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Ras Inhibition Reduces BMP4/Erk/Smad Signaling

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Ras Inhibition Reduces BMP4/Erk/Smad Signaling Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1087 Molecular Cancer Therapeutic Discovery Therapeutics Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling Batya Barkan1, Yoel Kloog1, and Marcelo Ehrlich2 Abstract À/– Neurofibromin-deficient (Nf1 ) malignant peripheral nerve sheath tumors (MPNST) are highly invasive, refractory to chemotherapy, and characterized by overactivated Ras. Ras activates mitogenic pathways and regulates morphogenic programs—such as those induced by bone morphogenetic proteins (BMP) and TGF-b. The role of such a cross-talk in determining the phenotype and transformation potential of MPNSTs is unknown. Here, we used MPNST cell lines and selective Ras inhibition with S-trans,trans-farnesylthiosa- licylic-acid (FTS; salirasib) in conjunction with specific inhibitors of TGF-b and BMP signaling. FTS perturbed signaling of BMP4 and TGF-b1 to Smad-dependent and Erk-dependent pathways. Furthermore, FTS inhibited motility and spreading, reduced the gelatinase secretion, eliminated the expression and activation of regulators of cell–matrix interaction, and altered gene expression. These phenomena are indicative of a phenotypic reversion of NF1-defficient cells by FTS. Inhibition of BMP4 and TGF-b by noggin and SB-431542, respectively, mimicked the FTS-mediated effects on adhesion, spreading, and cell morphology. This strongly suggests that a cross-talk among TGF-b superfamily ligands and Ras plays a significant role in the À transformation of NF1 /– MPNSTs. Our results support the therapeutic potential of FTS, in conjuncture with BMP and TGF-b pathway inhibitors, toward the inhibition of mitogenic and morphogenic signaling pathways and the alleviation of NF1 symptoms. Mol Cancer Ther; 10(8); 1317–26. ’2011 AACR. Introduction TGF-b and bone morphogenetic proteins (BMP), essen- tial in embryogenesis, induce EMT in development and Phenotypic alterations and dedifferentiation of cancer disease and stimulate metastasis (4, 5). BMPs regulate cells are critical in malignancy and increase the invasive/ neural crest migration (6), alter cell morphology, and metastatic attributes of tumors (1). Chronically active Ras increase cell motility and invasiveness in colon, prostate, drives tumor progression through deregulation of pro- ovarian, and pancreatic cancers (7, 8). TGF-b, despite its liferation and phenotypic transformation (2). Altered initial role as a tumor suppressor, establishes an undif- phenotypic regulation of cancers cells, such as the epithe- ferentiated phenotype and associates with progressed lial-to-mesenchymal transition (EMT) of epithelial stages of tumorigenesis (9). tumors, results from the integration of developmental BMPs and TGF-bs bind and activate heteromeric com- or morphogenic signaling pathways with those induced plexes of receptor serine/threonine kinases, and lead to by deregulated Ras (3). the phosphorylation/activation of pathway-specific Smad proteins (Smads2/3 for TGF-b and Smads1/5/8 for BMPs). Activated Smads oligomerize with Smad4, Authors' Affiliations: Departments of 1Neurobiology and 2Cell Research translocate to the nucleus, and mediate transcriptional and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv activation and/or repression (10, 11). TGF-b and BMP University, Tel Aviv, Israel receptors activate additional kinase cascades, such as Note: Supplementary material for this article is available at Molecular those regulated by Ras (10). Ras activation may promote, Cancer Therapeutics Online (http://mct.aacrjournals.org/). inhibit, or modify TGF-b and BMP signaling, depending Y. Kloog is the incumbent of the Jack H. Skirball Chair in Applied Neurobiology. on cellular context and signal intensity (3, 11). Overactivation of Ras stems from gene mutations, This work partially fulfills the requirements for a PhD thesis (Tel Aviv University) by B. Barkan. overactivation of receptors that activate Ras, and/or Corresponding Author: Yoel Kloog, Department of Neurobiology, Tel reduced activity of Ras-GTPase activating proteins Aviv University, Tel Aviv, 69978 Israel. Phone: 9723-640-9699; Fax: 9723- (Ras-GAP; ref. 12). A prototypical example of the latter 640-7643; E-mail: [email protected] occurs in neurofibromatosis type 1 (NF1), an autosomal doi: 10.1158/1535-7163.MCT-10-1087 inherited disease with a 1:3,000 prevalence (13). The ’2011 American Association for Cancer Research. hallmark of NF1 is the neurofibroma, a peripheral nerve www.aacrjournals.org 1317 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1087 Barkan et al. benign tumor comprised of transformed Schwann cells phalloidin–labeled cells (imaged with Nikon ECLIPSE (14). Neurofibromas undergo transformation into aggres- TE2000-S, CoolSNAPHQ, Â60 magnification; Image-Pro sive and chemotherapy-resistant malignant peripheral Plus) was determined with ImageJ software (NIH). nerve sheath tumors (MPNST), which are prone to live-threatening metastasis (15). Loss of neurofibromin Spreading assay Ras-GAP activity is associated with increased Ras-GTP Cells, treated with FTS or inhibitors (see legends), were and overactivation of Ras effectors (16), leading to NF1 harvested by trypsinization, washed, left to recover (30 (17, 18). The role of Ras in NF1-based malignancy sug- minutes, 37C), replated on fibronectin-coated 35–mm gests that Ras inhibitors, such as S-trans,trans-farne- plates (20 mg/mL fibronectin, 1 Â 105 cells per plate), sylthiosalicylic acid (FTS; salirasib) that interferes with fixed, stained (phalloidin/Hoechst), and imaged (ran- Ras-membrane anchorage (19), are an efficient therapeu- domly selected fields, Zeiss 200M microscope, Â10 lens, tic approach. Importantly, FTS interferes with the trans- HQ2-Photometrics camera, SlideBook software). Cells formed phenotype of NF1-associated MPNST cell lines in were identified by intensity based segmentation (phalloi- vitro and in vivo (20). din fluorescence), defined as objects, and measured (area, The tumorigenicity of NF1 cells is influenced by modi- SlideBook software). fications to Ras/Raf/Erk signaling (21), the absence of axon–Schwann cell interactions (21), and by the infiltration Wound-healing assay þ À of TGF-b–secreting NF1 / mast cells to their micro- Confluent cells (24 hours postplating) were treated environment (22). However, the cross-talk between Ras with FTS [18 hours, 10 mmol/L, 0.5% fetal calf serum and TGF-b superfamily signaling in NF1-deficient shwan- (FCS)], mytomycin C (18 hours, 10 mg/mL, 0.5% FCS), or nomas is unexplored. We report here that FTS reduced the vehicle [0.1% dimethyl sulfoxide (DMSO)]. Cell mono- spreading and motility of NF1-deficient MPNSTs, altered layers were scratched (3 scratches per plate), washed, and their transcriptome and the expression of Schwann-cell imaged by phase contrast microscopy (Â10 objective; markers, reduced BMP4 expression, and inhibited the after 0, 8, and 24 hours). Each condition was measured basal and ligand-mediated activation of Smad1/5/8 and at 9 different areas. Experiments were in duplicates, Smad3; all in accord with an FTS-induced phenotypic yielding 18 sets of distance measurements. Gap widths reversion of invasive NF1-deficient schwannomas via were measured with ImageJ. alterations to Ras and TGF-b superfamily signaling. Zymography Materials and Methods Cells [0.75 Â 106 cells; Dulbecco’s Modified Eagle’s Medium (DMEM)/5% FCS] were treated with FTS (48 Cell culture hours, 75 mmol/L) or 0.1% DMSO. Media from equal ST88-14, T265P21, and STS26T cells were from Dr. numbers of cells were separated on nonreducing 8% SDS- Nancy Ratner [Cincinnati Children’s Hospital and Med- PAGE/0.1% gelatin. Gels were washed with 2.5% Triton ical Center, University of Cincinnati; received in 2002; X-100, 50 mmol/L, and Tris-HCl; incubated with 50 immediately frozen, used at a low passage number (less mmol/L Tris-HCl, 0.02% azide, and 10 mmol/L CaCl2 than 4 months in culture)]. They were routinely checked (48 hours, 37C); and stained with Coomassie Blue R250. for Mycoplasma, maintained, and genetically analyzed [exon-specific PCR amplification, denaturing high-per- Gene expression profiling formance liquid chromatography, and sequencing (20)]. Cells treated with FTS (75 mmol/L and DMEM/5% Plating was at 0.75 Â 106 cells (10-cm dish), 15 Â 104 cells FCS) or DMSO (0.1% DMEM/5% FCS) were lysed (TRI- (6-well plate), and 7,500 cells (18-mm glass coverslip). zol) and analyzed with Affymetrix Human Genome U133A according to the manufacture instructions. Hybri- Rac-GTP assay dization (10 mg cRNA; overnight) was carried out with Rac-GTP was pulled down with glutathione–sepharose spike controls. The Mas5 algorithm was used for gene transferase conjugated Rac-binding domain of PAK1 analysis. When comparing FTS-treated and control cells, (GST–PBD; Rac-binding domain of PAK1-conjugated P 0.05 was considered significant. beads; ref. 23). Cell lysates [500 mg protein in 25 mmol/L HEPES (pH 7.5) 150 mmol/L NaCl, 1% NP40, 10% glycerol, Results 10 mmol/L MgCl2, 1 mmol/L EDTA, and protease inhibitors] were incubated with glutathione–sepharose The Ras inhibitor FTS reverses the beads/GST–PBD (1 hour, 4C, and 500 mg), separated by migratory/invasive phenotype of ST88-14 cells SDS-PAGE, and analyzed by immunoblotting (24). The effects of FTS (10 mmol/L, overnight) on ST88-14 cells’ motility was assayed by following
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