IL13 Receptor A2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis Ruben� A

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. 2015 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- 2015 American Association for Cancer Research. tion in a mouse model.

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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 of the invasion chamber was filled with medium pended in 2 Laemmli buffer, boiled for 5 minutes, centrifuged and subsequently loaded onto SDS-PAGE gels, which were ana- containing IL13 or with medium alone. After 22 hours at 37 C, lyzed by Western blot analysis. As a control, we incubated the noninvading cells were removed from the upper surface of the fi fi fi lysates with an unrelated IgG coupled to Sepharose beads to lter, and cells that migrated through the lter were xed with 4% discard unspecific proteins. paraformaldehyde (Sigma-Aldrich), stained with crystal violet, and counted under a microscope.

Mass spectrometry of immunoprecipitated proteins For proteomic analysis, 10 mg of cell lysates were immuno- Survival assays precipitated as before, and the proteins were loaded in SDS-PAGE, Cells were incubated with 1 mmol/L H2O2 for 16 hours without which were divided in three slices for in-gel digestion with trypsin. serum in the presence or absence of IL13, antibodies or inhibitors. Peptides were trapped onto a C18-A1 2 cm precolumn (Thermo Cells were detached and incubated with FITC labeled-Annexin V Scientific) and separated on a Biosphere C18 column (Nano- (Miltenyi Biotec Inc.) and propidium iodide according to the fl Separations) using a flow rate of 250 nL/minute in a 100-minute manufacturer's instructions, and analyzed by cyto uorometry gradient from 0% to 95% Buffer B (0.1% formic acid in aceto- (Coulter Epics XL). nitrile) on a nanoEasy HPLC coupled to a nanoelectrospay ion source (Proxeon). Mass spectra were acquired in a linear ion trap Flow cytometry quadrupole (LTQ) Orbitrap Velos in data-dependent mode with Cells were detached with 2 mmol/L EDTA in PBS, incubated at an automatic switch between mass spectrometry and MS/MS 4C with primary antibodies (10 mg/mL) for 30 minutes, washed scans using a top 15 method. Full scans were acquired in the and incubated with Alexa 488 labeled-secondary antibodies Orbitrap with a mass range of 400 to 1,200 Th, a target value of 106 (Dako). Fluorescence was analyzed in a Coulter Epics XL ions and a resolution of 3 104 (at m/z 400). The 15 most intense cytofluorometer.

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IL13Ra2 A 158 66 B CMTM6 EPHB4 39 85 66 5 PDZD8 22 7 ACTB FAM120A

Proteins that may interact in Proteins that interact with vivo with IL13Ra2 IL13Ra2 Signaling Proteins of unknown location Proteins involved in and function transport Membrane receptors Actin cytoskeleton Proteins within inaccessible Proteins that interact with compartments multiple proteins

C D kDa FAM120A 130 kDa 48 FAM120A 130 IL13Rα2 RHOGDI 35 IP: anti-IL13Rα2 IP: antibody Control antibody

E F Normal tissue

50 70 IL13Ra2+ * 60 40 IL13Ra2– 50 30 40 20 30 20 % of Patients of % 10 10 % of Patients of % with 0

recurrency at 18 months 0

FAM120A– FAM120Alo FAM120Ahi Colorectal cancer

Figure 1. FAM120A associates with IL13Ra2. A, classiﬁcation of IL13Ra2-coimmunoprecipitated proteins detected by proteomics assays in KM12SM cells according to their location (left) and function (right). B, protein interaction network for IL13Ra2. C, veriﬁcation of FAM120A–IL13Ra2 interaction by immunoprecipitation and Western blot analysis. D, expression of FAM120A in the indicated human colorectal cancer cell lines by Western blotting. E, by IHC, 55% of patients with colon cancer (n ¼ 119) expressed FAM120A in tumor samples [25% showed low/moderate expression (FAM120Alo) and 30% high expression (FAM120Ahi)]. IL13Ra2 was positive in 65% of patient samples (left). Representative images of FAM120A expression intensity in colorectal cancer samples and adjacent normal colon tissue of three patients obtained by IHC (right). F, patients with tumors coexpressing IL13Ra2 and high levels of FAM120A showed an increased risk of recurrence at 18 months (, P < 0.05).

Immunofluorescence Technologies). Then, samples were mounted with Mounting KM12SM cells were cultured onto Matrigel-coated coverslips, Fluorescence Medium (Dako) and images were captured using treated with or without IL13 (10 ng/mL) for 5 minutes, and fixed a TCS-SP5-AOBS confocal microscope (LEICA) with a 100 oil with 1% paraformaldehyde in PBS containing 0.1% Triton X-100. immersion objective. Cells were washed three times with PBS and incubated 30 minutes with primary antibodies (Supplementary Table S1), washed again Metastasis experiments in nude mice and incubated 25 minutes with secondary antibodies labeled with The Ethical Committee of the Consejo Superior de Investiga- Alexa-468 or Alexa-568, and 40,6-diamidino-2-phenylindole (Life ciones Científicas (Madrid, Spain) approved the protocols used

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for experimental work with mice. Swiss nude mice (Charles River; (FAM120Alo) to intense (FAM120Ahi)] in 55% of tumor samples, n ¼ 10 per condition) were inoculated in spleen with 106 whereas adjacent normal tissue samples showed a very weak KM12SM cells in 0.1 mL PBS. After 22 hours, mice were subjected expression (Fig. 1E). Patients with tumors expressing high levels to removal of the spleen. Mice were daily inspected for signs of of both, IL13Ra2 and FAM120A, showed a significantly increased disease, such as abdominal distension, locomotive deficit, or risk of recurrence at 18 months (Fig. 1F) compared with the rest tumor detectable by palpation. When signs were visible, mice of patients. These results indicate a significant overexpression were euthanized, subjected to necropsy, and inspected for metas- of FAM120A in colon cancer, and the association of such over- tasis in liver. expression with a higher risk of early recurrence in combination For liver colonization assessment, mice were inoculated as with IL13Ra2. before and euthanized at various times after cells inoculation (2, 8, 24, and 56 hours). RNA was isolated from liver using TRIzol Identification of FAM120A protein networks (Invitrogen), retrotranscribed and 0.3 mg cDNA subjected to PCR To identify protein networks containing FAM120A, KM12SM with Taq DNA polymerase (Invitrogen) to amplify human lysates were subjected to immunoprecipitation using anti- GAPDH as previously described (14). As a control, a 20 cycles- FAM120A antibodies followed by mass spectrometry. After amplification of murine b-actin was performed. discard proteins coimmunoprecipitated with control beads and those placed in inaccessible place for a cytosolic protein, 222 Statistical analyses proteins were found to interact with FAM120A (Fig. 2A). As Data were analyzed by one-way ANOVA followed by the before, we removed 131 proteins based on their protein-bind- Tukey–Kramer multiple comparison test. The significance of the ing functions (i.e., ribosomal, chaperones, etc.). From the difference for survival curves was estimated with the log-rank test, remaining 91 proteins, 48 were involved in protein transport. whereas the differences in early recurrence were analyzed by the c2 This large number of proteins involved in vesicle trafficking, not test. In all analyses, the minimum acceptable level of significance commonly associated with cytosolic proteins, suggests an addi- was P < 0.05. tional role for FAM120A in protein transport (Supplementary Table S5). Results The remaining 43 proteins associated with FAM120A (Supple- mentary Table S6) were subjected to GO analysis (Fig. 2B). Identification of protein partners for IL13Ra2 FAM120A interactors were involved in actin cytoskeleton dynam- To characterize the protein interaction network of IL13Ra2, cell ics, cell signaling, and apoptosis and cell adhesion. According to lysates of the highly metastatic cell line KM12SM were immuno- STRING interaction analysis and data mining, FAM120A forms precipitated using anti-IL13Ra2 monoclonal antibody coupled to part of different protein complexes, such as focal adhesion com- Sepharose beads. An irrelevant antibody was used as a control. plexes, G-protein–coupled receptors, and TNFRSF10B receptor Precipitated proteins were fractionated by SDS-PAGE followed by (also known as TRAILR2; Fig. 2C). A selection of the proteins mass spectrometry analysis. We identified 158 proteins coimmu- coimmunoprecipitated with FAM120A, together with other pro- noprecipitated exclusively with anti-IL13Ra2 antibody, using teins involved in signaling, cell adhesion, cell death, and actin Percolator with q value 0.01 (Fig. 1A). Proteins within a location cytoskeleton dynamics (FAK, Src, HCK, RAC, RHOA, PI3K, AKT, biologically inaccessible for a membrane protein (such as nuclear, RAF1, ERK1/2, and CASP8) was confirmed for association with mitochondrial, and peroxisomal) or those proteins involved in FAM120A by immunoprecipitation and Western blot analysis irrelevant protein-binding functions as ribosomal proteins, cha- (Fig. 2D). No association was found between the adaptor protein perons, and common background proteins (15) were removed FAM120A and IL13Ra1or Ras, in agreement with data obtained from further analysis. At the end, five proteins (Supplementary by mass spectrometry. Table S3) were selected as the best candidates for specific inter- To study subcellular localization of IL13Ra2 and FAM120A, we action with IL13Ra2. In addition, we identified other 22 proteins carried out confocal microscopy analysis (Fig. 2E). IL13Ra2 involved in vesicle trafficking from Golgi and endoplasmatic showed an intracellular distribution, with some surface staining, reticulum to cell membrane (Supplementary Table S4). One of whereas FAM120A staining was cytosolic and associated to mem- the five proteins, FAM120A (Family with sequence similarity brane. In addition, we tested AKT as surrogate for signaling 120A), a scaffold protein, was related to the Src family kinases interaction. There was a clear colocalization of IL13Ra2 with (16), which were already known to be involved in IL13Ra2 FAM120A and AKT, showing a slight increase for AKT after 5 signaling (2). Amongst the other four identified proteins were minutes of IL13 treatment. EPHB4, a receptor tyrosine kinase that mediates ephrin signaling; Together, these results confirm the colocalization of IL13Ra2 CMTM6, an unknown transmembrane protein; and PDZD8 and with FAM120A and other signaling molecules. ACTB proteins, which are involved in cytoskeletal organization (Fig. 1B). IL13 signaling through IL13Ra2 requires FAM120A The association of FAM120A with IL13Ra2 was confirmed by We explored the kinetics of IL13 signaling through IL13Ra2in coimmunoprecipitation in four human colorectal cancer cell lines KM12SM cells. IL13 induced the activation of mTOR, FAK, Src and further detection by Western blot analysis (Figs. 1C and 2D). family kinases, AKT, and ERK1/2, along with a decrease of p53 Expression of FAM120A was demonstrated in a panel of nine levels (Fig. 3A). Then, we studied the interaction of cell signaling human colorectal cancer cell lines. All of them were positive, proteins with FAM120A upon treatment with IL13 (Fig. 3B). FAK, except Caco2 (Fig. 1D). To study the clinical relevance of PI3K, Src, Fyn, AKT, and p53 showed a quick association with FAM120A, we carried out immunochemical analysis of 119 FAM120A at 5 minutes after treatment with IL13 and further paired colorectal cancer patient samples. We observed cyto- dissociation at 60 minutes. ERK1/2 MAP kinases showed also plasmic staining of FAM120A [ranging from low/moderate association to FAM120A after 5 minutes, but remained associated

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A 222 BC43

13 131 43 19 G-protein–coupled receptor complex 2 Killer receptor complex 3 6 48 Cell protrusion proteins Signaling of several complexes

Proteins that interact with Signaling IL13Rα2 FAM120A Actin cytoskeleton Membrane receptors Proteins involved in transport Cell adhesion Proteins that interact with multiple proteins Cell cycle and apoptosis Actin polymerization ITGB1 regulation ADAM9 TSPAN8

TLN1 PLEK2 LCP1 FAK VCL SRC ARHGEF7 PXN SLC9A3R1 FYN IL13Rα2 VASP CDC42 TNFRSF10B NCKAP1 RAC1 ARHGEF1 PI3K RHOA TRIP10 CTNNB1 IL13Rα2 IL13Rα2 PLP2 GPR56 TPM1 IL13Rα2 PFN1 RAP1B FAM120A FSCN1 CFL1 ARHGDIA PLCB3 CDK1 RAF1 PLCB3 GNG5 PI3K FAM120A PDLIM1 CALM1 PRKCI PDE8A SRC FAK PI3K PDE8B PRKCI MAPK2K2 SRC GNB4 FYN FYN ARHGEF1 PLS1 MAPK4K4 MAPK1 ARHGEF7 FAM120A CALM1 FAM120A FLNA MAP4K4 RHOA CTNNB1 CDC42 RHOA ACTB MAP2K2 CDK1 CALM1 RAC1 CORO1C MAPK1 CASP8 PPM1B TP53

D IP: IP: IP: IP: IP: kDa kDa kDa kDa kDa 48 β IL13Rα2 28 1-integrin 180 RAC PI3K (P85) 75 Caspase-8 48 28 63 IL13Rα1 48 RHOA P53 Paxillin 63 AKT 48 28 28 100 Talin Src 63 CDC42 RAS β-Catenin 180 FAK 130 HCK 63 RHOGDI 35 RAF1 75 FAM120A 130 63 48 FYN JNK 48 ERK1/2

E IL13Rα2 FAM120A Merge IL13Rα2 AKT Merge

Medium

IL13

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ABIL13 (min) IL13 (min)

0 1 2.5 51015 30 60 kDa 0 0 5 60 kDa p-Ser2448-mTOR 48 180 IL13Rα2 180 mTOR 180 FAK 130 130 p-Tyr397-FAK PI3K (P85) 75 130 FAK Src 63 75 FYN 63 p-Tyr416-SFK 63 AKT 63 63 Src P53 48 63 HCK ERK1, ERK2 48 Figure 3. FYN 63 RHOA 28 Signaling triggered by IL13 is partly 63 controlled by FAM120A. A, KM12SM p-Ser473-AKT 28 RAC cells were exposed to IL13 for the AKT 63 130 indicated times, lysed, and the FAM120A extracts were resolved in SDS-PAGE p-Ser15-P53 48 and analyzed with the indicated P53 IP: IP: 48 Control FAM120A antibodies by Western blot analysis. p-Thr202-ERK1 48 RHOGDI was used as loading control. p-Tyr204-ERK2 B, Western blot analysis showing the ERK1 48 results of KM12SM cells treated with ERK2 C IL13 for 0, 5, and 60 minutes and RHOGDI 35 kDa subjected to immunoprecipitation FAM120A 130 using anti-FAM120A or control RHOGDI 35 antibodies. C, Western blot analysis showing the results of KM12SM cells DEControl FAM120A transfected with FAM120A or control siRNA siRNA PP3 PP2 siRNAs. D, knocked down KM12SM IL13 (min): 0 5 60 0 5 60 kDa IL13 (min): 0 5 60 0 5 60 kDa cells or control cells were exposed to p-Ser2448-mTOR p-Ser2448-mTOR 180 IL13 for the indicated times, lysed, and 180 mTOR analyzed as in A. E, Western blot mTOR 180 180 analysis showing the results of cells p-Tyr397-FAK 130 p-Tyr397-FAK 130 treated with mock- and Src-inhibitors, 130 130 PP3 and PP2, respectively, and IL13 FAK FAK for the indicated times. 75 75 p-Tyr416-SFK 63 p-Tyr416-SFK 63 63 Src 63 Src FYN 63 FYN 63

p-Ser473-AKT 63 p-Ser473-AKT 63 63 AKT 63 AKT

p-Ser15-P53 p-Ser15-P53 48 48 P53 48 P53 48 p-Thr202-ERK1 p-Thr202-ERK1 48 48 p-Tyr204-ERK2 p-Tyr204-ERK2 ERK1 48 ERK1 48 ERK2 ERK2 RHOGDI 35 RHOGDI 35

for longer times. Although RHOA decreased, RAC increased with To study the role of FAM120A in IL13 signal transduction in time, supporting the high invasive capacity of these cells. In colon cancer, KM12SM cells were FAM120A silenced with speciﬁc contrast, IL13Ra2 remained permanently associated with siRNAs or treated with scrambled siRNAs (Fig. 3C). Silencing FAM120A. caused a loss of mTOR, FAK, and AKT activation after IL13

Figure 2. Proteins coimmunoprecipitated with FAM120A. A, classification of FAM120A-coimmunoprecipitated proteins detected by proteomics assays in KM12SM cells. B, function of proteins that interact with FAM120A. C, representation of FAM120A-coimmunoprecipitated proteins according to different functional clusters (cell protrusion, GPCR, and death receptor). D, confirmation of FAM120A-coimmunoprecipitated proteins by immunoprecipitation and Western blot analysis. E, KM12SM cells treated or not with IL13 were used to determine IL13Ra2, FAM120A, and AKT localization by immunofluorescent confocal microscopy. IL13Ra2 showed membrane and intracellular expression with a clear colocalization with FAM120A and AKT.

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A 60 KM12SM SW620 treatment. In contrast, activation of Src family kinases and ERK1/2 50 *** were not affected by FAM120A silencing (Fig. 3D). To examine the 50 ** 40 effect of Src in IL13 signaling, we used the PP2 inhibitor in 40 ◊◊◊ KM12SM cells treated with IL13. PP2 caused a marked inhibition ◊◊◊ 30 ◊◊◊ ◊◊◊ 30 ◊◊◊ of mTOR, AKT, and ERK1/2, but not FAK (Fig. 3E). In contrast, 20 PP3, a mock inhibitor, did not affect Src pathway activation. 20 ◊◊◊◊ Together, these results indicate that IL13 signaling requires Src

Wound closure (%) Wound closure (%) 10 10 Wound closure (%) activation for AKT/PI3K activation together with FAM120A. How-

0 0 ever, Src activation was FAM120A independent.

FAM120A is required for IL13-induced cell migration, invasion, and survival To characterize FAM120A in cell invasion and metastasis induced by IL13, we tested two metastatic cell lines, KM12SM and SW620. After FAM120A silencing, KM12SM or SW620 cells knocked down for FAM120A or scrambled were treated with B KM12SM SW620 250 ** 160 IL13 alone or combined with Src, PI3K, FAK, and MEK inhibitors. * As a control, we performed the experiments in the presence of 200 140 anti-IL13Ra2 or control antibodies competing with IL13 for 120 ◊ IL13Ra2 binding (17). IL13 provoked a strong increase in the 150 100 migratory and, in particular, invasive capacity of KM12SM and ◊◊ ◊◊ 80 ◊ 100 SW620 cells (Fig. 4A and B). This increase was abolished after 60 Invasive cells

◊◊ Invasive cells FAM120A silencing. In SW620, the silencing of FAM120A inhib- ◊◊ 40 50 ited also basal migration, indicating that the presence of this 20 protein is required for cell motility. Treatment with Src, PI3K and, 0 0 at a minor extent, FAK inhibitors caused a similar effect to FAM120A silencing and similar to anti-IL13Ra2 blocking. Regarding cell survival, IL13 induced a significant increase in viable KM12SM cell number in control cells, but not in FAM120A-silenced cells. IL13-mediated increase in cell survival was inhibited by all the inhibitors and, more strongly, by PI3K C and FAK inhibitors (Fig. 4C). In SW620 cells, IL13 also promoted 30 KM12SM 90 SW620 cell survival; but this effect was more moderate, probably because *** 80 * ◊ SW620 cells had a strong basal resistance to oxidative stress. 70 ◊ ◊◊ Collectively, these data indicate that FAM120A, Src, PI3K, and 20 ◊◊◊ ◊◊◊ 60 FAK are critical mediators in IL13-induced cell migration, inva- 50 sion, and survival. 40 10 ◊◊◊ 30 ◊◊◊ a 20 FAM120A controls IL13R 2 location in cell membrane Nonapoptotic cells (%) (%) Nonapoptotic cells Nonapoptotic cells (%) Nonapoptotic cells (%) fi 10 We also investigated the role of FAM120A in protein traf cking fl 0 0 and location. By ow cytometry, KM12SM and KM12C cells knocked down for FAM120A displayed lower levels of IL13Ra2 in cell membrane (Fig. 5A). However, the total amount of IL13Ra2 was identical in FAM120A-silenced or control cells by Western blot analysis (Fig. 5B). These results suggest that Control siRNA fi a FAM120A siRNA FAM120A participates in the traf c and location of IL13R 2in D cell surface and, therefore, in the functionality of this receptor. To

siRNA: kDa provide further evidence, we verified the association of FAM120A FAM120A 130 with some of the identified proteins involved in protein traffic, RHOGDI 35 such as MYH14, COPE, or Rab1A/B GTPase by Western blot analysis (Fig. 5C). In addition, treatment of KM12SM cells with Figure 4. anti-IL13Ra2 antibody showed that anti-IL13Ra2 promoted FAM120A controls IL13-triggered cell migration, invasion, and survival. IL13Ra2 degradation (Fig. 5D), probably by a mechanism of cell A–C, KM12SM and SW620 cells were transfected with control or FAM120A- targeting siRNAs and subjected to wound-healing assays (A), cell invasion internalization mediated by lysosomes. This effect could contrib- assays through Matrigel (B), and survival to oxidative stress induced ute to the blocking capacities of the anti-IL13Ra2 antibody. by H2O2 (C). In addition, KM12SM cells were treated with anti-IL13Ra2 or control antibodies (5 mg/mL) and the indicated inhibitors. Cell FAM120A plays a key role in liver metastasis of colorectal cancer migration/invasion/survival was significantly increased by addition of IL13 Finally, we studied the effect of FAM120A in cancer metastasis ( , P < 0.05; , P < 0.01; , P < 0.001) and decreased by treatment with in vivo the indicated inhibitors or antibodies (^, P < 0.05; ^^, P < 0.01; . FAM120A-silenced or control KM12SM cells, in the pres- a ^^^, P < 0.001). D, Western blot analysis showing the results of SW620 ence of anti-IL13R 2 or control antibodies, were inoculated in the cells transfected with FAM120A or control siRNAs. spleen of nude mice. Spleens were removed 24 hours after

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ABKM12C KM12SM C IP: KM12C KM12SM 0.94 1.48 siRNA: Control Control kDa kDa siRNA siRNA 130 FAM120A αTubulin 48 1.00 0.13 1.00 0.07 MYH14 180 Cell number IL13Rα2 48 COPE 35 0.44 0.77 RHOGDI 35 RAB1A/B 28 FAM120A FAM120A 130 siRNA siRNA FAM120A

Cell number D Anti-IL13Rα2 Control antibody 0 100 101 102 1030 100 101 102 103 2h 4 h 6 h 8 h 2h 4 h 6 h 8 h Fluorescence intensity IL13Rα2 48 Control antibody 1.00 1.02 0.58 0.39 0.11 0.96 0.97 1.05 1.07 Anti-IL13Rα2 RHOGDI 35

Figure 5. FAM120A regulates IL13Ra2 expression levels in cell membrane. A, ﬂow cytometric-analysis showing the expression of IL13Ra2 in the surface of KM12C and KM12SM cells FAM120A-silenced or control. B, Western blot analysis was performed to detect the lack of changes in total IL13Ra2 in the same cells after FAM120A silencing. RHOGDI was used to assess total protein content. C, veriﬁcation of FAM120A-interacting proteins involved in protein transport after immunoprecipitation and Western blot analysis. D, Western blot analysis showing the results of KM12SM cells treated with anti-IL13Ra2 or control antibody for the indicated times. A marked decrease of IL13Ra2 after antibody treatment was observed after 6 hours.

inoculation, to prevent the formation of tumors in spleen. About through IL13Ra2 for the migration and invasion of colon cancer 60% of mice inoculated with control cells and control antibody cells. FAM120A was identified as a scaffold protein that interacts showed severe signs of disease and were euthanized (Fig. 6A). with IL13Ra2 using mass spectrometry and coimmunoprecipita- Subsequent necropsy revealed macroscopic metastasis in liver tion experiments. FAM120A is an abundant cytosolic protein that (Fig. 6B). In contrast, mice inoculated either with FAM120A showed association with, at least, three protein networks involved knocked down cells or treated with anti-IL13Ra2 antibodies did in focal adhesion, GPCR and TRAIL receptors. The expression of not develop disease, except for a mouse inoculated with FAM120A was required for the IL13-triggered Src and PI3K/AKT/ FAM120A knocked down cells, which developed a tumor in mTOR signaling pathway activation (Fig. 7). Besides its scaffold peritoneal cavity without liver metastasis (Fig. 6A). After 120 activity, FAM120A was associated with proteins involved in motor days postinoculation, surviving mice did not show macroscopic proteins (kinesins) involved in cargo movement that could mod- metastasis in liver (Fig. 6B). ulate the presence of IL13Ra2 on the cell surface or its secretion In addition, time-assays were performed to analyze the behav- (Fig. 7). Finally, the blocking of IL13Ra2 with specific antibodies ior of tumor cells in the first 3 days after inoculation. For that or the silencing of FAM120A strongly decreased metastatic growth purpose, mice were euthanized between 2 hours and 56 hours and dissemination in colon cancer cells, providing two powerful after inoculation and RNA was isolated from the livers. After PCR independent strategies for metastasis control. amplification, human GAPDH, as a surrogate marker, was The binding of IL13 triggers the recruitment of several mole- detected 2 hours after inoculation in all livers at similar levels, cules to the IL13Ra2–FAM120A complex, such as FAK, PI3K, AKT, independently of the treatment. This quick detection was prob- Src, p53, ERK1/2, and changes in the small GTPases associated to ably due to fluid flow and cell travelling via the portal circulation the complex (RAC instead of RHOA). FAM120A recruits PI3K and to the liver within minutes. After 8 hours, PCR amplification works as a scaffold protein for PI3K and the Src family kinases showed a marked reduction, except for control mice. Moreover, (SFK), enabling phosphorylation and activation of PI3K (16). bands were barely discernible at 24 or 56 hours after inoculation Silencing of FAM120A impaired the activation of PI3K, AKT, and for cells transfected with FAM120A siRNAs or treated with anti- mTOR in the metastatic cells. However, it did not alter the IL13Ra2 antibodies, except for control siRNAs and control anti- activation of SFKs as the activation levels, based on its phosphor- bodies (Fig. 6C). These results suggest that FAM120A and ylation status, are similar in FAM120A-silenced cells than in IL13Ra2 are required for IL13-mediated engraftment or coloni- control siRNA transfected cells. The activation of AKT was depen- zation of colorectal cancer cells, increasing tumor cell adhesion dent of Src kinase, confirming that FAM120A is the scaffold and survival in the liver after cell arrival. required for Src-mediated PI3K activation, as previously reported (16). Along this line, a protector role to guard cells from oxidative Discussion stress-induced apoptosis by activation of SFKs was given to FAM120A (16). In addition, FAM120A could recruit FAK into In this report, we describe a key role for FAM120A, also known the IL13Ra2 complex to facilitate their mutual activation. Prob- as OSSA or C9orf10 (16), in the signal transduction of IL13 ably, transient FAK dimerization leads to autophosphorylation in

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Bartolome et al.

A 100 ** **

80 *

Control siRNA + control antibody 40 FAM120A siRNA + control antibody Percent survival Control siRNA + anti-IL13Rα2 20 Figure 6. FAM120A siRNA + anti-IL13Rα2 FAM120A and IL13Ra2 are required for liver homing and metastasis. A, 0 Kaplan—Meier survival results for 0 102030405060708090100110120 nude mice inoculated in spleen with Days after inoculation KM12SM cells previously transfected with FAM120A or control siRNAs and treated with anti-IL13Ra2 or control B antibody at 5 mg/mL. Either silencing 80 of FAM120A or treatment with anti- IL13Ra2 antibody signiﬁcantly 60 increased mice survival (, P < 0.05; , P < 0.01). B, percentage of mice 40 with macroscopic metastasis in liver siRNA Control + control antibody was signiﬁcantly reduced in mice metastasis

macroscopic macroscopic 20

% of Mice with % of Mice with inoculated with FAM120A-silenced or anti-IL13Ra2–treated KM12SM cells 0 (left; P < 0.01). Right, images of livers siRNA: Control FAM120A Control FAM120A of mice treated as in A showing Antibody: α Control Anti-IL13R 2 metastasis. C, at the indicated times, RNA was isolated from livers of siRNA FAM120A + siRNA control + siRNA FAM120A + inoculated mice and subjected to control antibody anti-IL13Rα2 anti-IL13Rα2 RT-PCR assays to detect human GAPDH as surrogate of cell colonization. Murine b-actin was C used as loading control.

hGAPDH Liver mβ-Actin

siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA control FAM120A control FAM120A control FAM120A control FAM120A

2 h 8 h 24 h 56 h

Tyr397, which increases the catalytic activity of FAK (18, 19). The FAM120A silencing would impair IL13Ra2 access to cell mem- presence of b1 integrin together with FAK in the FAM120A brane, avoiding the interaction with IL13 and precluding signal- network favors metastasis and cancer progression due to the ing pathway activation. activation of the integrin signaling pathway that promotes tumor Regarding other protein networks, FAM120A was also asso- growth (20, 21). Activation of both pathways by IL13 in colon ciated to TNFRSF10B (a proapoptotic death receptor for TRAIL) cancer cells required the presence of FAM120A. and caspase-8. The binding of TRAIL to TNFRSF10B leads to Scaffold proteins modulate cell signaling by tethering individ- caspase activation and initiation of apoptosis (24). Further inves- ual pathway components together and isolating different path- tigation is required to clarify whether IL13Ra2 contributes to ways from one another. Scaffold proteins can establish connec- resistance to apoptosis and survival by sequestering FAM120A tions between pathways in order to distribute signals (22). Fur- from the TRAIL complex in addition to the activation of thermore, many scaffold proteins interact with kinesin proteins PI3K. On the other hand, TRAIL signaling promotes a proinﬂam- and are involved in cargo movement (23). In our proteomic matory pathway (25) that can establish a relationship with a analysis, we identiﬁed a large number of proteins involved in similar IL13 activity. Finally, FAM120A showed an association transport, including kinesin proteins (KIF). Knocking down with GPR56, a G-protein–coupled receptor for type III collagen, FAM120A decreased IL13Ra2 presence on cell surface, without which activates the RHOA pathway (26). RHOA overexpression altering total IL13Ra2 expression. Therefore, FAM120A would is usually associated with elevated lethality and aggressive pro- play a dual role: (i) IL13Ra2 transport and location and (ii) liferation (27, 28). Therefore, IL13 might have different roles, bringing together IL13Ra2 with FAK, PI3K, and SFKs. Because through FAM120A, in the activation of different pathways that FAM120A modulates IL13Ra2 expression on cell surface, not only promote cell migration and invasion, but also adhesion,

2442 Cancer Res; 75(12) June 15, 2015 Cancer Research

IL13Ra2 Signaling in Colorectal Cancer Metastasis

ITGB1

IL13 IL13Rα2 IL13Rα2

P P FAK SRC PI3K P FAM120A FAM120A

Figure 7. ACTB MAPK2K2 P Dual role of FAM120A in IL13Ra2- ACTB P ERK1/2 driven metastasis of colorectal cancer cells. FAM120A showed a dual role in MYH9 IL13Ra2-driven metastasis. On the one MYH11 + IL13 hand, it promoted IL13Ra2 location in the cell membrane, and, then, after activation of the receptor by IL13 RAB14 FAM120A binding, it recruited other cell signaling RAB11A proteins to promote different biologic KIF events leading to liver colonization MIGRATION/ IL13RA2 SURVIVAL and survival. INVASION

TUBA1B TUBA1C COPE COPG1 TUBB2B TUBB3

METASTASIS

survival, and proliferation. These events facilitate the colonization Acquisition of data (provided animals, acquired and managed patients, by tumor cells and the subsequent formation of metastasis. The provided facilities, etc.): R.A. Bartolome, I. García-Palmero, S. Torres, identification of IL13Ra2 signaling should help to understand the M. Lopez-Lucendo, I.V. Balyasnikova Analysis and interpretation of data (e.g., statistical analysis, biostatistics, signaling events of IL13 in other cancers, asthma, ulcerative colitis, computational analysis): R.A. Bartolome, I. García-Palmero, S. Torres, J.I. Casal and other diseases. Writing, review, and/or revision of the manuscript: R.A. Bartolome, In summary, FAM120A is a scaffold protein required for the I.V. Balyasnikova, J.I. Casal proper IL13Ra2-trigger signaling, which leads to colon cancer cell Administrative, technical, or material support (i.e., reporting or organizing activation and liver metastasis. FAM120A modulates IL13Ra2 data, constructing databases): M. Lopez-Lucendo, J.I. Casal location and transport between the cell membrane and intercel- Study supervision: J.I. Casal lular compartments as well as the activation of different signaling Grant Support pathways. Our results with a metastatic mouse model suggest that R.A. Bartolome was supported by a contract to established research the silencing of FAM120A is as effective as the treatment with anti- groups of the Asociacion Espanola~ Contra el Cancer (AECC). I. García- a IL13R 2 blocking antibodies (17) in preventing liver coloniza- Palmero was supported by a contract from Comunidad de Madrid. S. Torres tion by colorectal cancer cells and the formation of macroscopic was a recipient of a Juan de la Cierva fellowship. M. Lopez-Lucendo was liver metastases. These results support the use of FAM120A as supported by a Proteored-ISCIII contract. This work was supported by potential target for colon cancer therapy and reinforce the ther- grants BIO2012-31023 from the Spanish Ministry of Economy and Com- apeutic value of IL13Ra2. petitiveness, grant to established research groups (AECC), grant S2011/ BMD-2344/(Colomics2) from ComunidaddeMadridandProteoRed- fl ISCIII platform. Disclosure of Potential Con icts of Interest The costs of publication of this article were defrayed in part by the No potential conflicts of interest were disclosed. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Authors' Contributions this fact. Conception and design: R.A. Bartolome, J.I. Casal Development of methodology: R.A. Bartolome, I. García-Palmero, S. Torres, Received December 12, 2014; revised March 30, 2015; accepted April 3, 2015; I.V. Balyasnikova published OnlineFirst April 20, 2015.

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2444 Cancer Res; 75(12) June 15, 2015 Cancer Research

IL13 Receptor α2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis

Rubén A. Bartolomé, Irene García-Palmero, Sofía Torres, et al.

Cancer Res 2015;75:2434-2444. Published OnlineFirst April 20, 2015.

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