Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis

G Bergers

R Brekken

G McMahon

T Vu

S Itohara+2 authors

^{Department of Biochemistry and Biophysics, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA}

^{Hormone Research Institute, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA}

^{Department of Vascular Biology, The Hope Heart Institute, 1124 Columbia Street, Seattle, Washington 98104, USA}

^{SUGEN Inc., 230 East Grand Avenue, South San Francisco, California 94080-4811, USA}

^{Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA}

^{Institute for Virus Research, Kyoto University, 53 Kawahara, Syogo-in, Sakyo-ku, Kyoto 606-8397, Japan}

^{Sankyo Co. Ltd, 2-58 Hiromachi 1-Chome, Shinagawa-Ku, Tokyo 140-8710, Japan}

^{Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75235-9111, USA}

^{Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan}

^{Department of Anatomy, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA}

^{UCSF Comprehensive Cancer Center, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA}

^{ude.fscu.mehcoib@hd}, Correspondence and requests for materials should be addressed to D.H.

^{Current address: Shionogi Institute for Medical Science, Shionogi & Co. Ltd, 12–4 Sagisu 5-Chome, Fukushima-ku, Osaka, 553-0002 Japan}

Abstract

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Abstract

Cancers arise through multistep pathways, acquiring necessary capabilities through changes in the programme of the cancer-cell genome, and by recruiting and mobilizing essential accessory cell types¹. Among the accessories are capillary endothelial cells, which are activated to produce new blood vessels; this capability for angiogenesis is critical for expansive tumour growth and metastasis²4.

In one approach to elucidating cancer mechanisms, we have studied a transgenic mouse model (RIP1-Tag2) of multistage carcinogenesis, in which every mouse develops islet tumours of the pancreas by 12–14 weeks of age as a result of expression of the SV40 T antigen (Tag) oncogene in insulin-producing β-cells⁵. Of the ~400 islets that express this oncogene, only 1–2% develop into adenomas and carcinomas, indicating that rate-limiting changes (steps) may be necessary for manifestation of the tumour stages⁵. The induction of angiogenesis is a discrete step in this multistage pathway⁶. Initially, hyperproliferative islets with quiescent vasculature emerge; these nodules (reaching 50% of all islets) have characteristics of in situ carcinoma lesions. Subsequently, a subset (~20%) of hyperproliferative islets switch on angiogenesis, as shown by endothelial sprouting, mitosis, microhaemorrhaging and vascular dilation in vivo, and by their ability to elicit an angiogenic response in vitro⁶. All tumours similarly show evidence of angiogenesis. The focal nature and statistics of the induction of angiogenesis in small precursor lesions indicate that the angiogenic switch may be governed by a discrete regulatory event.

Although a popular hypothesis for angiogenesis induction involves upregulation of angiogenic genes in response to hypoxia or expression of oncogenes⁷9, our analysis of angiogenic genes reveals a surprising conundrum —VEGF and acidic fibroblast growth factor (aFGF) are both constitutively expressed in normal islet β-cells of control mice, and in all stages of the RIP1-Tag2 islet carcinogenesis pathway¹⁰; similarly, the two VEGF receptors flk-1 (VEGF-R2) and flt-1 (VEGF-R1) are constitutively expressed in the islet vasculature before and after the angiogenic switch¹¹. Expression of such pro-angiogenic molecules in this neuroendocrine tissue may partly serve to control maintenance and homeostasis of the dense network of fenestrated endocrine capillaries that facilitates glucose homeostasis¹². How, then, is angiogenesis facilitated during islet carcinogenesis? Here we show that the switch from vascular quiescence to angiogenesis involves a matrix metalloproteinase (MMP), gelatinase B/MMP-9, which is upregulated in angiogenic islets and tumours, rendering VEGF more available to its receptors. Remarkably, MMP-9 is not expressed in tumour cells, but rather in a small number of cells that are proximal to the vasculature.

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