Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Yan Ding,Rick V Hay,James H Resau,Zhongfa Zhang,Arthur E Frankel,Nicholas S Duesbery,Eric Kort,John J Young,Kyle Furge,Elissa A Boguslawski,Kim Hardy,Bree D Berghuis

doi:10.1158/1535-7163.mct-07-2229

Abstract

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.

Highlights

Mitogen-activated protein kinase (MAPK) kinase (MKK) signal transduction pathways are critical for many aspects of normal cell function including cell cycle progression and differentiation [1]
To test whether MKK signaling was required for the release of angioproliferative growth factors from fibrosarcoma-derived cell lines, we examined the effects of lethal toxin (LeTx) on cytokine release from these cells in vitro using ELISA
Our analysis revealed that both LeTx and U0126 decreased the release of a number of angioproliferative factors, including basic fibroblast growth factor (bFGF), IL-8, and vascular endothelial growth factor (VEGF), the same cytokines that have been noted as strong correlates of disease-free and overall survival in other tumors such as melanoma [26], but this effect was far from global as the release of several cytokines including a-fetoprotein and tumor necrosis factor-a was unaffected

Summary

Introduction

Mitogen-activated protein kinase (MAPK) kinase (MKK) signal transduction pathways are critical for many aspects of normal cell function including cell cycle progression and differentiation [1]. Activated MAPK or elevated MAPK expression has been detected in a variety of human tumor, where it is believed they promote tumor growth and metastasis [2]. These observations have spurred the development of drugs that target the MKK pathways as potential cancer therapeutics [3]. Elevated levels of active MAPK have been detected in vitro in cell lines derived from fibrosarcoma (4 – 6), rhabdomyosarcoma [7], osteosarcoma [8], and Kaposi’s sarcoma [9, 10], little is known of their activities in vivo. Based on our earlier work with transformed fibroblasts [11], we hypothesized that signaling through multiple MKK pathways is essential for the growth and vascularization of xenograft sarcomas

Methods

Results

Conclusion