The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

Abstract

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Introduction

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a critical event in many physiological and pathological processes such as tumor growth and metastasis [1]. In fact, in order to grow, a tumor needs to develop new blood vessels once it reaches the dimension of 2 mm³ [2]. Angiogenesis is a multi-step process which involves endothelial cell proliferation, survival, and migration. Several molecules implicated in angiogenesis have been identified including growth factors (e.g. vascular endothelial growth factor, fibroblast growth factors and angiopoietins), integrins, notch receptors and their ligands, as well as molecules involved in mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathways [3], [4], [5]. Since angiogenesis plays a key role in tumor growth, targeting tumor angiogenesis represents a promising approach in cancer therapy. Accordingly, targeting the vascular endothelial growth factor (VEGF) has shown clinical efficacy and has been approved for the treatment of various cancers [6]. However, the benefits of anti-angiogenic therapies are transitory and most of the tumors eventually progress under therapy. Therefore, a strong need exists to design new therapeutic strategies that confer enduring anti-angiogenic effects.

mTOR is a key regulator of cell growth, proliferation, and survival as being part of two distinct complexes, mTORC1 and mTORC2. While mTORC1 is composed of five different components: mTOR, raptor, mLST8, PRAS40 and deptor, mTORC2 consists of mTOR, rictor, mSin1, mLST8, deptor and protor-1 [7]. mTORC1 phosphorylates, among others, S6K1 and 4E-BP1 resulting in the regulation of translation initiation and protein synthesis. mTORC2 phosphorylates Akt, SGK and PKC and is involved in cell proliferation, survival and cytoskeletal organization [8]. Numerous studies demonstrated that blocking mTOR with rapamycin reduces tumor angiogenesis [9]. Indeed, rapamycin inhibits the functions of endothelial cells relevant to angiogenesis in vitro and reduces angiogenesis in several models in vivo [10], [11], [12]. However, emerging evidence has shown that targeting mTOR also stops a negative feedback loop which results in the activation of proliferative signals such as Akt or MAPK that reduce the growth inhibitory properties of mTOR inhibitors [13]. The effect of mTOR inhibition on MAPK activity in endothelial cells as well as its relevance to angiogenesis has however not been determined. In this study, we evaluated the consequences of mTOR inhibition either by ATP-competitive inhibitors of mTOR or by rapamycin on MAPK activity in endothelial cells. We also explored the anti-angiogenic effects of mTOR inhibition in combination with MAPK inhibition both in vitro and in vivo.