Clinical Neuroendocrinology and Neuroendocrine Tumors

Background/Aim: Tumors exhibiting constitutively activated PI(3)K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3)K/Akt/mTOR signaling. Methods: BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. Results: RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. Conclusion: In sumReceived: November 16, 2006 Accepted after revision: January 18, 2007 Published online: February 19, 2007 Christoph J. Auernhammer, MD Department of Internal Medicine II, Grosshadern Ludwig-Maximilians University of Munich, Marchioninistrasse 15 DE–81377 Munich (Germany) , Tel. +49 89 70950, Fax +49 89 700 4418 E-Mail [email protected] © 2007 S. Karger AG, Basel 0028–3835/07/0851–0054$23.50/0 Accessible online at: www.karger.com/nen Antiproliferative Effects of RAD001 on Neuroendocrine Tumor Cells Neuroendocrinology 2007;85:54–60 55 tidylinositol-3-dependent kinase1 (PDK1). Activated Akt promotes resistance to apoptosis and unrestricted cell growth through coordinated phosphorylation of multiple downstream targets. One major effector of Akt signaling is the serine/threonine protein kinase mammalian target of rapamycin (mTOR). Complexed with regulatory-associated protein of mTOR (raptor) and LST8 in mTORC1, mTOR acts as a key integrator of signals from growth factors and nutrients (e.g. amino acids and glucose) [1, 2] . Two well-characterized mTOR substrates are eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), both regulating transcription and translation initiation of critical growth genes. Activated p70S6K also mediates phosphorylation and subsequent proteasomemediated degradation of IRS-1, the major substrate of the IGF-I/insulin receptors. Thus, p70S6K forms part of a powerful negative feedback mechanism of PI(3)K/Akt/ mTOR signaling [3] . Activating mutations in one or another PI(3)K/Akt/ mTOR pathway component, e.g. PTEN, occur in a wide range of tumors, contributing to resistance to apoptosis and unrestricted cell growth. Significantly, tumors harboring such mutations are hypersensitive to mTOR inhibitors [4, 5] . The rapamycin-derivative RAD001 (40O-(2-hydroxyethyl)-rapamycin, Novartis Pharma) is a potent, orally bioavailable mTOR inhibitor. RAD001 induces growth inhibition in a variety of tumor cell lines in vitro and a range of animal models of cancer [6–9] . Moreover, RAD001 has been shown to sensitize tumor cells to conventional therapeutic antitumor agents and radiation [7, 10–12] . RAD001 is presently being investigated in clinical phase II trials in various tumor entities, including NETs. NETs represent a rare and heterogeneous category of tumors. The majority of NETs show already local or distant metastasis at the time of diagnosis [13, 14] . Advanced stages are characterized mainly by hepatic metastases with a 5-year survival rate of less than 50% [14–16] . Currently available antiproliferative strategies against NETs of the GEP are only of moderate efficacy. While well-differentiated NETs of pancreatic origin demonstrate modest sensitivity to conventional chemotherapy regimens, no established chemotherapy protocol exists for NETs of midgut origin [15, 17–19] . Since lost expression of the PI(3)K antagonist PTEN occurs in 54% of poorly differentiated neuroendocrine carcinomas and 76% of all NETs display constitutive Akt phosphorylation [20, 21] , a majority of NETs is likely to be accessible to targeted antiproliferative therapy with mTOR inhibitors. Here, we evaluate the in vitro effects of RAD001 on BON cells. This cell line, derived from a human pancreatic neuroendocrine tumor, exhibits constitutive Akt/mTOR phosphorylation due to an autocrine IGF-I loop [22–24] . We demonstrate that RAD001 induces potent antiproliferative effects due to G0/G1 cell cycle arrest and apoptosis. In summary, our data indicate the novel mTOR inhibitor RAD001 to be a promising agent for targeted antiproliferative NET therapy. Materials and Methods Reagents DMEM/F12 (1: 1) medium and penicillin/streptomycin were from Gibco/Invitrogen (Karlsruhe, Germany). Amphotericin B and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany). RAD001 was kindly provided by Novartis Pharma (Basel, Switzerland). Antibodies against pp70S6 Kinase (#9234), p70S6 Kinase (#9202), pAkt (#9271), Akt (#9272), pGSK3 (#9331), GSK3 (#9315), cyclin D1 (#2926), p27Kip1 (#2552), caspase-3 (#9662) and PARP (#9542) were from Cell Signaling (Beverly, Mass., USA). Antibody against -actin (#A5441) was from Sigma-Aldrich (St. Louis, Mo., USA). Horseradish peroxidase-conjugated secondary antibodies to mouse (#31432) or rabbit (#31452) IgG and chemiluminescent substrate SuperSignal West Dura Extended Duration Substrate were from Pierce (Rockford, Ill., USA). Cell Culture Human pancreatic neuroendocrine BON tumor cells were kindly provided by R. Göke (Marburg, Germany). BON cells were cultured in DMEM/F12 (1: 1) medium supplemented with 10% FCS, 1% penicillin/streptomycin and 0.4% amphotericin B in a 5% CO 2 atmosphere. Cell Proliferation Assay For proliferation assays, BON cells were seeded into 96-well plates at a density of 2000 cells/well and grown for 24 h. The next day, medium was replaced by serum-rich medium (10% FCS) or serum-depleted medium (1% FCS) containing various concentrations of RAD001 (25, 30, 35, 40, 45, 50, 55 n M or 5, 10, 15, 20, 25, 30, 35 n M ) for 72 h. The cell proliferation rate was measured with Cell Titer 96 kit (Promega, Madison, Wisc., USA) according to the manufacturer’s instructions. Following 3 h of incubation with Cell Titer 96 solution, absorbance at 492 nm was determined using an ELISA plate reader. Measurement of Apoptosis and Cell Cycle Analysis Apoptosis and cell cycle distribution were analyzed using flow cytometry according to Nicoletti et al. [25] . Cells were scraped with a rubber policeman, washed with PBS and incubated in staining buffer containing 0.1% sodium citrate, 0.1% Triton X-100 (Sigma) and 50 g/ml propidium iodide overnight. Sub-G1 events and cell cycle distribution were measured in a fluorescence-activated cell sorter, FACSCalibur (Becton Dickinson, Franklin Lakes, N.J., USA), using Cell Quest software. Nuclei to the left