asxl3 is a novel pluripotency factor in human respiratory...

Therapeutics, Targets, and Chemical Biology

ASXL3 Is a Novel Pluripotency Factor in HumanRespiratory Epithelial Cells and a PotentialTherapeutic Target in Small Cell Lung CancerVivek Shukla1, Mahadev Rao1, Hongen Zhang2, Jeanette Beers3, Darawalee Wangsa2,DannyWangsa2, Floryne O. Buishand2,Yonghong Wang2, Zhiya Yu4, Holly S. Stevenson2,Emily S. Reardon1, Kaitlin C. McLoughlin1, Andrew S. Kaufman1, Eden C. Payabyab1,Julie A. Hong1, Mary Zhang1, Sean Davis2, Daniel Edelman2, Guokai Chen3,Markku M. Miettinen4, Nicholas P. Restifo5, Thomas Ried2, Paul A. Meltzer2, andDavid S. Schrump1

Abstract

In this study, we generated induced pluripotent stem cells(iPSC) from normal human small airway epithelial cells(SAEC) to investigate epigenetic mechanisms of stemness andpluripotency in lung cancers. We documented key hallmarks ofreprogramming in lung iPSCs (Lu-iPSC) that coincided withmodulation of more than 15,000 genes relative to parentalSAECs. Of particular novelty, we identified the PRC2-associatedprotein, ASXL3, which was markedly upregulated in Lu-iPSCsand small cell lung cancer (SCLC) lines and clinical specimens.

ASXL3 overexpression correlated with increased genomic copynumber in SCLC lines. ASXL3 silencing inhibited proliferation,clonogenicity, and teratoma formation by Lu-iPSCs, anddiminished clonogenicity and malignant growth of SCLC cellsin vivo. Collectively, our studies validate the utility of theLu-iPSC model for elucidating epigenetic mechanisms contrib-uting to pulmonary carcinogenesis and highlight ASXL3 asa novel candidate target for SCLC therapy. Cancer Res; 77(22);6267–81. �2017 AACR.

IntroductionLung cancer is the leading cause of cancer-related mortality

worldwide and currently accounts for approximately 160,000deaths annually in the United States (1). Approximately 85% ofpulmonary carcinomas are non–small cell lung cancers (NSCLC)comprised primarily of adeno-, squamous cell, and large cell undif-ferentiated carcinomas with distinct histologic and molecular fea-tures (2). The remaining 15% are small cell lung cancers (SCLC),which exhibit varyingdegrees of neuroendocrinedifferentiation andare typicallywidelymetastatic at presentationwithahighpropensityfor recurrence despite initial, often dramatic, responses to chemo-therapy (3). Whereas CT screening, targeted therapies, and immunecheckpoint inhibitors have recently improved outcomes for some

NSCLC patients (1), there have been no advances in detection ortreatment of SCLC in over 30 years (3). As such, SCLC has beendesignated an NCI priority (http://www.lungcanceralliance.org/News/SCLC%20Congressional%20Response%206-30-14%20FINAL%20with%20appendices.pdf).

Although lung cancers may occasionally arise in never-smokers, cigarette smoking remains the predominant riskfactor for NSCLC (4); virtually all SCLCs arise in active orformer smokers with substantial tobacco exposure (3). Despiteextensive studies, the genetic and epigenetic mechanisms bywhich cigarette smoke mediates initiation and progression oflung cancers have not been fully elucidated (5). Previously, wehave demonstrated that cigarette smoke condensate mediatestime- and dose-dependent epigenomic alterations in normalrespiratory epithelial cells (NREC), including perturbations ofthe histone code, activation of oncomirs and cancer-germlinegenes, and epigenetic repression of tumor suppressor genesand miRNAs (6–8). These tobacco-induced alterations inNRECs mirror those observed in lung cancer cells and primarytumors (9). A central theme emerging from these studies isthat via complementary mechanisms, including DNA methyl-ation, polycomb repressive complexes (PRC), and noncodingRNAs, cigarette smoke induces stem-like phenotypes thatcoincide with progression to malignancy in NRECs, as wellas enhanced growth and metastatic potential of lung cancercells (6–8, 10–12).

Reprogramming of somatic cells into induced pluripotent stemcells (iPSC) by induction of a defined set of transcriptional factorshas opened a new era in regenerativemedicine and cancer biology(13–15). iPSCs exhibit numerous similarities to embryonic stem

1Thoracic Epigenetics Section, Thoracic and Gastrointestinal Oncology Branch,Center for Cancer Research, NCI, Rockville, Maryland. 2Genetics Branch, Centerfor Cancer Research, NCI, Rockville, Maryland. 3NHLBI iPSC Core, NIH, Bethesda,Maryland. 4Laboratory of Pathology, Center for Cancer Research, NCI, Rockville,Maryland. 5Surgery Branch, Center for Cancer Research, NCI, Rockville,Maryland.

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

Current address for G. Chen: University of Macau, China.

Corresponding Author: David S. Schrump, NCI, Building 10, Rm. 4-3942, 10Center Drive, Bethesda, MD 20892-1502. Phone: 301-496-2128; Fax: 301-451-6934; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0570

�2017 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 6267

on July 9, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 21, 2017; DOI: 10.1158/0008-5472.CAN-17-0570

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-0570&domain=pdf&date_stamp=2017-11-3

http://www.lungcanceralliance.org/News/SCLC%20Congressional%20Response%206-30-14%20FINAL%20with%20appendices.pdf



http://cancerres.aacrjournals.org/

cells (ESC), including morphology, gene expression profiles,in vitro proliferation, and in vivo teratoma formation (13, 16). Assuch, iPSCs have emerged as important models with which tostudy epigenetic mechanisms contributing to pluripotency innormal cells, as well as stemness, chemoresistance, andmetastaticphenotypes of cancer cells (17).

In an effort to further delineate epigenetic mechanisms con-tributing to the pathogenesis of lung cancers and possibly identifynovel targets for lung cancer therapy, we sought to develop aniPSC model that might reflect epigenetic transitions from normalto malignant respiratory epithelia. Herein, we describe repro-gramming of normal human respiratory epithelial cells to plur-ipotency and demonstrate that Additional Sex Combs Like-3(ASXL3) is a novel factor critical for maintenance of pluripotentrespiratory epithelial cells, and a potential therapeutic target inSCLC.

Materials and MethodsCell lines

All lung cancer lines were available in repositories at the NCI(Rockville, MD), or were purchased from ATCC, and cultured asrecommended. cdk-4/h-TERT immortalized human bronchialepithelial cells (HBEC) were a generous gift from John Minna(UT-Southwestern) andwere cultured as described previously (8).Cell lineswere tested formycoplasma regularly (tested the latest inJuly, 2017) using a kit from Sigma (cat. no. MP0025) and werevalidated by HLA typing relative to original stocks.

Primary cell cultureNormal human bronchial epithelial cells (NHBE) as well as

SAECs derived from a 57-year-old Hispanic female nonsmokerwere purchased from Lonza and cultured as recommended by thevendor. STEMCCAKit (Millipore, cat. no. SCR545)was purchasedfrom Millipore and used as instructed. Irradiated mouse embry-onic fibroblasts (MEF) were obtained from NHLBI core facility,and Matrigel plates (cat. no. 354230) were purchased from BDBiosciences. Normal foreskin fibroblasts (CCD-1079Sk, ATCCcat. no.CRL-2097) and inducedpluripotent cells (ND1.2)derivedfrom foreskin fibroblasts were obtained from the NHLBI corefacility and were grown in DMEM and stem cell medium, respec-tively. 8medium (Life Technologies; cat. no. A1517001) andRho-associated kinase (Rock) inhibitor (Y-27632; Tocris; cat. no.1254) were used to culture the stem cells.

Generation of iPSCs from SAECsThe STEMCCA vector (Supplementary Fig. S1A) and protocol

described by Beers and colleagues (18) were used to reprogramSAECs to pluripotency. Briefly, 2.5 � 105 SAECs were plated ineachwell of a 6-well plate.Once the cells were approximately 70%confluent, they were transduced with STEMCCA lentivirus usingpolybrene and left to incubate overnight. The transduced cellswere then maintained in reprogramming medium with mediumchanged on alternate days. Six days after transduction, the cellswere trypsinized and replated on irradiated MEFs (feeder cells).From this day forward, the cells were maintained in reprogram-ming medium supplemented with basic FGF. Medium was chan-ged on alternate days, and cell colonies grew in size. On day 25after initial transduction, granular stem like cells were detachedfrom the feeder layer using a P20 pipette and transferred toMatrigel-coated petri dishes for expansion and further analysis.

Flow cytometry and alkaline phosphatase stainingLu-iPSCs were trypsinized with TryPLE Express (Thermo Fisher

Scientific), and the reaction was stopped by adding medium. Thecells were centrifuged, and the pellets resuspended in 4% para-formaldehyde for 10 minutes at room temperature to fix them.The cells were washed with PBS and then permeabilized with0.2% Tween-20 in PBS for 10 minutes at room temperature.Flow cytometry analysis (FACS) of pluripotency markers wasperformed using anti-OCT4-Alexa fluor 488 (Millipore#FCMAB113A4), anti-SSEA4-FITC (BioLegend #330410), anti-NANOG-Alexa Fluor 488 (Millipore #FCABS352A4), and anti-Tra-1-60-FITC (Millipore #FCMAB115F). Nonimmune control(Millipore #MABC006F) was used at 0.5 mL per 50 mL reaction.All the FACS analyses were performed on a MACSQuant FlowCytometer. The iPSC colonies were stained with alkaline phos-phatase (BCIP/NBT Alkaline Phosphatase Substrate Kit IV, VectorLaboratories #SK-5400).

Immunofluorescence stainingExpression of pluripotent marker proteins was assessed by

immunofluorescence techniques using a Zeiss 780 confocalmicroscope, optimized for automated imaging. Briefly, cells werefixed in 4% paraformaldehyde and later permeabilized in PBSwith 0.2% Triton X-100. After washing, the cells were blocked in3% BSA. The cells were stained with primary antibodies (SSEA3,SSEA4, TRA-1-60, and TRA-1-81; Supplementary Table S1). Alexa488 (mouse, rabbit) and 555 (mouse, rabbit) were used assecondary antibodies at 1:1,000 dilutions. DAPI was used asinternal control for immunofluorescence.

TaqMan quantitative RT-PCRTotal RNA was isolated using TRIzol (Invitrogen). cDNA syn-

thesis was performed using iScript cDNA Synthesis (Bio-Rad#170-8891). Quantitative analysis of gene expression was carriedout in triplicates using an ABI PRISM7500 Sequence DetectionSystem and primers are listed in Supplementary Table S1. Copynumbers were calculated as described previously (10) usingappropriate controls for specific genes.

HLA typingHLA typing was performed in the NIH Clinical Center HLA

laboratory.

Spectral karyotypingSpectral karyotyping (SKY) was performed as described previ-

ously (19). Briefly, metaphases were prepared in culture byincubating for approximately 1 hour in 0.02 mg/mL colcemid(Invitrogen). Cells were incubated in hypotonic solution (0.075mol/L KCl) for 20 minutes before being fixed in methanoland acetic acid (3:1). SKY probes were generated in-house andconsisted of a specific combination of labeled chromosomepainting probes that were hybridized simultaneously onto meta-phase chromosomes. Protocols used for SKY can be accessedat https://ccr.cancer.gov/Genetics-Branch/thomas-ried?qt-staff_profile_tabs¼9#qt-staff_profile_tabs. SKY imaging was per-formed on a Leica DMRXE microscope equipped with a Xenonlamp and Spectracube (Applied Spectral Imaging). For each cellline, 15 to 20 metaphases were imaged and analyzed using theSkyView software. Each metaphase was scored for numericaland structural aberrations according to human chromosome

Shukla et al.

Cancer Res; 77(22) November 15, 2017 Cancer Research6268



https://ccr.cancer.gov/Genetics-Branch/thomas-ried?qt-staff_profile_tabs=9#qt-staff_profile_tabs




nomenclature rules from ISCN (International Standing Commit-tee on Human Cytogenetic Nomenclature 2009).

Array comparative genomic hybridizationIsolated DNA was labeled using the Genomic DNAULS Label-

ing Kit (Agilent) and subsequently hybridized on Agilent Sure-Print G3Human CGHMicroarrays 4� 180 K (Agilent) accordingto the manufacturer's protocol version 3.5. Briefly, 1 mg DNA ofeach cell line and 1 mg normal genetic reference DNA (Promega)were differentially labeled with ULS-Cy5 and ULS-Cy3 (bothAgilent), respectively. After hybridizing and washing accordingto the manufacturer's instructions, slides were scanned withmicroarray scanner G256BA (Agilent), and images were analyzedby Feature Extraction software version 10.7.1.1 (Agilent). Thearray comparative genomic hybridization (aCGH) data werevisualized and analyzed using Nexus Copy Number softwareversion 7.5 (BioDiscovery). The correlation between averageCGHcopy number and average gene expression was performed usingPearson correlation with aCGH log2 ratio probe mean values asthe x-axis versus ASXL3 expression log2 ratios as the y-axis. Thesignificance threshold was P < 0.05 (two-sided t test).

Teratoma assayHuman iPSCswere grown on BDMatrigel plates. The cells were

collected using EDTA method and resuspended in cold iPSCmedium containing Matrigel and 10 mmol/L ROCK inhibitor(Tocris). Approximately 3–4 � 106 cells from each iPSC colonywere injected intramuscularly in the hind leg quadriceps along itslong axis of SCID mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Stocknumber-00557, The Jackson Laboratory) as described previously(18). At the same time, 3 � 106 to 5 � 106 of parental SAEC cellswere injected intramuscularly into other SCID mice. Approxi-mately 15weeks postinjection of cells, teratomaswere excised andfixed in 10% buffered formalin. Teratomas were embedded inparaffin and sectioned. Hematoxylin and eosin (H&E) staining aswell as immunostaining was performed to identify three germlayers. a-1-fetoprotein (endoderm, A000829, Dako), a-smoothmuscle actin (SMA; mesoderm, Sigma #A5228), and NeuronalClass III b-tubulin (TUJ1; ectoderm; MMS-435P Covance) wereanalyzed.

In vitro Matrigel clonogenic assayClonogenic assay was performed as described previously (8)

with minor modifications. Briefly, 6-well plates were prepared byresuspending Matrigel (BD Biosciences #354230) in cell culturemedium without serum and antibiotics. The medium containingMatrigel was evenly distributed in the wells and kept at roomtemperature in the biosafety cabinet for 30 minutes. Single-cellsuspension was prepared and plated on theMatrigel plate. After 7days, colonies were stained with Crystal violet and colonies werecounted.

In vivo tumorigenicity assayTumor formation assay was performed as described previously

(10).

Western blot analysisCell pellets were lysed with RIPA buffer containing protease

inhibitors. Thirty micrograms of proteins was loaded on 4%to 14% SDS-PAGE and electroblotted on PVDF membranes.

Membraneswere blocked in 3%BSA/PBS, followedby incubationwith primary antibodies in 3% BSA/PBS overnight at 4�C. Mem-branes were probed using primary antibodies and horseradishperoxidase–conjugated anti-rabbit or mouse IgG (Millipore) sec-ondary antibodies as listed in Supplementary Table S1.

DNA methylation arrayDNA was isolated using the QIAamp DNA Mini Kit (Qiagen

#51304) and treated with bisulfite using the EpiTect Bisulfite Kit(Qiagen).DNAmethylation profileswere assessed using InfiniumHumanMethylation450 BeadChip techniques (Illumina) asdescribed previously (8). The raw data file "FinalReport.txt"generated from Illumina "GenomeStudio" (Illumina) was nor-malized with "Simple Scaling Normalization" (SSN) methodimplemented in the "lumi" R package following the color-biasadjustment. Two files were produced, one with b value forindividual target and another one with corresponding "M" valuesfor the beta values. The file with "M" values was used for statisticalanalysis with FDR <0.05 (adjusted on the basis of Benjamini–Hochberg procedure) as significant cutoff unless otherwise indi-cated, and also, the absolute beta value difference greater than 0.2from the same contrast comparisonwas applied. PartekGenomicsSuite (Partek Inc.), R packages of lumi, methlumi, and otherrelated R packages were used for data processing, analyzing, anddata presentation. Data have been deposited to: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE102726.

Pyrosequencing and MSPGenomicDNAwas isolated from SAECs, iPSCs, and fromother

control cell lines using QIAamp DNAMini Kit (Qiagen #51304).TheDNAwas treated with sodium bisulfite using EpiTect Bisulfitekit (Qiagen #59104). Pyrosequencing analysis of LINE-1, NBL2,D4Z4, NANOG, OCT4, and ASXL3 was performed as describedpreviously (8) using primers listed in Supplementary Table S1.Methylation-specific PCR (MSP) analysis of the ASXL3 promoterwas performed using primers listed in Supplementary Table S1.

RNA sequencingTotal RNA was isolated using the RNeasy Mini Kit (Qiagen

#74104), followed by removal of ribosomal RNA (rRNA) usingbiotinylated, target-specific oligos combined with Ribo-ZerorRNA removal beads. The RNA was fragmented into small pieces,and the cleaved RNA fragments were copied into first-strandcDNA using reverse transcriptase and random primers, followedby second-strand cDNA synthesis using DNA Polymerase I andRNaseH. The resulting double-strand cDNAwas used as the inputto a standard Illumina library prep with end-repair, adapterligation, and PCR amplification being performed to yield a librarythat would go to the sequencer. The HiSeq Real Time Analysissoftware (RTA 1.12) was used for processing image files, theIllumina CASAVA_v1.8.2 was used for demultiplex and convert-ing binary base calls and qualities to fastq format. The sequencingreads were trimmed adapters and low quality bases using Trim-momatic (version 0.3), the trimmed reads were aligned to humanhg19 reference genome (GRCh37/UCSC hg19), and Ensemblannotation version 70 using TopHat_v2.0.8 software. RNA-Seqreads mapping and feature count: bam files were generated fromfastq files by alignment short reads to hg19 genome (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/) with STAR version2.4.0a (20). RNA expressionwas summarized frombam files withfeatureCounts tools provided by subread software (21) at gene

ASXL3, Lu-iPSC, and SCLC

www.aacrjournals.org Cancer Res; 77(22) November 15, 2017 6269



https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102726



http://hgdownload.soe.ucsc.edu/goldenPath/hg19/

http://hgdownload.soe.ucsc.edu/goldenPath/hg19/


level defined by gene annotation (gencode.v19.annotation.gtf)from GENCODE Project (http://www.gencodegenes.org/releases/current.html). Differential gene expression analysis wasperformed with R limma package (22). RNA sequencing (RNA-Seq) feature counts of all samples were quantile normalized firstto log2-counts per million (logCPM) and differentially expressedgene list was selected with adjusted P value (FDR) from linear-model fit between different groups. All graphics were generatedwith R.Scatter plot: mean logCPM of different cell lines wereplotted with R ggplot2 package and coefficients between two celllines were calculated with fitting linear model in R stat package.Heatmaps of differentially expressed genes were generated with Rgplots package and scaled by gene. Coverage of a single gene: rawshort read counts covering a specific gene in different sample bamfiles were plotted with R Gviz package. Circos plot was generatedwith R RCircos package. Gene lists were selected from linearmodel fitting between Lu-iPSC cell lines and SAEC cell lines(11,629 genes, adjusted P value < 0.05) and heatmap was gen-erated with mean logCPM of each cell line. Data have beendeposited to http://www.ncbi.nlm.nih.gov/bioproject/398652.

Quantitative ChIP and ChIP-SeqCell lines were treatedwith paraformaldehyde and sonicated to

generate DNA fragments. Immune complexes were formed witheither nonspecific IgG or chromatin grade antibodies recognizingH3K4me3 and H3K27me3. DNA was eluted and purified fromcomplexes.Quantitative chromatin immunoprecipitation (ChIP)assay was performed as described previously (10) using primerslisted in Supplementary Table S1.

ChIP-Seq was performed as described previously (23). Briefly,purified DNA after chromatin immunoprecipitation was sent toFrederick National Laboratory for Cancer Research. For sequenc-ing, the fragments were blunt-end ligated to the Illumina adap-tors, amplified, and processed by using Hi-Seq 2000 sequencer(Illumina). Sequencing files in Bam format (bam file) wereprovided by Frederick National Laboratory for Cancer Research,and all bam files were mapped to hg19 human genome withsoftware bowtie2. For peak enrichment in each sample, originalbam files were directly used. For finding overlapping and differ-entially bound peaks, bam files from triplicates were merged firstwith samtools software. Peaks enrichment and annotations wereall processed with software HOMER, version 4.7.2, and hg19genome. R gviz package was used to visualize peaks aroundspecific genes. Data have been deposited to http://www.ncbi.nlm.nih.gov/bioproject/398652.

ResultsGeneration and characterization of Lu-iPSCs from SAECs

The overall method of reprogramming SAEC to pluripotency isdepicted in Supplementary Fig. S1A. Figure 1A (a–f) depictsparental SAECs and various stages of reprogramming followinglentiviral transductionof Yamanaka factors.No iPSC-like colonieswere observed in nontransduced SAECs after 30 days of culture inreprogramming medium (Fig. 1A; g). Many lung-iPSC (Lu-iPSC)clones were generated, of which Lu-iPSC6 and Lu-iPSC7 wererandomly selected for further expansion and analysis.

Characterization of Lu-iPSCsA variety of methods were used to characterize Lu-iPSCs.

Initially, histochemical techniques were used to examine alkaline

phosphatase (AP) expression, which is typically high in iPSCs(24). Lu-iPSCs had positive staining for AP within 10 minutes ofexposure of cells to AP substrate, whereas parental SAECs showedno staining at 24 hours (Supplementary Fig. S1B). Immunoflu-orescence experiments demonstrated increased surface expressionof stage-specific embryonic antigen (SSEA)-3 and SSEA-4, as wellas TRA-1-60 and TRA-1-81 in Lu-iPSCs relative to parental SAECs(Fig. 1B; Supplementary Fig. S1C); these antigens are known to beexpressed at high levels in undifferentiated human ESCs, iPSCs,embryonal carcinoma, and embryonic germ cells (13, 24). Similarfindings were observed in ND1.2 (control iPSCs established fromdermal fibroblasts; Supplementary Fig. S1D). qRT-PCR as well asimmunoblot experiments confirmed upregulation of pluripo-tency-related genes in Lu-iPSCs as well as ND1.2 relative to SAECs(Fig. 1C and D). Upregulation of the stemness genes coincidedwith repression of numerous differentiation-associated genes,including KRT5A, KRT6A, MT2A, CXCL1, HOXA1, and HOXB2in Lu-iPSCs (Supplementary Fig. S2).

Additional experiments were performed to ascertain if thephenotypic changes observed in Lu-iPSCs were due to persistentexpression of the reprogramming genes. qRT-PCR analysis dem-onstrated expression of the transduced genes in SAEC 6 daysfollowing lentiviral transduction; however, all four transgeneswere silenced in Lu-iPSC6 and Lu-iPSC7 (Fig. 1E).

The initial stages of reprogramming of SAECs required the useof irradiated MEF feeder layers. These feeder cells did not prolif-erate and remained adherent to the culture dish while the Lu-iPSCs were harvested. Despite the low likelihood of hybridomaformation or simultaneous outgrowth of human and murinecells, additional experiments were performed to confirm that theLu-iPSCs were pure derivatives of SAECs, and ascertain if the Lu-iPSCs exhibited any genomic aberrations. PCR-based HLA typing(assays performed in the Department of Transfusion Medicine,Clinical Center, NIH, Bethesda, MD) demonstrated identicalresults for Lu-iPSC clones 6 and 7 and parenteral SAECs (Sup-plementary Fig. S3A), indicating no contamination with murineDNA. SKY experiments (Supplementary Fig. S3B) revealed nochromosomal abnormalities in Lu-iPSCs relative to parentalSAECs. In contrast, numerous chromosomal aberrations wereobserved in A549 andH1299 lung cancer cells. Collectively, theseexperiments confirmed that the Lu-iPSCs were derived only fromSAECs and that the reprogramming had not induced majorchromosomal alterations.

Lu-iPSCs form teratomas in SCID miceAdditional experiments were performed to ascertain whether

the reprogrammed respiratory epithelial cells formed teratomas inimmunodeficient SCID mice, the "gold standard" for pluripo-tency (13, 25). Briefly, Lu-iPSC clones 6 and 7 or parental SAECswere implantedwithMatrigel bilaterally into the superficial layersof the gastrocnemiusmuscles (5mice/group). Allmice implantedwith Lu-iPSCs had tumors evident approximately 12 weeks afterinoculation, which were collected for analysis approximately3 weeks later. In contrast, no tumors were detected in miceinoculated with parental SAECs following 20 weeks of observa-tion (results summarized in Fig. 2A; top). Histologic examinationof Lu-iPSC–derived tumors demonstrated immature teratomas,containing neural elements (ectoderm), cartilage and skeletalmuscle (mesoderm), and kidney and intestinal tissue(endoderm; Fig. 2A; bottom, and Supplementary Fig. S4A). Sub-sequent immunofluorescence experiments demonstrated

Shukla et al.




http://www.gencodegenes.org/releases/current.html

http://www.gencodegenes.org/releases/current.html

http://www.ncbi.nlm.nih.gov/bioproject/398652




Figure 1.

Generation of Lu-iPSCs from SAECs. A, a, SAECs before reprogramming. b, SAECs 6 days after transduction with STEMCCA. c, Emergence of Lu-iPSCcolonies after 10 to 15 days of reprogramming. d, Lu-iPSCs in ES cell medium after 20 days of reprogramming. e, Lu-iPSC colonies before dissociation after25 days of reprogramming. f, Established Lu-iPSCs. g, Nontransduced SAECs cultured for 30 days in reprogramming medium. B, Immunofluorescence analysisof SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression in Lu-iPSC6 relative to SAECs. Scale bars, 100 mm. C, qRT-PCR analysis of stemness-associated genesin Lu-iPSC6, Lu-iPSC7, and SAECs. ND1.2 was used as a positive control. D, Immunoblot analysis of pluripotency-related proteins in Lu-iPSCs, SAECs, and ND1.2.b-Actin was used as a control. E, RT-PCR analysis of OCT4, SOX2, KLF4, and C-MYC transgenes in SAEC 6 days after transduction with STEMCCA and Lu-iPSCs.






Figure 2.

Characterization of Lu-iPSCs. A, Top left, summary of xenograft experiments. Tumors were observed following inoculation with Lu-iPSCs but not parentalSAECs; top right, representative photograph of teratoma formation by Lu-iPSC6; bottom, hematoxylin and eosin staining of teratomas derived from Lu-iPSC6demonstrating ectoderm (neural tissues), mesoderm (cartilage and skeletal tissues), and endoderm (kidney and intestinal tissues). �� , P < 0.0001. B,Immunofluorescence staining for different germ layermarkers in Lu-iPSC6–derived teratomas (bIII tubulin for ectoderm,a-SMA formesoderm, anda-fetoprotein forendoderm). Scale bars, 100 mm. C, Left, qRT-PCR analysis of NY-ESO-1, MAGE-A1, and MAGE-A3 expression in Lu-iPSCs and SAECs. Right, qChIP analysis ofH3K27me3 and H3K4me3 levels within the NY-ESO-1, MAGE-A1, and MAGE-A3 promoters. D and E, Pyrosequencing (left) and ChIP-seq analysis (right)demonstrating that NANOG (D) and OCT4 (E) promoters are hypomethylated with increased H3K4me3 levels in Lu-iPSCs. U, unmethylated; M, methylated.

Shukla et al.





expression of bIII tubulin (ectodermal origin), a-SMA (meso-dermal origin), and a-fetoprotein (endodermal origin) in all ofthe harvested teratomas (Fig. 2B; Supplementary Fig. S4B).

Epigenetic alterations in Lu-iPSCsPreviously, we have demonstrated genomic hypomethylation

as evidenced by DNA demethylation of LINE-1, NBL2, and D4Z4repetitive elements in association with upregulation of cancer-testis (cancer germline) genes in lung cancer cells, as well asimmortalized respiratory epithelial cells following cigarettesmoke exposure (8). As such, pyrosequencing and qRT-PCRexperiments were performed to examine DNA methylation ofLINE-1, NBL2, and D4Z4 elements, as well as expression status ofseveral cancer-germline genes typically derepressed in lung cancercells. Pyrosequencing did not demonstrate demethylation ofthese repetitive elements in Lu-iPSCs compared with SAECs,possibly due to the limited number of CpG sites interrogatedwith these assays (Supplementary Fig. S4C). qRT-PCR experi-ments demonstrated very modest increases in NY-ESO-1,MAGE-A1, and MAGE-A3 expression in Lu-iPSCs relative toparental SAECs (Fig. 2C; left). Consistent with these findings,quantitative ChIP (qChIP) experiments (Fig. 2C; right) demon-strated decreased levels of the repressive histonemarkH3K27me3without corresponding increases inH3K4me3 (histone activationmark) in the NY-ESO-1, MAGE-A1, and MAGE-A3 promoters,indicative of incomplete derepression of these genes followingreprogramming. In contrast, pyrosequencing and ChIP-seqexperiments demonstrated that theNANOG andOCT4promoterswere significantly hypomethylated, with decreased H3K27me3and increased H3K4me3 levels in Lu-iPSCs relative to primarySAECs (Fig. 2D and E). These findings were consistent with resultsof aforementioned qRT-PCR and immunoblot experiments.

To more comprehensively examine DNA methylation changesassociated with reprogramming of respiratory epithelial cells,microarray experiments were performed using Illumina Infinium450 arrays (InfiniumHumanMethylation 450 BeadChip), whichcan interrogate 480,000 CpG sites throughout the genome (99%of RefSeq genes and 96% of CpG islands). This analysis demon-strated significant differences in DNA methylation profiles in Lu-iPSCs compared with SAECs with a shift toward a hypermethy-lated genome in the reprogrammed cells (Fig. 3A). Additionalanalysis was performed to ascertain whether this hypermethyla-tion was related to location of CpG sites (i.e., gene body, pro-moter, CpG island vs. non-CpG island). CpG targets in nonpro-moter regions were hypermethylated in Lu-iPSCs relative toSAECs, with a significant percent appearing to be fullymethylated(Fig. 3B). Targets in promoter regions were also hypermethylatedin Lu-iPSCs relative to SAECs, with some sites appearing to behemimethylated, whereas others exhibited biallelic methylation(Fig. 3C). A similar phenomenon was observed for CpG sites inCpG islands (Fig. 3D). Finally, CpG sites in CpG islands withinpromoters were also hypermethylated in Lu-iPSCs comparedwithSAECs, with many of these sites being hemimethylated (Fig. 3E).

To ascertain whether the hypermethylated phenotype wasunique to Lu-iPSCs, unsupervised analysis was performed using20,769 CpG targets with variance greater than 0.01 in severaliPSCs and ESCs relative to normal somatic cells in the GSE31848database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE31848). This analysis revealed a shift to a hypermethy-lated phenotype in iPSCs and ESCs relative to normal somaticcells. A similar phenomenon was observed for the same 20,769

targets in Lu-iPSCs relative to SAECs (Fig. 3F). Subsequent exam-ination of non-CPG methylation (CHH) sites demonstrated asimilar shift to a more methylated phenotype in Lu-iPSCs relativeto parental SAECs (Fig. 3G), consistent with what was observedfollowing analysis of pluripotent cell line data in the publicdatabase.

Because PRC2 has been shown to be a critical mediator ofpluripotency in normal as well as cancer stem cells (26, 27),additional experiments were performed to examine expression ofcore components of this complex. qRT-PCR and immunoblotexperiments (Fig. 4A and B) demonstrated significant increases inEZH2, EED, and SUZ12 expression in Lu-iPSCs relative to SAECs.Consistent with these findings, qRT-PCR and immunoblot experi-ments (Fig. 4C and D) demonstrated downregulation of DKK1,CDKN1A, and CDKN2A, which are well established PRC2 targetsthat are known to be repressed in iPSCs as well as ESCs and arefrequently downregulated in lung cancer cells. DNA methylationarray and RNA-seq analysis demonstrated that upregulation ofPRC2 components coincided with marked alterations in DNAmethylation and expression of numerous PRC2 target genes in Lu-iPSCs (representative results are depicted in Fig. 4E; full DNAheatmap and corresponding list of PRC2 targets are provided inSupplementary Fig. S4D and S4E; Supplementary Table S2,respectively). DNA methylation tended to be associated withrepression of the respective PRC2 targets genes, although thiswas not observed in all cases (Fig. 4E).

ASXL3 is upregulated and contributes to pluripotency inLu-iPSCs

Additional RNA-Seq analysis of more than 33,000 factorsrevealed that 18,983 genes were significantly altered in Lu-iPSCclone 6, whereas 18,068 genes were altered in Lu-iPSC clone 7relative to parental SAECs (Fig. 5A). RNA-Seq data showed that15,453 genes were commonly altered in Lu-iPSC6 and Lu-iPSC7(significance adj. P < 0.05; Fig. 5A); 1,851 genes were commonlyaltered more than 4-fold in Lu-iPSCs relative to SAECs (signifi-cance adj. P < 0.05; Fig. 5B). Differences in gene expressionbetween the two Lu-iPSC clones, which were derived from thesame SAEC stock, may be related to sites of transgene integrationand plasticity of the reprogrammed cells.

CIRCOS and scatter plot analysis of whole-genome data dem-onstrated that ASXL3, a mammalian homolog of DrosophilaAdditional Sex Combs (Asx; refs. 28, 29), was highly upregulatedin Lu-iPSCs relative to SAECs (Fig. 5C). RNA-Seq and ChIP-seqdata pertaining to ASXL3 in Lu-iPSCs and SAECs are depictedin Fig. 5D and E. qRT-PCR and immunoblot experiments dem-onstrated overexpression of ASXL3 in Lu-iPSCs and their respec-tive teratomas relative to SAECs (Fig. 5F). DNA methylationarrays, as well as pyrosequencing and MSP experiments, whichinterrogated three regions of a potential CpG island within theASXL3 promoter revealed no significant differences in DNAmethylation in Lu-iPSCs compared with SAECs (SupplementaryFig. S5A); consistent with these findings, deoxyazacytidine expo-sure was insufficient to upregulate ASXL3 in SAECs or HBECs(Supplementary Fig. S5B).

Additional experiments were performed to examine whetherASXL3 contributes to pluripotency in Lu-iPSCs. Briefly, Lu-iPSC7cells were transduced with lentiviral shRNAs targeting ASXL3.qRT-PCR and immunoblot experiments demonstrated that twooffive lentiviral constructs mediated 50% to 70% reductions ofASXL3 expression (Fig. 5G). The phenotypic effects of ASXL3









Figure 3.

DNA methylation array analysis of Lu-iPSCs andSAECs. A, Unsupervised analysis of b-valuedistribution for targets with variance greater than 0.1as specified. DNA methylation profiling for targets onbody (B), on promoters (C), in islands (D), andcollectively in islands and promoters (E),demonstrating hypermethylation in Lu-iPSCs relativeto parental SAECs. DNA methylation changes in Lu-iPSCs are similar to those observed following analysisof a public database. F, Unsupervised analysis ofb-value distribution for CpG targets with variancegreater than 0.01 from downloaded dataset ofGSE31848 (left) and the corresponding b valuedistribution of the same targets in our dataset (right).G, b-value distributions of all 2994 CHH targetsdesigned on the H450K methylation array fromdownloaded dataset (GSE31848, left) and ourdataset (right).

Shukla et al.





Figure 4.

PRC2 genes are upregulated and tumor suppressors are downregulated in Lu-iPSCs relative to SAECs. A, qRT-PCR analysis demonstrating upregulationof EZH2, EED, and SUZ12 in Lu-iPSCs relative to SAECs. B, Immunoblot analysis of EZH2, EED, and SUZ12 in Lu-iPSCs and SAECs. C, qRT-PCR analysis of DKK1,CDKN1A (p21), and CDKN2A (p16) in Lu-iPSCs and SAECs. D, Immunoblot analysis of DKK1, CDKN1A, and CDKN2A expression in Lu-iPSCs and SAECs. E,Representative comparison of DNA methylation array and RNA-seq analysis of PRC2 targets in Lu-iPSCs compared with parental SAECs. � , P < 0.05.






Figure 5.

ASXL3 is upregulated in Lu-iPSCs. A, Venn diagram showing overlap of genes between Lu-iPSC6 and 7 relative to SAECs [RNA-seq after normalizing readcounts (log2/million)]. B, Upon more in-depth analysis, 1,851 genes were altered in Lu-iPSCs compared with SAECs. C, Circos plot showing overexpression ofASXL3 in Lu-iPSCs, which is represented by red line. D, Normalized RNA read counts for ASXL3 in Lu-iPSCs and SAECs. E, Histone modifications peak readcounts of for H3K4me3 (left) and H3K27me3 (right) within the ASXL3 promoter. F, qRT-PCR and immunoblot showing overexpression of ASXL3 in Lu-iPSCs(left) and their teratomas (right). � , P < 0.05. G, Validation of ASXL3-shRNA lentiviral particles showing reduction of ASXL3 expression at transcriptionaland protein levels. H, Knockdown of ASXL3 reduces growth and alters morphology of Lu-iPSC7. I, Effects of ASXL3 knockdown on soft agar colony formationby Lu-iPSC7. J, Results of tumorigenicity assays demonstrating that knockdown of ASXL3 decreases number and size of teratomas induced by Lu-iPSC7 in SCIDmice. � , P < 0.05.

Shukla et al.





knockdown in Lu-iPSCs were evident within 72 hours followinglentiviral transduction; relative to Lu-iPSCs transduced with con-trol lentivirus, ASXL3 knockdown cells grewmore slowly and lostpluripotent morphology (Fig. 5H). Knockdown of ASXL3 signif-icantly decreased soft agar colony formation by Lu-iPSCs (Fig. 5I).Furthermore, knockdown of ASXL3 decreased the number andsize of teratomas in immunodeficient mice (Fig. 5J). Notably,ASXL3 expression levels in teratomas fromASXL3 knockdown Lu-iPSCs were similar to control teratomas (Supplementary Fig.S5C), suggesting that ASXL3 expression is indispensable formaintenance of pluripotency in respiratory epithelial cells.

ASXL3 functions as an oncogene in lung cancer cellsExamination of the Oncomine (https://www.oncomine.

org), GENT (http://medicalgenome.kribb.re.kr/GENT/search/g570_gsymbol_ASXL3_20161224_2OQ275_tissue.html), andCancer Cell Line Encyclopedia (CCLE; http://software.broadinstitute.org/software/cprg/?q¼node/11) databases revealedthat ASXL3 is expressed in some human lung cancers. As such,qRT-PCR and immunoblot experiments were performed toexamine expression of ASXL3 in a panel of NSCLC and SCLCcell lines relative to normal or immortalized human bronchialepithelial cells. As shown in Fig. 6A, ASXL3 was not expressedin SAECs, short-term NHBEs, or SV-40 or cdk4/h-TERT–immortalized respiratory epithelial cells (BEASs and HBECs,respectively). Furthermore, ASXL3 expression was either absentor very low in NSCLC lines. In contrast, ASXL3 was expressedin virtually all of the SCLC lines examined, with some linesexhibiting very high ASXL3 mRNA as well as protein levels.Consistent with these findings, IHC analysis demonstratedsignificantly higher ASXL3 expression in SCLC relative toNSCLC or normal lung (Fig. 6B and C); ASXL3 immunoreac-tivity in SCLC was predominantly nuclear (Fig. 6B).

Additional experiments were performed to ascertain the poten-tial relevance ofASXL3 expression in lung cancer cells. Briefly,H69SCLC cells were transduced with either lentiviral shRNA targetingASXL3 or control lentiviral vector. qRT-PCR and immunoblotanalysis demonstrated that lentiviral shRNA mediated 40% to60% knockdown of ASXL3 in H69 cells (Fig. 6D). Knockdown ofASXL3 significantly decreased soft agar colony formation (Fig. 6E)and markedly diminished growth of H69 xenografts in athymicnude mice (Fig. 6F and G), suggesting that ASXL3 functions as anoncogene in SCLC.

Influence of genomic imbalances on ASXL3 expression in SCLClines

Previously, it has been demonstrated that gene expressionlevels are directly dependent on chromosomal aneuploidies incolorectal and other carcinomas (30–32). Whereas the strongestcorrelations have been found between genomic copy number andaverage chromosome-wide expression levels, the expression ofindividual genes has also correlated with genomic copy numbers(33). Because SCLC exhibit a variety of amplifications and chro-mosomal fusions that directly influence expression levels of genessuch as MYC family members and SOX2 (34, 35), CGH array(aCGH) experiments were performed to examine whether ASXL3mRNA expression levels in SCLC were associated with alterationsin gene copy number. As shown in Fig. 6H, a significant corre-lation was observed between genomic copy number and ASXL3expression levels in SCLC lines (R¼ 0.78420, P¼ 0.007). Similaranalysis revealed no increase in ASXL3 copy number in Lu-iPSC6

and Lu-iPSC7 relative to parental SAECs. Collectively, these datasuggest that genomic amplification contributes to ASXL3 over-expression in SCLC.

DiscussionElucidation of epigeneticmechanismsmediating aberrant gene

expression during initiation and progression of lung cancers mayhasten thedevelopment of novel therapies for these neoplasms. Inthe current study, we developed iPSCs from short-term normalhuman SAECs using well-established techniques (18). TheLu-iPSCs exhibited similarities to other pluripotent cell linesreported in the literature (13), including surface antigen and stemcell gene expression profiles, in vitro growth, and teratoma for-mation. Although previously reported to be activated in stem cells(36, 37), cancer-germline genes commonly upregulated in lungcancers remained transcriptionally repressed in Lu-iPSCs. Theseunexpected findings are consistent with a report by Loriot andcolleagues (38) demonstrating no upregulation of 18 cancer-germline genes in human ESCs, mesenchymal stem cells, oradipose derived stem cells. In addition, Lu-iPSCs exhibited ahypermethylated genome relative to parental SAEC. Whereasthese unanticipated findings could be indicative of incompletereprogramming, DNA hypermethylation profiles in Lu-iPSCswere also observed following analysis of databases pertaining topreviously published ESCs and iPSCs. Using a microarray plat-form identical to what was used in our studies, He and colleagues(39) observed significant increases in DNA methylation in twohuman ESCs, as well as two episomally derived iPSCs and fourvirally derived iPSCs relative to twohumanfibroblast lines used togenerate the iPSCs (�12% vs. 1.1% of all CpG sites, respectively).Our analysis, which appears more extensive than that performedby He and colleagues (39), demonstrated that DNA hypermethy-lation in Lu-iPSCs is not restricted to CpG islands in promoterregions but insteadoccurs throughout the genome. As yet,wehavenot directly compared methylation signatures in our Lu-iPSCswith other iPSCs reported in the literature. Additional studies arerequired to more comprehensively characterize epigenomicalterations during reprogramming of human respiratory epithe-lial cells and ascertain their relevance with respect to lung cancerbiology.

In addition to complex alterations in DNA methylation, ourexperiments demonstrated that more than 15,000 genes werecommonly regulated in Lu-iPSCs relative to parental SAECs.Notably,ASXL3, one of threemammalian homologs ofDrosophilaAsx (28, 29), was markedly upregulated in Lu-iPSCs. Analysis ofpreviously published RNA-seq data summarized by Liu andcolleagues (40) revealed almost no expression of ASXL3 in iPSCsgenerated from different fibroblasts, suggesting that ASXL3 acti-vation may be context dependent, and possibly reflect transcrip-tional memory of the somatic cells undergoing reprogramming(41, 42). Subsequent experiments demonstrated marked upregu-lation of ASXL3 in SCLC; ASXL3 did not appear to be expressed inNSCLC lines. Interestingly, SCLC is the highest expresser ofASXL3among a large panel of cancer lines of diverse histologies in theCCLE database. Knockdown of ASXL3 decreased teratoma for-mation by Lu-iPSCs and inhibited growth of SCLC in vitro and invivo. To the best of our knowledge, these experiments are the firstdescription of reprogramming of human airway epithelial cells topluripotency, and the first demonstration that ASXL3 is a poten-tial therapeutic target in SCLC.





https://www.oncomine.org

https://www.oncomine.org

http://medicalgenome.kribb.re.kr/GENT/search/g570_gsymbol_ASXL3_20161224_2OQ275_tissue.html

http://medicalgenome.kribb.re.kr/GENT/search/g570_gsymbol_ASXL3_20161224_2OQ275_tissue.html

http://software.broadinstitute.org/software/cprg/?q=node/11




Figure 6.

ASXL3 is an oncogene in SCLC. A, qRT-PCR and immunoblot analyses of ASXL3 expression in lung cancer lines and primary or immortalizedrespiratory epithelial cells. B and C, IHC analysis demonstrating higher nuclear expression of ASXL3 in SCLC relative to NSCLC or normal lung. D, Effects oflentiviral knockdown of ASXL3 on ASXL3 mRNA and protein levels in H69 cells. E, Effects of ASXL3 knockdown on soft agar clonogenicity of H69 cells. F and G,Effects of ASXL3 knockdown on growth (F) and weight (G) of H69 xenografts in athymic nude mice. Scale bars, 100 mm. H, Correlation of genomic copynumbers of ASXL3 by CGH array with ASXL3 mRNA expression levels by qRT-PCR. A significant correlation was observed for genomic copy number andASXL3 expression levels in SCLC lines, suggesting adirect effect of gene copyon relativemessage levels (R¼0.78420,P¼0.007). Upregulation ofASXL3 in Lu-iPSCswas not associated with an increase in genomic copy number. � , P < 0.05. NS, nonsignificant.

Shukla et al.





The translational relevance of these findings may be inferredfrom observations that ASXL proteins physically interact withEZH2 and SUZ12 (core components of PRC2) to target this"initiation" complex to polycomb response elements throughoutthe genome (28, 29). ASXL proteins also interact with BRCA-1Associated Protein-1 (BAP1), a ubiquitin hydrolase that mediatesdeubiquitination of H2AK119Ub, the repressive mark placed bythe "maintenance" PRC1 (43). BAP1 does not interact with PRC2proteins (29). As such, ASXL–BAP1 complexes may function tomitigate repressive activities of ASXL-PRC2. Particularly germaneto SCLC, ASXL3 interacts with LSD1 (KDM1A), a histonedemethylase that colocalizes with OCT4 and NANOG to regulateexpression of pluripotency genes, and which together withPRC2 is required for maintenance of bivalent chromatin withinpromoters of lineage-specific genes in undifferentiated stemcells (44).

Among a large panel of normal tissues in the CCLE database,ASXL3 expression is highest in testis and to a lesser extent, ovary;ASXL3 is expressed at much lower levels in brain, but does notappear to be expressed in normal lung ormany other organs. Thishighly restricted expression suggests that ASXL3 expression con-tributes to germ cell/stem cell homeostasis and neural develop-ment. With the exception of one report indicating that an evolu-tionarily conserved retrotransposonmodulates ASXL3 expressionduring neural development by encoding an alternatively splicedexon, leading to nonsense mediated decay of the transcript (45),there are no published data pertaining to mechanisms regulatingASXL3 expression in normal cells. Furthermore, whereas truncat-ing mutations of ASXL3 have been associated with autism andBainbridge–Ropers syndrome (46), as well as a low percentage ofmelanomas (29, 47), there are no published data regardingmechanisms and clinical implications of ASXL3 overexpressionin cancers. ASXL3 is located on 18q11, in a region not previouslyassociated with genomic abnormalities by cytogenetic analysis inSCLC (48). Our preliminary experiments have demonstrated noinduction of ASXL3 in lung cancer cells following exposure todecitabine, suggesting that ASXL3 upregulation in SCLC is notsimply a manifestation of genomic demethylation. Instead, ouraCGH experiments strongly suggest that ASXL3 overexpression inSCLC is attributable, at least in some cases, to genomic amplifi-cation. This mechanism does not account for induction of ASXL3in Lu-iPSCs. Our array, as well as pyrosequencing and MSPexperiments did not demonstrate any changes in DNA methyl-ation within the ASXL3 promoter in Lu-iPSCs relative to parentalSAECs; these findings as well as results of our ChIP-seq experi-ments suggest that ASXL3 is repressed in normal respiratoryepithelia by polycomb-mediated mechanisms (as evidenced byH3K27me3 levels within the ASXL3 promoter in SAEC relative toLu-iPSCs). These findings are consistent with our previouslypublished observations pertaining to repression of DKK1 in lungcancer cells by PRC independent of DNA hypermethylation (7).On the other hand, our data donot rule out the possibility of focalDNA demethylation coinciding with the observed histone altera-tions that facilitate upregulation of ASXL3 during reprogram-ming. Conceivably, upregulation of ASXL3 in Lu-iPSCs is due toactivation of one or more highly restricted stem cell transcription

factors during reprogramming of parental SAECs; such mechan-isms might also contribute to overexpression of ASXL3 in SCLC.Experiments are under way using DNase hypersensitivity-seq andChIP-seq techniques to comprehensively examinemechanisms ofASXL3 upregulation in Lu-iPSCs and SCLC.

In light of the fact that overexpression of EZH2 (the catalyticcore component of PRC2), as well as upregulation of LSD-1 havebeen implicated in SCLC (49, 50), our findings pertaining toASXL3 overexpression in these neoplasms suggest that perturba-tions of ASXL3, PRC2, and LSD1 expression/activities are centralthemes of small cell lung carcinogenesis; as such, epigenomiclandscapes and clinical phenotypes of these malignancies may beestablished by specific and potentially opposing mechanismsdysregulating expression, stoichiometry, and targeting of theseepigenetic modifiers. Experiments using inducible overexpres-sion/knockdown techniques are in progress to examine theseissues. Collectively, our findings support further analysis of themechanisms and clinical implications ofASXL3 overexpression inSCLC, and the evaluation of novel pharmacologic regimenstargeting ASXL3 for SCLC therapy.

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

Authors' ContributionsConception and design: V. Shukla, M. Rao, D.S. SchrumpDevelopment of methodology: V. Shukla, D. Wangsa, D. Wangsa,M.M. Miettinen, D.S. SchrumpAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): V. Shukla, M. Rao, J. Beers, D. Wangsa, D. Wangsa,F.O. Buishand, Z. Yu, H.S. Stevenson, E.S. Reardon, K.C. McLoughlin,A.S. Kaufman, E.C. Payabyab, J.A. Hong, M. Zhang, D. Edelman, G. Chen,M.M. Miettinen, P.A. MeltzerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Shukla,M. Rao,H. Zhang, F.O. Buishand, Y.Wang,Z. Yu, E.S. Reardon, S. Davis, M.M.Miettinen, N.P. Restifo, T. Ried, P.A. Meltzer,D.S. SchrumpWriting, review, and/or revision of the manuscript: V. Shukla, M. Rao,H. Zhang, D. Wangsa, E.S. Reardon, A.S. Kaufman, J.A. Hong, D. Edelman,M.M. Miettinen, N.P. Restifo, P.A. Meltzer, D.S. SchrumpAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.A. Hong, M. Zhang, D. Edelman, N.P. RestifoStudy supervision: D.S. Schrump

AcknowledgmentsThe authors wish to thank Drs. Beverly Teicher and Yves Pommier for

assistance with access to cell lines and the CCLE database, Madeline Kim forassistance with editing the manuscript, and Jan Pappas as well as Joanna Lamotfor administrative assistance.

Grant SupportThis work was supported by NCI Intramural grants ZIA BC 011122 (to D.S.

Schrump) and ZIA BC 011418 (to D.S. Schrump), and the Stephen J. SolarzMemorial Fund (to D.S. Schrump).

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 February 23, 2017; revised July 28, 2017; accepted September 7,2017; published OnlineFirst September 21, 2017.

References1. Rajan A, Schrump DS. Precision therapy for lung cancer: tyrosine kinase

inhibitors and beyond. Semin Thorac Cardiovasc Surg 2015;27:36–48.2. Zheng M. Classification and pathology of lung cancer. Surg Oncol Clin N

Am 2016;25:447–68.






3. KalemkerianGP, Schneider BJ. Advances in small cell lung cancer. HematolOncol Clin N Am 2017;31:143–56.

4. Schrump DS, Carter D, Kelsey CR, Marks LB, Giaccone G. In: DeVita V,Lawrence TS, Rosenberg SA eds. Cancer principles and practice of oncology.Philadelphia, PA: Lippincott Williams Wilkins; 2011. p. 799–847.

5. Dong N, Shi L, Wang DC, Chen C, Wang X. Role of epigenetics in lungcancer heterogeneity and clinical implicatio. Semin Cell Devel Biol2017;64:18–25.

6. Hussain M, Rao M, Humphries AE, Hong JA, Liu F, Yang M, et al. Tobaccosmoke induces polycomb-mediated repression of Dickkopf-1 in lungcancer cells. Cancer Res 2009;69:3570–8.

7. Xi S, YangM, Tao Y, XuH, Shan J, Inchauste S, et al. Cigarette smoke inducesC/EBP-beta-mediated activation of miR-31 in normal human respiratoryepithelia and lung cancer cells. PLoS One 2010;5:e13764.

8. Liu F, Killian JK, Yang M, Walker RL, Hong JA, Zhang M, et al. Epigenomicalterations and gene expression profiles in respiratory epithelia exposed tocigarette smoke condensate. Oncogene 2010;29:3650–64.

9. SchrumpDS. Targeting epigeneticmediators of gene expression in thoracicmalignancies. Biochim Biophys Acta 2012;1819:836–45.

10. Xi S, Xu H, Shan J, Tao Y, Hong JA, Inchauste S, et al. Cigarette smokemediates epigenetic repression of miR-487b during pulmonary carcino-genesis. J Clin Invest 2013;123:1241–61.

11. Zhang M, Mathur A, Zhang Y, Xi S, Atay S, Hong JA, et al. Mithramycinrepresses basal and cigarette smoke-induced expression of ABCG2 andinhibits stem cell signaling in lung and esophageal cancer cells. Cancer Res2012;72:4178–92.

12. Portal-Nunez S, Shankavaram UT, Rao M, Datrice N, Atay S, Aparicio M,et al. Aryl hydrocarbon receptor-induced adrenomedullin mediates ciga-rette smoke carcinogenicity in humans and mice. Cancer Res2012;72:5790–800.

13. Takahashi K, Yamanaka S. A decade of transcription factor-mediatedreprogramming to pluripotency. Nature Rev Mol Cell Biol 2016;17:183–93.

14. Xie N, Tang B. The Application of human iPSCs in neurological diseases:from bench to bedside. Stem Cells Int 2016;2016:6484713.

15. Lim KL, Teoh HK, Choong PF, Teh HX, Cheong SK, Kamarul T. Repro-gramming cancer cells: overview & current progress. Expert Opin Biol Ther2016;16:941–51.

16. Fan J, Robert C, Jang YY, Liu H, Sharkis S, Baylin SB, et al. Human inducedpluripotent cells resemble embryonic stem cells demonstrating enhancedlevels of DNA repair and efficacy of nonhomologous end-joining. MutatRes 2011;713:8–17.

17. Papapetrou EP. Patient-derived induced pluripotent stem cells in cancerresearch and precision oncology. Nat Med. 2016;22:1392–401.

18. Beers J, GulbransonDR,GeorgeN, Siniscalchi LI, Jones J, Thomson JA, et al.Passaging and colony expansion of human pluripotent stem cells byenzyme-free dissociation in chemically defined culture conditions. NatProtoc 2012;7:2029–40.

19. Schrock E, duManoir S, Veldman T, Schoell B, Wienberg J, Ferguson-SmithMA, et al.Multicolor spectral karyotyping of human chromosomes. Science1996;273:494–7.

20. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR:ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15–21.

21. Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purposeprogram for assigning sequence reads to genomic features. Bioinformatics2014;30:923–30.

22. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powersdifferential expression analyses for RNA-sequencing and microarray stud-ies. Nucleic Acids Res 2015;43:e47.

23. Roychoudhuri R, Hirahara K, Mousavi K, Clever D, Klebanoff CA, BonelliM, et al. BACH2 represses effector programs to stabilize T(reg)-mediatedimmune homeostasis. Nature 2013;498:506–10.

24. Brambrink T, Foreman R,WelsteadGG, Lengner CJ,WernigM, SuhH, et al.Sequential expression of pluripotency markers during direct reprogram-ming of mouse somatic cells. Cell Stem Cell 2008;2:151–9.

25. Smith KP, Luong MX, Stein GS. Pluripotency: toward a gold standard forhuman ES and iPS cells. J Cell Physiol 2009;220:21–9.

26. Collinson A, Collier AJ, Morgan NP, Sienerth AR, Chandra T, Andrews S,et al. Deletion of the polycomb-group protein EZH2 leads to compromisedself-renewal and differentiation defects in human embryonic stem cells.Cell Rep 2016;17:2700–14.

27. Kadoch C, Copeland RA, Keilhack H. PRC2 and SWI/SNF chromatinremodeling complexes in health and disease. Biochemistry 2016;55:1600–14.

28. Katoh M. Functional and cancer genomics of ASXL family members. Br JCancer 2013;109:299–306.

29. KatohM. Functional proteomics of the epigenetic regulators ASXL1, ASXL2and ASXL3: a convergence of proteomics and epigenetics for translationalmedicine. Expert Rev Proteomics 2015;12:317–28.

30. Grade M, Hormann P, Becker S, Hummon AB, Wangsa D, Varma S, et al.Gene expression profiling reveals a massive, aneuploidy-dependent tran-scriptional deregulation and distinct differences between lymph node-negative and lymph node-positive colon carcinomas. Cancer Res 2007;67:41–56.

31. Habermann JK, Paulsen U, Roblick UJ, Upender MB, McShane LM, KornEL, et al. Stage-specific alterations of the genome, transcriptome, andproteome during colorectal carcinogenesis. Genes Chromosomes Cancer2007;46:10–26.

32. Camps J, Grade M, Nguyen QT, Hormann P, Becker S, Hummon AB, et al.Chromosomal breakpoints in primary colon cancer cluster at sites ofstructural variants in the genome. Cancer Res 2008;68:1284–95.

33. Ried T, Hu Y, Difilippantonio MJ, Ghadimi BM, Grade M, Camps J. Theconsequences of chromosomal aneuploidy on the transcriptome of cancercells. Biochim Biophys Acta 2012;1819:784–93.

34. Hwang DH, SunH, Rodig SJ, Hornick JL, Sholl LM.Myc protein expressioncorrelates with MYC amplification in small-cell lung carcinoma. Histopa-thology 2015;67:81–9.

35. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS,et al. Comprehensive genomic analysis identifies SOX2 as afrequently amplified gene in small-cell lung cancer. Nat Genet 2012;44:1111–6.

36. Cronwright G, Le BK, Gotherstrom C, Darcy P, Ehnman M, Brodin B.Cancer/testis antigen expression in humanmesenchymal stem cells: down-regulation of SSX impairs cell migration and matrix metalloproteinase 2expression. Cancer Res 2005;65:2207–15.

37. Lifantseva N, Koltsova A, Krylova T, Yakovleva T, Poljanskaya G, GordeevaO. Expression patterns of cancer-testis antigens in human embryonic stemcells and their cell derivatives indicate lineage tracks. Stem Cells Int 2011;2011:795239.

38. Loriot A, Reister S, Parvizi GK, Lysy PA, De Smet C. DNA methylation-associated repression of cancer-germline genes in human embryonic andadult stem cells. Stem Cells 2009;27:822–4.

39. He WY, Kang XJ, Du HZ, Song B, Lu ZY, Huang YL, et al. Definingdifferentially methylated regions specific for the acquisition of pluripo-tency and maintenance in human pluripotent stem cells via microarray.PLoS One 2014;9:e108350.

40. Liu Y, Cheng D, Li Z, Gao X, Wang H. The gene expression profiles ofinduced pluripotent stem cells (iPSCs) generated by a non-integratingmethod are more similar to embryonic stem cells than those ofiPSCs generated by an integrating method. Genet Mol Biol 2012;35:693–700.

41. Kim K, Zhao R, Doi A, Ng K, Unternaehrer J, Cahan P, et al. Donor celltype can influence the epigenome and differentiation potential ofhuman induced pluripotent stem cells. Nat Biotechnol 2011;29:1117–9.

42. Ohi Y, Qin H, Hong C, Blouin L, Polo JM, Guo T, et al. Incomplete DNAmethylation underlies a transcriptional memory of somatic cells in humaniPS cells. Nat Cell Biol 2011;13:541–9.

43. Daou S, Hammond-Martel I, Mashtalir N, Barbour H, Gagnon J, Iannan-tuono NV, et al. The BAP1/ASXL2 histone H2A deubiquitinase complexregulates cell proliferation and is disrupted in cancer. J Biol Chem2015;290:28643–63.

44. Adamo A, Sese B, Boue S, Castano J, Paramonov I, Barrero MJ, et al. LSD1regulates the balance between self-renewal and differentiation in humanembryonic stem cells. Nat Cell Biol 2011;13:652–9.

45. Lowe CB, Bejerano G, Salama SR, Haussler D. Endangered species holdclues to human evolution. J Hered 2010;101:437–47.

46. Hori I, Miya F, Ohashi K, Negishi Y, Hattori A, AndoN, et al. Novel splicingmutation in the ASXL3 gene causing Bainbridge-Ropers syndrome. Am JMed Genet A 2016;170:1863–7.

47. Micol JB, Abdel-WahabO. The role of additional sex combs-like proteins incancer. Cold Spring Harb Perspect Med 2016;6:pii:a026526.

Shukla et al.





48. Ashman JN, Brigham J, Cowen ME, Bahia H, Greenman J, Lind M, et al.Chromosomal alterations in small cell lung cancer revealed by multi-colour fluorescence in situ hybridization. Int J Cancer 2002;102:230–6.

49. Poirier JT, Gardner EE, Connis N, Moreira AL, de Stanchina E, Hann CL,et al. DNA methylation in small cell lung cancer defines distinct disease

subtypes and correlates with high expression of EZH2. Oncogene 2015;34:5869–78.

50. Mohammad HP, Smitheman KN, Kamat CD, Soong D, Federowicz KE,Van Aller GS, et al. A DNA hypomethylation signature predictsantitumor activity of LSD1 inhibitors in SCLC. Cancer Cell 2015;28:57–69.






2017;77:6267-6281. Published OnlineFirst September 21, 2017.Cancer Res Vivek Shukla, Mahadev Rao, Hongen Zhang, et al. Lung CancerEpithelial Cells and a Potential Therapeutic Target in Small Cell ASXL3 Is a Novel Pluripotency Factor in Human Respiratory

Updated version

10.1158/0008-5472.CAN-17-0570doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2017/09/21/0008-5472.CAN-17-0570.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/77/22/6267.full#ref-list-1

This article cites 49 articles, 9 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/77/22/6267To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-0570

http://cancerres.aacrjournals.org/content/suppl/2017/09/21/0008-5472.CAN-17-0570.DC1

http://cancerres.aacrjournals.org/content/77/22/6267.full#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/77/22/6267


asxl3 is a novel pluripotency factor in human respiratory...

Documents