New microtubulin inhibitor MT189 suppresses angiogenesis via the JNK-VEGF/VEGFR2 signaling axis

Highlights

• MT189 inhibits angiogenesis in vitro, ex vitro and in vivo.

• MT189 abrogates the VEGFR2-Src pathway by reducing VEGF expression and secretion.

• MT189 leads to cell-cell junction potentiation.

• MT189 attenuates focal adhesion turnover.

• MT189 suppresses angiogenesis via the JNK-VEGF/VEGFR2 signaling axis.

Abstract

The microtubulin inhibitor MT189 possesses anticancer activity and has been shown to overcome multidrug resistance. Here, we report that MT189 also inhibits angiogenesis. MT189 inhibited the proliferation, migration and differentiation of endothelial cells, with or without VEGF stimulation, and suppressed microvessel formation ex vivo and in vivo. MT189 reduced VEGF expression and secretion in both tumor and endothelial cells, under either hypoxic or normoxic conditions. The activation of VEGFR2 and downstream Src was thus abrogated in the MT189-treated endothelial cells. MT189 subsequently stabilized endothelial cell-cell junctions consist of VE-cadherin, β-catenin, vinculin, and actin. MT189 also disrupted endothelial cell-matrix junctions by inhibiting the turnover of focal adhesions containing FAK, paxillin, vinculin, and actin. Inhibition of JNK reversed MT189-mediated inhibition of endothelial migration and differentiation, JNK activation, the reduction of VEGF expression and secretion, and the decrease of Src and FAK phosphorylation. These results indicate that MT189 suppresses angiogenesis by reducing endothelial proliferation, migration, and differentiation via the JNK-VEGF/VEGFR2 signaling axis. Together with our previous report showing that MT189 exhibited anticancer activity via the JNK-MCL-1 pathway, these new findings further support MT189-based drug development for cancer therapy.

Introduction

Angiogenesis inhibitors are utilized clinically in cancer therapy because angiogenesis promotes tumor progression by inducing rapid expansion, invasion and metastasis [[1], [2], [3]]. Tubulin inhibitors have been reported to effectively inhibit tumor angiogenesis [2,3]; however the mechanisms behind this effect remain unknown.

Previously, we reported that the MT compounds including MT7, MT119, and MT189 are novel tubulin inhibitors, among which MT189 displayed the most potent anti-proliferative activity against cancer cells [[4], [5], [6], [7], [8]]. Treatment with MT189 resulted in similar levels of cell death in multidrug-resistant cancer cells and their respective parental cells. Moreover, MT189 elicited in vivo anticancer effects at a dose that had no obvious effect on body weight in the tested animals [8]. Importantly, MT189 has been shown to activate the MEKK1/TAK1- MKK4-JNK signaling pathway [8]. In previous studies, we demonstrated that the activation of JNK drove the phosphorylation of the transcription factor c-Jun, which resulted in a reduction of non-phosphorylated c-Jun [9]. Consequently, the key angiogenesis factor hypoxia-induced factor 1α (HIF-1α), was degraded, leading to inhibition of angiogenesis [[9], [10], [11]]. Therefore, this study aimed to determine the anti-angiogenic properties of MT189 and the mechanisms behind its effects.

Vascular endothelial cells play a critical role in tumor angiogenesis. Signaling initiated by vascular endothelial growth factor (VEGF), which is secreted from both tumor and endothelial cells, and its corresponding receptors (VEGFR1, 2 and 3; primarily VEGFR2) drives proliferation, migration, and differentiation of endothelial cells during the production of new blood vessels [[1], [2], [3]]. In particular, the binding of VEGF leads to the phosphorylation and activation of VEGFR2, subsequently activating Src kinase. Activated Src promotes focal adhesion (FA) turnover by activating FA complexes consisting of focal adhesion kinase (FAK) and paxillin [12]. In addition, activated VEGFR2 and Src cause the internalization of VE-cadherin and thus disrupt adherens junctions, weakening cell-cell junctions [13,14]. Consequently, the Src signaling triggered by the binding of VEGF to VEGFR2 stimulates migration of endothelial cells, facilitating angiogenesis.

In this study, we first investigated the inhibition of angiogenesis by MT189 in in vitro, ex vivo, and in vivo models [3]. We then examined the effects of MT189 on VEGF-stimulated angiogenesis, the expression and secretion of VEGF, and signal transduction. Further, we focused on the effects of MT189 on F-actin cytoskeletal rearrangement and endothelial cell adhesion via the VEGFR2-Src signaling. Finally, we explored whether and how the MT189-driven activation of JNK contributed to its anti-angiogenic effects. Our results revealed the potent anti-angiogenic activity of MT189 and its possible mechanisms of action.