stratiﬁn inhibits scf formation and blocks ubiquitination ...stratiﬁn inhibits scffbw7 formation...

Translational Cancer Mechanisms and Therapy

Stratifin Inhibits SCFFBW7 Formation and BlocksUbiquitination of Oncoproteins during the Courseof Lung AdenocarcinogenesisAya Shiba-Ishii1, Jeongmin Hong2, Takatsugu Hirokawa3,4, Yunjung Kim1,Tomoki Nakagawa2, Shingo Sakashita1, Noriaki Sakamoto1,Yukinori Kozuma5,Yukio Sato6,and Masayuki Noguchi1

Abstract

Purpose:Aberrant overexpressionof SFN(stratifin)plays anoncogenic role in lung adenocarcinoma. We have shownpreviously that SKP1, an adapter component of E3 ubiquitinligase forming an SCF complex, is a unique SFN-bindingprotein in lung adenocarcinoma cells.

Experimental Design: In silico simulation and in vitromuta-genesis analysis were performed to identify the SFN-bindingdomain on SKP1. We examined expression, localization, andstability of SKP1 after knockdown of SFN using lung adeno-carcinoma cells including A549. In silico library screening andexperimental validation were used for drug screening. Dailyoral administration of each candidate drugs to A549-injectedtumor-bearing mice was performed to evaluate their in vivoantitumor efficacy.

Results: Suppression of SFN upregulated the stability ofSKP1 and accelerated its cytoplasm-to-nucleus translocation.Consistently, IHC analysis revealed that cytoplasmic expres-

sion of SKP1 was significantly associated with SFN positivity,tumor malignancy, and poorer patient outcome. After SFNsuppression, ubiquitination of oncoproteins, includingp-cyclin E1, p-c-Myc, p-c-Jun, and cleaved Notch 1, which aretarget proteins of SCFFBW7, was strongly induced. These resultsindicate that SFN–SKP1 binding results in SCFFBW7 dysfunc-tion and allows several oncoproteins to evade ubiquitinationand subsequent degradation. Because inhibition of SFN-SKP1binding was expected to have antitumor efficacy, we nextsearched for candidate SFN inhibitors. Aprepitant and tica-grelor were finally selected as potential SFN inhibitors thatdose dependently reduced SFN-SKP1 binding and tumorprogression in vivo.

Conclusions: As overexpression of SFN is detectable inmost adenocarcinoma, we believe that SFN inhibitorswould be novel and promising antitumor drugs for lungadenocarcinoma.

IntroductionLung adenocarcinoma is the most common subtype of non–

small cell lung cancer (NSCLC), accounting for about 50% of allsuch cases. Although driver mutations and translocations inEGFR, EML4–ALK, and BRAF and several other specific oncogeneshave been identified in advanced adenocarcinoma, the mecha-nism that triggers its early progression has remained unclear.

Noguchi and colleagues have demonstrated that adenocarcinomain situ (AIS), which has an extremely favorable prognosis, pro-gresses in a stepwisemanner to early but invasive adenocarcinoma(eIA), with a 5-year survival rate of 75% (1). Previously, weidentified stratifin (SFN, 14-3-3 sigma) as a differentiallyexpressed gene that shows aberrant overexpression in most casesof invasive adenocarcinoma, whereas normal lung epitheliumand AIS show negative or very low expression (2). We subse-quently clarified that the abnormality of SFN expression wastriggered by DNA demethylation in the promoter region ofSFN (3).

14-3-3 is a highly conserved, ubiquitously expressed proteinfamily associated with many cellular processes. Among the 7human 14-3-3 isoforms (beta, epsilon, eta, gamma, tau, zeta,and sigma), 14-3-3 sigma (SFN) has a distinct function and hasbeen linked to cancer most directly. SFN was originally identifiedas a negative regulator of the cell cycle, particularly in response top53-sensitive DNA damage (4, 5). Recently, it was also reportedthat SFN negatively regulated the tumor-promoting metabolicprogram by enhancing c-Myc ubiquitination and subsequentdegradation (6). On the other hand, many previous reports haveindicated that SFN is a positive mediator of cell proliferation. Inbreast cancer, SFN induces G1/S progression by increasing theexpression of cyclin D1 (7). Moreover, its overexpression issignificantly associated with poorer prognosis in patientswith breast cancer (8), hepatocellular carcinoma (9), colorectal

1Department of Diagnostic Pathology, Faculty of Medicine, University of Tsu-kuba, Ibaraki, Japan. 2Graduate School of Comprehensive Human Sciences,University of Tsukuba, Ibaraki, Japan. 3Molecular Profiling Research Center forDrug Discovery, National Institutes of Advanced Industrial Science and Tech-nology, Tokyo, Japan. 4Department of Chemical Biology, Faculty of Medicine,University of Tsukuba, Ibaraki, Japan. 5Department of Medical Technology,Faculty of Health Science, Kumamoto Health Science University, Kumamoto,Japan. 6Department of Thoracic Surgery, Faculty of Medicine, University ofTsukuba, Ibaraki, Japan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A. Shiba-Ishii and J. Hong contributed equally to this article.

Corresponding Author: Aya Shiba-Ishii, Faculty of Medicine, University ofTsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Phone/Fax: 81-29-853-3350; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-3631

�2019 American Association for Cancer Research.

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carcinoma (10), and endometrial carcinoma (11).Our in vitro andin vivo functional analysis has also indicated that SFN regulatescell-cycle progression in a positive manner and promotes theprogression of lung adenocarcinoma (12). Importantly, 30% ofSFN-transgenic mice with lung-specific expression of human SFNspontaneously developed lung tumors, suggesting that SFN is apotent oncogene. Moreover, because SFN is known to be ananchor protein that binds to various intercellular molecules, wepreviously screened SFN-specific binding partner(s) in lung ade-nocarcinoma cells using pulldown assay and LC-MS/MS analysisin order to clarify the molecular mechanism by which SFNfacilitates tumor progression (13). Among the listed proteins, inthe present studywe focusedon S-phase kinase-associated protein1 (SKP1), which is an adaptor part of the SCF E3 ubiquitin ligasecomplex, as our previous results had shown that suppression ofSFN reduced the S-phase subpopulation, implying an associationof SFN with S-phase of the cell cycle (12).

SCF ubiquitin ligases constitute a subset of the cullin ring ligasefamily and are the largest family of E3 ubiquitin ligases inmammals. Each individual SCF E3 ligase consists of an adaptorprotein SKP1, a scaffold protein cullin-1, an F-box protein from a69-member family that serves as a substrate-determining com-ponent, and Rbx1, which contains the RING (Really InterestingNew Gene) finger domain for recruitment of the E2 enzyme (14).Combinations of these components make up a large number ofSCF E3s, including SCFFBW7, SCFSKP2, and SCFb-TrCP1, which playcrucial roles in numerous cellular processes by promoting ubi-quitination and subsequent proteasome-mediated degradationof diverse substrates involved in cell-cycle regulation, signaltransduction, and transcription (15). Particularly, SCFFBW7 reg-ulates the turnover—and thereby the activity—of important cel-lular regulatory proteins including cyclin E1 (16), c-Myc (17),c-Jun (18), and Notch1 (19). FBW7 recognizes substrate proteinsonly when they are phosphorylated (20). Additionally, the FBW7gene is frequently mutated in a broad spectrum of human malig-nancies (21). This is easily explained by the fact that many targetsof SCFFBW7 are oncoproteins that are expected to accumulate toabnormal levels when expression of FBW7 is lost or reduced.

However, in lung adenocarcinoma, mutation of FBW7 seems tobe rare. On the basis of this background, we speculated thatanother mechanism for inactivation of SCFFBW7 function mayexist in lung adenocarcinoma, probably in association with SFN.

The aim of the present study was to clarify the molecularmechanism of tumor progression in lung adenocarcinoma asso-ciatedwith SFNbinding to SKP1.Wehypothesized that SFNbindsto SKP1 and specifically blocks SCFFBW7 function to enhance theubiquitination of oncoproteins such as cyclin E1, c-Myc, c-Jun,and Notch 1. Because our previous study had shown that deple-tion of SFN led to a significant reduction of cell growth and tumorprogression in lung adenocarcinoma, we speculated that inhibi-tion of SFN might have potential efficacy against lung adenocar-cinoma. Here, we revealed the molecular mechanism of SFN-SKP1binding and showed that it is one of the key events occurringin lung adenocarcinogenesis. Finally, using in silicodrug screening,we identified 2 small molecules that inhibit SFN-SKP1 bindingand could have potential therapeutic application.

Materials and MethodsCell lines and culture conditions

The A549 cell line was purchased from RIKEN Cell Bank,HCC827 was obtained from the ATCC, and PL16T and PL16Bwere established in our laboratory from human lung AIS andhuman bronchial epithelium, respectively (22). A549 cells weremaintained in D-MEM/F12 (Thermo Fisher Scientific) supple-mented with 10% fetal bovine serum (FBS; Sigma-Aldrich).HCC827 cells were cultured with RPMI-1640 (Thermo FisherScientific) with 10% FBS. PL16B and PL16T cells weremaintainedin MCDB153HAA (Wako) supplemented with 2% FBS (Sigma-Aldrich), 0.5 ng/mL human-EGF (Toyobo), 5 mg/mL humaninsulin (Wako), 72 ng/mL hydrocortisone (Wako), 40 mg/mLhuman transferrin (Sigma-Aldrich), and 20 ng/mL sodium sele-nite (Sigma-Aldrich). All cells were cultured in a 5% CO2 incu-bator at 37�C and passaged every 3 days.

Patients and sample collectionWe used all 191 specimens of human lung adenocarcinomas

that had been surgically resected at the University of TsukubaHospital between 1999 and 2014. Follow-up information for allof the corresponding patients was obtainable from the medicalrecords. All of the patients provided informed consent for use oftheir materials. The study was approved by the institutional ethicsreview committee, and the lung adenocarcinoma cases wereclassified according to the UICC TNM classification of malignanttumors (seventh edition) and the World Health Organizationclassification of malignant tumors (fourth edition; refs. 23, 24).

Computational modeling of the SFN–SKP1 complexThe proposed model for the SFN–SKP1 complex was con-

structed using protein–protein docking. The X-ray structures ofSFN (Protein Data Bank ID code: 3IQU) and SKP1 (Protein DataBank ID code: 1FS1) were used as starting models. Both PDBentries were refined for protein–protein docking simulationsusing the Protein Preparation Wizard (25). ClusPro version2.0 (26) was used to generate docked conformations with thelowest docking energies and clustering properties. During theprotein–protein docking procedure, phosphorylation sites wereutilized as constraints for residues (Lys49, Arg56, Arg129, Tyr130of SFN, and Thr131 of SKP1) involved in complex formation at

Translational Relevance

This study demonstrates SFN (stratifin, 14-3-3 sigma) as adruggable potent oncogene that blocks the formation of E3ubiquitin ligase SCFFBW7 and induces abnormal stabiliza-tion of several oncoproteins, including cyclin E1 and c-Myc.Using in silico drug screening, we identified 2 potentiallypromising candidate SFN inhibitors, aprepitant and ticagre-lor, which are already approved as antiemesis or antiplateletagents, respectively. Drug repositioning is an efficientapproach to develop new treatments using preexisting drugswithout the possibility to fail during preclinical trials.Although the large numbers of early-stage adenocarcinoma,referred to as ground glass nodules (GGN), are beingdetected by low-dose CT screening and GGNs can be sur-gically resected, this can be dangerous or sometimes impos-sible in patients with multiple GGNs, or in those who areelderly or complicated by chronic interstitial lung disease.SFN inhibitors would provide a novel chemotherapeuticstrategy for early-stage lung adenocarcinoma.

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Clin Cancer Res; 25(9) May 1, 2019 Clinical Cancer Research2810




the protein–protein interaction interface. Putative druggable sitesat the interaction interface of the SFN–SKP1 complex resultingfromprotein–protein docking were also detected and representedby small dummy atoms with the PLB index (27) incorporated inthe SiteFinder program of MOE (Chemical Computing GroupULC).

In silico library screeningWe carried out in silico library screening based on the putative

druggable sites of SFN as well as the interaction interface withSKP1 using molecular docking against 7,133 bioactive com-pounds from the DrugBank database ver. 5.0 (28) and 9,101bioactive compounds from the Namiki bioactive compoundlibrary set (Namiki Shoji Co., Ltd).

For all compounds with molecular weights ranging from 200Da to 800 Da, ionization and energy minimization were per-formed by the OPLS3 force field in the LigPrep Script of Maestro(Schr€odinger, LLC). These minimized structures were used asinput structures for docking simulations. Docking simulationswere performed using the Glide (29, 30) SP docking program(Schr€odinger, LLC) with a grid box defined by small dummyatoms at putative druggable sites on SFN from computationalmodeling of the SFN–SKP1 complex step.

In vivo xenograft experimentsAnimal experiments were carried out humanely in accordance

with the Regulations for Animal Experiments of the University ofTsukuba and the Fundamental Guidelines for Proper Conduct ofAnimal Experiments and Related Activities in Academic ResearchInstitutions under the jurisdiction of the Ministry of Education,Culture, Sports, Science, and Technology of Japan and withapproval from the Institutional Animal Experiment Committeeof our university (#17-112).

First, 6-week-old female immune-deficient BALB/cAJcl-nu/numice were purchased from CLEA Japan, Inc., and 2.5 � 106 A549cells in 0.1 mL PBS were subcutaneously injected into the rightflank. From the following day (day 1), mice were treated dailywith the compounds (aprepitant, ticagrelor, ezetimibe, and chlor-hexidine) dissolved in 5% DMSO and 95% corn oil at 80 mg/kg/day. The control group received 200 mL of 5%DMSOþ 95% cornoil. Tumor growth was controlled and measured daily, and micewere checked for their vital status and weight. Tumor volume wascalculated using the formula: mm3¼ 1/2� L�W2. All mice weresacrificed at the end of the experiment.

Secondly, 2.5 � 106 A549 cells in 0.1 mL PBS were subcuta-neously injected into the right flank. Mice were randomized intogroups once the tumors had attained a volume of 100 mm3. Thetreatment groups were treated daily with 3 doses of aprepitant orticagrelor solution dissolved in 5% DMSO and 95% corn oil. Allof the mice were sacrificed at the end of the experiment. Resectedtumors were partially fixed with formalin for histologic analysisand frozen in liquid nitrogen for molecular analysis.

ResultsSFN binds to SKP1 at Thr131

Our previous pulldown assay and LC-MS/MS analysis hadidentified SKP1 as an SFN-binding partner in A549 lung adeno-carcinoma cells (13). Here, we also confirmed binding of theirendogenous proteins using immunoprecipitation (IP) and West-ern blotting (Supplementary Fig. S1A). Moreover, among 14-3-3

members, only SFN (14-3-3 sigma) showed binding to SKP1(Supplementary Fig. S1B). To exclude the possibility that a lack ofassociation between other 14-3-3 members and SKP1 was due tolow expression of endogenous 14-3-3 proteins, we overexpressed14-3-3 epsilon, zeta, or sigma and subsequently performed IPwith SKP1. As a result, even overexpressed 14-3-3 epsilon or zetashowed nobindingwith SKP1 (Supplementary Fig. S1C). Becausebinding of SFN with SKP1 has not yet been reported, we carriedout in silico docking simulation to identify the binding surface ofeach protein. The results indicated that Lys49, Arg56, Arg129, andTyr130 in SFN and Thr131 in SKP1 were important residues forbinding (Supplementary Fig. S2A). We created a mutant SKP1(T131V) with a Halo-tag and performed a pulldown assay usingHalo-tag and subsequent Western blotting with SFN antibodies.Although WT SKP1 bound with SFN, T131V impaired the inter-actionwith SFN (Supplementary Fig. S2B), indicating that Thr131is an SFN-binding site critical for SFN–SKP1 binding. Moreover,we detected phosphorylated SKP1 in A549 cells using antipho-sphorylated threonine (pThr) antibodies (SupplementaryFig. S2C), and T131V decreased expression of phosphor-SKP1(Supplementary Fig. S2D). Phosphorylated threonine at codon131 is supposed to be associated with SFN–SKP1 binding. Wethen generated a quadruplemutant of SFN includingK49A, R56A,R129A, and Y130A using a Flag-tag and subsequently performedIP with anti-Flag antibodies. The quadruple mutant of SFN wasfound to have lost its ability to bind with SKP1 (SupplementaryFig. S2E). Moreover, each single mutant of SFN also showedimpaired binding with SKP1 (Supplementary Fig. S2F), suggest-ing that the residues Lys49, Arg56, Arg129, and Tyr130 form thesurface for binding of SKP1 with SFN.

SFN alters the protein stability and subcellular localization ofSKP1

In order to investigate the influence of SFN on SKP1 protein, aspecific siRNA against SFN (siSFN) and overexpression of SFNwere used. Although both suppression and overexpression of SFNdonot show conspicuous change of SKP1mRNA (SupplementaryFig. S3A), suppression of SFN led to an increase of SKP1 proteinexpression, whereas overexpression of SFN conversely reduced it(Supplementary Fig. S3B). The cycloheximide chase assay indi-cated that the half-life of SKP1 protein wasmarkedly longer in thecells transfected with siSFN than in the controls (Fig. 1A). Addi-tionally, ubiquitination of SKP1 was upregulated by overexpres-sion of SFN (Fig. 1B), suggesting that SFNmight regulate the half-life or stability of SKP1. Next, various cell lines such as PL16B(immortalized normal bronchial epithelial cells), PL16T (immor-talized AIS cells), HCC827, and A549 (advanced lung adenocar-cinoma cells) were subjected to immunofluorescence (IF) stain-ing. Although PL16B showed no SFN expression and only nuclearSKP1 expression, 3 adenocarcinoma cell lines displayed coloca-lization of SFN and SKP1 in the cytoplasm (Supplementary Fig.S3C). Furthermore, we found that depletion of SFN in A549 orHCC827 cells reduced the expression of cytoplasmic SKP1 andincreased that of nuclear SKP1 (Fig. 1C, top; Supplementary Fig.S3D). Conversely, overexpression of SFN in PL16B cells increasedthe cytoplasmic expression of SKP1 (Fig. 1C, bottom). We alsoperformed fractionation of subcellular components and sub-jected the fractions toWestern blotting. Consistently, transfectionwith siSFN increased the expression of SKP1 in the nucleus andreduced it in the cytoplasm (Fig. 1D). These data suggest that SFNalters the protein stability and subcellular localization of SKP1.

SFN Blocks Ubiquitination of Oncoproteins in Lung Cancer

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Overexpression of SKP1 in the cytoplasm of human lungadenocarcinoma

To clarify the association of SFN with SKP1 in human lungadenocarcinoma tissue, SKP1 IHCwas performed using 191 casesof lung adenocarcinoma. We scored nuclear and cytoplasmicSKP1 positivity separately. Consequently, 92 cases showed highSKP1 expression in the cytoplasm. Although SFN-negative casestended to show SKP1 positivity only in the nucleus, most SFN-positive cases showedpositivity not only in the nucleus but also inthe cytoplasm (Fig. 2A). H-score for cytoplasmic SKP1 was sig-nificantly higher in SFN-positive tumors than in those withSFN negativity (Fig. 2B). Moreover, we found that cytoplasmicexpression of SKP1 was significantly associated with gender, theNoguchi classification for small adenocarcinomas of the lung(2 cm or less in diameter), pathologic subtype, pathologic stage,lymphatic permeation, and vascular invasion (SupplementaryTable S1). Furthermore, cytoplasmic SKP1 positivity was signif-icantly associated with a poorer outcome relative to cytoplasmicSKP1-negative cases (P < 0.001; Fig. 2C), whereas nuclear SKP1

positivity was not (P ¼ 0.132, data not shown). We also per-formed multivariate survival analysis using Cox proportionalhazards model using 6 factors selected on the basis of the resultsof univariate analysis: sex (female vs. male), vascular invasion(negative vs. positive), lymphatic permeation (negative vs. pos-itive), pathologic stage (p stage I, II vs. III, IV), smoking history(never-smoker vs. smoker), and cytoplasmic SKP1 H-score (lowvs. high). The results showed that vascular invasion, lymphaticpermeation, and pathologic stage but not cytoplasmic SKP1expression were independent prognostic factors (SupplementaryTable S2). Taken together, although cytoplasmic SKP1 expressionwas not an independent prognostic factor, our findings suggestthat cytoplasmic SKP1 expression is significantly associated withSFN expression and malignancy of lung adenocarcinoma.

Interaction of SFN with SKP1 is competitive with FBW7Although 69 types of F-box protein have been reported, FBW7,

SKP2, and b-TrCP1 are the best-characterized members andtherefore were studied further. We expected that the SFN-binding

Figure 1.

SFN alters the protein stability and localization of SKP1. A, Cycloheximide chase assay to examine the half-life of SKP1 protein using A549 cells. Suppression ofSFN prolonged the half-life of SKP1. We performed 3 independent experiments and quantified each band to show SKP1 expression as a line graph afternormalization of each by ACTB (b-actin). B, Ubiquitination assay for SKP1. A549 cells were transfected with siRNA-resistant pSFN for 24 hours followed byUb-HA for 24 hours. The cells were then treated with 10 mmol/L MG132, a proteasome inhibitor, for 6 hours at 37�C in a 5% CO2 incubator. The total protein wasextracted using IP Lysis Buffer containing 10 mmol/L N-ethylmaleimide (NEM), a deubiquitinating enzyme inhibitor. Ubiquitinated proteins in the cell lysate werecollected by co-IP and then subjected toWestern blotting. Overexpression of SFN induced ubiquitination of SKP1. C,A549 and PL16B cells transfected with siSFNor pSFN were subjected to immunofluorescence (IF) using SKP1 or SFN antibodies. Suppression of SFN in A549 cells led to translocalization of SKP1 from thecytoplasm to the nucleus. On the other hand, overexpression of SFN in PL16B cells induced translocalization of SKP1 from the nucleus to the cytoplasm. Scale bar,50 mm. D,Western blotting using subcellular fractionated protein extracted from A549 cells. Consistent with the IF results, depletion of SFN increased thenuclear expression of SKP1 and reduced the cytoplasmic expression. Lamin B was used as a nuclear protein marker, and a-tubulin as a cytoplasmic proteinmarker. NU; nuclear fraction, CY; cytoplasmic fraction.

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domain might overlap with the FBW7-binding domain on theSKP1 protein. IP with anti-SKP1 antibodies and subsequentWestern blotting indicated that suppression of SFN increasedSKP1–FBW7 binding but not SKP1–SKP2 or –b-TrCP1 binding(Supplementary Fig. S4A), suggesting that SFNmay compete withFBW7 for binding with SKP1. In silico docking simulation analysisalso supported this finding (Fig. 3A). In PL16B cells, which do notexpress SFN, SKP1 and FBW7were colocalized only in the nucleus(Supplementary Fig. S4B, left). However, inHCC827 cells, whichhave high SFN expression, SKP1 but not FBW7 was partiallylocalized in the cytoplasm (Supplementary Fig. S4B, right),implying that SFN might sequester SKP1 in the cytoplasm sothat SCFFBW7 does not form in the nucleus. SCFFBW7 specificallydegrades several phosphorylated oncoproteins that function incellular growth, including cyclin E1, c-Myc, c-Jun, and Notch1.Therefore, we analyzed the expression of target proteins ofSCFFBW7, SCFSKP2, and SCFb-TrCP1 after transfection with siSFN.The results showed that target proteins of SCFFBW7 such asp-cyclin E1, p-c-Myc, p-c-Jun, and NICD (Notch intracellulardomain) were downregulated after suppression of SFN, whereasp-p27 (target of SCFSKP2) and p-IkBa (target of SCFb-TrCP1) wereunaffected (Fig. 3B). Additionally, we found that suppression ofSFN clearly increased the ubiquitination of p-cyclin E1, p-c-Myc,p-c-Jun, and NICD (Fig. 3C). These results indicate that SFNmight obstruct formation of the SCFFBW7 complex and inhibitubiquitination of target oncoproteins.

We had previously found that depletion of SFN decreasedcell growth in vitro and tumor progression or developmentin vivo, suggesting that SFN might be a promising therapeutic

target for lung adenocarcinoma (12). Here, we revealed thatSFN enhances tumor progression through binding with SKP1and blocking of oncoprotein ubiquitination. Thus, it is alsoexpected that inhibition of SFN–SKP1 binding might haveantitumor efficacy through recovery of normal ubiquitinationand degradation of oncoproteins.

Aprepitant and ticagrelor are potential SFN inhibitorcandidates

To identify potential SFN inhibitor(s), we focused on preexist-ing low-molecular-weight drugs, because SFN is an intracellularmolecule. In silico simulation revealed a druggable pocket (PLBscore 2.86, exceeding the druggable threshold of 2.0) around theSKP1 binding site in SFN protein (Fig. 4A). In order to identifycompounds that can specifically bind to the pocket and inhibitSFN-SKP1 binding, we performed in silico screening using Drug-Bank (https://www.drugbank.ca/) and theNamiki bioactive com-pounds library (Namiki Shoji Co., Ltd). From a total of 7,133drugs, we extracted 5,597 whose molecular weights were 200 to800 and subjected them to in silico screening. Among 5,597 drugs,we obtained 46 available compounds that had high ranking(Supplementary Table S3). We then carried out experimentalvalidation of these 46 compounds using the WST-8 proliferationassay and IP/Western botting to analyze SFN–SKP1 dissociationusing lung adenocarcinoma cells after treatment with each com-pound. Among these compounds, 4 (aprepitant, ticagrelor, eze-timibe, and chlorhexidine) reduced both cell proliferationand SFN–SKP1 binding in a dose-dependent manner and weretherefore subjected to further analysis (Fig. 4B and C). Moreover,

Figure 2.

SKP1 shows overexpression in the cytoplasm of human lung adenocarcinoma. A, Image of SFN or SKP1 IHC for representative cases showing SFN positivity ornegativity in IHC.Whereas the SFN-negative case showed SKP1 expression only in the nucleus and not in the cytoplasm, the SFN-positive case had both nuclearand cytoplasmic SKP1 expression in the tumor cells. B, Scatter diagram for H-score of cytoplasmic SKP1 expression. SFN-positive tumor showed significantlyhigher expression of cytoplasmic SKP1 than negative tumors (P < 0.001). C,Disease-free survival depicted as Kaplan–Meier curves showing the correlationbetween outcome and cytoplasmic SKP1 expression. Positive expression of cytoplasmic SKP1 was associated with a poorer outcome than the case for negativeexpression (P < 0.001).





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in silico analysis indicated that the patterns of aprepitant andticagrelor binding to the SFN druggable pocket were similar,broadly blocking the SKP1-binding surface on the SFN protein(Fig. 4Da–b).However, ezetimibe lacked a polar functional groupand chlorhexidine had only few interactions with the pocket(Fig. 4Dc–d). Therefore, we considered aprepitant and ticagrelorto be the final SFN inhibitor candidates. Even though the originaltargets of these 2 compounds are completely different, they usecommon pharmacophores to link with the druggable pocket onthe SFNprotein (Fig. 4De). Additionally, these 2 compoundswereconfirmed to suppress cell growth in a dose-dependentmanner inanother 4 lung adenocarcinoma cell lines (Fig. 4E). However,importantly, Calu-6 (large cell lung carcinoma cells) and PL16B(immortalized bronchial epithelial cells), which have no SFN

expression, did not reduce cell number even after administrationof aprepitant or ticagrelor (Fig. 4E), indicating that their effects aremediated by inhibition of SFN.

Next, the in vivo antitumor effect was evaluated using 2 types ofexperimental schedule in a tumor-bearingmousemodel. First, wesubcutaneously injected A549 cells into nude mice (day 0) andstarted daily oral administration of each compound from thefollowing day (day 1) to mimic the effect of adjuvant chemo-therapy. Surprisingly, aprepitant completely blocked tumor for-mation, and ticagrelor markedly reduced tumor progression(Supplementary Fig. S5). These results are consistent with ourin silico data indicating that aprepitant and ticagrelor broadlyblocked SKP1 binding surface and were more appropriate candi-dates than the others.

Figure 3.

Interaction of SFNwith SKP1 is competitive with FBW7. A, Structural models of (a) the F-box/SKP1/Cul1 complex, (b) the SFN/SKP1/Cul1 complex, and (c) anoverlapped view of the F-box/SKP1/Cul1 complex with SFN. The F-box/SKP1/Cul1 complex was constructed using heterodimer complexes from a Cul1–Rbx1–Skp1-F boxSkp2 SCF ubiquitin ligase complex (Protein Data Bank ID code: 1LDK) and a FBW7–SKP1–Cyclin E1 complex (Protein Data Bank ID code: 2OVR) bystructural alignment with SKP1. The SFN/SKP1/Cul1 complex was constructed using a predicted model of SFN/SKP1 in this work and a Cul1–Rbx1–Skp1–F boxSkp2SCF ubiquitin ligase complex (Protein Data Bank ID code: 1LDK). Filled yellow circle indicates the area of structural overlap between the F-box and SFN (c).B,Western blotting using A549 cells transfected with 3 kinds of siSFN (1–3). Substrate proteins of FBW7 such as p-cyclin E1, p-c-Myc, p-c-Jun, and NICD (Notchintracellular domain) were suppressed after knockdown of SFN, although p-p27 (substrate protein of SKP2) and p-IkBa (substrate protein of b-TrCP1) showedno change in expression. C, Ubiquitination assay for substrate proteins of FBW7. A549 cells were transfected with siSFN for 24 hours followed by Ub-HA for 24hours. The cells were then treated with 10 mmol/L MG132, a proteasome inhibitor, for 6 hours at 37�C in a 5% CO2 incubator. The total protein was extracted usingIP Lysis Buffer containing 10 mmol/L N-ethylmaleimide (NEM), a deubiquitinating enzyme inhibitor. Ubiquitinated proteins in the cell lysate were collected by co-IP and then subjected toWestern blotting. Suppression of SFN induced marked ubiquitination of p-cyclin E1, p-c-Myc, p-c-Jun, and NICD.

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For the second in vivo validation procedure, we subcutane-ously injected A549 cells into nude mice. The mice wererandomized into 7 groups when tumors had attained a volumeof 100 mm3 (day 1) and started on daily oral administration ofeach compound at 3 different doses for 21 days. It was found

that both aprepitant and ticagrelor significantly suppressedtumor progression in a dose-dependent manner (Fig. 5Aand B). Histologically, the resected tumors were poorly differ-entiated adenocarcinomas with a medullary appearance. Nota-bly, tumors that developed in the mice treated with ticagrelor

Figure 4.

Aprepitant and ticagrelor as candidate SFN inhibitors. A, Putative druggable sites at the interaction interface of the SFN–SKP1 complex from protein–proteindocking and small dummy atoms (red¼ hydrophilic, white¼ hydrophobic) determined using the SiteFinder program of MOE (Chemical Computing Group ULC).The PLB score at this site was 2.86. B, IP with SFN antibodies after treatment with each compound (10 mmol/L). #1, aprepitant; #2, ticagrelor; #3, ezetimibe; #4,chlorhexidine. All 4 compounds inhibited the binding of SFN to SKP1. As a negative control, cells treated with DMSOwere used. C,WST-8 cell proliferation assayafter treatment with each compound. As a negative control, cells treated with DMSOwere used. Semi-log graph was used to show cell viability. All 4 compoundsreduced cell growth dose-dependently. D, Predicted interactions between 4 hit ligands and the SFN druggable pocket (a)–(d) and a pharmacophore model foraprepitant and ticagrelor on the SFN druggable pocket (e). Circles with dashed lines in (c) and (d) indicate the interaction gaps between the ligands (ezetimibeand chlorhexidine) and key residues of the SFN druggable pocket. Three-feature pharmacophore model generated for aprepitant and ticagrelor using MOE:hydrogen bond acceptor (cyan feature), hydrophobic region (dark green feature), and hydrophobic region or aromatic ring center (green feature; e).E,WST-8 cell proliferation assay after treatment with aprepitant or ticagrelor. PC9, H1975, HCC827, PL16T, PL16B, and Calu-6 were used. As a negative control,cells treated with DMSOwere used. Semi-log graph was used to show cell viability. In accordance with the previous pharmacokinetics reports on aprepitant andticagrelor, which indicated that their degree of plasma protein binding is 99.6% and 99.9%, respectively, we calculated the unbound drug concentration fromthe loading concentration of inhibitors. Both aprepitant and ticagrelor reduced the cell growth dose-dependently in PC9, H1975, HCC827, and PL16T. However,Calu-6 or PL16B cells, which have no SFN expression, did not reduce cell number.






had acellular spaces, and those administered with aprepitantalso had spaces filled with lymphocytes but not tumor cells,apparently attributable to regression of tumor cells after treat-ment. Moreover, the tumor cells surrounding these spacesshowed lower expression of Ki67, and even SFN, in comparisonwith those from mice treated with vehicle alone (Fig. 5C). Thissuggested that aprepitant and ticagrelor may exert an antitumoreffect by reducing tumor cell proliferation.

Using the collected subcutaneous tumors, we extracted thetotal proteins and subjected them to IP with SKP1 antibodiesand subsequent Western blotting for SFN, and vice versa. Thetumors treated with aprepitant or ticagrelor showed reduced

association of SFN with SKP1 (Supplementary Fig. S6A). Addi-tionally, Western blotting indicated that expression of p-cyclinE1, p-c-Jun, p-c-Myc, and NICD was lower in tumors from micetreated with aprepitant or ticagrelor than in those from vehicle-treated mice (Supplementary Fig. S6B), suggesting that theymay exert its antitumor effect through inhibition of SFN–SKP1binding, leading to ubiquitination and degradation of onco-proteins. Because those 2 compounds suppressed cell growtheven in PL16T cells (Fig. 4E), which had been established fromAIS, we considered that aprepitant and ticagrelor might showantitumor efficacy against not only advanced, but also early-stage lung adenocarcinoma.

Figure 5.

Aprepitant and ticagrelor suppressed tumor progression in vivo. A and B, Nude mice were given a subcutaneous injection of A549 cells and randomly dividedinto groups, each comprising 8 individuals. Three different doses of aprepitant or ticagrelor dissolved into 5% DMSOþ 95% corn oil were administered orallyevery day when the tumors had attained a volume of 100mm3. 5% DMSOþ 95% corn oil was administered in the vehicle group. Tumor volume was measuredwith a caliper at the indicated time points. Both aprepitant and ticagrelor decreased tumor growth dose-dependently. C, HE and IHC for SFN and Ki-67 wereconducted using the collected subcutaneous tumors. Red arrows indicate spaces filled with only lymphocytes or acellular spaces inside the tumors. Green linesindicate the tumor cells surrounded by acellular spaces. �P < 0.05, ��P < 0.01.

Shiba-Ishii et al.





DiscussionIn the present study, we revealed that SFN bound with SKP1

and inhibited formationof the SCFFBW7 complex, allowing severaloncoproteins to evade ubiquitination. Our data showed thatSKP1 bound only with SFN and not with other 14-3-3 familymembers (Supplementary Fig. S1B). This might be because SFNhas unique amino acids (Met202, Asp204, and His206) that maybe responsible for binding to particular ligands that are notrecognized by other 14-3-3 members (31). Because Lys49, Arg56,Arg129, and Tyr130 in the SFN protein are reportedly residuesimportant for bindingwith phosphorylated binding partners, andwe confirmed that SKP1 also used the same interface for bindingwith SFN (Supplementary Fig. S2E and S2F), we had speculatedthat SKP1 might require phosphorylation for binding to SFN.Based on the previous phosphoproteomics study that hadrevealed phosphorylated threonine at residue 131 of SKP1 (32),we clarified that the Thr131 residue in SKP1 protein was indeedphosphorylated and acted as a binding site for SFN (Supplemen-tary Fig. S2B–S2D), suggesting that the binding of SKP1 to SFNmight be phosphorylation dependent.

14-3-3 proteins are known to be anchor proteins that alter thestability, activity, or subcellular localization of binding part-ners (33). We also found that SFN reduced the stability of SKP1protein and accelerated its ubiquitination (Fig. 1A and B). Basedon these data, we expected that interactionwith SFNmight inducea conformational change in the SKP1 protein structure, leading torapid ubiquitination and degradation. Our data also indicatedthat SFN binding to SKP1 altered the latter's subcellular locali-zation (Fig. 1C and D; Supplementary Fig. S3D). SFN has beenreported to bind with constitutive photomorphogenic 1 (COP1),a p53-specific E3 ubiquitin ligase involved in regulating the levelof p53 protein, resulting in redistribution of COP1 to the cyto-plasm in colon cancer cells (34). It is assumed that SFN similarlybindswith newly-synthesized SKP1proteins in the cytoplasm andblocks the translocation of SKP1 to the nucleus, thus preventingformation of the SCF complex. We expect that SFN might haveparticular function to regulate the activity or subcellular locali-zation of various E3 ubiquitin ligases.

Our present IHC analysis showed that cytoplasmic SKP1expression was significantly associated with tumor malignancyor poor prognosis (Supplementary Table S1; Fig. 2). Although thephysiologic locationof SKP1 is the nucleus, cytoplasmic SKP1wasshown to be more associated with tumor progression. From ourin vitro results, we speculate that SKP1 expression in the cytoplasmmight result from sequestration of SKP1by SFN. These IHC resultsmight allow our in vitro findings to be generalized to humantumor tissue. As we have previously reported that expression ofSFN is related to poor prognosis (13), SKP1 expression in thecytoplasm might also serve as a prognostic marker for lungadenocarcinoma.

SFN appears to compete with only FBW7, and not SKP2 orb-TrCP1, for binding with SKP1 protein (Fig. 3; SupplementaryFig. S4). There is increasing evidence to suggest that cyclin E1, oneof the FBW7 target proteins, is physiologically activated in late G1

phase to facilitate G1/S transition, and degraded in S-phase bySCFFBW7. On the other hand, p27, the target substrate of SKP2, isknown to be degraded in late G1 phase by SCF

SKP2. These findingsindicate that each E3 ubiquitin ligase may function at a distincttime point in the cell cycle. Here, we showed that SFN selectivelyinhibited SCFFBW7 by competing with FBW7, suggesting that SFN

might bind with SKP1 in S-phase but not in G1 phase. In supportof this, a previous report has indicated that SFN enhances G1/Sprogression through upregulation of cyclin D1 in breast can-cer (7). On the other hand, the specificity of SFN for FBW7mightalso be related to the difference in localization of each F-boxprotein. Although we showed that FBW7 was located in thenucleus of lung adenocarcinoma cells (Supplementary Fig.S4B), SKP2 and b-TrCP1 function mainly in the cytoplasm. Asmentioned above, because SFN is thought to bind with newly-synthesized SKP1 protein in the cytoplasm and inhibit its nucleartransport, formation of the SCFFBW7 complex in the nucleusmightbe blocked more efficiently than that of the SCFSKP2 or SCFb-TrCP1

complex in the cytoplasm.Recently, Reiterer and colleagues reported that psuedopho-

sphatase STYX suppressed SCFFBW7 function by competing withSKP1 for binding to the F-box motif of FBW7, resulting inupregulation of FBW7 substrates including c-Myc, and cyclinE1 in HeLa cells (35). Additionally, various upstream factors suchas USP29, ERK and PLK1, which induce deubiquitination orautoubiquitination of FBW7, have been reported to modulatethe stability and activity of FBW7 (36). However, because indirectregulation of FBW7 through blocking of SCFFBW7 complex for-mation by binding with SKP1 has not yet been confirmed, ourfindings provide insight into a new mechanism for regulation ofFBW7.

In our recent report, we identified an SFN-binding partner otherthan SKP1, ubiquitin-specific protease 8 (USP8) (13). USP8 is adeubiquitination enzyme specific for receptor tyrosine kinases(RTK) including EGFR and MET, contributing to the proliferativeactivity of many human cancers including NSCLC. We found thatSFN stabilizes RTKs through activation of USP8 in lung adeno-carcinoma. Because 14-3-3 proteins are anchor proteins that canbind with various molecules, it is natural that SFN would havemultiple binding partners. Based on these facts, inhibition of SFNis thought to lead to ubiquitination of not only oncoproteins butalso RTKs. Because SFN is related tomultiple pathways, we believethat inhibition of SFN would simultaneously block several path-ways that contribute to lung adenocarcinogenesis, thus exertinghigh antitumor efficacy.

Our results indicate that inhibitors of SFN–SKP1 binding couldbe potential targeted drugs for treatment of lung adenocarcinoma.In order to screen potent inhibitors, we adopted an in silicoapproach utilizing structural data for protein–protein interaction(PPI) targets and a database of known bioactive compounds, astrategy known as "in silico drug repositioning." This drug-repo-sitioning strategy makes it possible to rationalize experimentalprotocols, prioritize the design of therapeutic compounds forPPIs, and speed up the development of promising drugs, whilereducing the risk of failure in clinical trials. Although the structureof the SFN–SKP1 complex has not been clarified experimentally, areliable computational model of the complex, which was con-sistent with the results of in vitro mutational experiments, andin silico detection of the druggable pockets overlapping at theSFN–SKP1 interaction interface led to the successful identificationof 2 independent drugs for this particular PPI: aprepitant andticagrelor.

Aprepitant is an NK-1R (neurokinin-1 receptor) antagonistalready approved as an antiemetic drug for cancer patients whoneed to take antitumor drugs that cause nausea or emesis. Apre-pitant has already been reported to suppress cell growth ofhepatoblastoma through inhibition of NK-1R (37). However, we






consider that the antitumor effect of aprepitant on lung adeno-carcinoma cells may not be due to inhibition of NK-1R bindingbut rather that of SFN–SKP1 binding, because lung adenocarci-noma cells do not express NK-1R (data not shown). It is thoughtthat aprepitant is a potential SFN inhibitor that could be used fortreatment for lung adenocarcinoma.On the other hand, ticagreloris a reversible P2Y12 inhibitor exerting an antiplatelet effect and isused clinically with aspirin for treatment of cardiovascular andcerebrovascular diseases. A phase III clinical study of ticagrelorshowed that 9.8% of patients who took daily ticagrelor andaspirin together suffered a bleeding event within 12 months.Although we confirmed that mice given a single dose of ticagrelorshowed a lower antiplatelet effect than was the case for coadmin-istration of ticagrelor and aspirin in terms of bleeding time andplatelet aggregation (Supplementary Fig. S7), we will need tocarefully verify the safety of ticagrelor as an antitumor drug.

Previous studies have shown that when aprepitant (MW534.43) was administered to humans as a 125-mg capsule, theCmax was 1,539 ng/mL (38), and the protein binding rate was99.6% (39), indicating that the plasma unbound drug concen-tration was approximately 11.52 nmol/L. Using the reportedplasma Cmax of aprepitant (3,535 ng/mL) when administered at30mg/kg tomice (39),we estimate that theplasmaunbounddrugconcentration of aprepitant after administration at 20 mg/kg tomice (Fig. 5A) could be approximately 17.64 nmol/L, which issimilar to that in humans (11.52 nmol/L). On the other hand, theCmax of ticagrelor (MW 522.57) when administered to humans ata single oral dose of 90 mg was reported to be 581 ng/mL, with aprotein binding rate of 99.9% (40), suggesting that the plasma

unbound drug concentration would be approximately 1.11nmol/L. Because administration of ticagrelor at 180 mg/kg tomicewas reported to result in a plasmadrug concentration of 0.99mmol/L (41), the dose of ticagrelor we used (100 mg/kg, Fig. 5B)would be expected to achieve a plasma unbound drug concen-tration of 0.56 nmol/L. This is lower than the aforementionedunbound drug concentration in humans, suggesting that the dosewould be tolerated in humans.

Many targeted drugs for treatment of lung adenocarcinoma,such as EGFR–TKI, ALK inhibitors, and BRAF inhibitors, areavailable. Unfortunately, however, these drugs do not significant-ly improve the patient survival rate. In the present study, wefocused on epigenetic changes in early-stage lung adenocarcino-ma to devise a new treatment strategy and were able to confirmthat SFN–SKP1 binding is one of the key events involved in themalignant progression of such cancers. As a result, 2 potentiallypromising candidate drugs, aprepitant and ticagrelor, were iden-tified. Recently, it has been confirmed that low-dose CT screeningis able to reduce the death rate of patients with early lungadenocarcinomas. Because this strategy is now being promotedin many countries, large numbers of small nodules, referred to asground glass nodules (GGN), are being detected. AlthoughGGNscan be surgically resected, this can be dangerous or sometimesimpossible in patients with multiple GGNs, or in those who areelderly or complicated by chronic interstitial lung disease. Inhi-bitors of SFN–SKP1 binding such as aprepitant and ticagrelor arethought to be desirable candidates for the treatment of early-stagelung adenocarcinoma. Because SFN is widely associated withtumor progression from an early stage, these drugs can be applied

Figure 6.

Molecular mechanism at a glance. SFN binds with SKP1 to sequester it in the cytoplasm, resulting in the obstruction of SCFFBW7 complex formation. Oncoproteinsincluding cyclin E1 and c-Jun subsequently evade from ubiquitination and proteasomal degradation and are stabilized due to lack of their specific E3 ubiquitinligase.

Shiba-Ishii et al.





without companion diagnosis, and used together with otherantitumor drugs such as immune-checkpoint inhibitors, even forneoadjuvant chemotherapy. Not only aprepitant and ticagrelor,but other small molecules might be effective for inhibition ofSFN–SKP1 binding, and efforts to identify new inhibitors withbetter potency would be justified.

In conclusion, we have revealed that SFN binds with SKP1 andinhibits the formation of SCFFBW7, resulting in stabilization ofoncoproteins (Fig. 6). Aprepitant and ticagrelor were identified ascandidate SFN inhibitors for suppression of abnormal SFN–SKP1binding and tumor progression. Our present findings suggest thatthese inhibitors may provide a novel chemotherapeutic strategyfor early-stage lung adenocarcinoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Shiba-Ishii, M. NoguchiDevelopment of methodology: A. Shiba-Ishii, J. Hong, T. HirokawaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):A. Shiba-Ishii, J. Hong, S. Sakashita, Y. Kozuma, Y. SatoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Shiba-Ishii, J. Hong, T. Hirokawa

Writing, review, and/or revision of the manuscript: A. Shiba-Ishii, J. Hong,T. Hirokawa, M. NoguchiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Kim, T. Nakagawa, S. Sakashita, N. SakamotoStudy supervision: M. Noguchi

AcknowledgmentsWe sincerely express our appreciation toDr. HidehitoHorinouchi, Dr. Shun-

ichiWatanabe, andDr.Masahiko Kusumoto (National Cancer CenterHospital)for their suggestion and support from the clinical point of view. Moreover, weare grateful to Professor Kenji Irie, Dr. Hiroyuki Suzuki, and Dr. Yuji Funakoshi(Faculty of Medicine, University of Tsukuba) for research support and kindadvice. This work was supported by JSPS KAKENHI grant number 17K15634(T. Hirokawa), the Platform Project for Supporting Drug Discovery and LifeScience Research [Basis for Supporting Innovative Drug Discovery and LifeScience Research (BINDS)] from AMED under grant number JP18am0101114(T. Hirokawa), and MEXT as "Priority Issue on Post-K computer" (BuildingInnovation Drug Discovery Infrastructure Through Functional Control ofBiomolecular Systems; hp160213; T. Hirokawa).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 5, 2018; revised December 11, 2018; accepted January17, 2019; published first February 6, 2019.

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2019;25:2809-2820. Published OnlineFirst February 6, 2019.Clin Cancer Res Aya Shiba-Ishii, Jeongmin Hong, Takatsugu Hirokawa, et al. Oncoproteins during the Course of Lung Adenocarcinogenesis

Formation and Blocks Ubiquitination ofFBW7Stratifin Inhibits SCF

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