An amino-bisphosphonate targets MMP-9–expressing macrophages and angiogenesis to impair cervical carcinogenesis
Enrico Giraudo, Masahiro Inoue, Douglas Hanahan
Enrico Giraudo, Masahiro Inoue, Douglas Hanahan
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Article Oncology

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.

Authors

Enrico Giraudo, Masahiro Inoue, Douglas Hanahan

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MMP-9 expression and activity is upregulated in macrophages during tumor...
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).