An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis

Abstract

A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA's actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an "unconventional" MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.

Figures

Figure 1

Stages of cervical carcinogenesis and angiogenic profile of HPV/E2 mice. (A) H&E staining shows the steps of tumor progression. (B) Blood vessels (red) were detected with anti–CD-31 Ab; nuclei were visualized by DAPI (blue). The number of vessels per field were: N/E2, 8.2 ± 1.2; CIN-1/2, 14.8 ± 1.1; CIN-3, 32.2 ± 1.7; SCC, 35.2 ± 1.8. Significant differences were observed between CIN-3/SCC and CIN-1/2 (P < 0.01), CIN-3/SCC and N/E2 (P < 0.01), and CIN-1/2 and N/E2 (P < 0.01), but not between CIN-3 and SCC (P = 0.309). (C) VEGF-A/VEGF-R2 complex was detected with GVM39 Ab (green) and ECs with anti–Meca-32 Ab (red). VEGF/VEGF-R2 complex colocalizes with most vessels in CIN-3 and SCC and with a subset in CIN-1/2 (arrows). Arrowheads in CIN-1/2 indicate vessels that do not bind GVM39 Ab. Minimal GVM39 staining was detected in N/E2 stroma. The percentage of VEGF-A/VEGF-R2–labeled vessels was quantitated: N/E2, 8.6 ± 1.2; CIN-1/2, 44.1 ± 2.9; CIN-3, 90.2 ± 3.1; SCC, 91.4 ± 3.4. Significant differences were observed between CIN-3/SCC and CIN-1/2 (P < 0.01), CIN-3/SCC and N/E2 (P < 0.01), and CIN-1/2 and N/E2 (P < 0.01). CIN-3 and SCC were not significantly different (P = 0.690). Values are mean ± SEM. P values were calculated using the Wilcoxon test. Scale bars in A and C: 50 μm (N/E2); 25 μm (CIN-1/2, CIN-3, and SCC). Scale bars in B: 50 μm. E, normal or dysplastic cervical epithelia; S, stroma; T, tumor cells.

Figure 2

MMP-9 expression and activity is upregulated in macrophages during tumor progression. (A) RT-PCR analysis revealed increased MMP-9 expression in CIN-3 and SCC as compared with CIN-1/2. No MMP-9 expression was detected in normal cervix (not shown) or in N/E2. MMP-2 was equally expressed at all stages. (B) Immunohistochemical analysis using an anti–MMP-9 Ab revealed no MMP-9 in N/E2 cervix and minimal expression in CIN-1/2 lesions (arrows); in contrast, MMP-9 was detected in the stroma proximal to CIN-3 lesions and tumors. (C) Zymography showing gelatinase activity in tissue lysates of different stages. Both pro–MMP-9 (inactive form, 105 kDa) and active MMP-9 (95 kDa) were upregulated in CIN lesions and tumors as compared with controls. pro–MMP-2 (72 kDa) was slightly increased during progression, but no active MMP-2 (62 kDa) was detected. (D) Gelatinase activity was measured using a fluorescin-gelatin assay in the absence or presence of the MMP inhibitor 1,10 Phe (4 mM). Statistically significant increases in gelatinase activity were observed in CIN lesions and tumors compared with controls: CIN-3/SCC versus CIN-1/2 (P < 0.01); CIN-3/SCC versus N/E2 (P < 0.01); CIN-1/2 versus N/E2 (P < 0.01); CIN-3 and SCC were not significantly different (P = 0.102). Values are mean ± SEM. P values were calculated using the Wilcoxon test. (E) Colocalization of MMP-9 (red) and macrophages (CD-68, green) was observed in the stroma underlying CIN-3 and surrounding SCC. Few MMP-9–expressing macrophages were detected in stroma adjacent to CIN-1/2 lesions (arrows). No MMP-9 expression was observed in macrophages in N/E2 mice (arrowheads). Scale bars: 50 μm (N/E2); 25 μm (CIN-1/2, CIN-3, and SCC).

Figure 3

BB94 reduces tumor incidence and tumor volume in HPV/E2 mice. Preclinical trials using the broad-spectrum MMPI BB94 (batimastat) in HPV/E2 mice. (A) Decrease of tumor incidence (51% reduction) in 7-month-old BB94-treated (n = 15) mice compared with control mice (n = 20). (B) Reduction of tumor volume in BB94-treated animals (63% reduction; **P < 0.01) compared with controls. Values are mean ± SEM. P values were calculated using the Wilcoxon test. Drug treatment, tumor volume, and histological scores were determined as described in Methods.

Figure 4

ZA inhibits angiogenesis and reduces tumor incidence and growth. (A) Perfusion of HPV/E2 control mice and ZA-treated mice (6 weeks of treatment; PT) with fluorescin-lectin revealed dramatic changes in the 3-dimensional organization of the vasculature proximal to CIN-3 lesions of treated mice at 5 months of age. (B) Vessels’ density, as assessed by Meca-32 immunostaining, was significantly reduced in ZA-treated mice as compared with controls (56% reduction). Results are mean ± SEM of five fields per mouse from a total of eight mice. (C) CIN-2/3 lesion-bearing mice (T0, beginning of treatment; 3.5 months old) treated with ZA or vehicle for 6 weeks (PT) showed a 55% reduction in the tumor incidence at the end of the treatment (T1; 5 months old) (n = 16 control, n = 10 ZA-treated). (D) Tumor volume of ZA-treated mice was reduced by 61% compared with untreated controls (PT; n = 16 control, n = 10 ZA-treated). (E) Mice bearing SCC (T0; 5 months old) treated with ZA for 1 month (T1) in a RT showed a 57% decrease in tumor volume (n = 15 control, n = 10 ZA-treated). Values are mean ± SEM. **P < 0.01; #P < 0.001. P values were calculated using the Wilcoxon test. Tumor volume and histological scores were determined as described in Methods. Scale bar: 50 μm.

Figure 5

ZA induces apoptosis in epithelial cells and ECs in tumors and CIN-3 lesions. (A–C) A significant increase in apoptosis in cervical carcinomas was observed in ZA-treated mice compared with controls (A) after 6 weeks of treatment (PT) as revealed by caspase-3 immunostaining (#P < 0.001, Wilcoxon test). Representative sections of tumors from treated and untreated mice are shown in B and C. (D–F) Similar increase in apoptosis was detected in CIN-3 epithelium of ZA-treated mice as compared with controls (D); treated versus untreated lesions are exemplified in E and F. Five fields per mouse were counted to assess the caspase-3–positive cells. Values are mean ± SEM. Increased apoptosis was observed in vessels in CIN-3 lesions of ZA-treated mice (G–I) compared with controls (J–L) as detected by colocalization of Meca-32 (green) with caspase-3 (red). Arrows indicate apoptotic ECs. Scale bars: 50 μm (B and E); 25 μm (C, F, and G–I).

Figure 6

ZA inhibits MMP-9 expression and activation in macrophages. (A) Reduced MMP-9 expression was detected in macrophages in the stroma adjacent to CIN-3 in ZA-treated mice compared with controls as revealed by colocalization of MMP-9 (green) and CD-68 (red). Arrows show MMP-9–expressing macrophages; arrowheads indicate macrophages that do not express MMP-9. (B) Quantification of double-labeled MMP-9+/CD-68+ cells (5 fields per mouse) revealed a 71% reduction in the PT (n = 8 control, n = 6 ZA-treated) and a 68% reduction in the RT (n = 10 control, n = 8 ZA-treated). Similar results were obtained with double-labeled MMP-9+/F4/80+ cells (not shown). #P < 0.001. (C) Gelatinase activity in tissue extracts was measured by incubation with fluorescin-conjugated gelatin in the absence or presence of the MMP inhibitor 1,10 Phe (4 mM). Gelatinase activity was lower in ZA-treated compared with control cervixes in both PT and RT. **P < 0.01 versus control. N/E2 indicates estrogen-treated normal cervix. (D) Zymography showing the pro- and active forms of gelatinases in tissue lysates from both control and ZA-treated mice. Pro- and active forms of MMP-9 and MMP-2 are indicated by arrows. Scale bar: 25 μm. Values are mean ± SEM. P values were calculated using the Wilcoxon test.

Figure 7

ZA inhibits the formation of VEGF/VEGF-R2 complexes on the neoplastic vasculature, phenocopying a MMP-9 gene KO. (A) Decrease in tumor incidence in 7-month-old HPV/E2-MMP-9–/– (66% reduction; n = 25) compared with HPV/E2-MMP-2–/– (n = 15) and control mice (n = 40). (B) Reduction of tumor volume in 7-month-old HPV/E2-MMP-9–/– (76% reduction; n = 25) compared with HPV/E2-MMP-2–/– and control mice. Tumor volume and histological scores were determined as described in Methods. (C–G) VEGF/VEGF-R2 complex was detected by immunohistochemistry using GVM39 Ab (green), while the ECs were visualized with the anti–Meca-32 Ab (red). Significant reduction in the VEGF/VEGF-R2 complex was observed on vessels in HPV/E2-MMP-9–/– (77% reduction compared with controls; n = 15) and ZA-treated (73% reduction compared with controls; n = 8) mice compared with HPV/E2-MMP-2–/– (n = 10) and controls (n = 20) (C). Representative analyses of tumors are shown in D–G. Arrows indicate the VEGF/VEGF-R2 complex associated to the vessels. Scale bar: 25 μm. Values are mean ± SEM. #P < 0.001. P values were calculated using the Wilcoxon test.