Ck2α' Drives Lung Cancer Metastasis by Targeting BRMS1 Nuclear Export and Degradation

Published OnlineFirst March 15, 2016; DOI: 10.1158/0008-5472.CAN-15-2888 Cancer Molecular and Cellular Pathobiology Research

CK2a' Drives Lung Cancer Metastasis by Targeting BRMS1 Nuclear Export and Degradation Yuan Liu1, Elianna B. Amin1, Marty W. Mayo2, Neel P. Chudgar1, Peter R. Bucciarelli1, Kyuichi Kadota1, Prasad S. Adusumilli1,3, and David R. Jones1,3

Abstract

Breast cancer metastasis suppressor 1 (BRMS1) is decreased speciﬁc small-molecule inhibitor CX4945 abrogates CK2a'- in non–small cell lung cancer (NSCLC) and other solid tumors, inducedcellmigrationandinvasionanddecreasesNSCLC and its loss correlates with increased metastases. We show that metastasis by 60-fold. Analysis of 160 human NSCLC speci- BRMS1 is posttranslationally regulated by TNF-induced casein mens conﬁrmed that tumor CK2a'andcytoplasmicBRMS1 kinase 2 catalytic subunit (CK2a') phosphorylation of nuclear expression levels are associated with increased tumor recur- BRMS1 on serine 30 (S30), resulting in 14-3-3e–mediated rence, metastatic foci, and reduced disease-free survival. Col- nuclear exportation, increased BRMS1 cytosolic expression, and lectively, we identify a therapeutically exploitable posttransla- ubiquitin-proteasome–induced BRMS1 degradation. Using our tional mechanism by which CK2a-mediated degradation of in vivo orthotopic mouse model of lung cancer metastases, we BRMS1 promotes metastases in lung cancer. Cancer Res; 76(9); 1– found that mutation of S30 in BRMS1 or the use of the CK2- 12. 2016 AACR.

Introduction in drug-resistant cancer cells, and clinical trials have demonstrated the antitumor activity of these compounds (12). Breast cancer metastasis suppressor 1 (BRMS1) has been impli- We (2) and others (13) have observed that although BRMS1 is cated in the suppression of breast, lung, and bladder cancer primarily a nuclear protein, it is also present in the cytosol in metastases without significantly affecting primary tumor growth cancer cells and primary human tumors. This suggests that the (1, 2). Specific to non–small cell lung cancer (NSCLC), BRMS1 function of BRMS1 may be modulated by intracellular compart- protein and transcript are differentially expressed in noncancer- mentalization. Although Rivera and colleagues identified a con- ous lung tissue (high) and tumors (low; refs. 2, 3). We previously served nuclear exportation motif in BRMS1 (14), the mechanisms demonstrated that transcriptional repression of BRMS1 occurs via through which BRMS1 undergoes nuclear export and the biologic RelA/p65-DNMT-1–mediated promoter methylation (4). Other significance of this process to the development of metastases are groups have indicated that BRMS1 is also regulated via the Cul3– unknown. SPOP complex (5). Herein, we report that BRMS1 protein is regulated by TNF- Casein kinase 2 (CK2) is a pleiotropic, highly conserved serine/ induced activation of CK2a'. CK2a'-mediated phosphorylation threonine kinase that consists of two a (a or a') catalytic and two b of BRMS1 at serine 30 (S30) promotes nuclear exportation of regulatory subunits (6). CK2 controls the stability of both IkB (7) BRMS1 via a 14-3-3–dependent mechanism. Using an orthotopic and the tumor suppressor PML (8) and regulates Snail 1–induced lung cancer model and CT visualization and quantification of epithelial–mesenchymal transition (EMT; ref. 9) and promotion tumor metastatic deposits, we show that CK2 phosphorylation of of NCoR-mediated cancer cell invasion (10). CK2 is a driver of S30 on BRMS1 results in a significant increase in metastatic cells. malignant progression and a classic example of nononcogene Moreover, we observed abnormally elevated levels of CK2a'in addiction in tumors with high levels of CK2 (11). More recently, human NSCLC specimens, compared with adjacent noncancer- CK2 has been added to the human "druggable kinome," as ous tissues. The increased expression of CK2a' was significantly evidence exists that selective CK2 inhibitors enhance apoptosis associated with nuclear exportation of BRMS1 and increased tumor recurrence. Collectively, our observations demonstrate that CK2-mediated phosphorylation of BRMS1 is an important posttranslational modification that regulates BRMS1 nuclear export 1 Department of Surgery, Memorial Sloan Kettering Cancer Center, New and protein stability. We also identify BRMS1 as a new target of York, New York. 2Department of Biochemistry & Molecular Genetics, University of Virginia, Charlottesville, Virginia. 3Weill Cornell Medical CK2 activity and provide mechanistic support and, thus, a strong College, New York, New York. rationale for the use of CK2-specific inhibitors in the treatment of Note: Supplementary data for this article are available at Cancer Research lung cancer. Online (http://cancerres.aacrjournals.org/). Corresponding Author: David R. Jones, Memorial Sloan Kettering Cancer Materials and Methods Center, 1275 York Ave, Box 7, New York, NY 10065. Phone: 212-639-6428; Fax: Cell culture, human NSCLC specimens, antibodies, and 212-639-6686; E-mail: [email protected] reagents doi: 10.1158/0008-5472.CAN-15-2888 Normal human bronchial epithelial (NHBE) cells and NSCLC 2016 American Association for Cancer Research. H1299, H1993, A549, and H157 cells were purchased from ATCC

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and grown as described in ref. 3. NHBE, H1299, H1993, and A549 peptide centered on phospho-Ser30, and the phosphospecific cells were used within 6 months after purchase. H157 cells were antibody affinity was purified with phosphopeptide. authenticated in December 2015 by PCR and genomic DNA sequencing. Mycoplasma was tested routinely. Low-passage Protein half-life analysis (<6) cell cultures were used for the experiments. Human NSCLC Cycloheximide blocking analysis was performed to determine specimens were obtained following written, informed consent the half-life of endogenous BRMS1. Cells were incubated with and approval by the Human Investigations Committee at Memo- cycloheximide (100 mmol/L) for various times, and endogenous rial Sloan Kettering Cancer Center (MSKCC). The antibodies used BRMS1 was detected by Western blot analysis. The densitometry were BRMS1, phospho-serine/threonine, Myc-tag, and His-tag of immunoblots for BRMS1 and tubulin was measured using the (Abcam); RNA Pol II, a-tubulin, CK2a', 14-3-3, and b-actin (Santa ChemiDoc MP System (Bio-Rad). The level of BRMS1 was quan- fi Cruz Biotechnology); and HA-epitope tag (BD Biosciences). The ti ed by normalization with tubulin, the percentage of remaining reagents used were human recombinant TNF, cycloheximide, BRMS1 was plotted on a logarithmic scale over time, and half-life and 4,5,6,7-tetrabromobenzotriazole (TBB; Sigma Aldrich); was determined using Prism 6.0 (GraphPad Software). CX4945 (Silmitasertib, Selleck Chemicals); MG132 (EMD Bios- For detection of the turnover rate of HA-BRMS1, pulse-chase ciences); human recombinant CK2 and substrate (Promega); 35S- assays were performed (18). In brief, cells transfected with HA- 35 labeled methionine/cysteine and [g-33P]-ATP (PerkinElmer); and BRMS1 were pulsed with 100 mCi of S-labeled methionine/ antibiotics (puromycin, geneticin, blasticidin, zeocin, and tetra- cysteine for 30 minutes and chased for various times. Immuno- cycline; Thermo Fisher Scientific). siRNA control and CK2a' pool precipitations were performed using antibody against HA-tag were purchased from Santa Cruz Biotechnology. (5 mg). Proteins were resolved by SDS-PAGE gel and visualized by autoradiography. Plasmid construction In vitro protein expression, purification, and kinase activity HA-tagged BRMS1, GST fusion BRMS1, His -tagged ubiqui- (6) assays tin, and shRNA BRMS1 were described previously (3, 15, 16). GST-fusion proteins were expressed and purified as described Site-directed mutagenesis (S!A) was performed using the previously (3). For in vivo kinase activity assays, endogenous QuikChange Mutagenesis Kit (Agilent). For pBabe-puro CK2a' was immunoprecipitated by CK2a' antibody (5 mg), fol- FLAG-tagged BRMS1, BRMS1 was amplified from pCMV HA- lowed by incubation with GST-fusion BRMS1 (20 mg) or CK2- tagged BRMS1 by PCR and inserted into BamHI/EcoRI sites. To specific substrate peptide (100 nmol) in the presence of [g-33P]- construct shRNA-resistant BRMS1, the shRNA-targeting ATP or regular ATP, respectively. For experiments using GST- sequence was synonymously mutated. Plasmids encoding sub- BRMS1 as substrate, phospho-GST-BRMS1 was resolved by units of CK2 were provided by Professor D. Litchfield (Univer- SDS-PAGE gel and visualized by autoradiography. For experi- sity of Western Ontario, Ontario, Canada). For construction of ments using CK2-specific substrate peptide as substrate, the CK2a' pcDNA 4/TO Myc/His -tagged CK2a' (Thermo Fisher Scien- (6) kinase activity was measured using the ADP-Glo Kinase Assay tific), CK2a' from pRC/CMV-HA- CK2a' was amplified by PCR (Promega) according to the manufacturer's instructions. andinsertedintoBamHI/EcoRI sites. pcDNA3-luciferase was For in vitro kinase assays, GST-BRMS1 (5 mg) was incubated with purchased from Addgene. recombinant CK2 with [g-33P]-ATP for 30 minutes at 30 C.

Preparation of cellular fractions Immunoprecipitation, Western blotting, and Nuclear and cytoplasmic extracts were isolated as described immunofluorescence previously (17). Immunoprecipitation, Western blotting, and immunofluorescence were conducted as described previously (3). Transfection Cultured cells were transfected using PolyFect Reagent for Ubiquitination assay plasmid transfection and Oligofectamine for siRNA transfection NSCLC cells were transfected with HA-CK2a', and ubiquitina- as described previously (3). tion assays were conducted as described previously (15). Virus production and stable cell generation Viruses were generated and H157 BRMS1 knockdown (KD) Invasion chamber assays cells were established as described previously (16). These H157 stable cells were pretreated with or without tetracycline BRMS1KD cells were cotransfected with pcDNA3-luciferase (1 mg/mL) for 48 hours. Invasion chamber assays were performed and Tet-on CK2a'. Stable clones were selected by geneticin as described previously (16). (400 mg/mL), blasticidin (5 mg/mL), and zeocin (150 mg/mL). Myc/His(6)- CK2a' expression was confirmed by immunoblot Orthotopic NSCLC xenograft model after treatment with tetracycline (1 mg/mL) for 48 hours. Then, All animal experiments were approved by the Animal Care and the H157 BRMS1KD/luciferase/Tet-on CK2a' cells were infected Use Committee at MSKCC (New York, NY; protocol #13-10-016). with pBabe-shRNA–resistant FLAG-BRMS1 wild-type, S30A H157 stable cells (1 106) suspended in 100 mL of DPBS were mutant, or empty vector and selected with puromycin (1 mg/mL). injected into the left lungs of forty 5-week-old female athymic Flag-BRMS1 expression was confirmed by Western blot analysis. nude mice (nu/nu, Taconic), including BRMS1KD/Tet-on CK2a'/ control (control; N ¼ 10); BRMS1KD/Tet-on CK2a'/FLAG-BRMS1 Generation of phosphospecific BRMS1 antibody S30A (BRMS1 S30A; N ¼ 10); and BRMS1KD/Tet-on CK2a'/FLAG- BRMS1 (pS30) antibody was generated by Open Biosystems. In BRMS1 wild-type (BRMS1 WT; N ¼ 20). The BRMS1 S30A group brief, two rabbits were immunized with KHL-conjugated BRMS1 and 10 mice from the BRMS1 WT group were administrated a

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doxycycline diet (0.625 g/kg; Harlan TEKLAD) on the day of half-life of BRMS1 in both NHBE and H1299 cells (Fig. 1B), injection. confirming the importance of TNF in the posttranslational regulation of BRMS1. Using H157 cells, which stably express In vivo fi imaging and quanti cation SR-IkB, a dominant negative inhibitor of NF-kB (H157I), or a fl Mice were anesthetized with 2.5% iso urane and imaged after vector control (H157V; ref. 4), subsequent experiments showed intraperitoneal injection of luciferin (150 mg/kg; Promega). that TNF-induced BRMS1 protein degradation is independent Imaging was performed with an IVIS Spectrum-CT System (Per- of NF-kB activation (Supplementary Fig. S1A). kinElmer) on day 8 after injection and repeated weekly (19). To Analysis of BRMS1 protein (http://scansite.mit.edu) indicat- determine the best time for imaging, a kinetic study was per- ed the presence of three evolutionarily conserved CK2 consen- formed by continuously imaging at 5-minute intervals for 40 sus sites (Fig. 1C). We therefore hypothesized that BRMS1 may minutes after luciferin injection. Three-dimensional reconstruc- be a substrate of CK2. As shown in Fig. 1D, GST-fusion BRMS1 tion was accomplished by the use of Living Image Software was phosphorylated by recombinant CK2 in vitro.Ofimpor- (version 4.2; Caliper). tance, introducing an S30A mutation in BRMS1 completely fi BRMS1KD For quanti cation, H157 /luciferase cells were seri- inhibited CK2-induced phosphorylation (Fig. 1D). These data ally diluted into 96-well plates. After the addition of luciferin indicate BRMS1 S30 is the primary site of CK2 phosphoryla- (15 mg/mL), the cells were continuously imaged at 2-minute tion. This is consistent with mass spectroscopy data identifying intervals for 30 minutes to catch the peak luciferase signal. The phosphorylation at S30, but not S45 or 46 (http://www.Phos- ¼ standard curve of regions of interest (unit radiance) versus cell phosite.org). numbers was drawn, and the cell numbers in primary tumors To elucidate whether BRMS1 S30 is the primary phosphoryla- and metastatic sites were calculated using Living Image Software. tion site in response to TNF, we developed a BRMS1 phosphospecific S30 (pS30) antibody. Stimulation with TNF significantly Tissue microarray and IHC increased BRMS1 (pS30) in a time-dependent manner (Fig. 1E). We created a tissue microarray (TMA) containing 160 NSCLCs Moreover, preincubation of lung cancer cells with a CK2-specific and matched adjacent noncancerous tissue (Supplementary Table pharmacologic inhibitor TBB blocked TNF-induced BRMS1 phos- S1). The immunohistochemical techniques used were described phorylation (Supplementary Fig. S1B). To confirm the specificity previously (2, 20). BRMS1 staining in the nucleus and cytoplasm of our BRMS1 (pS30) antibody, we expressed either HA-tagged were evaluated separately. The BRMS1 and CK2a' antibodies were BRMS1 wild-type or S30A mutant in NSCLC cells. The level of used at a 1:200 dilution for 30 minutes. BRMS1 (pS30) protein was robustly increased in cells expressing wild-type HA-BRMS1 following TNF treatment. However, no Statistical analysis (pS30) protein was detected in cells expressing mutant BRMS1 The results of all experiments represent the mean SEM of (S30A; Fig. 1F). These data confirm that TNF induces phosphor- three separate experiments performed in triplicate. Statistical ylation of BRMS1 at S30 through a CK2-dependent pathway. In analysis was performed using Prism. Student t test, one-way addition, similar to TNF, TGFb and IL6 (but not EGF) are able to ANOVA, Wilcoxon matched pairs signed rank test, Mann–Whit- induce BRMS1 (pS30) (Supplementary Fig. S1C), suggesting that ney test, and Spearman correlation were used. Progression-free multiple inflammation-related cytokines in the tumor microen- survival (PFS) was defined as the time from surgery to the vironment are involved in CK2-induced BRMS1 phosphorylation. development of metastasis and was assessed using the Kaplan–Meier method and compared using the log-rank test. CK2a' is responsible for TNF-induced degradation of BRMS1 The CK2a' immunoreactivity score cut-off value (3.3; P ¼ 0.002) To address whether CK2 participates in BRMS1 degradation, was determined by ROC curve analysis using maximum sum of we treated NSCLC cells with TNF and/or CX4945, a specific specificity and sensitivity. A two-sided P < 0.05 was considered to inhibitor of CK2 (23) currently in clinical trials. Treatment with indicate statistical significance for all calculations. TNF alone significantly reduced levels of BRMS1 protein. How- ever, pretreatment with CX4945 abrogated TNF-induced phos- Results phorylation and degradation of BRMS1 (Fig. 2A). These data TNF promotes CK2-mediated BRMS1 phosphorylation at S30 indicate that CK2 activity is required for TNF-induced degra- We observed that BRMS1 protein levels were decreased 5 to dation of BRMS1. 10 times more than transcript in NSCLC, compared with NHBE To determine which subunit of CK2 is involved in BRMS1 cells or adjacent noncancerous tissues (3). This suggests that degradation, wild-type and kinase-dead forms (CK2a K68/m and BRMS1 is posttranscriptionally regulated. To assess the stability CK2a' K69/m; ref. 24) of CK2 subunits were individually of endogenous BRMS1 protein in NSCLC cells, we performed expressed in H1299 cells. Endogenous BRMS1 was clearly cycloheximide blocking assays. As shown in Fig. 1A, BRMS1 decreased in cells with overexpression of CK2a' wild-type, but protein had a significantly shorter half-life in A549 and H1299 not in cells expressing other isoforms (Fig. 2B). Coimmunopre- cellsthaninNHBEcells(P < 0.0001). To exclude the possibility cipitation assays revealed that BRMS1 physically interacts with that a reduction of BRMS1 was secondary to transcriptional ectopic and endogenous CK2a', but not with CK2a (Fig. 2C and repression (4), we evaluated the half-life of ectopic HA-BRMS1. Supplementary Fig. S2A). TNF failed to induce BRMS1 degrada- The half-life of HA-BRMS1 was also shorter in H1299 cells than tion following siRNA knockdown of CK2a' (Fig. 2D). Although in NHBE cells (P < 0.0001; Fig. 1B). Given the contributions of CK2a and a' share an 86% similarity in their catalytic domain the tumor microenvironment and the associated proinflamma- (25), Western blots indicated that only a', not a, was specifically tory cytokines to tumorigenesis and metastases (21, 22), we knocked down by siRNA (Fig. 2D). Collectively, these results asked whether TNF accelerates BRMS1 protein degradation. indicate that CK2a' is the key catalytic subunit mediating BRMS1 TNF treatment resulted in a nearly 50% reduction in the degradation.

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NHBEC A549 H1299 ABNHBEC H1299 CHX (h) 0 246 8120 12345 0123 45 BRMS1 No TNF (h) 0 36 01 3 6 Tubulin HA-BRMS1

t1/2 = 6.41 h t1/2 = 3.46 h t1/2 = 3.12 h t1/2 = 7.11 h t1/2 = 4.37 h 15 15 8 6 TNF (h) 03 6 01 3 6 10 10 4 HA-BRMS1 t = 3.78 h t = 2.41 h 5 5 2 1/2 1/2 0 0 0 Density of BRMS1 0 2 4 6810012345012345 CHX (h) CHX (h) CHX (h) CD25 50 GST-BRMS1 NC

GST WT S30A S45A S46A S45/46A p-GST-BRMS1

MWM (KDa) 50

25 Coomassie blue stain gel EF HA-BRMS1 WT S30A H157 H1299 TNF–+ –+ TNF (h) 0 0.5 1 2 0 0.5 1 2 BRMS1 (pS30) IP : HA BRMS1 (pS30)

BRMS1 HA-BRMS1 Input

Figure 1. CK2 phosphorylates BRMS1 at S30. A, BRMS1 protein has a shorter half-life in NSCLC than in NHBE cells. NHBE and A549 and H1299 cells were treated with cycloheximide (CHX; 100 mmol/L) for the indicated times. BRMS1 expression was probed by immunoblot (top). The half-life (t1/2) of BRMS1 protein was calculated (bottom). B, TNF promotes BRMS1 degradation. NHBE and H1299 cells were transfected with HA-tagged BRMS1. Thirty-six hours posttransfection, pulse- chase analysis was performed with or without TNF (20 ng/mL) at the indicated times. BRMS1 was visualized by autoradiography, and t1/2 was calculated. C, schematic representation of BRMS1 protein. Orange rectangles, coiled-coil domains; red characters, putative CK2-phosphorylated sites. N, N-terminus of BRMS1; C, C-terminus of BRMS1. D, BRMS1 is phosphorylated by CK2 in vitro. In vitro phosphorylation assays were performed by incubating GST-BRMS1 with recombinant CK2 and subjection to SDS-PAGE gel. Phospho-GST-BRMS1 was visualized by autoradiography. MWM, molecular weight marker. E, TNF induces BRMS1 phosphorylation at S30. H157 and H1299 cells were treated with TNF (20 ng/mL) for the indicated times. BRMS1 (pS30) was detected by immunoblot. F, S30A mutant abrogates TNF-induced BRMS1 phosphorylation. H157 cells were transfected with HA-BRMS1 wild-type or S30A mutant and stimulated with or without TNF (20 ng/mL) for an additional 2 hours. Immunoprecipitation (IP) assays were performed using anti-HA antibody, and BRMS1 (pS30) was detected.

To determine whether the ubiquitination–proteasome pathway and was completely blocked following siRNA knockdown (Fig. is involved in CK2a'-mediated degradation of BRMS1, in vivo 3D). To confirm that TNF induces phosphorylation of BRMS1 at ubiquitination assays were performed. The proteasome inhibitor S30 through activation of CK2a' at a single-cell level, immuno- MG132 was used to block ectopic CK2a'–induced BRMS1 degra- fluorescence analysis was performed. Stimulation with TNF sig- dation. Ectopic CK2a'significantly enhanced BRMS1 polyubiqui- nificantly increased levels of BRMS1 (pS30) in NSCLC cells (Fig. tination in NSCLC cells (Fig. 2E). In addition, treatment with 3E). Interestingly, unlike BRMS1, the majority of BRMS1 (pS30) is MG132 rescued CK2a'-induced BRMS1 degradation (Supplemen- located in the cytoplasm, suggesting that phosphorylation alters tary Fig. S2B). The introduction of mutant BRMS1 (S30A) abrogated subcellular localization of BRMS1. Preincubation with CX4945 CK2a'-induced BRMS1 degradation (Fig. 2F). Thus, phosphoryla- abrogated TNF-induced BRMS1 (pS30) but did not affect the tion of BRMS1 at S30 is required for CK2a'-induced degradation. expression of CK2a', suggesting that CX4945 inhibits CK2a' kinase activity, not expression. Similar to a recent study (26), we TNF activates CK2a' to phosphorylate BRMS1 observed that TNF increased CK2a' protein levels (Fig. 3C–E). To confirm that TNF induces CK2a' kinase activity in NSCLC, TNF increases the phosphotransferase activity of CK2a', resulting we demonstrated that TNF induces the phosphotransferase activ- in phosphorylation of BRMS1 at S30. ity of CK2a' in a time-dependent manner (Fig. 3A and B). Importantly, pretreatment with CX4945 significantly decreased TNF-induced phosphorylation promotes 14-3-3«–dependent both basal and TNF-induced CK2a' kinase activity (Fig. 3B). In BRMS1 nuclear export addition, TNF-induced activation of CK2a' resulted in robust BRMS1 is primarily a nuclear protein, whereas ubiquitin- phosphorylation of BRMS1 in vitro and in vivo (Fig. 3C and D) dependent degradation occurs within the cytoplasm (27). To

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α α′ Figure 2. ABHA-CK2 HA-CK2 CK2 catalytic subunit a' is required for TNF-induced BRMS1 degradation. A, CX4945 – –++ CX4945 abrogates TNF-induced BRMS1 TNF –+– + Control WT K68/m WT K69/m CK2β degradation. H1299 cells were BRMS1 pretreated with or without CX4945 (30 BRMS1 4.85 3.96 7.08 0.47 7.24 3.81 mmol/L) for 2 hours, followed by TNF (20 ng/mL) for 16 hours. The indicated BRMS1 (pS30) HA-Epitope proteins were evaluated. B, CK2a' CK2α′ induces BRMS1 degradation. H157 cells Myc-CK2β were transfected with the indicated CK2 Actin subunits. Protein levels of BRMS1 and Tubulin CK2s were evaluated. BRMS1 expression was quantified by densitometry (normalized with tubulin) and labeled C D HA-CK2 siRNA Control CK2α′ under each corresponding band. C, CMV αα′ CK2a', but not a, interacts with BRMS1. TNF (h) 0 4 8 0 4 8 H1299 cells were transfected with BRMS1 IP : HA HA-tagged CK2a, a', or empty vector. BRMS1 Input Coimmunoprecipitation (IP) assays were performed following treatment HA-Epitope CK2α′ with MG132 (5 mmol/L) for 16 hours, and BRMS1 was detected. CMV, BRMS1 cytomegalovirus. D, knockdown CK2α CK2a' inhibits TNF-induced BRMS1 degradation. H1299 cells were A549 H1299 E Tubulin transfected with siRNA CK2a' or HA-CK2α′ –+ –+ scramble and treated with TNF MG132 ++ ++ (20 ng/mL). The indicated proteins were evaluated. E, CK2a' enhances BRMS1 F polyubiquitination. NSCLC cells were BRMS1 HA-BRMS1 WT S30A transfected with HA-CK2a' and treated Ubn HA-CK2α′ ––++ with MG132 (5 mmol/L) for 16 hours. Ubiquitination (Ubn) assays were HA-BRMS1 conducted. F, S30A mutation abrogates CK2a'-induced BRMS1 degradation. BRMS1 HA-CK2α′ H1299 cells were cotransfected with HA-CK2α′ HA-BRMS1 wild-type or S30A and HA-CK2a'. The indicated proteins Actin Tubulin were assessed. explore whether TNF alters the subcellular localization of BRMS1, addition, we observed that 14-3-3e knockdown blocked TNF- we isolated nuclear and cytosolic extracts from NSCLC cells after induced nuclear export of BRMS1 (Fig. 4F). Collectively, these stimulation with TNF. In the absence of stimulus, BRMS1 was data suggest that phosphorylation of BRMS1 by CK2 enhances 14- primarily nuclear (Fig. 4A). Following TNF stimulation, there was 3-3e interaction, which promotes BRMS1 nuclear export and a significant shift of BRMS1 protein from the nucleus to the degradation. cytosol of cells in a time-dependent manner (Fig. 4A). However, preincubation with CX4945 completely blocked TNF-induced BRMS1 S30A mutant tumors have a lower rate of metastases nuclear exportation of BRMS1 (Fig. 4A). Subsequent immuno- To determine the significance of CK2a'-mediated degradation fluorescence assays showed that stimulation with TNF dramati- of BRMS1 in NSCLC, we created H157 cell lines in which endog- cally increased cytosolic BRMS1, compared with vehicle treatment enous BRMS1 was stably knocked down. These cells were then (Supplementary Fig. S3A). Moreover, the BRMS1 S30 mutant was used to establish H157 lines that ectopically expressed shRNA- primarily intranuclear and did not undergo nuclear exportation, resistant BRMS1 wild-type or S30A mutant. In addition, these cells even with TNF stimulation (Fig. 4B). We therefore conclude that stably expressed Myc–CK2a' construct (Tet-on-Myc-CK2a') CK2-induced phosphorylation is required for the nuclear expor- under the control of the tetracycline-responsive operator. Treat- tation of BRMS1. ment with tetracycline induced expression of Myc-tagged CK2a' The 14-3-3 family of proteins regulates the subcellular local- and reduced the level of BRMS1 wild-type (Supplementary Fig. ization of multiple proteins via direct binding. Isoforms e and z S4A). Cells expressing BRMS1 wild-type had significantly less are the most abundant in lung tissue (28). As shown in Supple- invasion of NSCLC cells, compared with BRMS1 knockdown mentary Fig. S3B, the 14-3-3 isoform e, and not z, endogenously (control) cells. As expected, the S30A mutation did not affect the associates with BRMS1. TNF significantly enhanced the interac- capacity of BRMS1 to repress invasion of NSCLC. Tetracycline- tion of BRMS1 and 14-3-3e (Fig. 4C), whereas the BRMS1 S30 induced CK2 expression significantly increased the NSCLC inva- mutant failed to bind to 14-3-3e (Fig. 4D). Furthermore, knock- sion in cells expressing BRMS1 wild-type, but not the S30A down of 14-3-3e not only blocked cytoplasmic translocation of mutant (Supplementary Fig. S4B). BRMS1 but also increased BRMS1 protein within the cell, suggest- To examine the contribution of CK2a'-mediated degrada- ing that 14-3-3e is required for BRMS1 nuclear export (Fig. 4E). In tion of BRMS1 to the progression of NSCLC in vivo,weused

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A H1299 H157 B 40 150 12 30 Figure 3. 100 8 TNF induces BRMS1 phosphorylation by 20 activation of CK2a'. A, TNF increases 50 4 CK2a' activity. H1299 and H157 cells were

CK2 α′ Activity 10 (RLU × 10,000) treated with TNF (20 ng/mL) for the CK2 α′ Activity (fold of vehicle) (fold 0 0 0 indicated times. Endogenous CK2a'was 000.51122 0.5 TNF – + – + immunoprecipitated, and kinase activity – – ++ assays were performed. , P 0.05 and , TNF (h) TNF (h) CX4945 < P < 0.01, compared with time 0. RLU, relative luciferase unit. B, CX4945 blocks C TNF D TNF-induced CK2a' activation. H1299 0 20 m 40 m 1 h 2 h cells were pretreated with or without siRNA α′ p-GST-BRMS1 Control CK2 CX4945 (30 mmol/L) for 2 hours, TNF (min) 0030 30 NHBE CK2α′ followed by TNF (20 ng/mL) for an p-BRMS1 IP : BRMS1 additional 2 hours. Endogenous CK2a' Tubulin kinase activity was determined. , P < BRMS1 p-GST-BRMS1 0.05 and , P < 0.01, compared with α′ CK2 vehicle; #, P < 0.01, compared with TNF H1299 α′ CK2 alone. C, TNF activates CK2 ' to induce Tubulin a Tubulin BRMS1 phosphorylation. Cells were treated with TNF (20 ng/mL) for the E BRMS1 (pS30) CK2α′ Merged (DAPI) indicated times. Immunoprecipitated CK2a' was incubated with GST-BRMS1, and phospho-GST-BRMS1 was visualized Vehicle by autoradiography. D, knockdown CK2a' abrogates TNF-induced BRMS1 phosphorylation. H1299 cells were transfected with siRNA CK2a' or scramble and treated with or without TNF TNF (20 ng/mL) for 30 minutes. Phospho- BRMS1 was assessed. E, pretreatment with CX4945 blocks TNF-induced phosphorylation of BRMS1. H157 cells were pretreated with CX4945 CX4945 (30 mmol/L) for 2 hours, followed by stimulation with TNF (20 ng/mL) for an additional 1 hour. Immunoﬂuorescence assays were performed using antibodies against BRMS1 (pS30; red)/CK2a' TNF+ (green)/DAPI (blue). CX4945

an orthotopic xenograft lung cancer model. The position of CK2a' correlates with intracellular BRMS1 localization and primary tumors in the lung tissue was confirmed by biolu- metastases in human NSCLC minescence IVIS Spectrum-CT scan (Supplementary Fig. S5A), Having demonstrated the importance of BRMS1 in suppressing and ectopic expression of BRMS1 and CK2a'wasverified by CK2a'-driven metastases in our in vivo model, we wanted to Western blot analysis (Supplementary Fig. S5B). No signifi- determine whether similar correlations exist in human lung cant difference was observed for growth of the primary tumors cancer. Immunohistochemical analysis was performed on a TMA among groups (Supplementary Fig. S5C). As expected, ani- of 160 human NSCLC and matched adjacent noncancerous mals that received H157 cells expressing BRMS1 wild-type tissues (Supplementary Table S1). As shown in Fig. 6A, CK2a' had significantly fewer metastases compared with the H157 is overexpressed in human NSCLCs compared with adjacent control group in which endogenous BRMS1 was reduced (Fig. noncancerous tissues (mean SD, 4.07 3.4 vs. 2.99 2.92; 5A–C). Administration of doxycycline induced Myc-CK2a' P ¼ 0.004). Importantly, CK2a' expression levels were signifi- expression, which abrogated BRMS1 wild-type–mediated sup- cantly higher in patients who developed distant metastases com- pression of metastasis but was unable to drive metastases in pared with patients who remained progression free (P ¼ the BRMS1 S30A–mutant group (Fig. 5A–C). Mutation of S30 0.022; Fig. 6B). We also observed that high expression of CK2a' in BRMS1 resulted in a 60-fold reduction of CK2-driven in NSCLC is an independent factor associated with decreased PFS metastatic tumor burden (Fig. 5B and C). Collectively, these (P ¼ 0.04; Fig. 6C). data show that BRMS1 suppresses NSCLC metastasis in vitro To explore the relationship between CK2a' activity and cyto- and in vivo and that CK2a'-mediated phosphorylation of S30 solic localization of BRMS1 in vivo, we examined the protein levels results in degradation of BRMS1 and a robust increase in of BRMS1 in the same TMA. IHC scores for cytosolic BRMS1 were metastasis. lower than those for nuclear BRMS1 (1.37 1.94 vs. 3.61 3.83;

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A TNF (min) 0 5 15 30 60 120 0 5 15 30 60 120 CX4945 –––– – – ++++ + + Cytoplasmic BRMS1 Nuclear BRMS1 Cytoplasmic tubulin Nuclear RNA polll

BCNE CE Veh. TNF HA-BRMS1 WT S30A WT S30A

TNF –+ –+ –+ –+ IP IgGBRMS1 IgG BRMS1 HA-BRMS1 14-3-3ε RNA Polll Input Veh. TNF Actin 14-3-3ε

BRMS1

D E HA-BRMS1 WT S30A shRNA Control 14-3-3ε 14-3-3ε IP : HA Input 14-3-3ε BRMS1 DAPI HA-BRMS1

RNA Polll

shRNA Control 14-3-3ε 14-3-3ε NE CE F shRNA Control 14-3-3ε Control 14-3-3ε BRMS1 TNF +–++–– – + Actin BRMS1

RNA Polll

Actin

Figure 4. TNF induces nuclear exportation of BRMS1 in a 14-3-3–dependent manner. A, CK2 mediates TNF-induced BRMS1 nuclear exportation. H1299 cells were pretreated with MG132 (5 mmol/L) alone or with CX4945 (30 mmol/L) for 2 hours, followed by TNF (20 ng/mL). Nuclear/cytosolic BRMS1 expression was probed. B, S30A mutant abrogates TNF-induced nuclear exportation of BRMS1. H157 cells were transfected with HA-tagged BRMS1 wild-type or S30A mutant. Transfected cells were treated with or without TNF (20 ng/mL) for 2 hours; HA-BRMS1 in nuclear extract (NE) or cytosolic extract (CE) was probed separately. C, TNF enhances the interaction of BRMS1 and 14-3-3e. H157 cells were pretreated with MG132 (5 mmol/L) for 2 hours and then treated with or without TNF (20 ng/mL) for an additional 2 hours. Coimmunoprecipitation (IP) assays were performed using the indicated antibodies. The presence of 14-3-3e was detected. D, S30A mutant abrogates BRMS1 binding to 14-3-3e. H1299 cells were transfected with HA-tagged BRMS1 wild-type or S30A. Coimmunoprecipitation assays using NE were performed, and the presence of 14-3-3e was probed. E, BRMS1 is primarily located in the nucleus following the loss of 14-3-3e. Immunoﬂuorescent merged micrographs show subcellular localization of BRMS1 (red)/DAPI (blue) in H1993 cells infected with shRNA 14-3-3e or scramble. Western blots indicate that 14-3-3e was speciﬁcally knocked down. F, knockdown 14-3-3e inhibits TNF-induced nuclear exportation of BRMS1. H157 cells were infected with shRNA 14-3-3e or scramble, pretreated with MG132 (5 mmol/L) for 2 hours, and then treated with or without TNF (20 ng/mL) for an additional 2 hours. HA-BRMS1 in nuclear extract or cytosolic extract was detected. Veh., vehicle.

P < 0.0001), confirming that BRMS1 was primarily nuclear in 0.31 (95% confidence interval, 0.145–0.452) P ¼ 0.0002]. These human tumors. Cytosolic BRMS1 was commonly found in data provide important correlative confirmation that CK2a' pro- tumors with higher levels of CK2a'(P ¼ 0.0001; Fig. 6D). motes nuclear exportation of BRMS1 in NSCLC and begin to lay Strikingly, protein levels of cytosolic BRMS1 positively correlated the foundation for the use of CK2a' inhibitors in selected indi- with increased levels of CK2a' protein in NSCLC [Spearman R ¼ viduals with lung cancer.

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Liu et al.

Control BRMS1WT BRMS1WT BRMS1S30A AB(N = 7) (N = 7) (N = 9) (N = 6) Doxy. – –+ + Control 108 BRMS1WT BRMS1WT + Doxy. 7 10 BRMS1S30A + Doxy. 106

105

14710 104

103

(number of cells) (number 102 Total metastasis burden Total 10

0 0 7142128 35 42 49 Postinjection (days) 25811 C BRMS1WT BRMS1S30A Control BRMS1WT +Doxy. +Doxy. 0 –7 –200 –400 –1,000

36912 Control –1,250 BRMS1WT BRMS1WT + Doxy. BRMS1S30A + Doxy. Fold changes over control changes over Fold –1,500

Figure 5. CK2a'-induced BRMS1 degradation promotes metastasis of NSCLC in vivo. A, induction of CK2a' promotes distant metastasis of NSCLC. H157 stable cell lines were orthotopically injected into the left lung of mice. Mouse bioluminescent CT images show the growth of tumor and metastatic sites in three representative mice in each group (1–3, control; 4–6, BRMS1WT without doxycycline; 7–9, BRMS1WT with doxycycline (Doxy.); 10–12, BRMS1S30A with doxycycline). The scale indicates the signal intensity cell quantiﬁcation. B, S30A mutant blocks CK2a'-induced metastasis of NSCLC. The logarithmic graph represents average total cell numbers of all metastatic sites in each group. $, P < 0.05, compared with control; #, P < 0.05, compared with BRMS1 wild-type; , P < 0.05, compared with BRMS1 wild-type with doxycycline. C, activation of CK2a' induces metastasis of tumors with BRMS1 wild-type. The graph represents fold changes of metastasis burden over control group 43 days postinjection.

Next, we examined the levels of CK2a' and BRMS1 (pS30) in ylates BRMS1 on S30 and initiates 14-3-3–mediated nuclear selected human NSCLC tumors. CK2a' is overexpressed and export and proteasome-mediated degradation of BRMS1. Using BRMS1 is decreased in tumors compared with adjacent noncan- an orthotopic NSCLC mouse model, we established the biologic cerous tissues. BRMS1 (pS30) was also overexpressed in most significance of CK2a-mediated regulation of BRMS1 as a major tumors compared with adjacent tissues (9/12; Fig. 6E). In more driver of increased metastases (Fig. 7A). than half of these tumors (7/12), levels of BRMS1 (pS30) were Whereas CK2 kinase activity is constitutively active, CK2 sub- consistent with elevated levels of CK2a' (Fig. 6E). In addition, we strate recognition is regulated by cytokines, inflammatory med- observed significantly higher CK2a' kinase activity in primary iators, and growth factors, such as EGF, TNF, and TGFb (29), as human NSCLC tumors than in adjacent tissues (Supplementary well as by p38 MAPK–mediated CK2 activation (12, 30). The role Fig. S6A). Tumors with nodal metastasis had higher increased played by CK2 in cancer is illustrated in multiple myeloma, where activity of CK2a' compared with tumors without nodal metastasis, CK2 phosphorylates the PIKK-regulatory proteins Tel2/Tti1, indicating that elevated CK2a' activity is a strong predictor of the which facilitates their proteasomal degradation by Fbxo9 in the nononcogene addiction observed in NSCLC (Supplementary Fig. mTORC1 complex. This CK2-initiated, Fbxo9-mediated deregu- S6B). Collectively, our data demonstrate that both protein levels lated ubiquitinylation is associated with myelomagenesis (31). and kinase activity of CK2a' are increased in NSCLC compared Other groups have shown that inositol pyrophosphates mediate with adjacent tissues. In addition, elevated levels of CK2a' strongly DNA-PK/ATM-p53 cell death via CK2 phosphorylation (32) and correlate with BRMS1 S30 phosphorylation, cytoplasmic localiza- that CK2-mediated phosphorylation of prostate apoptosis tion, increased metastases, and poor clinical prognosis. response-4 reduces its apoptotic function and increases cell survival (33). These reports, as well as our own observations regarding BRMS1, provide evidence that CK2 is a potential therapeutic Discussion target for tumors with high CK2 activity. Herein, we have shown that BRMS1 is regulated posttransla- Protein expression of the CK2 catalytic subunit a or regulatory tionally via TNF-induced activation of CK2a', which phosphor- subunit b is elevated in various cancers, and overexpression of

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CK2-Mediated Degradation of BRMS1

Tumor Adjacent P = 0.022 A 15 P = 0.004 (N = 160) B 15

10 10 5

5 0 CK2 α′ Score Scores of CK2 α′ 0 –5 Tumor Adjacent –10 No Local Distant (N = 118) (N = 4) (N = 22) Recurrence C D Patients 100 15 P = 0.0001 1 2

10 50 PFS (%) Low N = 62 P = 0.04 5

High N = 84 CK2 α′

0 Scores of CK2 α′ 0 12 24 36 48 60 0 Number at risk Months Negative Positive Low 62 42 29 15 3 1 N = 93 N = 49 High 84 55 40 19 1 0 Cytoplasm BRMS1 BRMS1 E Lymph node Positive Negative Positive Negative Patient 1 2 3 4 5 6 7 8 9 10 11 12 N TNTNTNTNTNT NTNTNTNTNTNT CK2α′ 36 3.2 0.4 0.3 22 2 4.1 6.7 3.6 1.9 1.1 2.7 BRMS1 (pS30) 2.6 1.2 0.4 8.1 8.8 1.7 4.1 36 55 4.3 6.8 7.5 BRMS1 2.5 0.2 0.8 0.2 2e–20.8 2e–30.2 4e–3 7e–2 7e–35e–3 Actin

Figure 6. CK2a' overexpression is associated with cytoplasmic localization of BRMS1 and metastases in human lung cancer. A, CK2a' is overexpressed in NSCLC. The graph represents IHC scores of CK2a' in tumors and adjacent noncancerous tissues in our NSCLC TMA (N ¼ 160; P ¼ 0.004). Photomicrographs show CK2a' staining in two pairs of representative samples. B, CK2a' is increased in patients with distant metastasis. IHC scores of CK2a' in patients with or without tumor recurrence (P ¼ 0.022, patients with distant metastasis compared with those without recurrence). C, high CK2a' is associated with poor PFS. Kaplan–Meier PFS plot based on tumor CK2a' protein levels (P ¼ 0.04). D, higher levels of CK2a' correlate with cytosolic BRMS1 staining. IHC scores of CK2a' in patients with positive (>0) and negative (0) cytosolic BRMS1 staining (P ¼ 0.0001). Photomicrographs show staining for CK2a' and BRMS1 in two representative patient samples. E, increased BRMS1 (pS30) in NSCLC, compared with matched adjacent tissues. The indicated proteins were detected in NSCLC (T) and adjacent noncancerous tissues (N): 1–6, squamous cell carcinoma; 7–12, adenocarcinoma. Immunoblot bands were quantiﬁed by densitometry and normalized with actin. Fold changes of T versus N are labeled under each corresponding blot.

these subunits is correlated with poor prognosis (34, 35). In significantly higher levels in lung cancer tissue (38). Although the NSCLC, we found that CK2a' led to BRMS1 degradation, affirm- functions of CK2a and b are well studied, the functional role of ing the specificity of select CK2 catalytic subunits (36). Prior CK2a' in tumor biology remains unclear. Herein, we show that studies demonstrated that the free form of CK2a' phosphorylates CK2a' significantly enhances distant tumor metastasis in vivo and NKX3.1 in prostate tumor cells (37). Recently, Turowec and that expression of BRMS1 S30A mutant significantly diminishes colleagues revealed distinct functions of CK2 isoforms in pro- CK2a'-enhanced metastasis in vitro and in vivo. moting cancer cell survival by identifying isoform-specific phos- Although BRMS1 is a predominantly nuclear protein, it does phorylation of caspase-3 by CK2a', not CK2a (25). Confirming shuttle between the nucleus and cytoplasm (14). Nuclear export our observations that CK2a' is overexpressed in human NSCLC, a of BRMS1 is not CRM-1 dependent (14), and 14-3-3 can transport recent Oncomine (www.oncomine.org) analysis of lung cancer proteins (e.g., HDAC7) independent of CRM1 (39). 14-3-3 pro- and normal lung tissue demonstrated that CK2a' was expressed at teins are capable of regulating intracellular localization of their

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Liu et al.

Figure 7. Putative therapeutic relevance of the mechanisms controlling BRMS1 phosphorylation, nuclear exportation, and degradation. A, in response to TNF, activated CK2a' phosphorylates BRMS1 at S30, which is required for the interaction with 14-3-3e, resulting in nuclear exportation and ubiquitin (Ub)-proteasome (P)–mediated degradation of BRMS1. This reduction of intratumoral BRMS1 promotes lung cancer metastasis. B, hypothetic model: treatment with a speciﬁc CK2 inhibitor (CX4945 or others) preserves BRMS1 expression and inhibits lung cancer metastasis.

binding partners, such as Raf, Cdc25, BAD, and FKHRL1 (40). We cytoplasmic ubiquitin–proteasome machinery. This occurs for observed that CK2a'-mediated phosphorylation induced nuclear many proteins, including the nuclear proteins p53 (41), cyclin kip1 exportation of BRMS1 in a 14-3-3e–dependent manner, which is D1 (42), p27 (43), and IkBa (44). There have been conflicting enhanced in the presence of TNF. Simultaneously, we observed observations regarding the clinical significance of cytosolic that CK2a'-mediated phosphorylation is required for nuclear BRMS1, with some reports suggesting it is a favorable prognostic export of BRMS1, as BRMS1 S30A mutant failed to interact with indicator (45) and others suggesting the opposite (13). We show 14-3-3e and undergo nuclear exportation, even in the presence of that increased CK2a' is observed in tumors with BRMS1 localized TNF. Importantly, our observation of a positive correlation to the cytoplasm; this was associated with increased metastasis, between the protein levels of CK2a' and cytoplasmic BRMS1 tumor recurrence, and reduced PFS in patients with NSCLC. expression in human NSCLC specimens confirms that phosphor- CX4945 is a potent and specific ATP-competitive inhibitor of ylation by CK2a' is a prerequisite for nuclear exportation of both CK2a and a'. As the first orally bioavailable small-molecule BRMS1. inhibitor of CK2, CX4945 exhibits antitumor activity in multiple Our data indicate that the fate of phosphorylated BRMS1/14-3- solid and hematopoietic tumor models, and its safety and efficacy 3 complex, once it enters the cytosol, is to be degraded. Whereas have been established by a phase I/II clinical trial (23). A recent in the ubiquitin–proteasome components can be found in the vitro study demonstrated that CX4945 suppresses EMT and metas- nucleus, full degradation of targeted proteins occurs through tasis in A549 lung cancer cells through inhibition of multiple

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CK2-Mediated Degradation of BRMS1

signaling pathways, including MAPK, AKT, FAK/Src, and MMPs Analysis and interpretation of data (e.g., statistical analysis, biostatistics, (46). Our study demonstrates that CX4945 abrogates TNF- computational analysis): Y. Liu, P.S. Adusumilli, D.R. Jones induced BRMS1 degradation through inhibition of CK2a'-medi- Writing, review, and/or revision of the manuscript: Y. Liu, E.B. Amin, M.W. Mayo, N.P. Chudgar, P.R. Bucciarelli, K. Kadota, P.S. Adusumilli, D.R. Jones ated phosphorylation. This suggests that treatment with CX4945, Administrative, technical, or material support (i.e., reporting or organizing potentially with the addition of a proteasome inhibitor, may data, constructing databases): Y. Liu, P.S. Adusumilli, D.R. Jones result in a robust suppression of NSCLC metastasis via inhibition Study supervision: Y. Liu, D.R. Jones of BRMS1 degradation (Fig. 7B). Moreover, mutation of S30 in BRMS1 prevents CK2a'-mediated phosphorylation and degrada- Acknowledgments tion and leads to a 60-fold reduction in metastases; this illustrates The authors thank Professor D. Litchfield (University of Western Ontario, the importance of BRMS1 as a primary regulator of metastases. Ontario, Canada) for providing CK2 constructs and Dr. Neal Rosen (Memorial Sloan Kettering Cancer Center, New York, NY) for helpful comments during the In summary, our work establishes a new, targetable mechanism manuscript preparation. through which BRMS1 is posttranslationally regulated. Given the increased expression of CK2a' and the correlative low levels of Grant Support BRMS1 in a number of different highly metastatic tumors, these This work was supported by grants R01 CA136705 (D.R. Jones), R01 observations may have broad implications in the clinical man- CA104397 (M.W. Mayo), R01 CA132580 (M.W. Mayo), U54 CA137788 agement of metastatic cancer. (P.S. Adusumilli.), and 5 T32 CA 9501-27 (P.R. Bucciarelli) from the NIH/ NCI. This work was also supported, in part, by NIH/NCI Cancer Center Support Disclosure of Potential Conflicts of Interest Grant P30 CA008748. 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 Authors' Contributions in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Conception and design: Y. Liu, M.W. Mayo, D.R. Jones Development of methodology: Y. Liu, K. Kadota, D.R. Jones Acquisition of data (provided animals, acquired and managed patients, Received October 16, 2015; revised January 29, 2016; accepted February 27, provided facilities, etc.): Y. Liu, E.B. Amin, K. Kadota, D.R. Jones 2016; published OnlineFirst March 15, 2016.

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CK2α' Drives Lung Cancer Metastasis by Targeting BRMS1 Nuclear Export and Degradation

Yuan Liu, Elianna B. Amin, Marty W. Mayo, et al.

Cancer Res Published OnlineFirst March 15, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-2888

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