(C) Three cases of 5 patients tested. of the cdk inhibitors p27kip1 and p21cip1. Under conditions that reproduce the biomechanical fluidic environment of the BM, CCI-779 is usually equally effective in inhibiting BM endothelial-cell proliferation. Finally, simultaneous blockade of mTOR and NF-B pathways synergize to significantly inhibit or abrogate the proliferative responses of BM endothelial cells to mitogenic stimuli. This study identifies mTOR as an important pathway for the proangiogenic stimulation of BM endothelium. Modulation of this pathway may serve as a valid therapeutic intervention in BM malignancies evolving in association with an angiogenic phenotype. Introduction The formation of new vessels in normal and pathologic conditions requires the activation of quiescent endothelial cells (ECs), a process brought R18 on by proangiogenic factors that are generally R18 elevated in cancer patients.1,2 Bone marrow endothelial cells R18 (BM-ECs) and their precursors play important functions in the neovascularization associated with malignancies developing in the bone marrow (BM)3-5 and seem to be implicated in cancers evolving in other tissues.6,7 Of interest, ECs purified from tumor-infiltrated BM exhibit an activated, angiogenic phenotype.8 Studies in a leukemia model showed that specific targeting of the EC markedly inhibited tumor development, suggesting a critical role for the BM endothelium in leukemia biology.9 More recently, it has been shown that endothelial microdomains in the BM play important roles in leukemia-cell homing and maintenance.10 Taken together, these studies suggest that BM endothelium plays an important role in the development and maintenance of tumors evolving in the BM, and that strategies targeting BM-ECs may provide a therapeutic advantage. A significant number of reports have evaluated the molecular events and pathways involved in EC responses to extrinsic stimuli. PI3K/Akt, MAPK/ERK, Jak/STAT, and small GTPases, as well as NF-B pathways,11-13 seem to play significant functions in the endothelial-cell responses to mitogenic stimuli and in the switch to an angiogenic phenotype. How these multiple, distinct signals are integrated within ECs needs further evaluation. Moreover, most signaling studies were performed in umbilical vein endothelial cells (HUVECs), and little is known around the signaling machinery activated in other ECs, particularly in BM-ECs. This is relevant, as ECs from different tissues/organs, and even within the same tissue, possess variable phenotypic, metabolic, and functional properties, including their responsiveness to extrinsic stimuli.14,15 For example, BM-ECs differ from HUVECs in their ability to support adhesion of hematopoietic progenitors16 and cancer cells.17 Also, ECs from different tissue beds respond differentially to biomechanical stimuli, 18 which translates into activation of distinctive transcriptional profiles and results in different functional phenotypes.19 The mammalian target of rapamycin (mTOR) pathway coordinates cell growth and cell-cycle progression by integrating growth Mouse monoclonal to p53 factor signals and nutrient availability,20,21 modulating the protein translation machinery through inhibition of 4E-BP1 and activation of S6K1 and its substrate S6 ribosomal protein (S6RibP). The mechanism(s) involved in growth factor stimulation of mTOR pathway are still a matter of controversy. However, recent studies indicate that mTOR nutrient sensing ability crosstalks with PI3K-regulated growth factor signaling. In this model, PI3K lays both upstream and in parallel to mTOR and shares common downstream targets.20,21 The mTOR-specific blocker rapamycin exerts antitumor activity by disrupting tumor angiogenesis.22,23 Also, mTOR blockade by rapamycin induces PKB/Akt degradation,24 whereas VEGF-induced activation of PI3K/Akt/mTOR stabilizes PKB/Akt, promoting EC survival. Here, we show that activation of BM endothelium by proangiogenic factors triggers mTOR, activating its downstream pathways 4E-BP1 and S6K1. Specific blockade of mTOR by rapamycin or CCI-779 abrogates the cytokine- or leukemia-promoted stimulation of mTOR pathway in BM-ECs and inhibits their proliferation by modulating crucial mediators of cell-cycle progression. The inhibitory effects of CCI-779 on BM-ECs are also observed under flow conditions that recapitulate the biochemical environment of the BM. Finally, simultaneous blockade of mTOR and NF-B pathways results in the synergistic inhibition of BM endothelium. Materials and methods Endothelial cells and.