Published OnlineFirst April 20, 2015; DOI: 10.1158/0008-5472.CAN-14-3650 Cancer Microenvironment and Immunology Research IL13 Receptor a2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis Ruben A. Bartolome1, Irene García-Palmero1, Sofía Torres1, María Lopez-Lucendo 2, Irina V. Balyasnikova3, and J. Ignacio Casal1 Abstract IL13 signaling through its receptor IL13Ra2 plays a critical involved in cargo movement along microtubules. IL13Ra2-trig- role in colon cancer invasion and liver metastasis, but the gered activation of the FAK and PI3K/AKT/mTOR pathways was mechanistic features of this process are obscure. In this study, mediated by FAM120A, which also recruited PI3K and func- we identified a scaffold protein, FAM120A (C9ORF10), as a tioned as a scaffold protein to enable phosphorylation and signaling partner in this process. FAM120A was overexpressed activation of PI3K by Src family kinases. FAM120A silencing in human colon cancer cell lines and 55% of human colon abolished IL13-induced cell migration, invasion, and survival. cancer specimens. IL13Ra2-FAM120A coimmunoprecipitation Finally, antibody blockade of IL13Ra2 or FAM120A silencing experiments revealed further signaling network associations precluded liver colonization in nude mice or metastasis. In that could regulate the activity of IL13Ra2, including FAK, SRC, conclusion, we identified FAM120A in the IL13/IL13Ra2signal- PI3K, G-protein–coupled receptors, and TRAIL receptors. In ing pathway as a key mediator of invasion and liver metastasis addition, FAM120A associated with kinesins and motor proteins in colon cancer. Cancer Res; 75(12); 2434–44. Ó2015 AACR. Introduction IL13 binding to IL13Ra2 induced a significant increase in cell adhesion, migration, and invasion capacity of colorectal cancer Interleukin 13 (IL13) has been associated to different pathologic cells (2). Cell signaling was independent of type II IL4 receptor conditions (asthma, autoimmune diseases, ulcerative colitis, and and IL13Ra1 receptor as KM12 cells did not express these others; ref. 1). More recently, we demonstrated that IL13 signaling alternative IL13 receptors on the cell surface (2). Moreover, through IL13 receptor subunit a-2 (IL13Ra2) plays a critical role in silencing of IL13Ra2 increased mice survival and provoked a colon cancer invasion and liver metastasis (2). We described the clear reduction in liver colonization in mouse xenograft models overexpression of IL13Ra2 in 66% of tumor samples from patients (2). Although initially was considered a decoy receptor (11), with colon cancer, which was associated tolatestages ofprogression there are multiple evidences that IL13Ra2 is functional and (metastasis in lymph nodes or liver) and poor outcome of patients induces the activation of several pathways and the transcription with colorectal cancer (2). IL13Ra2 is overexpressed in a variety of factor AP-1, inducing the expression of TGFb (2, 12, 13). human tumor types, such as colon, glioblastoma, renal cell carci- IL13Ra2 cytoplasmic domain is very short, consisting of only noma, pancreatic, melanoma, head and neck, mesothelioma, and 14 amino acids residues, which does not contain any recog- ovarian, where it has been proposed as biomarker and potential nizable motif and makes difficult its interaction with other therapeutic target (2–10). In glioblastoma multiforme, IL13Ra2 proteins. Nevertheless, this receptor participates in signal trans- expression occurs in 75% of tumors and is associated with poor duction, triggering the activation of several signaling proteins, prognosis (3). A similar situation occurs for head and neck squa- such as PI3K and Src kinases (2, 12, 13). Little was known about mous cell carcinoma (8) and ovarian cancer (9). the signaling mechanisms of IL13 through IL13Ra2inmetas- tasis and cancer progression. fi a 1Department of Cellular and Molecular Medicine, Centro de Investiga- Here, we have identi ed the molecular partners of IL13R 2 ciones Biologicas (CIB-CSIC), Madrid, Spain. 2Proteomics Facility, and the mechanisms of signal transduction by using immuno- Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain. 3The precipitation experiments combined with a proteomic Brain Tumor Center, University of Chicago, Chicago, Illinois. approach. We identified the adaptor FAM120A as the scaffold Note: Supplementary data for this article are available at Cancer Research protein necessary for IL13Ra2-mediated signaling. FAM120A, Online (http://cancerres.aacrjournals.org/). also known as c9orf10 or OSSA (oxidative stress-associated Src Corresponding Author: J. Ignacio Casal, Functional Proteomics Laboratory, activator), was required for the activation and recruitment of Centro de Investigaciones Biologicas (CIB-CSIC), Ramiro de Maeztu, 9, 28040 FAK, Src, PI3K, and most of the proteins involved in IL13Ra2 Madrid. Spain. Phone: 34-91-8373112; Fax: 34-91-5360432; E-mail: signaling, providing an overall picture of IL13 signaling in [email protected] colorectal cancer cells and its relevance in liver metastasis. doi: 10.1158/0008-5472.CAN-14-3650 FAM120A and/or IL13Ra2 targeting abolished liver coloniza- Ó2015 American Association for Cancer Research. tion in a mouse model. 2434 Cancer Res; 75(12) June 15, 2015 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 20, 2015; DOI: 10.1158/0008-5472.CAN-14-3650 IL13Ra2 Signaling in Colorectal Cancer Metastasis Materials and Methods ions were submitted to collision-induced dissociation in the LTQ using normalized collision energy of 35% and a target value of 104 Cell culture and reagents ions. Dynamic exclusion was enabled with a repeat count of one KM12C and KM12SM human colon cancer cells were obtained and exclusion duration of 30 seconds. Mass spectra were searched from I. Fidler's laboratory (MD Anderson Cancer Center. Hous- using SEQUEST search engine with Proteome Discoverer (Thermo ton, TX). These cell lines were authenticated by short tandem Scientific) against the Uniprot Database. Search parameters repeat analysis. Other cell lines were obtained directly from the included precursor and fragment mass tolerances of 10 p.p.m. ATCC. These cell lines were passaged fewer than 6 months after and 0.8 Da, respectively, a maximum of two missed cleavages purchase for all the experiments. All cell lines were cultured in allowed, a fixed modification of carbamidomethyl cysteine and a DMEM (Gibco-Life Technologies) containing 10% FCS (Sigma- variable modification of methionine oxidation. Identified pep- Aldrich) and antibiotics at 37Cina5%CO-humidified 2 tides were validated using Percolator algorithm with a q-value atmosphere. threshold 0.01. IL13 was used at 10 ng/mL and purchased from PeproTech. PP2 (used at 3 mmol/L), PP3 (3 mmol/L), and UO126 (5 mmol/L) IHC analysis inhibitors were from Calbiochem. LY294002 (25 mmol/L) was Paraffin samples were obtained from 119 patients diagnosed from Sigma-Aldrich and FAK inhibitor 14 (10 mmol/L) from Santa and treated of colorectal adenocarcinoma between 2001 and Cruz Biotechnology. All antibodies used in this article are listed in 2014 in Fundacion Jimenez Díaz (Madrid, Spain). Clinicopatho- Supplementary Table S1. logical data are shown in Supplementary Table S2. Each sample was deparaffinated for antigen retrieval using citrate sodium siRNAs transfections buffer at pH 6.0 for 20 minutes and subsequent incubation with For transient transfections, siRNAs targeting specifically the primary antibody against FAM120A. The reaction was FAM120A (SASI-Hs01-00149752, Sigma-Aldrich) or control revealed using DAB as chromogen and hematoxylin for counter- siRNAs were transfected with JetPrime (Polyplus Transfection) staining. In all cases, sections from normal colonic mucosa distant according to the manufacturer's instructions. from the tumor site were used as negative controls. Western blot analysis and immunoprecipitation assays Wound healing Cells were detached, washed, and lysed with protease and Cells were cultured to confluence in Matrigel-coated plates phosphatase inhibitors in lysis buffer (1% Igepal, 100 mmol/L (0.4 mg/mm2). A 1-mm wide wound was done across the mono- NaCl, 2 mmol/L MgCl , 10% Glycerol in 50 mmol/L Tris-HCl). 2 layer. Cells were incubated in serum-free medium containing Protein extracts were separated in SDS-PAGE, transferred to nitro- IL13, inhibitors, and antibodies. Pictures were taken either imme- cellulose membranes, which were incubated with primary anti- diately (0 hour) or after 48 hours in culture at 37 C after the injury. bodies (Supplementary Table S1) followed by incubation with Migration was quantified as a percentage of wound closure. either HRP-anti-mouse IgG (Thermo Scientific) or HRP-anti-rab- bit IgG (Sigma-Aldrich). Specific reactive proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate Invasion assays  4 (Thermo Scientific). For Matrigel invasion assays, 6 10 KM12C or KM12SM cells For immunoprecipitation, cells were lysed and 500 mg of cell were resuspended in invasion medium (serum-free DMEM con- m fi lysate were incubated with the indicated antibodies. The immu- taining 0.4% BSA) and loaded onto 8 m pore-size lters coated m nocomplex was captured by adding 100 mL of Protein G-sephar- with 35 L of Matrigel (1:3 dilution; BD Biosciences) in Transwell ose beads (Sigma-Aldrich). After washing, samples were resus- plates (Costar) in presence of inhibitors or antibodies. The lower  compartment