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Cancer Biology and Translational Studies

Targeting Notch1 and IKKa Enhanced NF-kBActivation in CD133þ Skin Cancer Stem CellsXin Xin Quan1, Nga Voong Hawk2,Weiping Chen3, Jamie Coupar1, Steven K. Lee1,David W. Petersen4, Paul S. Meltzer4, Andrew Montemarano5, Martin Braun6,Zhong Chen1, and Carter Van Waes1

Abstract

Cancer stem–like cells are hypothesized to be the majortumor-initiating cell population of human cutaneous squa-mous cell carcinoma (cSCC), but the landscape of molecularalterations underpinning their signaling and cellular phe-notypes as drug targets remains undefined. In this study, wedeveloped an experimental pipeline to isolate a highlyenriched CD133þCD31�CD45�CD61�CD24� (CD133þ)cell population from primary cSCC specimens by flowcytometry. The CD133þ cells show enhanced stem–likephenotypes, which were verified by spheroid and colonyformation in vitro and tumor generation in vivo. Geneexpression profiling of CD133þ/� cells was compared andvalidated, and differentially expressed gene signatures andtop pathways were identified. CD133þ cells expressed arepertoire of stemness and cancer-related genes, includingNOTCH and NOTCH1-mediated NF-kB pathway signaling.

Other cancer-related genes from WNT, growth factor recep-tors, PI3K/mTOR, STAT pathways, and chromatin modifierswere also identified. Pharmacologic and genetic targeting ofNOTCH1, IKKa, RELA, and RELB modulated NF-kB trans-activation, the CD133þ population, and cellular and stem-ness phenotypes. Immunofluorescent staining confirmedcolocalization of CD133þ and IKKa expression in SCCtumor specimens. Our functional, genetic, and pharmaco-logic studies uncovered a novel linkage between NOTCH1,IKKa, and NF-kB pathway activation in maintaining theCD133þ stem SCC phenotypes. Studies investigating mar-kers of activation and modulators of NOTCH, IKK/NF-kB,and other pathways regulating these cancer stem gene sig-natures could further accelerate the development of effectivetherapeutic strategies to treat cSCC recurrence and metasta-sis. Mol Cancer Ther; 17(9); 2034–48. �2018 AACR.

IntroductionHuman cutaneous squamous cell carcinoma (cSCC) has been

increasing over the past several decades, with more than 700,000cases in the United States annually (1, 2). Despite surgery,radiation, and chemotherapy, SCC cells can escape treatment andreform tumors and metastasis, increasing morbidity and mortal-ity. This gives prominence to the existence of a subpopulation ofSCC cells capable of tumor initiation and therapeutic resistance,and the importance of characterizing and targeting the molecularalterations mediating their maintenance for cancer preventionand treatment.

The cancer stem cell (CSC) hypothesis holds that tumors area hierarchical organ derived from cell subpopulation(s) capa-ble of self-renewal, called cancer stem–like cells or tumor-initiating cells (TIC; ref. 3). TICs exhibit stem–like andtumor-initiating properties, including increased self-renewal/colony formation, tumor-forming capacity, as well as alteredmigration, differentiation, and therapeutic sensitivity. Evidencefor the existence of TICs has been obtained in different types ofhuman solid tumors through identification of subpopulationsenriched for surface determinants or enzymatic markers, suchas CD133, CD44, CXCR4, and ALDH1 (3). We previouslydemonstrated that cell membrane protein CD133 (also calledprominin-1) is specifically expressed by cSCC cells enrichedfor a TIC phenotype, and not by CD45 and other non-epithelial subpopulations overlapping CD44 (4). CD133þ

cSCC cells exhibited long-term proliferative ability, self-renew-al, sphere formation, regeneration of differentiated SCC tumorcells, and enriched tumor-initiating capacity for xenografts inimmunodeficient mice. CD133þ has also been identified as abiologically and clinically relevant marker for subpopulationsof lung and head and neck SCC (HNSCC), where it is associatedwith tumor-initiating capacity, aggressive clinical features, andresistance to cytotoxic therapies (5, 6).

Recent studies of cSCC, HNSCC, and lung SCC tumors haveidentified significant genetic alterations involving componentsof several common and distinct pathways important in cellgrowth, deathor survival,migration, and epithelial/mesenchymaldifferentiation (7–10). These alterations are implicated in inac-tivation or activation of several canonical pathways, including

1Tumor Biology Section, Head and Neck Surgery Branch, National Institute onDeafness and Other Communication Disorders, NIH, Bethesda, Maryland.2Experimental Transplantation and Immunology Branch, NCI, NIH, Bethesda,Maryland. 3Microarray Core Facility, National Institute of Diabetes and Digestiveand Kidney Diseases, NIH, Bethesda, Maryland. 4Cancer Genetics Branch, NCI,NIH, Bethesda, Maryland. 5Skin Cancer Surgery Center, Bethesda, Maryland.6Braun Dermatology Associates, Washington, District of Columbia.

Z. Chen and C. VanWaes contributed equally to, and share senior authorship of,this article.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthors: Zhong Chen, NIDCD/NIH, Building 10, Room 7N240, 10Center Drive, Bethesda, MD 20892. Phone: 301-435-2073; Fax: 301-402-1140;E-mail: [email protected]; and Carter Van Waes, [email protected]

doi: 10.1158/1535-7163.MCT-17-0421

�2018 American Association for Cancer Research.

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NOTCH, WNT, HEDGEHOG, NF-kB, growth factor receptors,RAS-mitogen–activated protein kinase, PI3K–Akt–mTOR, andTP53, but their expression and role in TIC versus other popula-tions in established cSCC have not been dissected. Among theseare numbers of genes linked to stem cell maintenance or differ-entiation (11–14). NOTCH signaling is important in epithelialdifferentiation and as a suppressor of tumor development in asubset ofHNSCC, but is activated inothers (8, 15). IKK andNF-kBsignaling has been implicated in promoting tumor cell survival,inflammatory, and angiogenesis responses (9). However, howthese signaling pathways and function of corresponding molec-ular components contribute to the regulation of phenotype ofthe CSC/TIC subpopulation in cSCC tumors are not well under-stood. Identification of deregulated components of pathwayscritical to maintenance of the CD133þ CSC phenotype couldpotentially help identify targets for precision medicine for pre-vention and therapy.

In this study, we integrated the molecular profiling, sig-naling pathway, mechanistic, and pharmacological studies ofthe CD133þ subpopulation in primary human cSCC tumorsand cell line models. Through cell sorting with multiplepositive and negative selection markers by flow cytometry,we successfully isolated live CD133þ cells that form spheroidcolonies in vitro and tumors in vivo. This small distinctCD133þ population differentially expresses stem-like andcancer gene signatures linked to NOTCH1-mediated NF-kBmodulation, NF-kB, and WNT pathways. Characterization ofthe landscape of gene signatures in these CD133þ stem cellsrevealed activation of multiple pathways, which were linkedto NOTCH and NF-kB signaling networks and showed sen-sitivity to genetic and pharmacologic inhibitors of NOTCHand NF-kB. Our functional, genetic, and pharmacologic stud-ies uncovered a linkage between NOTCH1, IKKa, and NF-kBpathway activation in maintaining the CD133þ populationand its self-renewal ability in established primary cSCC andcell lines.

Materials and MethodsHuman tumor tissue samples

Deidentified primary human skin cSCC tissue samples wereobtained under an exemption from IRB approval by the Office ofHuman Subjects Research, National Institutes of Health, fromPotomac Ambulatory Surgery Center, Bethesda, MD, and BraunDermatology Associates, Washington, DC.

Preparation of single-cell suspension from cSCC specimensTumor tissues were gently chopped into small pieces

and incubated with collagenase III and dispase, thenfurther dissociated using GentleMACS Dissociator. Thedissociated tissues were spun down, incubated with tryp-sin and EDTA and filtered, spun down, and the cSCCsingle-cell suspension was then ready for staining andFACS sorting for sphere, tumorigenicity assays, and expres-sion profiling.

Flow cytometry analysis and FACS sortingcSCC single-cell suspensions were labeled with multiple anti-

bodies and sorted by BD FACSAria-II following standard protocolas detailed in Supplementary Methods. Analysis was performedwith BD LSR II and FlowJo7.6.5.

Sphere formation assayFifteen cSCC samples were collected and used for sphere

experiments after preparation of single-cell suspension. The cSCCsingle-cell suspension of unsorted or sorted CD133þ or CD133�

cells was seeded on NIH 3T3 fibroblast cells feeder layer andcultured for 14 to 21 days. Spheres �50 mm were counted underthe microscope.

In vivo mouse modelAnimal studies were performed under a protocol approved by

the Institutional Animal Care andUse Committee of theNationalCancer Institute. Six- to 8-week-old nude mice were obtainedfromNCI Frederick. To establish aniche, 106humanendotheliumand fibroblast cells with 120 mL Matrigel were injected into 0.5�0.5 cm GelFoam sponges implanted subcutaneously 2 weeksprior to tumor cell transplantation. Freshly obtained, dissociated,and sorted CD133þ, CD133�, and unsorted cells at doses of106, 105, 104, 103, or 102 cells were mixed with 106 humanendothelium and fibroblast cells in Matrigel, for injection intothe established GelFoamniche. Each dose level included 15mice,with 5 mice each that received freshly isolated primary sortedCD133þ, CD133�, or unsorted SCC tumor cells. To obtain106 rare CD133þ and matched CD133� cells, we sorted dissoci-ated suspensions from pools of 5, 5, 6, 6, and 6 (28 total) freshtumors for inoculation of 5 pairs of mice. CD133þ and CD133�

cells sorted from 5 tumors were sufficient to inoculate 5 pairedrecipients at 102, 103, 104, and 3 pairs at 105, and cells pooledfrom 3 and 4 additional tumors each (12 total) were required tosort 105 CD133þ and CD133� cells for 2 additional pairs. Fivetumors were freshly dissociated and 106, 105, 104, 103, and 102

cells from each were inoculated into paired recipients as unsortedcontrols for each dose level.

Affymetrix genechip human gene 1.0 ST arrayTotal RNAwas isolated after FACS sorting, and 15 sampleswere

used with RNA integrity numbers equal to or greater than 7.0,as the control for RNA quality. Single-stranded cDNA was gen-erated, fragmented, labeled, and hybridized with standard Affy-metrix protocol. Data were scanned with an Affymetrix GeneChipScanner 3000 and analyzed with Partek and GeneGo. The micro-array data have been submitted to NCBI with GEO submissionnumber (GSE84588).

Nanostring nCounter gene expression assayTotal RNAs (100 ng) from sorted CD133þ and CD133� cells

from seven cSCC samples were hybridized, purified, and immo-bilized in the Prep Station. The images weremeasured and furtheranalyzed with nSolver.

NF-kB family transcription factor DNA-binding assaysNuclear fraction of SCC13 cells was placed on a plate coated

with immobilized NF-kB consensus oligonucleotides. Activa-ted NF-kB is recognized by antibodies, followed by the HRP-conjugated second antibody, and quantified by spectrophotometry.

Drugs and treatmentThe following compounds were used for inhibition of

gamma-secretase-NOTCHand inhibitor-kappaB kinase in experi-ments: DAPT (https://pubchem.ncbi.nlm.nih.gov/compound/dapt), catalog D5942, Sigma Aldrich); RO4929097 (https://pubchem.ncbi.nlm.nih.gov/compound/49867930#section¼

Targeting NOTCH1 and IKKa/NF-kB in CD133þ Skin Cancer

www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 2035



https://pubchem.ncbi.nlm.nih.gov/compound/dapt

https://pubchem.ncbi.nlm.nih.gov/compound/dapt

https://pubchem.ncbi.nlm.nih.gov/compound/49867930&num;section=Top



Top), catalog No. ADV465749148, Sigma Aldrich); wedelo-lactone (https://pubchem.ncbi.nlm.nih.gov/compound/Wedelolactone#section¼Top, catalog #56639, Sigma Aldrich).

SCC13 cells were treated with two different NOTCH inhib-itors, DAPT or R04929097, in different doses and measured byFACS analysis. Wedelolactone (10 mmol/L) was used to treatSCC13 cells for 24 hours. Cells then harvested and stained withCD133 antibody for FACS analysis. Spheres or colonies werecounted after 5 days of drug treatment. Medium with drug waschanged every 3 days.

Luciferase reporter gene assaysSCC cells were cotransfected with NF-kB Luciferase and b-Gal

and then treated with wedelolactone and TNF-a at different timepoints. Relative luciferase activity was normalized to the cellswithout treatment.

Western blottingWhole cell lysates (15 mg) were used for electrophoresis, then

protein was transferred onto a Nitrocellulose membrane, andincubated with first and second antibodies following standardprotocol.

Statistical analysisMean and standard deviation (SD)were calculated for different

experiments, and the statistical significance was calculated usingStudent t test. P value <0.05 was considered a statistically signif-icant difference. To analyzemicroarray data, a two-way analysis ofvariance (ANOVA) for the paired samples was used.

Additional information about human SCC specimens, anti-bodies, and instrumentation settings is presented in the Supple-mentary Information.

ResultsClinical characteristics of human primary cutaneous squamouscell carcinomas (cSCC)

Eighty-two primary cSCC were collected fresh after Mohsmicrographic surgery from patients. The diagnosis and thetumor's grade was confirmed by a dermatopathologist, and theclinical characteristics are summarized in Supplementary TableS1. The majority of subjects were men (78%), and tumors werepredominantly located on sun-exposed skin from head (62.2%)and limbs (34.2%). Most tumors were well or moderately differ-entiated (85.4 þ 12.2 ¼ 97.6%) cSCC of less than 2 cm2 in size.The collected tumors were freshly delivered from the clinics andprocessed on the same day, to isolate stem cell subpopulationsfor in vitro and in vivo characterization of purity, stemness phe-notype, molecular profiling, and functional validation studies(Supplementary Table S1; Supplementary Fig. S1). The clinicalcharacteristics of tumor samples used in different experimentalstudies below are summarized Supplementary Table S2.

CD133þCD31�CD45�CD61�CD24� cells isolated fromcSCC exhibit enriched CSC/TIC features by in vitro andin vivo assays

New protocols to isolate, dissociate, and separate cSCC cellsfrom other populations in tumor specimens were optimized, assummarized in Supplementary Methods and SupplementaryFig. S1. Each cSCC sample wasmicrodissected to carefully removeexcess surrounding stroma, and regions with >90% malignant

epithelia were obtained. We developed protocols for cell sortingand improved purification of CD133þ and CD133� tumor cellsafter gating out differentiating keratinocytes (CD24), stromalendothelial cells (CD31), leukocytes (CD45), and fibroblasts(CD61). The percentage of cells expressing these CD markers incSCC varied among individual tumors by FACS analysis (Fig. 1A).CD133þ cells represent a rare population that resides withina relatively stable range from 0.14% to 1.7% (mean0.62%; Fig. 1A and B) and displayed a slight increase withtumor size that was not significant (R2 ¼ 0.037, P > 0.05).Sorted CD133þCD31�CD45�CD61� CD24� (hereafter calledCD133þ) cells have a purity as high as 95% as detected by FACSanalysis (Fig. 1C). More than 99% of FACS sorted CD133þ cellsare pan-Keratin positive (Fig. 1D), indicating that the CD133þ

subpopulation represents highly purified CD133þ keratinocytesfrom cSCC tissues. Thus, we isolated highly purified CD133þCSCand compared with those relatively pure CD133� keratinocyteswithout stemness characteristics using this improved sorting andselection protocol with the five different cellular markers.

To confirm whether the CD133þ population exhibits TICfeatures including self-renewal and tumorigenic ability estab-lished previously (3), we compared them with both CD133�

and unsorted cells for sphere formation efficiency in vitro andtumor formation in vivo. Unlike normal human keratinocytesgrowing as monolayer colonies, dissociated cSCC cells grew astethered spheres on NIH 3T3 feeder layers (Fig. 1E). Further, 1 �103 sorted CD133þ cells formed 55 � 8 spheres, while the samenumber of unsorted andCD133� cSCC cells grewonly 23� 3 and23 � 4 spheres, respectively (Fig. 1F). The sphere formation inCD133þ was significantly increased (P < 0.0001) compared withunsorted cSCC or CD133� cells, whereas there is no differencebetween the unsorted and the CD133� tumor cell groups. Thesphere numbers increased corresponding to increases inthe seeded cell numbers, indicating that cSCC cell aggregationalone is unlikely to account for sphere formation.

To confirm the enrichment of TICs within the purified CD133þ

population, we used the cSCC tumor xenograft assay in a mousemodel established previously (3), modified to replenish thenormal human fibroblasts and endothelial cells depleted bysorting. Varying concentrations of sorted CD133þ, CD133�, andunsorted cells were injected subcutaneously, to analyze tumorformation frequency by limiting dilution assay. A minimum of105 unsorted cSCC cells was required for initiating tumor in thismodel (Fig. 1G, blue bar). By contrast, as few as 100CD133þ cellsgenerated tumors in vivo (Fig. 1G, green bars). At the same time,the CD133� subpopulation did not form any visible tumors withas high as 106 cells (Fig. 1G, yellow bar). Thus, the estimatedtumor-initiating efficiency of the CD133þ subset is 1/245, whichis dramatically enriched �340-fold compared with unsortedcSCC cells (1/83,333).

Gene expression profiling of CD133þ cells displays geneenrichment for stem cell markers and NOTCH and NF-kBsignaling pathways

To investigate genes differentially expressed in the CD133þ

cSCC population, we compared the whole genome expressionprofile between sorted CD133þ and CD133� cells after gating forthe CD31�CD45�CD61�CD24� population from 15 fresh cSCCtumor samples by using Affymetrix Whole Genome RNA micro-array. The microarray data were normalized by the robust multi-chip average method and analyzed using the ANOVA paired test

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Figure 1.

Isolated CD133þCD61�CD31�CD24�CD45� cell subpopulation exhibits CSC features in human cutaneous SCC (hsSCC). A, Distribution of cell surface markersin human SCC cells isolated from tumor specimens. B, FACS detection of CD133þ CSC population in hsSCC. The red square gate was used to quantify the CD133þ

population after gating out the other four CD marker–positive cells. C, Purity detection after FACS sorting for CD133þ cells after sorting from CD133� cells,and gated CD61�CD31�CD24�CD45� cell subpopulation from hsSCC. D, Cytokeratin staining for CD133þ cell subpopulation. CD133þ cells (99%) werepan-cytokeratin positive as shown in the right square gate on the right. E, Human SCC CD133þ cells are enriched for spheroid colony formation in vitro.Normal human keratinocytes grew as adherent monolayer on the culture dishes with NIH 3T3 feeder layer (left top, 40�), whereas the dissociated cSCC cellsgrew as tethered spheres on the NIH 3T3 feeder layer (right top, 40�). The lower two panels show enlarged spheroid images under microscope (100�). F, CD133þ

cells exhibit statistically significantly greater spheroid formation numbers than CD133� (P < 0.05) or unsorted SCC cells (P < 0.05) when seeded the same numbercells (1 � 103). G, In vivo tumor formation assay in nude mice for comparison of transplant of different numbers of primary cSCC cells. Each group includes15 mice with 5 mice each that received the indicated number of primary SCC tumor cells from unsorted (green bars), sorted CD133� (yellow bars) and CD133þ

(blue bars) cells, as described in Materials and Methods.






in PartekGenomics Suite,withP value�0.05 and expression levelchange > 1.5-fold. There were 2,089 genes significantly differen-tially expressed in CD133þ when compared with CD133� tumorcells. We further compared this gene list with 247 stem-cell–related genes frommajor publications of human stem cell micro-array data (2, 11–14).We identified 80 overlapped genes between2,089 genes identified in this study versus 247 stem-cell–relatedgenes from major publications (Supplementary Fig. S2A). Aftersubtraction of the 80 genes, the Venn diagramof pink circle showstotal of 2,009 genes with significantly altered expression inCD133þ cells compared with CD133� cells. There are 167 stemgenes from the literature after subtraction of 80 genes (greencircle). Our data indicate that the sorted CD133þ cells areenriched for a stem cell gene expression signature. Moreover, aheat map of 80 signature genes shows that the genes are mainlydistributed in two clusters (Fig. 2A). Cluster I includes 32 upre-gulated stem-related genes in CD133þ cells, including severalNOTCH signaling pathway genes NOTCH1, JAG2, and MAML1;WNT pathway genes WNT3, FZD1, and CTNNB1; and SonicHedgehog pathway genesGLI2, SUFU, PTCH1, andGCNF (GermCell Nuclear Factor). Cluster II shows 48 downregulated genesin the CD133þ group, including NOTCH signaling-relatedgenes JAG1, NOTCH4, and downstream components RBPJ, andNUMB;Wnt signaling negative regulator gene GSK3B and hedge-hog signaling regulator HHIP. Unexpectedly, we also detectedincreased expression of hematopoietic cell determinant CD8A inseveral CD133þ samples. CD8ahas been associatedwithCD133þ

lymphomyeloid stem cells and cytotoxic T lymphocytes, whichcould potentially infiltrate cSCC and be detected among a subsetof samples (16–19).

GeneGo analysis of this 80-stem gene signature revealed theNOTCH pathway, NOTCH1-mediated NF-kB pathway, WNTpathway, Hedgehog, and NOTCH-mediated EMT signalingpathways are the top five most significantly ranked pathways inCD133þ cSCC cells (Supplementary Fig. S2B; SupplementaryTable S3). To further investigate what kinds of signaling pathwayscontributed in the CD133þ population, we analyzed pathwaysranking for all 2,089 significantly different expressed genes basedon the ANOVA paired test results (Supplementary Fig. S2C). Thebroader analysis showed that molecular mechanisms of cancer,regulation of EMT pathways, PI3K/AKT, mTOR, NF-kB, ERK/MAPK signaling, and RAR activation are within the top 20 path-ways in the CD133þ cell subset.

Because NOTCH, NOTCH1-mediated NF-kB, and WNT sig-naling appeared in top-ranked pathways in both stem cellsupervised and unsupervised analyses, we generated heat mapsfor genes included in the top-ranked signaling/stem gene sig-natures from the 2,089 differentially expressed genes in cSCC(Fig. 2B and C; Supplementary Fig. S2D). The heat map of theNOTCH pathway showed two distinct clusters (Fig. 2B): clusterI displayed relative increase in gene expression, of ligand JAG2,receptors NOTCH1, NOTCH2, NOTCH3, and downstreamtranscription factor MAML1; cluster II and III revealed down-regulated gene expression, including ligands JAG1, DLL1, recep-tors NOTCH4, glycosylation modification factor POFUT2, anddownstream transcription factor component RBPJ. CD133þ

cSCC did not exhibit significant differential expression ofcanonical NOTCH target genes HES and HEY1, consistent witha recent study that reported defective transcription of HES andHEY1 in subsets of human HNSCC with wild-type NOTCHgenes (15).

We next examined the profile of NF-kB–related pathwaygenes in cSCC based on our hypothesis that Notch and othermediators could contribute to regulation of the NF-kB signalingnetwork (20–23). The clustering data revealed increased geneexpression of several key signal and transcription factor sub-units for the NF-kB pathway in CD133þ cells (Fig. 2C). Theseinclude IL1R1, IRAK2/4, TRAF2/3, TRAF3IP2, CHUK (IKKal-pha), IKBKB, REL, NFKB2, RELB, as well as AKT1/2, implicatedin NF-kB activation by PI3K–Akt signaling. In addition, theheat map of the WNT pathway showed increase in WNT3,DVL2, and CTNNB1 and decrease in expression of the repres-sors such as AXIN2 and GSK3B, in CD133þ SCC cells (Sup-plementary Fig. S2D).

We hypothesized that the NOTCH, NF-kB, and WNT path-ways could form interactive networks in cSCC CD133þ CSCpopulations and used Ingenuity Pathway Analysis (IPA) toreveal the potential regulatory relationships annotated amongthese three pathways. The results showed experimental evi-dence for altered expression and annotated links between NF-kB with the NOTCH (Fig. 2D) or WNT pathway (Fig. 2E). Thenetwork map suggests the hypothesis that noncanonicalNOTCH1 signaling could contribute to the activation of RELand RELB in cSCC (Fig. 2D). The interaction of NF-kB with theWNT–b-catenin pathway is more indirectly through othernodes and pathways (Fig. 2E).

Independent validation of stem and pathway signaturegene expression using Nanostring in a different set of cSCCtissue samples

To independently validate the microarray results, we verifiedgene expression levels with sorted CD133þ and CD133� cellsfrom seven additional SCC tumors, using the Nanostring nCoun-ter Gene Expression assay, for 149 selected genes from the stem-and top-ranked cancer-related pathway gene signatures. Notchsignalingpathway componentsNOTCH1,NOTCH2, andMAML1geneswere confirmed tohave higher expression levels, while JAG1and EP300 showed decreased expression (Fig. 3A). The genesinvolved in the NF-kB pathway also showed upregulated expres-sion, including IL1R1, IRAK2/4, CHUK, and RELB, as well asFAS, TLR6, PRKACA, MMP8, and ICAM4. In the WNT pathway,increasedWNT3 and DVL2 expression and decreased GSK3B andDVL3 expression in CD133þ cells were independently verifiedusing Nanostring.

Furthermore, genes in several other signaling pathways dis-playing genomic or expression alterations and implicated inpathogenesis of SCC (8–10) were confirmed by Nanostringvalidation (Fig. 3B and C). In CD133þ cells, increased geneexpression was observed in EGFR/ERBB, IGF1R, AKT, and mTORand STAT family genes, as well as those implicated in FGF, MAPK,and TGF-b signaling pathways (Fig. 3B). Among other validatedgenes, cell cycle–related genes FBXW11 and PPP2R5C are over-expressed, while YAP1 and CTNNA1 related to cell growth anddifferentiation are decreased (Fig. 3C). Additionally, chromatinregulatory factors including DNMT1 and EZH2 genes weredecreased, whileHDAC6 is overexpressed. The differential expres-sion of mRNA for several of these genes in CD133þ versusCD133� cells is not always congruent with the predicted effectsof gene mutations (NOTCH1), copy-number alterations (YAP1),or overall expression (EZH2) observed in SCC subsets versusnonmalignant epithelia (7–9, 19, 20), underscoring the impor-tance of functional validation.

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Figure 2.

Characteristics of the transcriptional profile of the CD133þ CSC subpopulation and its stem gene expression signature. A, Hierarchical clustering of alteredexpression of 80 upregulated or downregulated stem gene signature in sorted CD133þ cells compared with CD133� cells after gating for theCD61�CD31�CD24�CD45� population from 15 tumor specimens using Affymetrix microarray (fold change >1.5, ANOVA analysis, P < 0.05). Section I:upregulated gene clustering and section II: downregulated gene clustering. Hierarchical cluster analysis of NOTCH (B) and NF-kB (C) pathways from 2,089significantly different expressedgenes in CD133þCSCs comparedwith CD133� tumor cells. IPA analysis of the network ofNF-kBandNOTCHpathway interaction (D),and NF-kB and Wnt pathway interaction (E). The solid line indicates direct interaction, and dotted line indicates indirect interaction. The pink color indicatesupregulated genes, and the blue color indicates downregulated genes. The different shapes indicate different function category of the molecules as indicated.






Figure 3.

Validation of differentially expressed genes in the CD133þ CSC population using Nanostring expression assay. Totally 149 significantly altered genes involvedin stem cell gene signature or signaling pathways were selected and validated using Nanostring nCounter gene expression assay. The consistently alteredgenes detected by both microarray and Nanostring are shown in NF-kB, NOTCH, WNT, and SHH pathways (A), EGFR/ERBB, FGF, MAPK, PI3K/AKT/MTOR,JAK/STAT, TP53/P73, and TGFb pathways (B), and cell cycle, chromatin regulation, and other function categories (C). Blue bar, microarray; pink bar,Nanostring nCounter assay.

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Inhibiting NOTCH reduces the CD133þ cell populationAs NOTCH signaling has been implicated in suppressing dif-

ferentiation or promoting growth in different SCC (7, 15, 21), wefirst examined the functional effects of two different g-secretaseinhibitors (DAPT and RO4929097) that inhibit NOTCH, uponthe CD133þ subset in the human cSCC cell line SCC13 (23). Weconfirmed that SCC13 exhibits a similar small percentage ofCD133þ cells varying between 0.2% and 6%, depending onculture conditions, and displays clonogenic and sphere-formingpotential, as observed by primary cSCC tumors. Three concentra-tions of DAPT (1, 5, 10 mmol/L) and RO4929097 (1, 3, 5 mmol/L)were selected based on ranges found to include IC50 for othertumor cell lines (24). FACS analysis shows that the CD133þ cellpopulation is significantly inhibited by 5 to 10 mmol/L DAPTand � 1 mmol/L RO4929097, supporting a functional role ofNOTCH1 in the maintenance of CD133þ cells (Fig. 4A). We nexttested if these two NOTCH inhibitors could affect CD133þ cellcolony formation of SCC13, as well as UM-SCC-46, an HNSCCline verified to express wtNOTCH1 and pathway genes by wholeexome and RNA sequencing (Hui Cheng, unpublished observa-tions). Colony formation of both SCC13 and UM-SCC46 cellswas significantly decreased byDAPTorRO4929097 (Fig. 4B; blue,red bars).

To independently examine the specificity of these findings forNOTCH1 or 2 differentially expressed in tumor CD133þ cellsin Fig. 3A, we examined the expression ofNOTCH1 and 2protein,and tested the effects of small interfering (si)RNAs silencingNOTCH1 and NOTCH2 on the CD133þ cell population in twocell lines. We confirmed that NOTCH1 protein of expected size isexpressed and knocked down efficiently by siRNA in both SCC13and UMSCC46 cells, while weak expression of NOTCH2 proteinis detected only with prolonged exposure and weakly inhibited(Fig. 4C, top). Consistent with the differential expression andknockdown of NOTCH proteins, the relative CD133þ cell num-ber decreased�40%of control with NOTCH1 knockdown alone,while no effect was attributable to the limited expression andknockdown ofNOTCH2 alone orwhen combinedwithNOTCH1siRNA (Fig. 4C, bottom). Together, the functional effects observedwith pharmacologic and genetic approaches suggest NOTCH1signaling contributes to the CD133þ population and colony-forming capability in a cSCC as well as an HNSCC cell lineexpressing wild-type NOTCH1.

Inhibiting NOTCH reduces NF-kB transcriptional and nuclearDNA-binding activity in cSCC cells

Because NOTCH1-mediated NF-kB activation is the secondtop-ranked GeneGo analysis signature for the CD133þ cell pop-ulation, and aberrant activation of NF-kB has been observed inSCC (8, 25), we explored the possibility that NOTCH signalingenhances NF-kB pathway signaling, by assay for NF-kB reportergene activity after NOTCH inhibition. As shown in Fig. 4D,inhibitors of g-secretase and Notch activation, DAPT andRO4929097, decrease TNF-a–induced NF-kB reporter activity inhuman SCC13 cells in a dose-dependent manner. To furtherconfirm the above results, siRNA was used to specifically knockdown NOTCH1 and/or NOTCH2, and nuclear DNA binding offive NF-kB nuclear transcription factor subunits was assayed andquantified. The results showed that after transfection withNOTCH1 siRNA, the DNA-binding activity of canonical NF-kBpathway subunit p65 (RelA), andnoncanonical pathway subunitsp52, and RELB, were significantly decreased to 64%, 80%, and

77% respectively (P < 0.001) compared with the control siRNAgroup (Fig. 4E, left). NOTCH2 siRNA showed similar but weakerinhibitory effects on nuclear DNA-binding activity of these threetranscription factor components, and combination of NOTCH2with NOTCH1 siRNA did not further inhibit NF-kB nuclearbinding activity (Fig. 4E, middle and right). These results provideevidence for NOTCH-MODULATED NF-kB pathway activity,which could potentially promote the CD133þ population incSCC cells.

IKK–NF-kB pathway signaling promotes the CD133þ cellpopulation in cSCC

As IKKa (CHUK) and other NF-kB pathway components weredifferentially overexpressed in CD133þ SCC tumor cells, wescreened SCC13 cells for the effects of wedelolactone, an inhibitorshown to inhibit IKKa and b kinases, and IKKa-dependent NF-kBactivation in SCC cells (25, 26). Wedelolactone inhibits TNF-ainducible NF-kB reporter activity assayed 24 and 48 hours aftertreatment in SCC13 cells (Fig. 5A). To determine the effects ofNF-kB inhibition on SCCCD133þ cells, we analyzed the CD133þ

cell population with or without wedelolactone treatment. TheFACS results showed lower concentrations of 1 to 5 mmol/Lwedelolactone reduce CD133þ cells, while 10 mmol/L wedelo-lactone significantly decreases the CD133þ population (Fig. 5B,P < 0.001). The effect of wedelolactone on the CD133þ popula-tion is not due to general cytotoxicity. The above results suggestthe NF-kB pathway contributes tomaintaining the cSCCCD133þ

CSC population in vitro.

Expression and activation of IKKa promote the CD133þ cellpopulation in cSCC

As both our microarray and Nanostring verification datashowed there is increased CHUK (IKKa) gene expression inCD133þ cells, and IKK inhibitor wedelolactone could decreasetheCD133þpopulation inhumanSCC13 cells,we independentlyinvestigated the specific role and function of IKKa for the main-tenance of CD133þ CSC in cSCC. To achieve this, we first testedthe effect of siRNA knockdown of endogenous IKKa on theCD133þ cells. Knockdown by siRNA efficiently inhibited cyto-plasmic and nuclear of IKKa (Fig. 5C) and reduced the CD133þ

population by�50% compared with the control group in SCC13cells (Fig. 5D). Similar effects were also observed in the HNSCCcell line UMSCC46. Next, we examined the effects of introducingIKKa wild-type (WT) or activated or inactivated genetic IKKamutants upon the CD133þ population. These include IKKa withserine phospho-acceptor sites substituted with constitutively acti-vating glutamate residues (EE); inactivating alanines (AA); orLysine44 substituted with alanine, which produces a catalyticallyinactive form (KA; ref. 25). Cells expressing IKKaWTorEEproteinsignificantly increased theCD133þpopulation 2.1- (P<0.01) and3.5-fold (P < 0.001) in SCC13 cells, whereas the inactive IKKaforms AA or KA exhibited no increase in the CD133þ subset (Fig.5E). Furthermore, we confirmed that the increase in the CD133þ

population by overexpression ofwild-type IKKa (WT) or its activeform (EE) can be significantly inhibited by IKKa siRNA knock-down (Fig. 5F).

To further examine the potential relationship between IKKaand CD133þ population in cSCC in vivo, we performed immu-nofluorescent (IF) staining of cSCC specimens. Figure 5G showscolocalization of IKKa and CD133þ immunostaining in cellsubpopulations in two different primary cSCC tissues. The tumor






Figure 4.

Blocking NOTCH inhibits the CD133þ cell population and reduces NF-kB reporter activity and p65, p52, and RELB nuclear activation. A, Relative CD133þ cellpopulation in human skin SCC13 cells was treated with two different NOTCH inhibitors, DAPT or R04929097, in different doses and measured by FACS analysis.B, The relative colony formation ratio in both SCC13 and UMSCC46 cells were examined after DAPT or R04929097 treatment. C, The specific NOTCH1 siRNAwere transfected either alone or in combination into SCC13 or UMSCC 46 cells for 24 hours, and proteins after knockdown were shown by Western blot (top).The CD133þ cell population of SCC13 was examined by FACS. Nontargeting siRNA control was used as the control, and its CDl33þ population was set as 100%(bottom). D, NF-kB reporter activity of SCCl3 cells was measured with NOTCH inhibitors DAPT or R04929097 treatment for 24, 48, and 72 hours. NF-kBactivitywas stimulatedwith TNF-a (10 ng/mL) and normalizedwithb-gal. E,SilencingNOTCH 1, NOTCH2, or combinedNOTCH1, and 2 siRNApartially decreases p65,p52, and RelB nuclear activation by binding assay. All results are shown asmeanþ SD; �, P < 0.05; �� , P <0.01; �� , P < 0.001. The experimentswere done in triplicate.

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specimens from 2 patients in Fig. 5G and Supplementary Fig. S3Cand S3D show merged staining of CD133 and IKKa, as well aswith DAPI, at the basal layer and leading edge of epithelial tumorcell nests shown in adjacent H&E sections. The costained popu-

lation of cells is seen along the interface of malignant epitheliawith intervening inflammatory (Supplementary Fig. S3C and 3D)or fibrovascular stroma (Fig. 5G). In addition, SupplementaryFig. S3A and 3B shows merged staining of CD133 and IKKa for

Figure 5.

ModulationofCD133þCSCs by IKK inhibitor andIKKa expression, which colocalized with CD133expression in cSCC tissues. A, IKK inhibitorwedelolactone inhibits NF-kB activity in SCC13cells. Cells were transfected with NF-kBluciferase reporter and b-Gal plasmids andtreatedwithwedelolactone and TNF-a alone orin combination for 24 and 48 hours. Red, noTNF-a; blue, TNF-a treatment. � , P < 0.05,compared with TNF-a-treated, non-wedelolactone group; #, P < 0.05, comparedwith non–TNF-a-treated group. B,Wedelolactone reduces CD133þ CSCpopulation in SCC13 cells. Cells were treatedwith wedelolactone in different doses for 24hours, stained with CD133-PE antibody, and theCD133þ population was analyzed with FACS. C,IKKa (CHUK) siRNA exhibited high knockdownefficiency in both cytoplasmic and nuclearcompartment of SCC13 cells. D, IKKa (CHUK)knockdown significantly decreased CD133þ

CSC population both in SCC13 and UMSCC46cells. E, Overexpression IKKa (CHUK) activeforms dramatically increases the CD133þ CSCpopulation in SCC13 cells. F, Decreased CD133þ

population caused by IKKa (CHUK) siRNAknockdown could be reversed by introducingWT IKKa or IKKa active forms (EE). Cells werecotransfected with different active forms ofIKKa (WT, EE, AA, and KA) plus IKKa siRNAwith control siRNA. CD133þ population wasanalyzed after 48 hours transfection. � ,P<0.05,compared withWT or EE control siRNA groups;#,P <0.05, comparedwith vector control siRNAgroup. G, The frozen sections of cSCC tissueswere coimmunostained with anti-IKKa andCD133 primary antibodies followed by Alexa-Fluor-488 (green) and Alexa-Fluor-555 (red)secondary antibodies. DAPI (blue) was used forvisualizing nuclei, and H&E staining is showingmorphology of tumor specimens.Magnification, 200�. All data represent meanand SD of one representative of threeindependent experiments, and each conductedin triplicate.






cells in two additional SCC tumors, over a wider region at 100�magnification. These data are consistent with the differentialcoexpression of these detected in CD133þ cells from multipletumors, and functional effects of IKKa genomic mutants onCD133þ cells in cell lines. Together, these observations supporta role for increased IKKa expression in the CD133þ population incSCC cells and tumors.

Knockdown of RELA and/or RELB inhibits the cSCC CD133þ

population and its colony/spheroid formation phenotypeIKKa can mediate downstream canonical and alternative

NF-kB pathway activation through RELA and RELB, which arefound to be overexpressed in CD133þ cells. Next, we examinedthe knockdown effects of RELA and/or RELB on the CD133þ

population in cSCC SCC13 and HNSCC UMSCC46 cells(Fig. 6A). The CD133þ cell populations were clearly reduced by54% with RELA siRNA, 43% with RELB siRNA, and 43% withcombined knockdown in SCC13 cells (Fig. 6B, blue bars). Similarresults were observed in UMSCC46 cells (Fig. 6B, green bars).Next, we quantified the colony/spheroid formation ability inSCC13 and UMSCC46 cells after knockdown of RELA and/orRELB. Clonogenic colonies from CD133þ cells were reduced to78%, 63%, and 55% by RELA or RELB siRNA knockdown alone,or in combination in SCC13 cells (Fig. 6C, pink bars). Spheroidformation in UMSCC46 cells also decreased to 76%, 69%, and68%, respectively (Fig. 6C, purple bars; Fig. 6D). Together, theabove data indicate both IKKa and NF-kB components RELA andRELB play an important role in maintaining the CD133þ popu-lation and its cancer stem–like features in human SCC.

DiscussionTo our knowledge, this is the first study to demonstrate the

CD133þ gene expression signature and its top-ranked signalingpathways in a purified cancer stem cell–like subset from primarycSCC tumors. There are four important findings from ourresearch. First, our optimized isolation and separation furtherenriched for high purity CD133þ cytokeratinþ cells, overcoming asignificant technical obstacle and enabling whole gene expressionprofiling detection and validation of stem and cancer genes in thesmall CD133þ population from clinical tumor samples. Second,our study provides a global overview of the gene signatures ofCD133þ CSC in the human cSCC. Using microarray, Nanostringvalidation and bioinformatics analyses, these data provide avaluable resource for hypothesis generation and testing for func-tional and clinical role of different signaling components andpathways in cSCC CD133þ CSC. Our stem gene expressionprofiling data identified �2,000 candidate genes differentiallyexpressed in CD133þ cSCC cells, including an 80-gene set ofknown stem cell signatures. Third, we demonstrated upregulatedexpression of genes of key signaling pathways and networks fromthe CSC population by analyzing the top-ranked pathwaysinvolved with GeneGO and IPA platform for the CD133þ tran-script signature. The CD133þ transcript signatures represent path-ways implicated in self-renewal and epithelial differentiation,such as NOTCH, WNT, SHH, and NF-kB, and cancer-relatedpathways PTEN, PI3K/AKT/mTOR, NGF, MAPK, EGFR/ERBB,FGF, JAK/STAT, TP53, and TGF-b. Genomics and experimentalstudies provide evidence supporting the biologic and clinicalsignificance of several of these pathways to SCC biology (7, 8).Complementing our observations, recent whole exome sequenc-

ing studies of cSCC and HNSCC provide evidence for gene copyalterations or mutations in components or coregulators of theNOTCH (NOTCH 1,2), WNT (AJUBA), cancer-related MAPK andPI3K growth (HRAS, RASA1, BRAF, PIK3CA), NF-kB prosurvivaland death pathways (FADD, BIRC2/3, CASP8, RIPK4). Lastly, ourfindings revealed a link between the NOTCH and NF-kB signalpathways and expression of several of their key components,NOTCH1, IKKa, RELA, and RELB, contributing to the mainte-nance of the CD133þ CSC population and its stem-like pheno-typic features (Fig. 6E).

We uncovered a NOTCH cSCC CSC gene signature andshowed that NOTCH plays a role in maintaining the CD133þ

cell population. Blocking NOTCH by either of two differentg-secretase inhibitors, or NOTCH1 by siRNA, reduced the per-centage of CD133þ SCC cells, and their capacity for clonogenicsphere formation, which are established surface and phenotypicmarkers of CSC/TIC (3). The NOTCH pathway plays a key rolein cell–cell communication via interaction ofmembrane-boundJAG ligands and NOTCH receptors (27). The NOTCH receptorfamily is composed of 4 family members (27). In HNSCC andcSCC, inactivating and missense mutations in NOTCH1 and2 have been observed in a subset of tumors, suggesting thatthe NOTCH may function as a tumor suppressor in thosecontexts (8, 9, 28, 29). Complexes of NOTCH receptor intra-cellular domains (NICD) with canonical NOTCH transcription-al cofactors can activate HES1/HEY1 repressors that triggerepithelial differentiation. Intriguingly, differential expressionof HES1/HEY1 was not detected among CD133þ cSCC over-expressing NOTCH1 and other signal components, and therewas decreased expression of key canonical transcription factorRBPJ. These findings are consistent with recent studies in humanHNSCC, where tumors expressing WT NOTCH1 included sub-sets without evidence of HES1/HEY1 activation, and whereNOTCH inhibitors reduced proliferation, through an unknownmechanism (15).

In this regard, we provide evidence that NOTCH can promotenoncanonical cross-activation of nuclear factor-kB (NF-kB/REL),a family of signal activated transcription factors that are oftenaberrantly activated in cSCC, HNSCC, and cervical SCC (25,30–33). The activation of canonical NF-kB1/RELA and alternateNF-kB2/RELB transcription factors is mediated by inhibitor-kappaB kinase (IKK)a/b/g or IKKa complexes, respectively(25). Our profiling of CD133þ cSCC cells unveiled increasedexpression of multiple components of the NOTCH and NF-kBsignaling pathways, including key components NOTCH1, IKKa(CHUK), RELA, and RELB (Fig. 6E).We showed that inhibition ofNOTCH by g-secretase or NOTCH1 by siRNA functionally par-tially inhibits NF-kB reporter and nuclear DNA binding of NF-kBp52, RELA and RELB along with the CD133þphenotype, and thatknocking down overexpressed IKKa, RELA, and RELB similarlyinhibited the CD133þ phenotype. The partial reduction of NF-kBactivity by inhibition of NOTCH signaling is expected, becauseNOTCH are not the only molecules that directly modulate NF-kBactivity in SCC (8, 25).

Interestingly, our study also establishes that IKKa is an impor-tant regulator for maintaining the CD133þ CSC population incSCC cells. Notably, NOTCH1 and IKKa have previously beenshown to physically and functionally interact in normal kerati-nocytes and cervical SCC cells (33, 34). NOTCH1 knockdownmodulated TNFa-induced NF-kB activity and promoter bindingin cervical SCC cells (33) similar to our findings for Notch

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Figure 6.

Knockdown NF-kB RelA and/or RelB inhibit CD133þ population and its colony/spheroid formation abilities, and the summary of NOTCH and NF-kB pathwayactivation.A,RELA and RELB siRNA exhibited high efficiency of siRNA knockdown.B,Knockdown of RELA and/or RELB inhibited CD133þ population. After 48 hoursof transfection, the CD133þ population was analyzed by FACS. C, Knockdown of RELA or RELB inhibited colony and spheroid formation abilities. After transfectionfor 24 hours, cells were seeded for colony formation of SCC13 cells (left) and spheroid formation of UMSCC46 cells (right). Results were shown as mean � SD,and data represent three independent experiments. � , P < 0.05; �� , P < 0.01 and �� , P < 0.001. D, The images of UMSCC46 spheroids with different siRNAknockdownafter 14 daysof culture (40�).E,Schematic illustrationof activatedNotch enhancesNF-kBpathwayactivation in cSCCCSCpopulation. TheCD133þ cSCCCSC gene signature is demonstrated with activated NOTCH signaling, which includes NOTCH1, NOTCH2, and JAG2 increased expressing. The activatedNOTCH pathway then enhances CHUK(IKKa), one of the key upstream components of NF-kB signaling pathway. The active CHUK(IKKa) forms complexeswith other IKKs and translocates to the nucleus, which further increased the NF-kB2 p52/RelB complexes activation and induces target gene expression.The increased NOTCH1 and NOTCH2 signal to the nucleus and regulate transcription factor MAML1 and RBPJ to control the downstream target gene expression.






g-secretase pharmacologic inhibition of TNF-induced NF-kBactivity in cSCC cells. In cervical SCC, NOTCH1 and IKKa alsopromoted chemotherapeutic resistance, a feature oftenattributed to CSC. Our data indicate that WT or activating phos-pho-acceptor site mutants for TNF-mediated IKKa activationincrease CD133þ cells, whereas inactivated IKKa mutants didnot, implicating the role of IKKa in the expansion of CD133þ

CSC. Further supporting this, IKKa colocalized inCD133þ cells inmalignant epithelia of cSCC tumors. Besides its role in NF-kBactivation, IKKamay also function as a cofactor to regulate otherkey transcription factor genes that control cell proliferation anddifferentiation, such as E2F1/BMI1 (35). Intriguingly, IKKa hasalso been reported to bind the promoter and reciprocally repressNotch canonical target gene HES1 (36), potentially helping toexplain the lack of HES1 induction among NOTCH-related geneswe detected, and the paradoxical role of NOTCH1 and IKKa inpromoting CSC expansion without triggering NOTCH-mediatedterminal differentiation.

Although the involvement of the IKK–NF-kB signaling path-way in promoting SCC has been demonstrated (25, 30–33), itsrole in the maintenance of a CD133þ CSC population in cSCChas not previously been reported. Our gene expression profilingand functional analysis reveals the important role of increasedNF-kB signaling pathway and key transcription factors RELAand RELB in promoting CD133þ CSCs in cSCC. Our findingthat elevated NF-kB signaling is important to maintain CSCsis supported by microarray expression profiling in humanembryonic stem cell (ESC), which demonstrates that RELA isessential to maintain ESC pluripotency (37).

The evidence for altered expression of components of thePI3K/AKT/mTOR, MAPK, FGF, ERBB, and STAT signaling oractivating pathways is consistent with evidence for frequentgenomic alterations affecting these pathways in HNSCC andcSCC (38). These pathways are also important for controllingthe self-renewal and growth of CSCs (39–43). PI3K and MAPKsignaling is implicated in SCC growth and CSC (44–48). Theregulatory role of the STAT pathway on CSCs self-renewal wasreported in HNSCC (49). Hence, our profiling data suggest thatmultiple signaling pathways may contribute to the stemnesssignature in cSCC. Consistent with this, we observed onlypartial inhibition of CSC phenotypes when blocking NOTCHand IKK–NF-KB signal. Thus, combination therapy inhibitingCSCs may be more effective, because pharmacologic inhibitionof one pathway may only partially reduce the CD133þ cellpopulation.

Several stemness makers have been studied in SCC and othercancers, including CD44, ALDH1, and CD133 (3, 4, 5, 50).Among these, CD44 and ALDH1 are expressed in relativelybroader populations in tumor and cell lines, and additionalmarkers or criteria are often used to enrich and characterize theCSC subpopulations. In our prior (3) and current study, theCD133þ population showed relatively lower expression of CD44,and <1.5-fold difference in ALDH1 compared with the CD133�

population. As ALDH1, CD44, and CD133 populations do not

always overlap, they may contain distinct stem cell subpopula-tions. Our prior and current study of skin SCC tumors establishedthat the relatively rare CD133þ population is a biologicallyand therapeutically relevant subset enriched for TIC andsphere-forming stem phenotypes. While our studies did notpreclude that there are CSC-bearing overlapping or distinct mar-kers, this study explores and experimentally validates a role forrelated NOTCH and IKK–NF-kB signaling in the CD133þ pop-ulation CSC phenotype. This study provides information aboutgene expression patterns and signaling pathways for this CSCpopulation in human primary cSCC, which could helpidentify potential drug targets for CSC to complement currenttherapies for cSCC.

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

Authors' ContributionsConception and design: X.X. Quan, Z. Chen, C. Van WaesDevelopment of methodology: X.X. Quan, N.V. Hawk, Z. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X.X. Quan, P.S. Meltzer, A. Montemarano, M. Braun,Z. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X.X. Quan, W. Chen, S.K. Lee, P.S. Meltzer, Z. Chen,C. Van WaesWriting, review, and/or revision of the manuscript: X.X. Quan, Z. Chen,C. Van WaesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X.X. Quan, S.K. Lee, D.W. Petersen, Z. ChenStudy supervision: X.X. Quan, Z. Chen, C. Van WaesOther (ran experiments, lab technician): J. Coupar

AcknowledgmentsThis study has been supported by the Intramural Research Program of the

National Cancer Institute and the National Institute on Deafness and OtherCommunication Disorders (X. Quan, J. Coupar, S.K. Lee, Z. Chen, and C. VanWaes were supported by intramural projects ZIA-DC-000016, ZIA-DC-000073, and ZIA-DC-000074, which were awarded to C. Van Waes). Weespecially thank Dr. Jonathan C. Vogel, who is deceased, for his guidance ininitiating this project, helpful discussions and comments on the purificationof the CD133þ cell subpopulation, and the design and setup of microarrayexperiment. We thank Dr. Mark Udey in the Dermatology Branch, NCI, NIH,Bethesda, Maryland, for many comments and strong support for the project.We thank Dr. Thomas Hornyak, in the Dermatology Department, UniversityofMaryland, School ofMedicine, Baltimore, Maryland, for his comments andsupport. We thank Dr. William G. Telford, in the Experimental Transplanta-tion and Immunology Branch, NCI, NIH, Bethesda, for his advice on cell flowcytometry experiments. This study utilized the high-performance computa-tional capabilities of the Biowulf Linux cluster at theNIH, Bethesda,Maryland(https://hpc.nih.gov/systems/).

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

Received May 18, 2017; revised January 7, 2018; accepted June 20, 2018;published first June 29, 2018.

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Quan et al.




2018;17:2034-2048. Published OnlineFirst June 29, 2018.Mol Cancer Ther Xin Xin Quan, Nga Voong Hawk, Weiping Chen, et al. Skin Cancer Stem Cells

+B Activation in CD133κ Enhanced NF-αTargeting Notch1 and IKK

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