Nuclear IGF1R Interacts with Regulatory Regions of ... · tumor cell survival and IGF-induced...

Molecular Cell Biology

Nuclear IGF1R Interacts with Regulatory Regionsof Chromatin to Promote RNA Polymerase IIRecruitment and Gene Expression Associatedwith Advanced Tumor StageTamara Aleksic1, Nicki Gray2, Xiaoning Wu1, Guillaume Rieunier1, Eliot Osher1,Jack Mills1, Clare Verrill3, Richard J. Bryant1,4, Cheng Han1,5, Kathryn Hutchinson1,Adam G. Lambert4, Rajeev Kumar4, Freddie C. Hamdy4, Ulrike Weyer-Czernilofsky6,Michael P. Sanderson6, Thomas Bogenrieder6,7, Stephen Taylor2, andValentine M. Macaulay1,5

Abstract

Internalization of ligand-activated type I IGF receptor(IGF1R) is followed by recycling to the plasma membrane,degradation or nuclear translocation. Nuclear IGF1R reportedlyassociates with clinical response to IGF1R inhibitory drugs, yetits role in the nucleus is poorly characterized. Here, we inves-tigated the significance of nuclear IGF1R in clinical cancersand cell line models. In prostate cancers, IGF1R was pre-dominantly membrane localized in benign glands, whilemalignant epithelium contained prominent internalized(nuclear/cytoplasmic) IGF1R, and nuclear IGF1R associatedsignificantly with advanced tumor stage. Using ChIP-seq toassess global chromatin occupancy, we identified IGF1R–binding sites at or near transcription start sites of genes includ-ing JUN and FAM21, most sites coinciding with occupancy byRNA polymerase II (RNAPol2) and histone marks of activeenhancers/promoters. IGF1R was inducibly recruited to chro-matin, directly binding DNA and interacting with RNAPol2 to

upregulate expression of JUN and FAM21, shown to mediatetumor cell survival and IGF-induced migration. IGF1 alsoenriched RNAPol2 on promoters containing IGF1R–bindingsites. These functions were inhibited by IGF1/II–neutralizingantibody xentuzumab (BI 836845), or by blocking receptorinternalization. We detected IGF1R on JUN and FAM21 pro-moters in fresh prostate cancers that contained abundantnuclear IGF1R, with evidence of correlation between nuclearIGF1R content and JUN expression in malignant prostaticepithelium. Taken together, these data reveal previously unrec-ognized molecular mechanisms through which IGFs promotetumorigenesis, with implications for therapeutic evaluation ofanti-IGF drugs.

Significance: These findings reveal a noncanonical nuclear rolefor IGF1R in tumorigenesis, with implications for therapeuticevaluation of IGF inhibitory drugs. Cancer Res; 78(13); 3497–509.�2018 AACR.

IntroductionGrowing evidence implicates the insulin-like growth factor

(IGF) axis in promoting risk of cancer and propensity for metas-

tasis, therapy resistance and cancer-related death (1–4). IGFssignal via cell surface type I IGF receptors (IGF1R), activatingmultiple effectors including AKT and ERKs (5). Until recently, theability of IGF1R to regulate transcription was thought to beexplained solely by these canonical signaling networks down-stream of cell surface IGF1Rs (5). This view was challenged whenour group and Larsson and colleagues showed that followingclathrin-dependent endocytosis, activated internalized IGF-1Rstraffic to the nucleus (6, 7). Furthermore, Larsson's group foundthat IGF1R import through the nuclear pore complex requiresIGF1R-b SUMOylation and activities of p150 Glued and impor-tin-b/RanBP2 (6, 8).We previously reported that nuclear IGF1R isa feature of preinvasive lesions and invasive cancers includingprostate, renal, and breast cancers, and identified associationbetween nuclear IGF1R and adverse prognosis in renal cancer(7). Subsequent data associate nuclear IGF1R with proliferation,tumorigenicity, resistance to EGFR inhibition, and clinicalresponse to therapeutic anti–IGF1R antibodies (7, 9–15), suggest-ing that IGF1R nuclear import requires strong IGF axis activationamounting to IGF dependence.

While nuclear IGF1R is known to interact with chromatin(6, 7), genomic-binding sites previously identified by chromatinimmunoprecipitation sequencing (ChIP-seq) in melanoma cells

1Department of Oncology, University of Oxford, Oxford, United Kingdom.2Computational Biology Research Group, University of Oxford, WeatherallInstitute of Molecular Medicine, Oxford, United Kingdom. 3Department ofCellular Pathology, Oxford University Hospitals NHS Foundation Trust, JohnRadcliffe Hospital, Oxford, United Kingdom. 4Nuffield Department of SurgicalSciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.5Oxford Cancer and Haematology Centre, Oxford University Hospitals NHSFoundation Trust, Churchill Hospital, Oxford, United Kingdom. 6BoehringerIngelheim RCV GmbH & Co KG, Vienna, Austria. 7Department of Urology,University Hospital Grosshadern, Ludwig-Maximilians-University, Marchioninis-trasse, Munich, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Valentine M. Macaulay, University of Oxford, Old RoadCampus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.Phone 44-1865-617337; Fax: 44-1865-617334; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-3498

�2018 American Association for Cancer Research.

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were predominantly intergenic (6), hence of uncertain signifi-cance. The aims here were to investigate whether nuclear IGF1R isrecruited to transcriptionally active regions of genomic DNA andprobe the significance of this phenomenon in clinical cancers. Wenow report that nuclear IGF1R associates with advanced tumorstage and is recruited selectively to regulatory regions of chroma-tin including JUN and FAM21A promoters. We identify JUN andFAM21A asmediators of cell survival and IGF-inducedmigration,properties that tumors require to attain advanced stage. Finally,we detect IGF1R on JUN and FAM21A promoters in tumors thatcontain nuclear IGF1R, and identify association between tumornuclear IGF1R content and JUN expression.

Materials and MethodsImmunohistochemistry

Formalin-fixed paraffin-embedded (FFPE) radical prostatecto-my (RP) sections were used for IHC using IGF1R antibody #9750(Cell Signaling Technology) as described (see SupplementaryMethods; refs. 16, 17). IGF1R was scored blinded by Uro-Pathol-ogist CV for intensity and percentage of tumor stained, generatingimmunoreactive scores (range, 0–12) formembrane, cytoplasmicand nuclear IGF1R, and also internalized (cytoplasmic/nuclear,0–24), and total (membrane/cytoplasmic/nuclear, 0–36) IGF1R.We utilized the same method and scoring system for JUNIHC on adjacent sections using antibody ab32137 (Abcam). Thestudy was approved by National Research Ethics ServiceCommittee Oxfordshire Committee C (study 07/H0606/120).All patients provided written informed consent to use of tissuein research.

Cell lines and reagentsDU145 prostate cancer (from Cancer Research UK Clare Hall

Laboratories) and SK-N-MC Ewing Sarcoma Family Tumor(ESFT) cells (from Professor Nicholas Athanasou, University ofOxford, United Kingdom) were cultured in RPMI 1640 mediumwith 10% fetal calf serum (FCS). Both were mycoplasma-freewhen testedwithMycoAlert (Lonza Rockland Inc.). Cultures wereused within 20 passages of authentication by STR genotyping(Eurofins Medigenomix Forensik GmbH). Xentuzumab (BI836845) was provided by Boehringer Ingelheim, bafilomycinA1 (BafA1) and long R3-IGF1 purchased from Sigma-Aldrich.

ChIP and ChIP-seqSerum-starved DU145 and SK-N-MC cultures (50 � 106 cells

per condition) were treated with 50 nmol/L IGF1 for 30 minutes,fixed, lysed, and subjected to ChIP using antibodies to IGF1R(#3027, Cell Signaling Technology), H3K4me1 (ab8895,Abcam), H3K4me3 (ab8580, Abcam), RNAPol2 (ab5095,Abcam), or IgG (Santa Cruz Biotechnology, negative control)and the ChIP Assay Kit (17-295, Millipore) according to themanufacturer's instructions (see Supplementary Methods). Inde-pendent replicate ChIP-DNAs underwent paired-end sequencing(HiSeq, Illumina). ChIP-Seq reads were mapped using Bowtie2(18) aligned to the human reference genome (hg19) from UCSC.Aligned reads were filtered against IgG DNA and analyzed withMACS2 for peak calling (19). These softwares reported peakswith assigned FDR values and P values that identify DNA regionswith statistically significant binding enrichment. ChIP-seq–iden-tified peaks were validated on triplicate independent samples byChIP-quantitative Polymerase Chain Reaction (qPCR).

Reverse transcription and qPCRRNAs were extracted and reverse transcribed using Pure Link

RNAMini RNA extraction kits (Ambion) and SuperScript III First-Strand Synthesis SuperMix (Invitrogen). ChIP DNAs and cDNAswere amplified using primers shown in Supplementary Table S1and Sybr Green PCRMix (Applied Biosystems) on a 7500 Fast RT-PCR System (Applied Biosystems).

Electrophoretic mobility shift assayChIP-seq datawere used to design 80 bpoligonucleotides, each

50 biotin end-labelled on the sense strand (Supplementary TableS1). After annealing (95�C for 5 minutes, cooling to 23�C over 2hours), biotinylated double-stranded (ds) oligonucleotides wereused in electrophoretic mobility shift assay (EMSA) with recom-binant human IGF1R residues 960-1397 (rhIGF-1R, ThermoFisher Scientific) using the EMSA assay kit (Active Motif), accord-ing to (20) and the manufacturer's protocol with minor mod-ifications. Each reaction used 100 pmol biotinylated oligonucle-otide probe with 0.2 mg rhIGF1R in the absence or presence of500-fold excess unlabeled probe.

Western blotting, immunoprecipitation, and immunofluores-cence were performed as previously with minor modifications(see Supplementary Methods; ref. 7).

JUN promoter reporterDU145 genomic DNA was used as a template to amplify

nucleotides –982 to þ394 of the JUN promoter (see Supplemen-tary Methods; ref. 21). The 1.4 kb PCR product was digested withXhoI and HindIII-HF (New England Biolabs), cloned into sim-ilarly digested pNLCol2 vector (Promega), and the sequenceconfirmed by DNA sequencing (Source Bioscience). DU145 cellswere transfected with pNLCol2-JUN or pNLCol2 empty vector(EV) using Lipofectamine 3000 (Invitrogen), selected with500 mg/mL hygromycin, and stable clones screened for promoteractivity in ONE-Glo EX Luciferase assays (Promega) on aPOLARstar Omega platereader (BMG Labtech). DU145 clonesincorporating EV or JUN promoter plasmid were serum starvedovernight, treated with 50 nmol/L IGF1 for 24 hours, andluciferase assays performed as above.

Assays for proliferation, cell survival, motility, and migrationwere performed as described in (2) and Supplementary Methods.

Statistical analysisT tests were used to analyze two groups, one-way or two-way

ANOVA for >2 groups, andWilcoxonmatched pairs signed ranktest for nonparametric data. We assessed the significance ofvariation in IGF1R with clinical parameters with c2, Mann–Whitney U tests and correlation analyses using Prism v6(GraphPad Software) and Stata package release 11.2 (StataCorporation). All tests were two sided and P value <0.05 wasconsidered significant.

ResultsNuclear IGF1R associates with advanced stage in clinicalprostate cancers

As a first approach to investigate the significance of IGF1Rsubcellular localization, we used IGF1R IHC to score IGF1R in themembrane, cytoplasm, and nucleus of 137 RPs from British menwith prostate cancer recruited to the Prostate Cancer Mechanismsof Progression and Treatment (ProMPT) study (Supplementary

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Table S2). IGF1R was detected in benign and malignant epithe-lium of all RPs, with a luminal-basal IGF1R gradient in benignepithelia that was lost in the cancers (Fig. 1A). Total IGF1R in thecancers was greater than in benign areas of the same RPs (Fig. 1B;Supplementary Fig. S1A), supporting our previous report of IGF1R

overexpression in primary prostate cancers (22). Malignantepithelium contained significantly more internalized (nuclear/cytoplasmic) IGF1R, while IGF1R was predominantly in theplasma membranes of benign glands (Fig. 1C; SupplementaryFig. S1B). This difference in subcellular localization is novel and

Figure 1.

Nuclear IGF1R is associated with advanced tumor stage. A, IGF1R IHC in radical prostatectomy. a, Benign epithelium showing membrane IGF1R, withcytoplasmic IGF1R in basal cells; b,Mixed Gleason 3 (gray arrow) and 4 (black arrow) cancer containing more IGF1R than benign epithelium, prominent cytoplasmicand nuclear IGF1R, and perineural invasion (white arrow). Scale bar, 20 mm. B, IGF1R IHC scored for total IGF1R (n ¼ 137 RPs). Graph, total IGF1R score (bars,mean � SEM, red) in benign and malignant epithelia. The cancers contained significantly more IGF1R than benign prostatic epithelium from the same RP(�� , P ¼ 0.001, Wilcoxon matched pairs signed rank test). C, IGF1R quantification in plasma membrane, cytoplasm, nucleus (n ¼ 137 RPs; �� , P < 0.001,Wilcoxon test). D, Stage pT3 prostate cancers contain more nuclear IGF1R than stage pT1-2 cancers (P ¼ 0.011).

Nuclear IGF1R Is Recruited to Regulatory Regions of DNA

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may reflect increased IGF1R activation in malignant versusbenign epithelium. Importantly, nuclear IGF1R associated withhigher pathological tumor stage (pT1–2 vs. 3, P ¼ 0.011; Fig.1D; Table 1). We also identified borderline association betweeninternalized (nuclear plus cytoplasmic) IGF1R and higher path-ological grade (primary Gleason grade 3 vs. 4–5, P¼ 0.057; Table1; Supplementary Fig. S1C).

Nuclear IGF1R undergoes IGF-induced recruitment totranscriptionally active regions of DNA.

Having identified association between nuclear IGF1R andadverse clinical factors in men with prostate cancer, we nextinvestigated nuclear IGF1R function by ChIP-seq in humanDU145 prostate cancer cells. We also performed ChIP-seqfor RNAPol2, and H3K4me1 and H3K4me3 that mark activeenhancers and promoters respectively (23). Of approximately 7to 14� 106 reads per sample,�85% were mapped to the humangenome (Supplementary Table S4). Peak calling identified16,239, 19,759, and 21,782 peaks of RNAPol2, H3K4me1, andH3K4me3 enrichment, respectively, consistent with findings inother cell lines, with a pattern of sharp peaks of RNAPol2 andH3K4me1 recruitment, and broader peaks of H3K4me3 (Supple-mentary Fig. S2A), reportedly associated with increased transcrip-tional consistency (23, 24). In contrast, we identified 62 regionswith a clear increase in IGF1R ChIP fragment depth comparedwith control (IgG) ChIP (Supplementary Fig. S2B–S2C). To testthe robustness of our data, we repeated IGF1R ChIP-seq in asecond model, SK-N-MC Ewing sarcoma cells, which like DU145showed nuclear IGF1R positivity and inhibitory response toIGF-neutralizing antibody xentuzumab (25) (SupplementaryFig. S2D–S2F). The genome of SK-N-MC cells contained 66IGF1R–binding peaks, of which 25 were shared with DU145(Supplementary Fig. S2C).

By comparison with peaks called in RNAPol2 andH3K4me1/3 ChIP-seq, we explored the genomic locations ofsites of IGF1R recruitment. Predictably, most RNAPol2 andH3K4me1/3 peaks were within 300 kb of the transcriptionstart site (TSS). Unexpectedly, given the intergenic location ofthe majority of IGF1R–binding sites reported by (6), ouranalysis showed that IGF1R peaks also clustered near a TSS(Fig. 2A). Supplementary Table S5 lists the coordinates ofIGF1R peaks, the distance from the nearest TSS and the identityof the nearest gene. Of the 62 unique regions of IGF1Rbinding, 59 (95%) were coincident with RNAPol2 peaks, 54(87%) with H3K4me1 peaks, and 31 (50%) with H3K4me3.We detected only two peaks in common with IGF1R peaksidentified by ChIP-seq in melanoma cells, on chromosome 8(Supplementary Fig. S2G; ref. 6).

We focused on IGF1R–binding sites within the JUN andFAM21A/C genes that coincided with RNAPol2 and H3K4me1peaks in bothDU145 and SK-N-MC cells (Fig. 2B; SupplementaryFig. S3A), suggesting conserved binding to active regulatoryregions. In EMSA, IGF1R bound directly to dsDNA probes repre-senting IGF1R binding regions of JUN and FAM21A promoters(lanes 2, 5; Fig. 2C). The specificity of this interaction is supportedby its abolition in reactions containing excess unlabeled probe(lanes 3, 6; Fig. 2C). We then performed ChIP-qPCR to validateChIP-seq–detected IGF1R binding, first confirming that IGF1activated IGF1R over 30 minutes (Supplementary Fig. S3B).IGF1R recruitment to JUN and FAM21A/C promoters wasenhanced by IGF1 and suppressed by xentuzumab (Fig. 2Dand E), supporting requirement for IGF1R activation. SimilarChIP-qPCR would be required to validate the additional IGF1Rpeaks we identified in ChIP-seq. Contrasting with data frombreast cancer and melanoma cells (26, 27), we did not detectIGF1R on CCND1 or IGF1R promoters (Supplementary Fig.S3C and S3D).

Nuclear IGF1R promotes RNAPol2 recruitment and expressionof protumorigenic genes.

Identification of nuclear IGF1R at the TSS of the JUN andFAM21A/C promoters (Fig. 3A) suggests regulatory function. Asan initial step to explore this hypothesis, we cloned the proximalJUN promoter, representing the peak of IGF1R recruitment(nucleotides –982 toþ394), into a luciferase reporter. In DU145cells stably transfected with JUN promoter reporter, we detectedluciferase activity significantly greater than that in empty-vectortransfectants, and in serum-starved cells, reporter activity wasenhanced by IGF1 (Fig. 3B). We noted that IGF1R–bindingregions of the JUN and FAM21 promoters contained GATA-2binding motifs, and the JUN promoter peak also contained aKU80-binding motif and AP-1 (FOS/JUN)-like site (Fig. 3A;Supplementary Fig. S4A). This prompted us to question whethernuclear IGF1R interactswith these transcriptional effectors. There-fore, after confirming IGF1R detection in DU145 nuclear extract(Fig. 3C), we performed reciprocal coimmunoprecipitation (co-IP) experiments, revealing evidence of interaction between IGF1Rand RNAPol2. This appeared to be IGF dependent when detectedby IGF1R IP but constitutive (i.e., present in serum-starved cells)in RNAPol2 IPs. We also detected ligand-independent interactionof IGF1R with KU80 and GATA-2 (Fig. 3D and E; SupplementaryFig. S4B). Noting the abundance of IGF1R in cytoplasmic extract(Fig. 3C), we also tested for interaction between RNAPol2 andcytoplasmic IGF-1R.However, RNAPol2was almost undetectable

Table 1. Nuclear IGF1R associates with advanced-stage prostate cancer

Internalized IGF1RIGF1R � 6 IGF1R > 6 P

StageStage pT1–2 40 27 0.293Stage pT3 35 34

GradeGleason grade 6 þ 7(3 þ 4) 59 39 0.057Gleason grade 7(4 þ 3) þ 8–9 16 22

PSA0–10 60 43 0.422

>10 15 15

Nuclear IGF1RIGF1R ¼ 0 IGF1R > 0 P

StageStage pT1–2 50 17 0.011Stage pT3 37 32

GradeGleason grade 6 þ 7(3 þ 4) 64 34 0.602Gleason grade 7(4 þ 3) þ 8–9 23 15

PSA0–10 67 36 0.612

>10 18 12

NOTE: IGF1R IHC was performed on 137 radical prostatectomies. Internalized(nuclear plus cytoplasmic) IGF1R showed borderline association with higherGleason grade tumors, and nuclear IGF1R was significantly associated withtumors of higher pathological stage (c2 test). There were no significant associa-tions between clinical parameters and total IGF1R or IGF1R in the plasmamembrane or cytoplasm (Supplementary Table S3).

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Figure 2.

IGF1R is recruited to regulatory regions of DNA. A, Distance from TSS for ChIP-seq-identified peaks. B, UCSC browser images. IGF1R–binding sites within JUNand FAM21A/C promoters in DU145 cells treated with or without IGF1 (dark/light gray, duplicate ChIPs), and for IgG, RNAPol2, and H3K4me1/3. H3K27Ac mark,often found near active regulatory elements, from ENCODE (https://genome.ucsc.edu/ENCODE/). C, EMSA. rhIGF1R retards mobility of dsDNA probescorresponding to IGF1R–binding peaks in promoters of JUN (lanes 1–3) and FAM21A (lanes 4–6). White arrow, mobility of free probes; black, biotinylatedprobes bound to rhIGF1R. Mobility shift abolished by excess unlabeled probes (lanes 3, 6), supporting specificity. No signal in absence of biotinylated probe (lane 7).D, Serum-starved DU145 cells treated with 50 nmol/L IGF1 for 5–30minutes, subjected to IGF1R ChIP-qPCR to amplify IGF1R peaks in JUN and FAM21A/C promoters.Graphs, mean � SEM fold enrichment over serum-starved controls. IGF1 increased IGF1R recruitment, peaking at 10 minutes (�� , P < 0.001). E, IGF1R ChIPperformed as in D on serum-starved cells treated with 50 nmol/L IGF1 for 10 minutes alone or with 1 hour 100 nmol/L xentuzumab pretreatment.Graphs, mean � SEM fold enrichment of IGF1R binding to promoters of JUN (left), FAM21A (center), FAM21C (right). � , P < 0.05; �� , P < 0.01.





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Figure 3.

Nuclear IGF1R interacts with RNAPol2. A, JUN and FAM21A/C promoters showing regions bound by IGF1R (dashed square bracket, coordinates of binding)that overlap TSS (arrow) and contain binding sites for GATA-2, KU80, and AP-1–like sites. B, Luciferase activity generated by stably integrated EVor JUN promoter reporter in DU145 cells (left); right, JUN promoter reporter in serum-starved DU145 cells treated with solvent or 50 nmol/L IGF1 for 24 hours (n¼ 3assays in each case; �� , P < 0.001). C, Serum-starved DU145 cells were treated with 50 nmol/L IGF1 for 30 minutes and cytoplasmic and nuclear extractswere analyzed by Western blot. D and E, Nuclear extracts were immunoprecipitated for IGF1R (D) and RNAPol2 (left), GATA2 (right; E). The same resultswere obtained in two independent experiments. F, IGF-treated DU145 cells were analyzed by RNAPol2 ChIP-qPCR to amplify IGF1R–binding regions of JUN andFAM21A/C promoters (mean � SEM of triplicate independent ChIPs). After 5 minutes, IGF1 enhanced RNAPol2 recruitment (�� , P < 0.01; �� , P < 0.001).

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in the cytoplasm, with no evidence of IGF1R co-IP (Supplemen-tary Fig. S4C).

Identification of IGF-induced interaction between nuclearIGF1R and RNAPol2 led us to speculate that IGF axis activationmight influence RNAPol2 recruitment to chromatin. We usedthree approaches to test the dependence of RNAPol2 recruitmenton nuclear IGF1R. Firstly, using RNAPol2 ChIP-qPCR, we foundthat IGF1 enhanced recruitment of RNAPol2 to JUN and FAM21A/C promoters (Fig. 3F). Secondly, we assessed RNAPol2 recruit-ment to the TSS of b2-microglobulin and FOS genes that lackIGF1R peaks. We detected RNAPol2 on these promoters butfound no enrichment of RNAPol2 binding upon IGF1 treatment(Supplementary Fig. S4D). Thirdly, to differentiate functions ofcell surface and nuclear IGF1Rs, we used BafA1, a vacuolar Hþ-ATPase inhibitor that blocks vesicular trafficking (28). Bothxentuzumab and BafA1 blocked IGF1R nuclear translocation;xentuzumab did this by inhibiting IGF1R activation, hence alsosuppressing downstream signaling, while BafA1 prevented IGF1Rinternalization without preventing ligand-induced activation ofcell surface IGF1Rs and their ability to signal via AKT and ERKs(Fig. 4A–C; Supplementary Fig. S4E). We found that IGF-inducedenhancement of RNAPol2 recruitment to the JUN and FAM21Apromoters was suppressed by both xentuzumab and BafA1 (Fig.4D). Furthermore, IGF1 upregulated expression of JUN andFAM21A; as with RNAPol2 recruitment, these effects were alsoinhibited by xentuzumab and BafA1, although only partially inthe case of IGF-induced JUN upregulation (Fig. 4E). While BafA1blocks internalization of many proteins (28), inhibition of IGF-induced recruitment and transcription does associate this effectwith IGF1R. We also detected IGF-induced RNAPol2 recruitmentto the FAM21C promoter, but this transcript was not upregulatedby IGF1 (Fig. 4D–E, right).

To assess the functional significance of JUN and FAM21Aupregulation, we tested effects of depleting these proteins (Fig.5A). Consistent with the known protumorigenic role of JUN (29),cell survival was reduced in JUN-depleted, although not FAM21A-depleted prostate cancer cells (Fig. 5B). Seeking a FAM21A-asso-ciated phenotype, we noted that FAM21 is a component of theWiskott-Aldrich syndrome protein and SCAR homolog (WASH)complex involved in endosomal trafficking (30). WASH-associ-ated proteins are reportedly required for actin polymerization andcell motility (31), leading us to speculate that FAM21A contri-butes to this process. Indeed, FAM21A depletion caused delay incell migration that was significant at 12 and 36 hours, while JUN-depleted cells showed a delay only at 12 hours (Fig. 5C and D;Supplementary Fig. S5A and S5B). To assess more specificallywhether JUN and FAM21A contribute to IGF-dependent migra-tion, we performed transwell assays in low serum (0.2% FCS),detecting enhancement of migration toward IGF1 (1.23 � 0.03fold,P<0.001) in control transfectants. This effectwas suppressedby both JUN and FAM21A depletion (Fig. 5E), supporting thehypothesis that these proteins contribute to promigratory effectsof IGF1. In controls, we observed greater enhancement of migra-tion using 10% FCS as stimulus (1.56 � 0.63 fold, P < 0.001),consistent with the presence in serum of promigratory factors inaddition to IGFs, and this effect was partially suppressed in JUN-depleted but not FAM21A-depleted cells (Supplementary Fig.S5C). To assess potentially confounding effects of proliferation,we also performed viability assays on parallel siRNA-transfectedcultures, finding no differences in proliferation over the 24-hourtime course of migration assays (Supplementary Fig. S5D).

Nuclear IGF1R is recruited to gene promoters in clinical cancersand associates with JUN expression

Having identified a transcriptional role for IGF1R in thenucleusof cultured prostate cancer cells, we used two approaches toinvestigate the significance of nuclear IGF1R in clinical cancers.First, we performed ChIP-qPCR on fresh-frozen primary prostatecancers and were able to detect IGF1R on JUN and FAM21Apromoters, with higher signal in tumors with abundant nuclearIGF1R (Fig. 6A), supporting the clinical relevance of IGF1R ChIP-seq findings in cultured cells. Finally, to further probe the rela-tionship between nuclear IGF1R and JUN expression, we per-formed IHC for JUN in adjacent FFPE sections of the radicalprostatectomies in whichwe had evaluated IGF1R expression andsubcellular localization (Fig. 1). After scoring JUN signal in themalignant epithelium, it was apparent that JUN showed signif-icant correlation with nuclear IGF1R (Fig. 6B and C; Supplemen-tary Fig. S6A). This correlation was not seen for total IGF1R(Supplementary Fig. S6B), supporting the importance of nuclearIGF1R in upregulating JUN. Taken together, these data highlightan important noncanonical nuclear role for IGF1R that promotesthe properties required to attain advanced tumor stage (Fig. 6D).

DiscussionTheprincipal findings of our study are that IGF1Rbinds directly

to DNA, interacts with key transcriptional regulators, contributesto RNAPol2 recruitment and gene expression, and associates withadvanced tumor stage. We identified IGF1R recruitment to regu-latory DNA sequences by performing parallel ChIP-seq for RNA-Pol2 and histone marks of active enhancers and promoters. Thisstrategy allowed us to locate regulatory regions of the genome andalso assess ChIP-seq efficiency. We compared RNAPol2 andH3K4me1/3 peak numbers with those reported in ref. 23, whichused ChIP-seq to study transcriptional regulators in LNCaP pros-tate cancer cells, obtaining 1.36–10.22 � 106 uniquely mappedreads, and reporting 7,028 binding sites for RNAPol2, 25,469 forH3K4me1, 24,921 for H3K4me3. Therefore, we identified moreRNAPol2-binding sites and similar numbers of H3K4me1/3 sites,supporting the ability of our ChIP-seq protocol to detect enrich-ment of regulatory proteins onDNA.We identified far fewer peaksof IGF1R binding, although two factors support the credibility ofthe identified peaks and their functional importance. Firstly, weidentified IGF1R–binding peaks in common between two cancercell lines, and we validated IGF1R binding by ChIP-qPCR. Sec-ondly, two previous studies had performed ChIP-seq using thesame IGF1R antibody, finding relatively few IGF1R–binding sitesin genomic DNA. Larsson's group was the first to use thisapproach, identifying 568 IGF1R–binding sites in melanomacells, of which 80% were intergenic and 3.4% (�20 sites) were�20 kb of a TSS (6). In immortalized corneal epithelial cells, Wuand colleagues identified nuclear IGF-1R:INSR hybrid receptors,reporting 88binding peaks for IGF1R and86 for INSR, assigned tonearest genes involved in proliferation, cell death, differentiation,cell adhesion, signal transduction, metabolism, and cell com-munication (32). Thus, there is support for the concept thatIGF1R binds to a limited subset of sites in the human genome.The location of these sites may be cell-type specific, possiblyrelated to differences in nuclear structure and chromatin orga-nization (33), given that the binding sites we identified appearto cluster selectively around the TSS, unlike the majority of sitesidentified in ref. 6.






Figure 4.

IGF axis blockade and inhibition of IGF1R internalization induce comparable suppression of IGF-induced RNAPol2 recruitment and gene expression.A–C, Serum-starved DU145 cells were incubated with 50 nmol/L IGF1 for 30 minutes alone or with 1 hour pretreatment with 100 nmol/L xentuzumab or 50 nmol/LBafA1. A, Representative IGF1R immunofluorescence images. Scale bar, 20 mm. B, Quantification of mean� SEM nuclear IGF1R as percentage of total cellular IGF1R(�� , P < 0.001). C,Western blot to assess IGF-induced activation of IGF1R, AKT, and ERKs. D and E, Serum-starved DU145 cells were treated with IGF1 alone or withxentuzumab or BafA1. D, ChIP-qPCR. IGF-induced RNAPol2 recruitment to JUN and FAM21A/C promoters was attenuated by xentuzumab and BafA1 (� , P < 0.05;�� , P < 0.01; �� , P < 0.001). E, JUN and FAM21A/C expression quantified by qRT-PCR, showing mean � SEM fold expression corrected for ACTB, relativeto serum-starved cells. IGF-induced JUN and FAM21A upregulation was inhibited by both xentuzumab and BafA1 (�� , P < 0.01; �� , P < 0.001). IGF1 did notupregulate FAM21C.

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In addition to clustering around a TSS, the majority of sites ofIGF1R recruitment we identified were coincident with peaks ofRNAPol2 and H3K4me1 enrichment and 50% coincided withH3K4me3 peaks. This identification of nuclear IGF1R–bindingsites at regulatory DNA regions is consistent with a model inwhich interaction of nuclear IGF1R with DNA regulates genetranscription, supporting previous reports identifying IGF1R onthe CCDN1 and IGF1R gene promoters (26, 27). The majordifference is that we identified specific promoters by ChIP-seq,while these previous reports were guided to theCCDN1 promoterby recognition that nuclear IRS-1 is also present at TCF/LEF sites ofthis promoter (27), and the IGF1R promoter by interest inregulation of IGF1R gene expression (26). We did not detectIGF1R on either promoter (Supplementary Fig. S3C and S3D),again suggesting cell-type–specific differences.

The concept that IGF1R binds directly to DNA is supported byour EMSA data using probes corresponding to ChIP-seq–identi-fied IGF1R–binding peaks. In these assays, recombinant IGF1Rprotein was the only component added to reactions in which

probe mobility was retarded (Fig. 2C). Such data have beenconsidered to provide evidence for direct protein:DNA interactionin EMSA characterizing DNA binding of other recombinant orhighly purified proteins (20, 34–36). Our co-IP data (Fig. 3D andE) indicate that within intact cells, nuclear IGF1R exists in proteincomplexes and may be recruited in this context to chromatin.Importantly, we report a hitherto-unrecognized interactionbetween nuclear IGF1R and RNAPol2. The functional implica-tions of this interaction are currently unclear, given the discrep-ancybetween the time course of the interaction, increasing over 30minutes (Fig. 3C), and more rapid (5 minutes) IGF-inducedRNAPol2 recruitment (Fig. 3F), which could suggest that thisresponse is independent of nuclear IGF1R. We considered prob-ing the contribution of nuclear IGF1R by manipulating its local-ization, by mutating a nuclear localization sequence (NLS) toblock trafficking via importins, or expressing SUMO-site mutantIGF1R (6). However, recent work detects SUMO-site mutantIGF1R in the nucleus, possibly via heterodimerization with INSR(37), and furthermore IGF1R lacks an identifiableNLS, andunlike

Figure 5.

Genes upregulated following nuclear IGF1R recruitment contribute to tumor cell survival and IGF-induced motility. A and B, DU145 cells were siRNAtransfected and the following daywere disaggregated and used for assessment of JUN and FAM21A expression by qPCR (n¼ 3 assays for JUN, n¼ 5 for FAM21A;A).B, Clonogenic survival assays showing representative plate and to right, graph of cell survival expressed as % survival of control transfectants (�� , P < 0.01;�� , P < 0.001). C and D, Control or FAM21A siRNA-transfected confluent monolayers were scratched and imaged. C, Representative images. Scale bar, 1 mm.D, Migration expressed as mean � SEM % defect remaining at 12 hours (left), 36 hours (right). �, P < 0.05; �� , P < 0.01; �� , <0.001. E, DU145 cells were transfectedwith siControl, siJUN_3, or siFAM21A_2, the following day seeded into upper wells of transwell plates in low-serum medium and migration toward 50 nmol/L IGF1quantified after 24 hours (� , P < 0.05; �� , <0.001 by 2-way ANOVA).






(8) we cannot detect IGF1R interaction with importin-b (7).Therefore, we adopted the strategy of comparing complete path-way blockade by xentuzumab with internalization inhibition

using BafA1. This approach generated evidence implicating nucle-ar IGF1R, by the suppression of IGF-induced RNAPol2 recruit-ment by BafA1 (Fig. 4D), which blocks receptor internalization

Figure 6.

Nuclear IGF1R is present onpromoters of clinical prostate cancers and correlateswith JUNexpression.A, Fresh-frozen prostate cancers underwent IGF1R or control (IgG)ChIP-qPCR for IGF1R–binding regions of JUN and FAM21A promoters. Top, relative IGF1R enrichment; center, representative IGF1R IHC from adjacent FFPEtumor (scale bar, 20mm); bottom,Gleasongrades and IGF1R scores inmembrane (M), cytoplasm(C), nucleus (N).B, IGF1R and JUN IHConadjacent RP sections, showingGleason 4 pattern gland (scale bar, 30 mm). C, Graph, total JUN immunoreactive scores in n¼ 80 RPs correlated with nuclear IGF1R (Spearman coefficient). D, NuclearIGF1R binds to DNA and interacts with transcriptional regulators, inducing expression of genes that promote tumor cell survival and migration.

Aleksic et al.





but not membrane signaling (Fig. 4A–C). Furthermore, IGF1 didnot influence RNAPol2 recruitment to promoters lacking IGF1R–binding sites (Supplementary Fig. S4C). The functional signifi-cance of IGF1R:RNAPol2 complex formation could be furtherexplored by identifying and disrupting the IGF1R domain(s)required for RNAPol2 interaction, using ChIP to test recruitmentof IGF1R and RNAPol2 to JUN and FAM21A promoters.

While nuclear IGF1R is detectable by immunohistochemistry,immunofluorescence, and subcellular fractionation, these tech-niques also detect abundant cytoplasmic IGF1R. It is unclear towhat extent nuclear IGF1R functions are related to/dependent oncytoplasmic IGF1R. Ligand-induced IGF1R activation promotesIGF1R internalization into the cytoplasm, but we were unable todetect interactionwith RNAPol2 in this subcellular compartment.However, internalized IGF1R is known to mediate sustainedsignaling to AKT, and/or reflects IGF1R that is en route to degra-dation, recycling to the plasma membrane or trafficking to thenucleus (6, 7, 38, 39).Given these considerations, it is plausible toconsider that plasmamembrane IGF1Rmight be inactive,withoutprotumorigenic function. In future, it will be interesting to assesstissue IGF expression, to determine whether nuclear IGF1R pos-itivity associates with, and is potentially a response to, highambient ligand levels.

It is increasingly recognized that multiple components of theIGF-insulin axis undergo nuclear translocation, including IGF andinsulin receptors, docking molecules and IGF-binding proteins(26, 40–43). Indeed, INSR reportedly undergoes insulin-stimulat-ed recruitment to the promoters of genes already known to beinsulin induced, contributing to glucose homeostasis (41). Similar-ly, IGF1 is known to promote tumor growth at least in part byupregulating JUN (44). FAM21 has not previously been linkedwith IGF signaling, but FAM21A is clearly upregulated here byIGF1, and IGF-induced JUN and FAM21A transcription is compa-rably inhibited by IGF-neutralizing antibody xentuzumab and bypreventing IGF1R internalization (Fig. 4E). The equivalence ofthese responses suggests that internalized/nuclear IGF1R contri-butes to IGF-induced transcription, although we acknowledge thatcanonical signaling, for example, via ERKs may also contribute tothis transcriptional effect. IGF1 does not upregulate FAM21C, sug-gesting either that IGF1R recruitment has no functional effect at thislocus, or is consistent with reports that transcription is initiated butnot completed in a large fraction of human genes (45).

Together with our earlier report of association with adverseoutcome in renal cancer (7), the finding here that nuclear IGF1Rassociates with advanced stage in prostate cancer supports a linkwith aggressive tumor behavior. While it is not possible to infer acausative association from these clinical findings, experimentaldata support functional relevance, linking nuclear IGF1R withincreased IGF-induced proliferation, gefitinib resistance, andenhanced tumorigenicity (9, 10, 13). These phenotypes could bemediated at least partly by FAM21, reported to promote chemo-resistance in pancreatic cancer (46), and JUN, which associateswith radioresistant prostate cancer in patients andmurinemodels(47, 48). In radiotherapy-treated prostate cancers, we recentlyreported that IGF1R upregulation associates with high Gleasongrade and risk of metastasis, and cytoplasmic and internalizedIGF1R with biochemical recurrence, although there were nospecific associations with nuclear IGF1R (49). However, nuclearIGF1R was recently found to interact with PCNA to influence theresponse to DNA damage (50). Finally, we identify JUN andFAM21 as mediators of IGF-induced migration (Fig. 5E).

Suppressed migration toward FCS in JUN-depleted but notFAM21A-depleted cells (Supplementary Fig. S5C) suggests thatJUNmediates migration induced by additional stimuli present inFCS, while FAM21A may more specifically mediate chemotacticresponse to IGF1.

Taken together, these data highlight an important noncanonicalnuclear role for IGF1R that associates with advanced tumor stageand reveal hitherto-unrecognized molecular pathways throughwhich IGFs promote tumor cell survival and motility. Given thereported association of nuclear IGF1R with clinical response toIGF1R inhibition (14, 15) and our demonstration that IGF-neu-tralizing antibody antagonizes nuclear IGF1R functions, thesefindings have implications for clinical evaluation of IGF-inhibitorydrugs.

Disclosure of Potential Conflicts of InterestT. Bogenrieder is clinical development leader in Boehringer Ingelheim. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: T. Aleksic, G. Rieunier, E. Osher, M. Sanderson,V.M. MacaulayDevelopment of methodology: T. Aleksic, G. Rieunier, E. Osher, J. Mills,R.J. Bryant, V.M. MacaulayAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Aleksic, X. Wu, R.J. Bryant, K. Hutchinson,F.C. HamdyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Aleksic, N.E. Gray, X. Wu, G. Rieunier, E. Osher,J. Mills, R.J. Bryant, C. Han, M. Sanderson, S. Taylor, V.M. MacaulayWriting, review, and/or revision of the manuscript: T. Aleksic, G. Rieunier,E. Osher, R.J. Bryant, U. Weyer-Czernilofsky, M. Sanderson, T. Bogenrieder,V.M. MacaulayAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Aleksic, N.E. Gray, A. Lambert, U. Weyer-Czernilofsky, T. Bogenrieder, S. TaylorStudy supervision: V.M. MacaulayOther (scoring of IHC and selection of tumor areas): C. VerrillOther (identified and provided samples of researchmaterial fromBiobankofprostate cancer material): A. Lambert, R. Kumar

AcknowledgmentsWe are very grateful to the prostate cancer patients who gave permission for

use of tumor tissue in research. We are also grateful for technical advice andsupport from SimonEngledow (High-ThroughputGenomicsGroup,WellcomeTrust Centre for Human Genetics Oxford), and Graham Brown (MicroscopyCore Facility, Department of Oncology, Oxford), and for comments on themanuscript from Eric O'Neill and Sovan Sarkar. This study was supportedby Prostate Cancer UK (G2011/20, G2012/25), Development Fund ofCancer Research UK Oxford Cancer Research Centre (CRUKDF 0715-VMTA),UCARE-Oxford (TA/VM-2016), National Institute for Health Research(NIHR) Research Capacity Funding (grant AC14/037), Breast Cancer Now(2014NovPR364), The Rosetrees Trust and John Black Charitable Foundation(M330-F1), and support to V.M. Macaulay from the NIHR Oxford BiomedicalResearch Centre. The ProMPT study is supported by the UK NIHR, CancerResearchUK and theMRC, and theCambridge andOxford Biomedical ResearchCentres. The funding source had no role in the design, conduct of the study,collection, management, analysis and interpretation or preparation, review, orapproval of the manuscript.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 10, 2017; revised March 28, 2018; accepted April 26,2018; published first May 7, 2018.






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2018;78:3497-3509. Published OnlineFirst May 7, 2018.Cancer Res Tamara Aleksic, Nicki Gray, Xiaoning Wu, et al. Associated with Advanced Tumor StagePromote RNA Polymerase II Recruitment and Gene Expression Nuclear IGF1R Interacts with Regulatory Regions of Chromatin to

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