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 À À HEK293A, HEK293T, MEF, triple knockout Src/Yes/Fyn SYF( / ) À/À 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- Ó2020 American Association for Cancer Research. son Cancer Center (Houston, TX; ref. 19). GSCs were maintained as AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 6, 2020; DOI: 10.1158/1541-7786.MCR-18-1370 Bunda et al. 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.