C-Src Phosphorylates and Inhibits the Function of the CIC Tumor

Published OnlineFirst February 6, 2020; DOI: 10.1158/1541-7786.MCR-18-1370

MOLECULAR CANCER RESEARCH | SIGNAL TRANSDUCTION AND FUNCTIONAL IMAGING

c-Src Phosphorylates and Inhibits the Function of the CIC Tumor Suppressor Protein Severa Bunda1, Pardeep Heir1, Annie Si Cong Li1, Yasin Mamatjan1, Gelareh Zadeh1,2,3, and Kenneth Aldape1,4

ABSTRACT ◥ Capicua (CIC) is a transcriptional repressor that counteracts Src family kinase inhibitor, dasatinib, which prevents EGF- activation of genes in response to receptor tyrosine kinase (RTK)/ mediated tyrosine phosphorylation of CIC and attenuates ele- Ras/ERK signaling. Following activation of RTK, ERK enters the vated ETV1 and ETV5 levels, reduces viability of GBM cells and nucleus and serine-phosphorylates CIC, releasing it from its glioma stem cells (GSC), but not of their control cells with targets to permit gene expression. We recently showed that ERK undetectable c-Src activity. In fact, GBM cells and GSC expres- triggers ubiquitin-mediated degradation of CIC in glioblastoma sing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity (GBM). In this study, we examined whether another important to dasatinib, further endorsing the effect of dasatinib on Src- downstream effector of RTK/EGFR, the non-RTK c-Src, affects mediated tyrosine phosphorylation of CIC. These ﬁndings elu- CIC repressor function in GBM. We found that c-Src binds and cidate important mechanisms of CIC regulation and provide the tyrosine-phosphorylates CIC on residue 1455 to promote nuclear rationale to target c-Src alongside ERK pathway inhibitors as a export of CIC. On the other hand, CIC-mutant allele (CIC- way to fully restore CIC tumor suppressor function in neoplasms Y1455F), that escapes c-Src–mediated tyrosine phosphorylation, such as GBM. remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors Implications: c-Src tyrosine-phosphorylates CIC exports to cyto- ETV1 and ETV5. Furthermore, we show that the orally available plasm and inactivates its repressor function in GBM.

Introduction findings validate the importance of CIC, the molecular mechanisms regulating CIC repressor function are not well defined, especially Capicua (CIC) is a high-mobility group (HMG)-box transcrip- in mammalian cells. Posttranslational events on CIC, including tional repressor that counteracts activation of genes downstream of ERK-mediated serine/threonine phosphorylation (1–3, 14–16) have receptor tyrosine kinase (RTK) Ras/ERK signaling and was first been shown to promote its inactivation by either degradation or described in Drosophila to be involved in EGFR-mediated devel- nuclear-to-cytoplasmic shuttling of CIC, preventing its ability to opmental patterning and cell fate (1–4). The importance of CIC in function as a transcriptional repressor. We recently showed that in mammalian cells emerged after the discovery of loss-of-function glioblastoma (GBM), CIC is degraded because of ERK-mediated mutations in CIC in tumors, such as oligodendrogliomas (5, 6), and serine (S173) phosphorylation of CIC, which promotes binding of gene fusions of CIC with either DUX4 or FOXO4 in round cell the E3 ligase PJA1 to initiate ubiquitin-mediated degradation sarcomas (7, 8). Subsequently, CIC mutations have been linked to of CIC (17). Given the importance of posttranslational modifica- other tumor types (9, 10) and connected to additional biological tionsofCIConitsrepressorandtumorsuppressorfunction,we processes, such as lung development, liver homeostasis, autoim- examineinthisreporttheroleoftyrosinephosphorylationonthe munity, and neurobehavioral processes (11). The oncogenic tran- function of CIC. scription factors ETV1, ETV4, and ETV5 (12), which mediate cell proliferation, motility, and invasion downstream of Ras (13), are the best-characterized CIC targets in mammalian cells. While these Materials and Methods Cells HEK293A, HEK293T, MEF, triple knockout Src/Yes/Fyn SYF( / ) / 1MacFeeters Hamilton Centre for Neuro-Oncology Research, Princess Margaret MEFs [referred to as MEF Src( ) throughout the article], U87, U251, Cancer Centre, Toronto, Ontario, Canada. 2Division of Neurosurgery, Toronto U118, A172, T98G, and GL261 were obtained from ATCC. Normal Western Hospital, Toronto, Ontario, Canada. 3Institute of Medical Science, human astrocytes (NHA) were described previously (18). Normal 4 Toronto, Ontario, Canada. Laboratory of Pathology, NCI, Bethesda, Maryland. mouse astrocytes were purchased from ScienCell Research Laborato- Note: Supplementary data for this article are available at Molecular Cancer ries. Cells were maintained in DMEM (Invitrogen) supplemented with Research Online (http://mcr.aacrjournals.org/). fi 10% heat-inactivated FBS (Wisent) at 37 C in a humidi ed 5% CO2 Corresponding Authors: Kenneth Aldape, NCI, Laboratory of Pathology, atmosphere. Six glioma stem cell (GSC) cultures (GSC 8-18, GSC 7-2, Bethesda, MD 20814. Phone: 301-480-8853; Fax: 713-253-5284; E-mail: GSC 7-11, GSC 28, and GSC 30) were derived from freshly operated [email protected]; and Gelareh Zadeh, MacFeeters-Hamilton Center for tumor samples from patients with GBM at the University of Texas MD Neuro-Oncology, Princess Margaret Cancer Center, Princess Margaret Cancer Anderson Cancer Center (Houston, TX) as per guidelines set by the Research Tower, 101 College Street, 14th floor, Room 601, Toronto, Ontario, Canada M5G 1L7. E-mail: [email protected] institutional review board and described previously (17). Each patient provided written informed consent for tumor tissues and this study Mol Cancer Res 2020;XX:XX–XX was conducted under protocol LAB03-0687, which was approved by doi: 10.1158/1541-7786.MCR-18-1370 the Institutional Review Board of the University of Texas MD Ander- 2020 American Association for Cancer Research. son Cancer Center (Houston, TX; ref. 19). GSCs were maintained as

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neurospheres in either defined DMEM/F12 media or neurobasal Mass spectrometry media (Gibco), respectively, in the presence of growth factors EGF HEK293 cells transfected with an empty plasmid control, (20 ng/mL), recombinant basic FGF (20 ng/mL; R&D Systems), and HA-CIC(WT) alone or HA-CIC(WT) and c-Src together were lysed B27 growth supplement with vitamin A (1:50 working concentration; and immunoprecipitated with anti-HA antibody. The immuno- Life Technologies) as described previously (17). Endogenously HA- precipitated protein was trypsin digested, followed by LC/MS-MS), tagged CIC in HEK293 cells was described previously (17). Briefly, the was performed at the SPARC BioCentre mass spectrometry facility following DNA constructs were transfected: pRNAT-H1.3(Hygro), of the Hospital for Sick Children (Toronto, Ontario, Canada). pX459-CICend, and double stranded donor DNA, 50-CCCCAGCC- CTCCCCCCCACCCCCAGGTCCCTCCACAGCTGCCACAGGCA- Luciferase assay GGTACCCCTACGACGTGCCCGACTACGCCTGAGGGACCCC- Cells were transfected in triplicate with pGL3-ETV5 plasmid, which TGAGAAGATGCCAGGACTTATAGTACCCCCTCAGGACATGG. contains four consensus CIC octameric motifs, and pRL-SV40 (Pro- Cells were selected with hygromycin and monoclonal lines were mega) Renilla luciferase control as previously described (17). screened. To generate GL261, U87, or GSC 7-2 cells that express Cells were transfected with the indicated plasmids and lysates were control, FLAG-CIC(WT), or FLAG-CIC(Y1455F) the following assayed for luciferase activity. A Dual-Luciferase Reporter Assay pMXs-GW-FLAG-IRES-BsdR transfer plasmids, along with pUMVC System (Promega) was used, and luminescence was measured (Addgene 8449) and pCMV-VSV-G (Addgene 8454) were used to using a GloMax 20/20 Luminometer (Promega). Relative light units generate retroviral supernatants as described previously (17). Cells from firefly luciferase were normalized against Renilla luciferase were selected in blasticidin. All cell lines were routinely tested for values. Mycoplasma infection using the PlasmoTest Kit (InvivoGen). Cell lines were not specifically authenticated and were used within 15 passages. Immunoprecipitation, oligonucleotide pull-down assay, and immunoblotting Plasmids Immunoprecipitation and Western blotting were performed as CIC cDNA was a kind gift from Paul Scotting (University of described previously (17). Cells were harvested in EBC lysis buffer Nottingham, Nottingham, England). The cDNA was prepared for (50 mmol/L Tris, pH 8, 120 mmol/L NaCl, and 0.5% NP-40) and Gateway system using a two-step PCR with primary gene specific supplemented with protease inhibitors (Roche). Specifically, lysates primers (50- CAAAAAAGCAGGCTCCACCATGTATTCGGCCCA- were immunoprecipitated using the indicated antibodies along with CAGGCCC-30;50-CAAGAAAGCTGGGTTTCACCTGCCTGTGG- protein A-Sepharose (Repligen). Bound proteins were washed five CAGCTGTG-30) and secondary AttB-specific primers (50- GGGGA- times in NETN buffer (20 mmol/L Tris, pH 8, 100 mmol/L NaCl, 1 CAAGTTTGTACAAAAAAGCAGGCTCCACC- 30;50-GGGGAC- mmol/L EDTA, and 0.5% NP-40), eluted by boiling in sample buffer CACTTTGTACAAGAAAGCTGGGTT-30). Mutations were intro- and resolved by SDS-PAGE. Proteins were electro-transferred onto duced using site-directed mutagenesis with KOD polymerase polyvinylidene difluoride membrane (Bio-Rad), blocked, and probed (Novagen, Merck). Primers used: R201W (50- ATGGGCCGCCA- with the indicated antibodies. For experiments in which the nuclear or GATGTGGTCCTTCTCCCGCTT-30;50-AAGCGGCACCAGGCC- cytoplasmic fractions were prepared the Nuclear Extraction Kit (Cay- CTGGTCCACCAGC-30) and R1515H (50- CGTGAGGTGCACCA- man Chemicals) was used according to the manufacturer's instruction GAAGATCATGCAGGCTGC- 30;50-GATCTTCTGGTGCACCT- and immunoblotting was performed as stated above. For denaturing CACGGATCTTCAACTG-30). The Y1455F plasmids were generated immunoprecipitations used to detect ubiquitylation lysates were sup- using overlap extension and the following four primers: CIC-Eco47III- plemented with 1% SDS and boiled for 5 minutes to denature proteins F: 50-CAG CAG AGCGCTTTGCTGAGTTGCCTG. CIC-Y1455F-R: and disrupt interactions. The boiled lysates were diluted so the final 50-GGGAGGAGTATGGCACCTTGTCAAACTCTAGCTCCCCAA- concentration of SDS was less than 0.1% and immunoprecipitation GCACGTCC CIC-Y1455F-F: 50-GGACGTGCTTGGGGAGCTA- was performed. Oligonucleotide pull-down assay was performed as GAGTTTGACAAGGTGCCATACTCCTCCC. NotI-CIC-R: 50-CT- described previously (17). The following oligos were annealed ETV5- GCTG GCGGCCGC TCACCTGCCTGTGGCAGCTGTG. The frag- WT octameric repeat: 50-Biotin-CGCGTTTTTTATGAATGAAAA- ment generated was digested with Eco47III and NotI and ligated into ACGTCCTTA and 50-TAAGGACGTTTTTCATTCATAAAAAAC- the parental plasmid that was digested with the same enzymes and gel GCG or for ETV5-Mutant octameric repeat the following oligos were purified. All variants were sequence verified, using standard Gateway annealed: 50-Biotin-CGCGTTTTTTATTAAAAGGAAACGTCCTTA sequencing primers and gene internal primers (50-TGCCCTACC- and 50-TAAGGACGTTTCCTTTTAATAAAAAACGCGC. Annealed CAAGGAACGG-30;50-CAGGCGCTACAGGAACTGACG-30;50- oligonucleotides (1 mg) was bound to streptavidin agarose (Thermo CGCCTGCTTCCTCCTCAGC-30;50-CCACACTTGGTGGCTGGA- Fisher Scientific) and mixed with lysate for 2 hours. Bound proteins CC-30;50-TCAGTTTCTCCCGTGCAGGC-30;50-GCACCCACCTC- were resolved as indicated above. CTCAGCACC-30;50-CAGAGACCTGGACTCCCACGG-30; and 50- CCCACCCTGCAGTCTCTGGC-30). Sequence was compared with Antibodies accession number NM_015125. Subsequently the CIC cDNA was The following antibodies were obtained from Cell Signaling subcloned into modified pMXs Vektors (FLAG-tag, Gateway-cassette, Technology: HA (C29F4) (1:6,000), c-Src (2108), pSrc(416) IRES, and BsdR) using the LR-reaction following the manufacturer's (2101), Lamin(A/C) (2032), pERK (4370), b-actin (8H10D10) protocol (Invitrogen) a generous gift from Dr. Stefan Pusch (Heidelberg (1:20,000), and a-tubulin (2144) (1:5,000). pERK (sc-7383 and University Hospital, German Cancer Center). pCMV5-HA-CIC sc-16982-R) and GFP (sc-9996) (1:6,000) were obtained from Santa (DU19108) and pcDNA5-FRT/TO-GFP-CIC (DU16689) were pur- Cruz Biotechnology. Phospho Ser/Thr (ab17464), capicua chased from MRC-PPU University of Dundee (Dundee, Scotland). (ab123822), and ETV1 (ab81086) were obtained from Abcam. The HA-ERK (8974), c-Src (42202), and c-Src (K295R Y527F) were pur- following antibodies were purchased from Millipore: capicua chased from Addgene. EGFR-GFP was a kind gift from Dr. Yi Wang (ABN446), capicua (MABN449), EGFR, and pTYR. FLAG-M2 (Radiation Biology and Health Branch, Chalk River ON, Canada). (F1804), b-actin (A5316) (1:10,000), vinculin (V9264) (1:30,000),

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ETV5 (WH0002119M2), and polyclonal ERK (M5670) antibodies Ingenuity pathway analysis (1:5,000) were obtained from Sigma. All antibodies were utilized at a To perform pathway analysis, we initially performed differential 1:1,000 dilution unless otherwise specified. expression analysis between GBM versus normal patients. We then selected significantly differentially expressed genes (n ¼ 6,668 genes) Chemicals and reagents that were passed from a criteria (FDR < 0.05, fold change > 2or PD98509 (P215) and dasatinib were obtained from Sigma. EGF was fold change < 2) based on the changes in expression between purchased from Gibco. Streptavidin-agarose resin was obtained from GBM versus normal patients. Ingenuity pathway analysis spring Thermo Fisher Scientific (20349). release (March 2018) of differentially expressed genes in GBM versus normal patients identified several significantly contributed Cell proliferation pathways. Equal numbers of cells were plated in triplicate in 96-well plates and cellular proliferation was assessed using Alamar Blue prolif- Statistical analysis eration assay as per the manufacturer's instructions (Invitrogen) or All experiments were performed at least in triplicate with mean and 5-bromodeoxyuridine (5-BrdU) cell proliferation assay as per the SEM reported. Log-rank statistics were performed on survival curves. manufacturer's instructions (BioVision) as described previous- For direct comparisons, an unpaired Student t test was carried out. ly (17). Trypan blue exclusion assay was performed by directly Significance was defined as , P < 0.05 unless specifically stated in figure counting cells plated in triplicate using the Beckman Coulter Vi- legends. CELL (12-Sample Carousel) Cell Viability Analyzer (Beckman Coulter) as described previously (17). For drug sensitivity assays, 5,000 cells were seeded in 96-well plates and 24 hours later the cells Results were treated with increasing concentrations of drugs and Alamar CIC is tyrosyl-phosphorylated in response to EGF stimulation Blue assay was performed. While earlier work investigated phosphorylation of serine and threonine residues on CIC in response to ERK downstream of EGFR Patient-derived tumor samples activation (17), tyrosine phosphorylation is an equally important Patient resected samples were obtained from Toronto Western posttranslational modification that regulates protein function. We Brain Tumor Bank and processed in accordance with a University asked whether CIC is posttranslationally modified via tyrosine Health Network Research Ethics Board–approved protocol. phosphorylation in response to EGFR activation. HEK293 cells were transfected with plasmids encodingHA-taggedCICandEGFR Xenograft GBM model and cell lysates were immunoprecipitated using an anti-HA anti- All animal procedures were carried out according to animal user body. The HA-CIC immunoprecipitate reacted with anti-pSer/Thr protocols approved ethically by the Institutional Animal Care antibody, as expected, but also exhibited marked tyrosyl phosphor- Committee under the guidelines of the Canadian Council on ylation in response to signaling in EGFR-activated cells (Fig. 1A). Animal Care and the University Health Network Research Ethics We confirmed this finding for mouse embryonic fibroblasts (MEF) Board as described previously (17). Briefly, NOD/SCID (male cotransfected with HA-CIC and EGFR (Fig. 1B). We next asked 8-week-old) were anaesthetized using 0.5 mg/g of intraperitoneal whether CIC tyrosyl phosphorylation can be detected in an endog- injection of 20 mg/mL Avertin (Sigma-Aldrich) and 5 mg/kg of the enous setting (in the absence of overexpression). CIC was tyrosyl- presurgical analgesic Anafen 1 mg/mL (Ketoprofen) was adminis- phosphorylated upon EGF treatment of HEK293 cells in which a C- tered subcutaneously. TearGel (Novartis) was applied to the eyes to terminal HA-epitope tag was genetically engineered onto endoge- prevent corneal dehydration and abrasion. Once a toe pinch no nous CIC using CRISPR/Cas9 (Fig. 1C). Likewise, endogenous CIC longer elicited a response, the scalp was cleaned and hair removed, in NHA was found to be tyrosyl-phosphorylated in response to EGF and a midline incision was made from the ears to the eyes. treatment (Fig. 1D). As expected, EGF treatment triggered dere- Underlying periosteum was frozen with 2% Lidocaine (Bimeda pression of CIC target ETV5 under these conditions (Fig. 1D). MTC) and removed with scissors. Mice were placed on a digital These results suggest that CIC can be posttranslationally modified stereotaxic frame and from bregma the cortex coordinates were by tyrosine phosphorylation in response to signaling downstream of identified (X:1.6, Y:1, Z:0.6). A high-speed dental drill with a 0.7- EGFR. mm adaptor (Fine Science Tools) was used to bore a whole through the skull. A total of 1 105 U87 cells resuspended in 10 mL of sterile c-Src binds to and tyrosine-phosphorylates CIC 1 PBS were injected 3 mm deep through the hole using a 10-mL c-Src is another important kinase that is activated in response to 30-G Hamilton microsyringe, over a 1-minute period. Mice were EGFR signaling. c-Src tyrosine-phosphorylates its targets both in the sutured and returned to a fresh sterile heated cage to recover and cytoplasm and in the nucleus. To explore the potential of c-Src to supplemented with 0.3 mg/ml enrofloxacin in the drinking water tyrosine-phosphorylate CIC, HEK293 cells were engineered to over- (Baytril Bayer/CDMV, catalog no.102207). express c-Src, which was able to bind and promote the tyrosyl phosphorylation of endogenous CIC (Fig. 2A). A kinase-dead c-Src The Cancer Genome Atlas data analysis (K295R, Y527F) mutant was deficient in both of these abilities mRNA dataset for GBM (n ¼ 143) and normal patients (n ¼ 5) were (Fig. 2A). We recently demonstrated that in GBM, endogenous CIC downloaded from the UCSC Cancer Genomics Browser (https:// is serine-phosphorylated and cleared by activated ERK (17). To genome-cancer.ucsc.edu). The gene expression datasets (RSEM nor- examine whether activated c-Src tyrosine-phosphorylates CIC, we malized count) were quantified using the Illumina Hiseq _RNASeqV2 stably expressed FLAG-CIC(WT) in U87 cells and cotransfected by the UCSC Cancer Browser team and log2 transformed. We eval- kinase activate and inactive c-Src. Like HEK293 cells, only kinase uated IDH wild-type GBM only and did not include any IDH-mutant active c-Src–bound and tyrosine-phosphorylated CIC in U87 cells GBM. (Fig. 2B). Furthermore, we found that dasatinib, a well-established

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Figure 1. EGFR activation promotes tyrosine-phosphorylation of CIC. A, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated (IP) with anti-HA antibody followed by immunoblotting with indicated antibodies. B, MEFs transfected with the indicated plasmids were lysed and immunoprecipitated with anti-HA antibody followed by immunoblotting with indicated antibodies. C, HEK293 cells endogenously tagged with HA-CIC, treated with or without EGF at indicated time points were lysed, and immunoprecipitated with anti-HA antibody followed by immunoblotting with indicated antibodies. D, NHA treated with or without EGF at indicated time points were lysed and immunoprecipitated with anti-CIC antibody followed by immunoblotting with indicated antibodies. WCE, whole-cell extract. Ctr, control.

inhibitor of c-Src kinase activity, prevented EGF-mediated tyrosyl pretreatment ameliorated this effect (Fig. 2D). To conﬁrm that EGFR phosphorylation of CIC (Fig. 2C), further substantiating that EGFR utilizes c-Src to tyrosine-phosphorylate CIC we obtained Src- utilizes c-Src to tyrosine-phosphorylate CIC. Importantly, we show knockout MEFs [MEF Src( / )] and showed that endogenous CIC that endogenous CIC is tyrosine phosphorylated in response to EGF was not tyrosine phosphorylated in response to EGF treatment in these treatment correlating with c-Src binding in NHA cells, while dasatinib cells lacking Src kinase activity (Fig. 2E). To localize the region within

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Figure 2. c-Src binds and tyrosine-phosphorylates CIC in response to EGFR activation. A, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated (IP) with anti-CIC antibody followed by immunoblotting with indicated antibodies. B, U87 stably expressing FLAG-CIC(WT) cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-CIC antibody followed by immunoblotting with indicated antibodies. C, U87 stably expressing FLAG-CIC(WT) cells were pretreated with or without 100 nmol/L of dasatinib for 1 hour prior to treatment with or without EGF were lysed and immunoprecipitated with anti-CIC antibody followed by immunoblotting with indicated antibodies. D, NHAs pretreated with or without 100 nmol/L of dasatinib for 1 hour prior to treatment with or without EGF were lysed and immunoprecipitated with anti-CIC antibody followed by immunoblotting with indicated antibodies. E, MEFs or Src(/) MEFs were treated with or without EGF and equal amounts of lysates were immunoprecipitated with anti-pTyr antibody and immunoblotted with the indicated antibodies. F, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-GFP antibody followed by immunoblotting with indicated antibodies. G, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-CIC antibody followed by immunoblotting with indicated antibodies. H, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-FLAG antibody followed by immunoblotting with indicated antibodies. WCE: whole-cell extract.

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CIC that c-Src recognizes, we utilized two CIC fragments one com- retained its transcriptional repressive activity was confirmed in addi- prising the N-terminal HMG-box DNA-binding domain CIC(1-450) tional cell lines, including patient-derived GSCs (Fig. 4D– G). Overall, and the other comprising the C-terminal C1 repressor domain CIC tyrosine phosphorylation at residue 1455 did not affect CIC's repressor (450-End). Following cotransfection with c-Src into HEK293 cells, activity and effect on proliferation in GBM cells. These findings only CIC(1-450) fragment showed an interaction with c-Src suggest that c-Src–mediated tyrosine phosphorylation negatively (Fig. 2F), but surprisingly, was not tyrosyl phosphorylated regulates CIC repressor function by promoting release of CIC from (Fig. 2G). Specific hotspot mutations of CIC, CIC(R201W) and its target genes in a manner similar to ERK-mediated serine/ CIC(R1515H) found in human gliomas, which fail to bind to threonine phosphorylation. We then used a biotinylated DNA DNA (20) bound strongly to c-Src and underwent tyrosyl phos- pull-down assay that encodes a portion of the ETV5 promoter phorylation (Fig. 2H), suggesting that phosphorylation of CIC is containing the octameric CIC-binding motif (TGAATGAA). With not dependent on its ability to bind to the DNA. Collectively, these this assay, we show that overexpression of c-Src promotes the findings show that c-Src binds and tyrosine-phosphorylates CIC in release of both wild-type CIC and the Y1455F phosphorylation– response to EGFR activation. defective mutant from the ETV5 DNA octamer (Fig. 4H), in a manner that was rescued by pretreatment with the specificERK Residue Y1455 of CIC is phosphorylated by c-Src inhibitor PD98509 (Fig. 4I). These findings indicate that c-Src– To examine the tyrosine residue(s) phosphorylated by c-Src, we induced tyrosine 1455 phosphorylation of CIC does not mediate the conducted mass spectrometry of CIC-enriched HEK293 lysates release of DNA-bound CIC. cotransfected with c-Src, which revealed tyrosine 1455 as the potential phosphorylation site (Fig. 3A), consistent with our findings showing Tyrosyl phosphorylation of CIC triggers nucleocytoplasmic that the 450-end region of CIC was tyrosine-phosphorylated (Fig. 2G). shuttling A cross-species alignment of CIC protein shows that Y1455 is a EGFR signaling triggers derepression of CIC from its target genes, conserved residue and that aspartic acid (D) or glutamic acid (E) which can be sustained by two mechanisms, protein degradation and residue immediately follows a tyrosine (Y) residue, and in humans is nuclear export. We have previously demonstrated that ERK activation consistent with a candidate YD c-Src phosphorylation motif triggers nuclear degradation of CIC through the E3 ubiquitin ligase (ref. 21; Fig. 3B). To validate that c-Src phosphorylates tyrosine PJA1 (17). Because c-Src–mediated tyrosyl phosphorylation of CIC 1455 on CIC, HEK293 cells were cotransfected with c-Src and a did not affect its DNA-binding ability (Fig. 4H and I), we hypothesized plasmid encoding a mutant [tyrosine (Y) to phenylalanine (F) at that c-Src mediates tyrosyl phosphorylation of CIC to influence residue 1455] to prevent Y1455 tyrosine phosphorylation. The nucleocytoplasmic shuttling. The close proximity of Y1455 to the Y1455F allele showed markedly reduced phosphorylation of CIC putative NES of CIC is consistent with this as a possible mechanism compared with wild-type CIC (Fig. 3C). To examine whether CIC (Fig. 3E). Ectopic expression of c-Src or EGFR, but not kinase dead is tyrosine phosphorylated on the same residue in GBM cells we stably c-Src(K295R, Y527F) triggered nuclear export of wild-type CIC expressed FLAG-CIC(WT) and FLAG-CIC(Y1455F) in patient- (Fig. 5A and B), demonstrating that active c-Src is required for derived GSCs that are otherwise devoid of CIC (17). Similar to nucleocytoplasmic shuttling of CIC to occur. To confirm these findings HEK293 cells, wild-type, but not tyrosine-defective CIC-Y1455F in an endogenous setting, HEK293 cells endogenously tagged with mutant was tyrosine phosphorylated in response to EGF treatment HA-CIC were treated with EGF in the presence and absence of either (Fig. 3D). Notably, Y1455 is located near the C1 repressor domain, the ERK inhibitor (PD98509) or Src kinase inhibitor (dasatinib). which is essential for the recognition and binding of the N-terminal Nuclear CIC was significantly reduced in EGF-treated cells that were HMG-box to target DNA, as well as nuclear localization signal (NLS) rescued in cells pretreated with PD98509 (Fig. 4C). This confirms our and predicted nuclear export signals (NES) of CIC (Fig. 3E) impli- previous findings that ERK promotes degradation of nuclear CIC cating the potential functional implication of this phosphorylation bound to the DNA (1). Importantly, we show for the first time that EGF event on CIC function. These results show that tyrosine 1455 is treatment concordantly promotes cytoplasmic accumulation of CIC phosphorylated on CIC in response to EGF treatment or c-Src that can be rescued by pretreatment with dasatinib (Fig. 5C). Similarly, expression. in MEFs lacking any Src kinase activity, CIC failed to accumulate in the cytoplasm following EGF treatment (Fig. 5D). These findings suggest Y1455 phosphorylation is not required for CIC's repressor that in addition to ERK-mediated degradation of nuclear CIC that is function bound to the DNA (1), EGFR also employs c-Src to clear nuclear CIC Residue 1455 is near the functional C1 domain, which mediates to the cytoplasm. In fact, we show that the two disease-causing CIC CIC's DNA-binding ability (20) and we therefore asked whether c-Src– mutants (R201W and R1515H) have a higher affinity to interact with mediated tyrosine phosphorylation at this residue influences CIC's c-Src, to be tyrosine phosphorylated (Fig. 2H) and are preferentially repressor function on its targets, including the oncogenic transcription accumulated in the cytoplasm compare with wild-type CIC (Fig. 5E). factors ETV1, ETV4, and ETV5 (12, 13). To test this, we first examined Because these disease-causing CIC mutants fail to bind to their DNA the role of tyrosine phosphorylation of CIC on its repressor function. target (17, 20), further supports our speculation that c-Src tyrosine- Using the Y1455F phosphorylation-defective CIC mutant we show phosphorylates nuclear CIC (that is not bound to DNA) for export to that it retains the ability to repress luciferase activity through the cytoplasm. To formally assess the role of tyrosine 1455 phosphory- octameric consensus motif (TGAATGAA) of ETV5, similar to levels of lation and nuclear export, we first transfected HEK293 cells with wild-type CIC (Fig. 4A). Consistent with this finding, we found that plasmids encoding HA-CIC(WT) or HA-CIC(Y1455F) in combina- cells stably expressing FLAG-CIC(Y1455F) suppressed ETV5 and tion with c-Src. Nuclear export of CIC(Y1455F) in response to c-Src ETV1 protein levels and reduced cellular proliferation similar to expression was drastically reduced compared with the wild-type wild-type controls. We note that this result is in contrast to cells that control (Fig. 5F). Similarly, EGF treatment failed to promote cyto- express the disease-associated mutation in residue 1515 (R1515H) or plasmic accumulation in GSCs stably expressing FLAG-CIC(Y1455F) 201 (R201W; Fig. 4B and C). The finding that the Y1455F CIC allele compared with FLAG-CIC(WT)-expressing cells (Fig. 5G). These

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Figure 3. c-Src phosphorylates CIC on tyrosine 1455 residue. A, Mass spectrometry analysis identiﬁes tyrosine phosphorylation on residue 1455 of CIC from HEK293 lysates cotransfected with HA-CIC and c-Src. CIC was immunoprecipitated (IP) using HA antibody, trypsin digest of the sample, followed by LC/MS-MS analysis was performed at the SPARC BioCentre mass spectrometry facility of the Hospital for Sick Children (Toronto, Ontario, Canada). Shown is an annotated representative CID spectrum for phosphorylated peptide containing Y1455. b ions are indicated in blue, and g ions are indicated in red. B, A multiple sequence alignment was performed using Clustal Omega (29). CIC tyrosine (Y) 1455 residues are conserved across species and are in yellow. Shown in blue are aspartic acid (D) residues or glutamic acid (E) that immediately follows a Tyr (Y) residue, making a candidate c-Src phosphorylation motif (21). C, HEK293 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-HA antibody followed by immunoblotting with indicated antibodies. D, GSC 7-2 stably expressing either FLAG-CIC(WT or Y1455F) cells were treated with or without EGF, lysed, and immunoprecipitated with anti-HA antibody followed by immunoblotting with indicated antibodies. E, Diagram depicting functional domains of CIC with the tyrosine (Y) 1455 residue. Ataxin 1 (ATXN1)-binding domain, DNA-binding HMG box domain, NLS, andthe predicted NES that were generated using the publically available NES prediction server, NESbase (24). Figure adapted with permission from Journal of Cell Science (12). Ctr, control.

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results suggest that c-Src–mediated tyrosyl phosphorylation of CIC on cells (Fig. 6I). To demonstrate that the specificity of dasatinib on residue 1455 preferentially promotes nuclear export of CIC that is not cell viability is through c-Src–mediated tyrosine-phosphorylation of bound to its target DNA (Fig. 5H), thereby preventing CIC from its CIC we assessed the effect of dasatinib in GL261, U87, and GSC function as a transcriptional repressor. stably expressing FLAG-CIC(WT) or FLAG-CIC(Y1455F). These results demonstrate that dasatinib had no significant effect on the Dasatinib affects the viability of GBM cells and not their normal viability of GL261 (Fig. 6J), U87 (Fig. 6K), or GSC 7-2 (Fig. 6L) counterparts cells expressing Flag-CIC(Y1455F), compared with cells expressing RTK signaling is a feature of GBM and promotes activation of wild-type CIC. Similar results were observed in U87 cells expressing other important signaling pathways including c-Src. To test wheth- CIC (R201W or R1515H) mutants (Supplementary Fig. S1D). These er c-Src is concordantly active in GBM, we assessed phosphoryla- findings further support the specificity of dasatinib treatment on c- tion of c-Src on tyrosine 416 residue, which is an established Src inhibition that concordantly affects CIC repressor function and marker of c-Src kinase activity (22). All five GBM cell lines test the expression of its ETV targets in GBM cells. (U87, U251, A172, U118, and T98G) displayed a greater proportion of active c-Src compared with control NHA cells (Fig. 6A). Intra- cranial xenografts of U87 cells in mice revealed active c-Src in the Discussion xenograft tissue, but not in the normal mouse brain tissue (Fig. 6B). In this article, we explored the regulation of CIC in a series of Compared with control neural stem cells (NSC), five GSC cultures experiments and validated its relevance in GBMs to further eluci- displayedincreaseinactivec-Src(Fig. 6C). A similar result was date its biological role in this tumor type. CIC is a transcriptional observed in 17 of 17 newly diagnosed GBM human tumors tested repressor that inhibits activation of genes downstream of RTK/Ras/ compared with four samples derived from nonneoplastic brain ERK signaling. This well-conserved signaling pathway controls tissue (Fig. 6D). As additional evidence, mouse glioma GL261 cells many different biological processes and is commonly activated in also had elevated pSrc(416) level compared with normal mouse cancer. We recently found that in the highly malignant and astrocytes (Fig. 6E). To validate the relevance of c-Src activation in aggressive GBM, which is characterized by hyperactive RTK sig- GBM, we compared differentially expressed genes in GBM versus naling, CIC protein levels are undetectable due to ERK-induced nonneoplastic brain and found c-Src activation as one of the top serine phosphorylation and proteasomal degradation of CIC (17). pathways upregulated in GBM in The Cancer Genome Atlas data We extend these findings by showing that the non-RTK c-Src (Supplementary Fig. S1A). regulates CIC repressor function by tyrosine-phosphorylating and Given that the c-Src pathway is active in GBM, we tested the exporting CIC to the cytoplasm. Our findings suggest that activa- effect of increasing concentrations of highly specific Src kinase tion of EGFR employs two parallel regulatory mechanisms to ensure inhibitor, dasatinib, using clinically relevant doses and lower, on CIC inactivation and derepression of EGFR-responsive genes. First, viability of GBM cells versus their normal counterparts. We show EGFR activation leads to ERK-mediated serine-phosphorylation that concentrations of dasatinib that are below the clinically rele- that induces degradation of DNA-bound CIC (17). Second, EGFR vant dose (100 nmol/L) significantly affect viability of GL261 activates c-Src, which in turn results in tyrosine-phosphorylation of (Fig. 6F), U87 (Fig. 6G), and GSC-7-2 (Fig. 6H). Notably, dasatinib nuclear CIC that is not DNA bound, promoting its nuclear export at 100 nmol/L did not affect viability of normal human astrocytes (Fig. 5H). Accordingly, it is likely that c-Src–mediated tyrosine- (NHA) (Supplementary Fig. S1B), NHA cells (Fig. 6G), or NSC phosphorylation of CIC ensures that free, unbound nuclear CIC is (Fig. 6H). NSC did not show a reduction in viability even at doses as rapidly exported to the cytoplasm and is inaccessible for additional high as 750 nmol/L (Supplementary Fig. S1C), further substanti- binding to its target DNA following degradation of formerly DNA- ating that dasatinib affects only the viability of cells with active c-Src bound CIC. such as in GBM. Importantly, we found that dasatinib treatment We found that c-Src specifically phosphorylates CIC at tyrosine reduces activation of c-Src, but not of ERK, in GL261, U87, and GSC 1455, which is located within the regulatory C-terminal region of the 7-2 cells (Fig. 6I). Consistent with our datashowinganinteraction protein (Fig. 3E). In addition to its close proximity to the C1 repressor of CIC and c-Src, we also found that dasatinib treatment correlated domain, which is essential for the binding of the N-terminal HMG-box with reduced levels of the key CIC targets ETV1 and ETV5 in these to its target DNA, Y1455 is also close to the ERK-binding domain and

Figure 4. Phosphorylation-defective CIC-Y1455F mutant retains strong repressor function. A, pGL3ETV5 was cotransfected with empty vector (mock), HA-CIC(WT), or HA- CIC(Y1455F)-mutant plasmid into HEK293 cells, and luciferase expression was measured. The bar graph depicts the relative levels of luciferase activity normalized to empty vector control. The data shown are the mean SEM from luciferase assays performed in triplicate. , P < 0.05 Student t test compared with mock. B, U87 control or stably expressing FLAG-CIC(WT, Y1455F, R201W, or R1515H) cells were lysed and immunoblotted with indicated antibodies. C, Equal number of U87 control or stably expressing FLAG-CIC(WT, Y1455F, R201W, or R1515H) cells were plated and cell proliferation was assessed by BrdU incorporation assay. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with control. D, GL261 cells stably expressing control, CIC- FLAG(WT), or CIC-FLAG(Y1455F) plasmids were lysed and immunoblotted with indicated antibodies. E, Equal number of GL261 cells stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were plated and Alamar blue (top) or trypan blue exclusion (bottom) assay was performed. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with GL261 control. F, GSC 7-2 stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were lysed and immunoblotted with indicated antibodies. G, Equal number of GSC 7-2 stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were plated and Alamar blue (top) or trypan blue exclusion (bottom) assay was performed. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with GSC 7-2 control. H, HEK293 cells transfected with indicated plasmids were lysed and an equivalent amount of lysate was incubated with Streptavidin agarose bound to biotinylated ETV5 oligonucleotides octameric motif, and bound proteins were detected by immunoblotting. I, HEK293 cells transfected with indicated plasmids were either pretreated with or without the ERK inhibitor, PD98509, lysed, and an equivalent amount of lysate was incubated with Streptavidin agarose bound to biotinylated ETV5 oligonucleotides octameric motif, and bound proteins were detected by immunoblotting. Ctr, control; strep-PD, Streptavidin pull-down; WCE, whole-cell extract.

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Figure 5. c-Src–mediated tyrosine 1455 phosphorylation regulates CIC nuclear export. A, HEK293 cells transfected with indicated plasmids were lysed and nuclear or cytoplasmic fractions were obtained and immunoblotted with indicated antibodies. B, U87 cells stably expressing CIC-FLAG(WT) transfected with indicated plasmids were lysed and nuclear or cytoplasmic fractions were obtained and immunoblotted with indicated antibodies. C, HEK293 parental cells or endogenously tagged with HA-CIC pretreated for 1 hour with either PD98509 or dasatinib prior to treatment with or without EGF were lysed and nuclear or cytoplasmic fractions were obtained and immunoblotted with indicated antibodies. D, MEFs or Src(/) MEFs treated with or without EGF were lysed and nuclear or cytoplasmic fractions obtained and immunoblotted with indicated antibodies. E and F, HEK293 cells transfected with indicated plasmids were lysed and nuclear or cytoplasmic fractions were obtained and immunoblotted with indicated antibodies. G, GSC 7-2 stably expressing FLAG-CIC(WT or Y1455F) treated with or without EGF were lysed and nuclear or cytoplasmic fractions were obtained and immunoblotted with indicated antibodies. H, Diagram depicting two parallel mechanism that regulate CIC function upon EGFR activation, the c-Src–mediated phosphorylation of tyrosine 1455 residue of nuclear CIC that is not DNA bound and ERK-mediated serine phosphorylation and degradation of DNA-bound CIC. Ctr, control.

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c-Src Tyrosine Phosphorylates CIC

Figure 6. Dasatinib affects viability of GBM cells with elevated c-Src activity but not normal cells with negligible Src activity. A, Human-derived GBM cell lines or NHAs were lysed and protein lysates were immunoblotted with indicated antibodies. B, Tumors or unaffected normal brains obtained from intracranial U87 xenograft from two different mice were lysed and protein lysates were immunoblotted with indicated antibodies. C, GSCs or NSCs were lysed and protein lysates were immunoblotted with indicated antibodies. D, Human operative GBM samples or normal derived brains (NB) were lysed and immunoblotted with indicated antibodies. E, GL261 cells or normal mouse astrocytes were lysed and protein lysates were immunoblotted with indicated antibodies. F, Equal number of GL261 cells were plated and treated with or without indicated concentrations of dasatinib and cell viability was assessed by an Alamar blue assay. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with control. G, Equal number of U87 or NHAs treated with or without indicated concentrations of dasatinib was plated and cell viability was assessed by an Alamar blue assay. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with control. H, Equal number of GSC 7-2, GSC 8-11, or NSCs treated with or without indicated concentrations of dasatinib were plated and cell viability was assessed by an Alamar blue assay. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with control. I, GL261, U87, or GSC 7-2 treated with or without dasatinib 100 nmol/L were lysed and immunoblotted with indicated antibodies. GL261 (J), U87 (K), or GSC 7-2 (L) control or stably expressing FLAG-CIC(WT or Y1455F) treated with or without indicated concentrations of dasatinib were plated and cell viability was assessed by an Alamar blue assay. Data represent mean SEM of three independent experiments performed in octuplet. , P < 0.05 Student t test compared with respective controls.

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the C-terminal NLS (Fig. 3E). Our work adds to the existing literature activation, two parallel mechanisms (ERK and c-Src activation) oper- on the regulation of localization of CIC. Specifically, prior work ate to negatively regulate CIC function. Because GBM is characterized suggests that ERK-mediated serine-phosphorylation of CIC prevents with hyper-activated EGFR signaling, our work suggests that dual ERK its nuclear import (23). Our study provides evidence that c-Src– and c-Src kinase inhibition may be required to fully restore CIC's mediated tyrosine phosphorylation also plays a role in the localization tumor suppressor function in GBM, and future studies will be aimed to of CIC by promoting its nuclear export. Furthermore, the publicly address this. available NES prediction server, NESbase, a database of proteins experimentally authenticated to identify leucine-rich NES (24), pre- Disclosure of Potential Conflicts of Interest – dicted one potential NES at the C-terminal region (1472 1475) of CIC No potential conflicts of interest were disclosed. (Fig. 3E). Although this requires further validation, the close prox- fi imity of the predicted NES to tyrosine 1455 reinforces our ndings that Authors’ Contributions c-Src–mediated tyrosine 1455 phosphorylation is important in the – Conception and design: S. Bunda, P. Heir, G. Zadeh, K. Aldape nuclear export of CIC, and is consistent with a role for c-Src mediated Development of methodology: S. Bunda, P. Heir, K. Aldape nuclear export of other transcription factors including YAP1 (25), Acquisition of data (provided animals, acquired and managed patients, provided FOXO (26), and RUNX3 (27). facilities, etc.): S. Bunda, P. Heir, A.S.C. Li c-Src has several important functional domains that regulate its Analysis and interpretation of data (e.g., statistical analysis, biostatistics, activity. For instance, c-Src typically recognizes its substrates using computational analysis): S. Bunda, P. Heir, A.S.C. Li, Y. Mamatjan, G. Zadeh, K. Aldape its SH3 domain and tyrosine phosphorylates its substrates using its Writing, review, and/or revision of the manuscript: S. Bunda, Y. Mamatjan, protein kinase domain (22). Consistently, we found that while c-Src G. Zadeh, K. Aldape tyrosine phosphorylates CIC at its C-terminal end, it bound to CIC Administrative, technical, or material support (i.e., reporting or organizing data, atitsN-terminus(Fig. 2F and G). Because an N-terminal proline- constructing databases): S. Bunda, K. Aldape rich region, close to the DNA-binding HMG box, has been iden- Study supervision: S. Bunda, K. Aldape tified (Fig. 3E), we speculate that c-Src recognizes this proline-rich Other (cosenior supervisor): G. Zadeh region via its SH3 domain to initiatetyrosine1455phosphorylation. This interplay is analogous to binding of CIC to DNA, which occurs Acknowledgments through the concerted effort of the C-terminal C1 repressor domain This work was supported by funding from the Canada Cancer Society Research and the N-terminal HMG-box (20). Institute Innovation Grant and the MacFeeters Hamilton Neuro-Oncology Program. The activation of c-Src is observed in a large proportion of tumors from multiple organ sites (28), and in this study we characterize a role The costs of publication of this article were defrayed in part by the payment of page for c-Src in GBM. We show that GBM cells are highly sensitive to the c- charges. This article must therefore be hereby marked advertisement in accordance Src inhibitor, dasatinib, even at low concentrations. Importantly, we with 18 U.S.C. Section 1734 solely to indicate this fact. show that dasatinib does not affect activation of ERK in GBM cells, but it results in derepression of ETV levels, which are known CIC Received May 6, 2019; revised August 28, 2019; accepted February 3, 2020; targets. Results from our study demonstrate that in response to EGFR published first February 6, 2020.

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AACRJournals.org Mol Cancer Res; 2020 OF13

c-Src Phosphorylates and Inhibits the Function of the CIC Tumor Suppressor Protein

Severa Bunda, Pardeep Heir, Annie Si Cong Li, et al.

Mol Cancer Res Published OnlineFirst February 6, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-1370

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