Inhibition of Transforming Growth Factor Signaling Reduces Pancreatic Adenocarcinoma Growth and Invasiveness

Transforming growth factor (TGF ) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGF pathway in tumor cells often leads to resistance to the antiproliferative effects of TGF while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGF receptor I kinase (TGF RI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGF -dependent Smad2 phosphorylation and expression of TGF -inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGF -regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGF -stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGF signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGF RI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer. Pancreatic cancer is the fifth leading cause of cancer-related deaths, resulting in approximately 31,000 deaths annually in the United States alone (Jemal et al., 2006). It is a highly metastatic cancer with an average survival of 3 to 8 months after diagnosis. Because of the aggressiveness of the cancer, difficulties in diagnosis, and lack of effective treatment, only 5% of patients who receive a diagnosis of pancreatic cancer survive longer than 5 years (Jemal et al., 2006). The current treatment, gemcitabine, confers only a modest survival advantage when used as a stand-alone treatment or in combination with other therapies (Eckel et al., 2006). A number of genetic and epigenetic alterations have been identified in pancreatic cancer. The most common are mutations that affect the activity or expression of K-ras, p15, p16, p53, and DPC4/Smad4. Activating point mutations in the K-ras oncogene are believed to occur early in progression to neoplasia (Hruban et al., 2000) and are found in 85 to 95% of pancreatic cancers (Friess et al., 1999). Mutations in cell cycle inhibitor genes p15 and p16 are found at a frequency of approximately 60 and 80%, respectively (Naumann et al., 1996; Villanueva et al., 1998). Aberrations in p53 and DPC4/ Smad4 are believed to occur late in tumor progression (Hruban et al., 2000) and are found in approximately half of pancreatic cancers (Schutte et al., 1996; Friess et al., 1999). Deletion of DPC4/Smad4, a key mediator of TGF signaling, has been associated with abnormal growth arrest by TGF (Yasutome et al., 2005). In addition to altered Smad4 expression, signaling from the Smad pathway may be disrupted by mutations that affect the expression of TGF receptors, Smad6, Smad7, and downstream genes (Friess et al., 1999). TGF signals through the Smad pathway and through Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.029025. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: TGF , transforming growth factor ; SMAD, mothers against dipeptidyl peptidase homolog; ECM, extracellular matrix; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; CTGF, connective tissue growth factor; PAI-1, plasminogen activator inhibitor, type 1; DMEM, Dulbecco’s modified Eagle’s medium; EMT, epithelial-to-mesenchymal transition; SD-208, 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase kinase; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; VEGF, vascular endothelial growth factor; IGF2, insulin-like growth factor 2; SB-431542, 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate. 0026-895X/07/7201-152–161$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 1 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 29025/3215635 Mol Pharmacol 72:152–161, 2007 Printed in U.S.A. 152 http://molpharm.aspetjournals.org/content/suppl/2007/04/02/mol.106.029025.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from Smad-independent pathways. Smad signaling is initiated upon binding of TGF to a type II receptor (TGF RII) followed by recruitment and transphosphorylation of TGF RI (Heldin et al., 1997). Activated TGF RI then phosphorylates regulatory Smads, Smad2 and Smad3. Once phosphorylated, Smad2 and Smad3 form a complex with Smad4 and translocate to the nucleus, where they activate the transcription of TGF -responsive genes. TGF signaling through the Smad pathway is tightly controlled by negative feedback loops involving Smad6 and Smad7. TGF can also signal through mitogen-activated protein kinase (ERK, JNK, p38) cascades and the phosphatidylinositol-3 kinase pathway (Derynck and Zhang, 2003; Elliott and Blobe, 2005). Cross-talk between these pathways and the TGF pathway coordinates proliferation, survival signals, and other signals. In normal epithelial cells, TGF acts as a tumor suppressor, mediating growth arrest through down-regulation of cMyc, and through transcriptional activation of cell cycle inhibitors p15 and p21 (Grau et al., 1997; Donovan and Slingerland, 2000; Adhikary and Eilers, 2005). In addition to regulating survival and proliferation, TGF signaling promotes angiogenesis, fibrosis, metastasis, and immune suppression (Elliott and Blobe, 2005). Alterations that affect the expression or activity of components of the TGF pathway can render cells insensitive to TGF -mediated growth arrest while enabling other responses that support tumor progression (Dumont et al., 2003; Nicolás and Hill, 2003). During neoplastic conversion, autocrine expression of TGF is believed to promote tumorigenesis. The cellular response is also influenced by other dysregulated pathways such as the Ras-MEK-ERK-signaling cascade (Ellenrieder et al., 2001) and by stromal cell interactions, growth factors, and cytokines in the tumor cell microenvironment. The pivotal role of TGF in promoting cellular processes that are important for tumor progression suggests that the pathway may be a good target for therapy. In this study, we investigated whether SD-208, a small-molecule inhibitor of TGF RI, can inhibit tumor progression in pancreatic cancer. We used PANC-1, a human pancreatic ductal carcinoma that harbors genetic alterations (K-ras, p15, p16, and p53) commonly found in pancreatic cancer (Villanueva et al., 1998; Moore et al., 2001). PANC-1 has also been reported to have altered TGF RI and Smad7 expression (Nicolás and Hill, 2003). It is an attractive model for the human disease because PANC-1 tumors are metastatic when grown orthotopically in nu/nu (nude) mice. Furthermore, PANC-1 secretes TGF , which is believed to promote tumor progression and desmoplasia in human pancreatic cancer. Our studies reveal that SD-208 abrogates TGF -mediated gene responses that may facilitate tumor growth and metastasis. We also demonstrate for the first time that a small-molecule inhibitor of TGF RI attenuates growth and metastasis of established tumors in an orthotopic xenograft model of pancreatic adenocarcinoma. Materials and Methods Reagents. Recombinant human TGF was purchased from R&D Systems (Minneapolis, MN). TGF 1 and VEGF ELISA kits were from Biosource International (Camarillo, CA). The TGF 2 ELISA was from R&D Systems. The PAI-1 ELISA was from American Diagnostica, Inc. (Stamford, CT). The rabbit polyclonal antibody for phospho-Smad2 (Ser465/467) was from Cell Signaling Technology (Danvers, MA). The mouse monoclonal antibody against vimentin was from Affinity Bioreagents (Golden, CO). HRP-conjugated donkey anti-rabbit secondary antibody was from Amersham (Pittsburgh, PA), and HRP-conjugated goat anti-mouse secondary antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). CTGF ELISA. CTGF-specific polyclonal antibodies, which were generated using peptides derived from the C terminus of the protein, were absorbed on a high-binding ELISA plate. After blocking, CTGF standards and cell-culture supernatants were added and incubated overnight at 4°C. Bound CTGF was detected via its heparin-binding site by incubation with biotinylated-heparin (Sigma-Aldrich, St. Louis, MO) and streptavidin-HRP (Chemicon International, Temecula, CA). Quantitation of bound CTGF was extrapolated from a standard curve generated with recombinant human CTGF. Cell Culture and Inhibitor Treatment. Human pancreatic cancer cell lines PANC-1 (CRL-1469) and BxPC-3 (CRL-1687) were acquired from American Type Culture Collection (Manassas, VA). PANC-1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum. BxPC-3 was cultured in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum. TGF RI kinase inhibitor SD-208 (Scios, Inc., Fremont, CA) was dissolved in DMSO (1000 stock). SD-208 has an IC50 value of 49 nM when measured by direct enzymatic assay of TGF RI kinase activity in vitro. It is 100-fold less specific for TGF RII and is more than 17-fold less specific for related kinases (Kapoun et al., 2006). Construction of PANC-1 Luciferase Cells. For constitutive expression of luciferase and the Zeocin-resistance gene, PANC-1 were cotransfected with pGL-3 (Promega, Madison, WI) and pSV40Zeo (Invitrogen, Carlsbad, CA) using FuGENE transfection reagent (Roche Applied Science, Alameda, CA). After selection with Zeocin (Invitrogen), a clone with stable luciferase expression and normal growth characteristics was selected for studies. Phospho-Smad2 Analysis. Cells were seeded in six-well plates at 2 10 cells/well and cultured in serum-containing medium. The next day, they were treated with SD-208 (31.25–1000 nM) for 15 min before the addition of TGF 1 (2 ng/ml). After 65 min, cell lysates were prepared and analyzed by Western blot as described previously (Kapoun et al., 2006). Measurement of Secreted Proteins. PANC-1 cells were seeded in six-well plates at 3 10 cells/well and cultured in serum-containing medium. The next day, medium was changed to serum-free DMEM containing 1 Insulin-Transferrin-Selenium (Gibco) and 0.2% bovine serum albumin. The medium also contained combinations of the following treatments: 0.1% DMSO vehicle control, 400 nM SD-208, and 5 ng/ml TGF 1. After 24 h, cell supernatants were collected and assayed for TGF , VEGF, CTGF, and PAI-1 by ELISA. Cells were harvested in MPER buffer (Pierce, Rockford, IL), and protein was quantitated by bicinchoninic acid assay (Pierce). Concentrations of secreted proteins were determined by ELISA and normalized to the total cell protein. Gene Expression Analysis. PANC-1 cells were seeded in DMEM containing 10% serum and grown to 70% confluence. The next day, cells were treated in complete medium with vehicle (0.1% DMSO), 400 nM SD-208, 2 ng/ml TGF 1, or a combination of TGF 1 and SD-208 for 24 h. Total RNA was extracted from cells using an RNeasy kit (QIAGEN, Valencia, CA). Real-time RT-PCR and cDNA microarray analysis were performed as described by Kapoun et al. (2006). Sequences of primers and probes for real-time RT-PCR can be found in Table 1. Invasion Assays. Cell invasion was analyzed in 24-well Matrigelcoated invasion chambers (BD Biosciences Discovery Labware, Bedford, MA) according to manufacturer’s directions with the following modifications: hydrated chambers were transferred to a new 24-well plate containing DMEM with 10% serum plus TGF 1 (2 ng/ml) and/or SD-208 (1 M) or DMSO (0.1%). Cells (5 10) were added to each chamber and incubated for 20 h. Cells that did not pass through Inhibition of TGF Signaling in Pancreatic Cancer 153 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from the filter were removed with a cotton swab before processing the filter with a Hema-3 staining kit (Fisher, Pasadena, CA) and mounting the filter on a microscope slide. Cells in five fields on the filter were photographed at 40 magnification and counted manually for