalization of VEGF (green) and CD11b (red). A strong reduction in the amount of VEGF produced by tumour cells was also observed in ZA-treated mice as compared to control mice. Arrowheads indicate some of the VEGF expressing tumour cells. Arrows indicate some of the VEGF expressing TAMs. Scale bars, 80 μm.

Fig 4

ZA’s antitumour effect is associated with its ability to reverse TAM polarization from M2 to M1, and is dependent on IFN-γ. Immunohistochemical analysis of representative mammary glands of 18-week-old control (A, C) versus ZA-treated (B, D) BALB-neuT mice. (A, B) Anti IL-10 staining (green) showed a clear reduction of IL-10 release in the tumour microenvironment of ZA-treated mammary tumours. (C, D) A significant enhancement of IFN-γ release (green) is observed in the microenvironment of ZA-treated mammary tumours. Scale bars, 40 μm. (E, F) ZA antitumour activity in BALB-neuT/IFN-γ KO. BALB-neuT/IFN-γ KO female mice received weekly i.v. courses of ZA (black line, n= 6) or saline (dotted grey line, n= 7). ZA-treated BALB-neuT/IFN-γ KO mice displayed the same tumour incidence (E) and tumour multiplicity (F) as control mice.

Fig 5

Modulation of macrophages functions by low-dose ZA. Cells were isolated from control BALB-neuT mice and left untreated (CTRL) or treated ex vivo for 24 hrs with 1 μM ZA (ZA 24 hrs), or isolated from ZA-treated mice (ZA) as reported in the ‘Materials and methods’ section. (A) Inhibition of FPP synthase. A dose-dependent reduction of intracellular FPP, ubiquinone and cholesterol (always *P < 0.01, Student’s t-test), together with a significant increase of IPP (*P < 0.05, Student’s t-test) were displayed by ZA 24 hrs and ZA macrophages as compared to CTRL macrophages. Results are shown as mean ± S.E.M. of three experiments. (B) Inhibition of Ras prenylation and Ras activity. Ras pull-down assay and band shift assay in CTRL, ZA 24 hrs and ZA macrophages. (C, D) Activation of NF-κB. (C) Western blot analysis of IκB and GAPDH in CTRL, ZA 24 hrs and ZA macrophages. N11 glial cells cultured with (LPS N11) or without (CTRL N11) LPS 20 μg/ml were used as controls. (D) NF-κB translocation detected by EMSA on nuclear extracts from cell preparations run in lanes labelled as in (C). (E, F) Induction of iNOS expression and nitrite production. (E) Western blot analysis of iNOS and GAPDH in protein lysates from cell preparations run in lanes labelled as in (C and D). Results shown in (B–E) are from one representative out of three experiments. (F) Nitrite secretion by CTRL, ZA 24 hrs and ZA macrophages (MAC). Untreated (N11 CTRL) or LPS-treated (N11 LPS) N11 glial cells were used as controls. Results are expressed as mean ± S.E.M. of three experiments. The difference between ZA 24 hrs and CTRL macrophages is statistically significant (*P < 0.01, Student’s t-test). (G) Nitrotyrosine expression (arrowheads) in the tumour stroma of ZA-treated (right panel) versus control (left panel) BALB-neuT mice. Scale bars, 40 μm.