Published OnlineFirst June 26, 2018; DOI: 10.1158/0008-5472.CAN-17-3858 Cancer Tumor Biology and Immunology Research Upregulation of E3 Ubiquitin Ligase CBLC Enhances EGFR Dysregulation and Signaling in Lung Adenocarcinoma Shiao-Ya Hong1, Yu-Rung Kao1, Te-Chang Lee1, and Cheng-Wen Wu1,2,3,4 Abstract CBLC (CBL proto-oncogene c) belongs to the CBL protein ubiquitinated and positively regulated aEGFR stability family, which has E3 ubiquitin ligase activity toward activated through the conjugation of polyubiquitin by K6 and K11 receptor tyrosine kinases. CBLC is frequently upregulated in linkages. This CBLC-mediated polyubiquitination promoted non–small cell lung cancer (NSCLC), yet very little is known either preferential recycling of aEGFR back to the plasma about the functions of CBLC in tumorigenesis. Here we show membrane or trafficking to the cell nucleus. IHC analyses that CBLC is an epigenetically demethylated target and its revealed a positive correlation between phospho-EGFR and expression can be upregulated in NSCLC after treatment with CBLC in lung adenocarcinoma. In summary, we demonstrate a the DNA methylation inhibitor 50-azacytidine. Depletion novel mechanism by which aEGFR escapes lysosomal degra- of CBLC significantly inhibited cell viability and clonogenicity dation in a CBLC/ubiquitin-dependent manner to sustain its in vitro and reduced tumor growth in a xenograft model. CBLC activation. Our work identifies CBLC as a potential diagnostic silencing further sensitized EGFR-mutated NSCLC cells to biomarker and also points to its utilization as a novel thera- treatment with tyrosine kinase inhibitors. Conversely, ectopic peutic target for NSCLC therapy. expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by Significance: This work demonstrates the role of CBLC expres- competing with CBL for EGFR binding. Analysis of ubiquitin sion as a diagnostic biomarker and potential therapeutic target in linkages on activated EGFR (aEGFR) revealed that CBLC lung adenocarcinoma. Cancer Res; 78(17); 4984–96. Ó2018 AACR. Introduction a linker region, and a RING finger motif. CBLC differs from CBL and CBLB in that the latter two both have a proline-rich region, The activation of tyrosine kinase receptors (RTK) engages a wide several regulatory tyrosine phosphorylation sites, and an ubiqui- range of signaling pathways that regulate cellular processes and tin-associated (UBA) domain in their C-termini (2). While CBL functions (1, 2). Aberrant activation of RTKs can be triggered by and CBLB are ubiquitously expressed, the expression of CBLC is genetic alterations (e.g., amplification, somatic mutation, and specifically restricted to normal epithelial cells (5). deletion/insertion) or evading lysosomal degradation. The EGFR, The ubiquitination mediated by CBL can negatively regulate an important member of RTKs, is frequently hyperactivated in a EGFR signaling through lysosomal degradation. Upon ligand variety of cancers (3). Dysregulation of EGFR signaling can lead to stimulation, CBL binds to activated EGFR (aEGFR) through its uncontrolled cell proliferation,tumorigenesis, and cancer progres- TKB domain (6). Subsequently, CBL-interacting protein of 85 kDa sion (3). (CIN85) and endophilin are recruited to EGFR complexes by The CBL family of E3 ligases has emerged as a key negative tyrosine phosphorylation of CBL. This process has been found to regulator of activated RTKs. CBL family proteins belong to the be required for EGFR internalization into endosomal sorting (7). RING finger class of E3 ligases, which catalyze the transfer of The ubiquitin modification mediated by CBL then can serve as a ubiquitin from E2 to substrates (4). The CBL family proteins (CBL, signal for directing aEGFR toward lysosomal degradation (8). CBLB, and CBLC in mammals) share a highly conserved N- However, accumulating evidence also links CBL protein family to terminus consisting of a tyrosine-kinase binding (TKB) domain, a potential role of oncogenic driver in cancer progression. Muta- tions in CBL are associated with approximately 5% of myeloid 1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. 2Institute of neoplasms. The mutant proteins fail to ubiquitinate RTKs but Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, retain the adaptor function to activate downstream PI3K pathway. 3 Taiwan. Institute of Microbiology and Immunology, National Yang-Ming CBL mutations in the RING finger motif lost its E3 activity, 4 University, Taipei, Taiwan. Institute of Clinical Medicine, National Yang-Ming assuming an oncogenic role in cancer progression (9). University, Taipei, Taiwan. In comparison with CBL, the genetic variations of CBLC are less Note: Supplementary data for this article are available at Cancer Research common and little is known about its functions in tumorigenesis. Online (http://cancerres.aacrjournals.org/). Although previous reports suggest that CBLC may as well serve as a Corresponding Author: Cheng-Wen Wu, Institute of Biomedical Sciences, negative regulator of aEGFR (1, 5), it has been revealed that CBLC Academia Sinica, 128 Section 2, Academia Road, Nankang, Taipei 11529, Taiwan. neither interacts nor colocalizes with CIN85 in mammalian cells Phone: 8862-2652-3015; Fax: 8862-2652-3075; E-mail: [email protected] due to the absence of the distal part of C-terminus found in CBL or doi: 10.1158/0008-5472.CAN-17-3858 CBLB proteins (10). Because CBL–CIN85–endophilin interaction Ó2018 American Association for Cancer Research. is required to initiate the endocytosis and degradation of EGFR, 4984 Cancer Res; 78(17) September 1, 2018 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst June 26, 2018; DOI: 10.1158/0008-5472.CAN-17-3858 CBLC Dysregulates EGFR Signaling the exact role of CBLC in regulating aEGFR turnover remains facturer's protocol. UPL probe-based qPCR (Roche) was carried elusive. Here, we identified that CBLC is an epigenetically out on a LightCycler 480 and analyzed using the manufacturer's demethylated target and its expression can be upregulated after software. The following primers and UPL probes were used for treatment with a DNA methylation inhibitor, 50-azacytidine in amplification: EGFR forward: 50-CATGTCGATGGACTTCCAGA- non–small cell lung cancer (NSCLC) cells. Our data revealed that 30, reverse: 50-GGGACAGCTTGGATCACACT-30, probe: 44; CBL CBLC knockdown renders EGFR-mutant NSCLC cells more sen- forward 50-TGACGTATGACGAAGTGAAAGC-30, reverse: 50- sitive to tyrosine kinase inhibitor (TKI) treatments, probably CAGCTCAGCCGGAAGATATAA-30, probe: 50; CBLB forward: through reducing the protein stability of aEGFR. We further found 50- GTGCACCTCTTGCCTTACG-30, reverse: 50-CCTTTTATTTCA- that CBLC overexpression reduces CBL-mediated ubiquitination CAACGACAGAAA, probe: 51; CBLC forward: 50-TGCTGAGAG- and degradation of aEGFR through the conjugation of ubiquitin CAACAAGGATG-30, reverse: 50-TCTGGCTGTCCGAGTGCT-30, K6 and K11 linkages. The CBLC-mediated stabilization of aEGFR probe: 71; b-actin forward: 50-AGAGCTACGAGCTGCCTGAC- is preferentially destined for the recycling program, leading to 30, reverse: 50-CGTGGATGCCACAGGACT-30, probe: 9. sustained EGFR activation in cancer cells. Immunoblotting Cultured cells were lysed with RIPA buffer (50 mmol/L Tris Materials and Methods HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% NP-40, 1% Database and statistical analyses sodium deoxycholate, 0.1% SDS) supplemented with protease The level of CBLC promoter methylation was extracted from and phosphatase inhibitors (Roche). The protein concentration TCGA and analyzed by GraphPad Prism. Other statistical analyses was determined using the Bio-Rad protein assay Kit (Bio-Rad). were also performed using GraphPad Prism. P values less than Lysates were separated in a 8%–12% SDS-PAGE, transferred to 0.05 were considered significant. nitrocellulose membranes, incubated with specific antibodies overnight, and developed by enhanced chemiluminescence. Cell lines, plasmids, and antibodies Images were acquired with the LAS-3000 system (Fujifilm). The human lung normal cells (BEAS2B, NL-20), lung cancer cells (A549, H358, H1437, H3255, H1975, HCC827, H2170, Genomic DNA preparation and bisulfite conversion PCR H226, H520), and HEK293T were from ATCC. All cell lines were Genomic DNA from cultured cells was isolated by using a authenticatedbycytogeneticanalysisfromthisestablishedprovider. commercially available DNA Extraction kit (Macherey-Nagel) They were maintained according to ATCC's instructions and used followed by the corresponding manufacturer's protocols. DNA within 6 months after resuscitation. H928 lung adenocarcinoma samples were bisulfite converted using EpiTect Bisulfite Conver- cells were authenticated and provided by the National Health sion Kit (Qiagen) following the manufacturer's instructions. The Research Institute (Taipei, Taiwan), and cultured in RPMI1640 resulting modified DNA was amplified by PCR using methylation- medium supplemented with 10% FBS and 1% penicillin/strepto- specific and unmethylation-specific primer sets. The primer mycin.AllcellswereroutinelydetectedasMycoplasma-freebytheEZ- sequences were as follows: methylated forward primer: 50- PCR Mycoplasma Test Kit (Biological Industries). CBLC cDNA was TTTTTTTCGGTTTTTTAGGATTCGT-30, methylated reverse primer: cloned into pCDNA3/HA or pEGFP-C2 vector. Rab family (RAB5, 50-TAAACCACCTCTCGAAACAACTACG-30; unmethylated for- RAB7, and RAB11)