Targeting Notch1 and Ikka Enhanced NF-Kb Activation in CD133 Skin

Published OnlineFirst June 29, 2018; DOI: 10.1158/1535-7163.MCT-17-0421

Cancer Biology and Translational Studies Molecular Cancer Therapeutics Targeting Notch1 and IKKa Enhanced NF-kB Activation in CD133þ Skin Cancer Stem Cells Xin 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 major Other cancer-related genes from WNT, growth factor recep- tumor-initiating cell population of human cutaneous squa- tors,PI3K/mTOR,STATpathways,andchromatinmodifiers mous cell carcinoma (cSCC), but the landscape of molecular were also identified. Pharmacologic and genetic targeting of alterations underpinning their signaling and cellular phe- NOTCH1, IKKa, RELA, and RELB modulated NF-kBtrans- þ notypesasdrugtargetsremainsundefined. In this study, we activation, the CD133 population, and cellular and stem- developed an experimental pipeline to isolate a highly ness phenotypes. Immunofluorescent staining confirmed þ þ þ enriched CD133 CD31 CD45 CD61 CD24 (CD133 ) colocalization of CD133 and IKKa expression in SCC cell population from primary cSCC specimens by flow tumorspecimens.Ourfunctional,genetic,andpharmaco- þ cytometry. The CD133 cells show enhanced stem–like logic studies uncovered a novel linkage between NOTCH1, phenotypes, which were verified by spheroid and colony IKKa,andNF-kB pathway activation in maintaining the þ formation in vitro and tumor generation in vivo. Gene CD133 stem SCC phenotypes. Studies investigating mar- þ expression profiling of CD133 / cells was compared and kers of activation and modulators of NOTCH, IKK/NF-kB, validated, and differentially expressed gene signatures and and other pathways regulating these cancer stem gene sig- þ top pathways were identified. CD133 cells expressed a natures could further accelerate the development of effective repertoire of stemness and cancer-related genes, including therapeutic strategies to treat cSCC recurrence and metasta- NOTCH and NOTCH1-mediated NF-kB pathway signaling. sis. Mol Cancer Ther; 17(9); 2034–48. 2018 AACR.

Introduction The cancer stem cell (CSC) hypothesis holds that tumors are a hierarchical organ derived from cell subpopulation(s) capa- Human cutaneous squamous cell carcinoma (cSCC) has been ble of self-renewal, called cancer stem–like cells or tumor- increasing over the past several decades, with more than 700,000 initiating cells (TIC; ref. 3). TICs exhibit stem–like and cases in the United States annually (1, 2). Despite surgery, tumor-initiating properties, including increased self-renewal/ radiation, and chemotherapy, SCC cells can escape treatment and colony formation, tumor-forming capacity, as well as altered reform tumors and metastasis, increasing morbidity and mortal- migration, differentiation, and therapeutic sensitivity. Evidence ity. This gives prominence to the existence of a subpopulation of for the existence of TICs has been obtained in different types of SCC cells capable of tumor initiation and therapeutic resistance, human solid tumors through identification of subpopulations and the importance of characterizing and targeting the molecular enriched for surface determinants or enzymatic markers, such alterations mediating their maintenance for cancer prevention asCD133,CD44,CXCR4,andALDH1(3).Wepreviously and treatment. demonstrated that cell membrane protein CD133 (also called prominin-1) is specifically expressed by cSCC cells enriched 1 for a TIC phenotype, and not by CD45 and other non- Tumor Biology Section, Head and Neck Surgery Branch, National Institute on þ Deafness and Other Communication Disorders, NIH, Bethesda, Maryland. epithelial subpopulations overlapping CD44 (4). CD133 2Experimental Transplantation and Immunology Branch, NCI, NIH, Bethesda, cSCC cells exhibited long-term proliferative ability, self-renew- Maryland. 3Microarray Core Facility, National Institute of Diabetes and Digestive al, sphere formation, regeneration of differentiated SCC tumor 4 and Kidney Diseases, NIH, Bethesda, Maryland. Cancer Genetics Branch, NCI, cells, and enriched tumor-initiating capacity for xenografts in 5 þ NIH, Bethesda, Maryland. Skin Cancer Surgery Center, Bethesda, Maryland. immunodeficient mice. CD133 has also been identified as a 6Braun Dermatology Associates, Washington, District of Columbia. biologically and clinically relevant marker for subpopulations Z. Chen and C. Van Waes contributed equally to, and share senior authorship of, of lung and head and neck SCC (HNSCC), where it is associated this article. with tumor-initiating capacity, aggressive clinical features, and Note: Supplementary data for this article are available at Molecular Cancer resistance to cytotoxic therapies (5, 6). Therapeutics Online (http://mct.aacrjournals.org/). Recent studies of cSCC, HNSCC, and lung SCC tumors have Corresponding Authors: Zhong Chen, NIDCD/NIH, Building 10, Room 7N240, 10 identified significant genetic alterations involving components Center Drive, Bethesda, MD 20892. Phone: 301-435-2073; Fax: 301-402-1140; of several common and distinct pathways important in cell E-mail: [email protected]; and Carter Van Waes, [email protected] growth, death or survival, migration, and epithelial/mesenchymal doi: 10.1158/1535-7163.MCT-17-0421 differentiation (7–10). These alterations are implicated in inac- 2018 American Association for Cancer Research. tivation or activation of several canonical pathways, including

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þ Targeting NOTCH1 and IKKa/NF-kB in CD133 Skin Cancer

NOTCH, WNT, HEDGEHOG, NF-kB, growth factor receptors, Sphere formation assay RAS-mitogen–activated protein kinase, PI3K–Akt–mTOR, and Fifteen cSCC samples were collected and used for sphere TP53, but their expression and role in TIC versus other popula- experiments after preparation of single-cell suspension. The cSCC þ tions in established cSCC have not been dissected. Among these single-cell suspension of unsorted or sorted CD133 or CD133 are numbers of genes linked to stem cell maintenance or differ- cells was seeded on NIH 3T3 fibroblast cells feeder layer and entiation (11–14). NOTCH signaling is important in epithelial cultured for 14 to 21 days. Spheres 50 mm were counted under differentiation and as a suppressor of tumor development in a the microscope. subset of HNSCC, but is activated in others (8, 15). IKK and NF-kB signaling has been implicated in promoting tumor cell survival, In vivo mouse model inflammatory, and angiogenesis responses (9). However, how Animal studies were performed under a protocol approved by these signaling pathways and function of corresponding molec- the Institutional Animal Care and Use Committee of the National ular components contribute to the regulation of phenotype of Cancer Institute. Six- to 8-week-old nude mice were obtained the CSC/TIC subpopulation in cSCC tumors are not well under- from NCI Frederick. To establish a niche, 106 human endothelium stood. Identification of deregulated components of pathways and fibroblast cells with 120 mL Matrigel were injected into 0.5 þ critical to maintenance of the CD133 CSC phenotype could 0.5 cm GelFoam sponges implanted subcutaneously 2 weeks potentially help identify targets for precision medicine for pre- prior to tumor cell transplantation. Freshly obtained, dissociated, þ vention and therapy. and sorted CD133 , CD133 , and unsorted cells at doses of In this study, we integrated the molecular profiling, sig- 106,105,104,103,or102 cells were mixed with 106 human naling pathway, mechanistic, and pharmacological studies of endothelium and fibroblast cells in Matrigel, for injection into þ the CD133 subpopulation in primary human cSCC tumors the established GelFoam niche. Each dose level included 15 mice, and cell line models. Through cell sorting with multiple with 5 mice each that received freshly isolated primary sorted þ positive and negative selection markers by flow cytometry, CD133 , CD133 , or unsorted SCC tumor cells. To obtain þ þ we successfully isolated live CD133 cells that form spheroid 106 rare CD133 and matched CD133 cells, we sorted dissoci- colonies in vitro and tumors in vivo.Thissmalldistinct ated suspensions from pools of 5, 5, 6, 6, and 6 (28 total) fresh þ þ CD133 population differentially expresses stem-like and tumors for inoculation of 5 pairs of mice. CD133 and CD133 cancer gene signatures linked to NOTCH1-mediated NF-kB cells sorted from 5 tumors were sufficient to inoculate 5 paired modulation, NF-kB, and WNT pathways. Characterization of recipients at 102,103,104, and 3 pairs at 105, and cells pooled þ the landscape of gene signatures in these CD133 stem cells from 3 and 4 additional tumors each (12 total) were required to þ revealed activation of multiple pathways, which were linked sort 105 CD133 and CD133 cells for 2 additional pairs. Five to NOTCH and NF-kB signaling networks and showed sen- tumors were freshly dissociated and 106,105,104,103, and 102 sitivity to genetic and pharmacologic inhibitors of NOTCH cells from each were inoculated into paired recipients as unsorted and NF-kB. Our functional, genetic, and pharmacologic stud- controls for each dose level. ies uncovered a linkage between NOTCH1, IKKa,andNF-kB þ pathway activation in maintaining the CD133 population Affymetrix genechip human gene 1.0 ST array and its self-renewal ability in established primary cSCC and Total RNA was isolated after FACS sorting, and 15 samples were cell lines. used with RNA integrity numbers equal to or greater than 7.0, as the control for RNA quality. Single-stranded cDNA was generated, fragmented, labeled, and hybridized with standard Affy- Materials and Methods metrix protocol. Data were scanned with an Affymetrix GeneChip Human tumor tissue samples Scanner 3000 and analyzed with Partek and GeneGo. The micro- Deidentified primary human skin cSCC tissue samples were array data have been submitted to NCBI with GEO submission obtained under an exemption from IRB approval by the Office of number (GSE84588). Human Subjects Research, National Institutes of Health, from Potomac Ambulatory Surgery Center, Bethesda, MD, and Braun Nanostring nCounter gene expression assay þ Dermatology Associates, Washington, DC. Total RNAs (100 ng) from sorted CD133 and CD133 cells from seven cSCC samples were hybridized, purified, and immo- Preparation of single-cell suspension from cSCC specimens bilized in the Prep Station. The images were measured and further Tumor tissues were gently chopped into small pieces analyzed with nSolver. and incubated with collagenase III and dispase, then further dissociated using GentleMACS Dissociator. The NF-kB family transcription factor DNA-binding assays dissociated tissues were spun down, incubated with tryp- Nuclear fraction of SCC13 cells was placed on a plate coated sin and EDTA and filtered, spun down, and the cSCC with immobilized NF-kB consensus oligonucleotides. Activa- single-cell suspension was then ready for staining and ted NF-kB is recognized by antibodies, followed by the HRP- FACS sorting for sphere, tumorigenicity assays, and expres- conjugated second antibody, and quantified by spectrophotometry. sion profiling. Drugs and treatment Flow cytometry analysis and FACS sorting The following compounds were used for inhibition of cSCC single-cell suspensions were labeled with multiple anti- gamma-secretase-NOTCH and inhibitor-kappaB kinase in experi- bodies and sorted by BD FACSAria-II following standard protocol ments: DAPT (https://pubchem.ncbi.nlm.nih.gov/compound/ as detailed in Supplementary Methods. Analysis was performed dapt), catalog D5942, Sigma Aldrich); RO4929097 (https:// with BD LSR II and FlowJo7.6.5. pubchem.ncbi.nlm.nih.gov/compound/49867930#section¼

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Top), catalog No. ADV465749148, Sigma Aldrich); wedelo- epithelia were obtained. We developed protocols for cell sorting þ lactone (https://pubchem.ncbi.nlm.nih.gov/compound/Wede and improved purification of CD133 and CD133 tumor cells lolactone#section¼Top, catalog #56639, Sigma Aldrich). after gating out differentiating keratinocytes (CD24), stromal SCC13 cells were treated with two different NOTCH inhib- endothelial cells (CD31), leukocytes (CD45), and fibroblasts itors, DAPT or R04929097, in different doses and measured by (CD61). The percentage of cells expressing these CD markers in FACS analysis. Wedelolactone (10 mmol/L) was used to treat cSCC varied among individual tumors by FACS analysis (Fig. 1A). þ SCC13 cells for 24 hours. Cells then harvested and stained with CD133 cells represent a rare population that resides within CD133 antibody for FACS analysis. Spheres or colonies were a relatively stable range from 0.14% to 1.7% (mean counted after 5 days of drug treatment. Medium with drug was 0.62%; Fig. 1A and B) and displayed a slight increase with changed every 3 days. tumor size that was not significant (R2 ¼ 0.037, P > 0.05). þ Sorted CD133 CD31 CD45 CD61 CD24 (hereafter called þ Luciferase reporter gene assays CD133 ) cells have a purity as high as 95% as detected by FACS þ SCC cells were cotransfected with NF-kB Luciferase and b-Gal analysis (Fig. 1C). More than 99% of FACS sorted CD133 cells þ and then treated with wedelolactone and TNF-a at different time are pan-Keratin positive (Fig. 1D), indicating that the CD133 þ points. Relative luciferase activity was normalized to the cells subpopulation represents highly purified CD133 keratinocytes þ without treatment. from cSCC tissues. Thus, we isolated highly purified CD133 CSC and compared with those relatively pure CD133 keratinocytes Western blotting without stemness characteristics using this improved sorting and Whole cell lysates (15 mg) were used for electrophoresis, then selection protocol with the five different cellular markers. þ protein was transferred onto a Nitrocellulose membrane, and To confirm whether the CD133 population exhibits TIC incubated with first and second antibodies following standard features including self-renewal and tumorigenic ability estab- protocol. lished previously (3), we compared them with both CD133 and unsorted cells for sphere formation efficiency in vitro and Statistical analysis tumor formation in vivo. Unlike normal human keratinocytes Mean and standard deviation (SD) were calculated for different growing as monolayer colonies, dissociated cSCC cells grew as experiments, and the statistical significance was calculated using tethered spheres on NIH 3T3 feeder layers (Fig. 1E). Further, 1 þ Student t test. P value <0.05 was considered a statistically signif- 103 sorted CD133 cells formed 55 8 spheres, while the same icant difference. To analyze microarray data, a two-way analysis of number of unsorted and CD133 cSCC cells grew only 23 3 and variance (ANOVA) for the paired samples was used. 23 4 spheres, respectively (Fig. 1F). The sphere formation in þ Additional information about human SCC specimens, anti- CD133 was significantly increased (P < 0.0001) compared with bodies, and instrumentation settings is presented in the Supple- unsorted cSCC or CD133 cells, whereas there is no difference mentary Information. between the unsorted and the CD133 tumor cell groups. The sphere numbers increased corresponding to increases in the seeded cell numbers, indicating that cSCC cell aggregation Results alone is unlikely to account for sphere formation. þ Clinical characteristics of human primary cutaneous squamous To confirm the enrichment of TICs within the purified CD133 cell carcinomas (cSCC) population, we used the cSCC tumor xenograft assay in a mouse Eighty-two primary cSCC were collected fresh after Mohs model established previously (3), modified to replenish the micrographic surgery from patients. The diagnosis and the normal human fibroblasts and endothelial cells depleted by þ tumor's grade was confirmed by a dermatopathologist, and the sorting. Varying concentrations of sorted CD133 , CD133 , and clinical characteristics are summarized in Supplementary Table unsorted cells were injected subcutaneously, to analyze tumor S1. The majority of subjects were men (78%), and tumors were formation frequency by limiting dilution assay. A minimum of predominantly located on sun-exposed skin from head (62.2%) 105 unsorted cSCC cells was required for initiating tumor in this þ and limbs (34.2%). Most tumors were well or moderately differ- model (Fig. 1G, blue bar). By contrast, as few as 100 CD133 cells entiated (85.4 þ 12.2 ¼ 97.6%) cSCC of less than 2 cm2 in size. generated tumors in vivo (Fig. 1G, green bars). At the same time, The collected tumors were freshly delivered from the clinics and the CD133 subpopulation did not form any visible tumors with processed on the same day, to isolate stem cell subpopulations as high as 106 cells (Fig. 1G, yellow bar). Thus, the estimated þ for in vitro and in vivo characterization of purity, stemness phe- tumor-initiating efficiency of the CD133 subset is 1/245, which notype, molecular profiling, and functional validation studies is dramatically enriched 340-fold compared with unsorted (Supplementary Table S1; Supplementary Fig. S1). The clinical cSCC cells (1/83,333). characteristics of tumor samples used in different experimental þ studies below are summarized Supplementary Table S2. Gene expression profiling of CD133 cells displays gene enrichment for stem cell markers and NOTCH and NF-kB þ CD133 CD31 CD45 CD61 CD24 cells isolated from signaling pathways þ cSCC exhibit enriched CSC/TIC features by in vitro and To investigate genes differentially expressed in the CD133 in vivo assays cSCC population, we compared the whole genome expression þ New protocols to isolate, dissociate, and separate cSCC cells profile between sorted CD133 and CD133 cells after gating for from other populations in tumor specimens were optimized, as the CD31 CD45 CD61 CD24 population from 15 fresh cSCC summarized in Supplementary Methods and Supplementary tumor samples by using Affymetrix Whole Genome RNA micro- Fig. S1. Each cSCC sample was microdissected to carefully remove array. The microarray data were normalized by the robust multi- excess surrounding stroma, and regions with >90% malignant chip average method and analyzed using the ANOVA paired test

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Figure 1. Isolated CD133þCD61CD31CD24CD45 cell subpopulation exhibits CSC features in human cutaneous SCC (hsSCC). A, Distribution of cell surface markers in 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 CD61CD31CD24CD45 cell subpopulation from hsSCC. D, Cytokeratin staining for CD133þ cell subpopulation. CD133þ cells (99%) were pan-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 cells grew 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 number cells (1 103). G, In vivo tumor formation assay in nude mice for comparison of transplant of different numbers of primary cSCC cells. Each group includes 15 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.

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in Partek Genomics Suite, with P value 0.05 and expression level We next examined the profile of NF-kB–related pathway change > 1.5-fold. There were 2,089 genes significantly differen- genes in cSCC based on our hypothesis that Notch and other þ tially expressed in CD133 when compared with CD133 tumor mediators could contribute to regulation of the NF-kB signaling cells. We further compared this gene list with 247 stem-cell– network (20–23). The clustering data revealed increased gene related genes from major publications of human stem cell micro- expression of several key signal and transcription factor sub- þ array data (2, 11–14). We identified 80 overlapped genes between units for the NF-kB pathway in CD133 cells (Fig. 2C). These 2,089 genes identified in this study versus 247 stem-cell–related include IL1R1, IRAK2/4, TRAF2/3, TRAF3IP2, CHUK (IKKal- genes from major publications (Supplementary Fig. S2A). After pha),IKBKB,REL,NFKB2,RELB,as well as AKT1/2, implicated subtraction of the 80 genes, the Venn diagram of pink circle shows in NF-kB activation by PI3K–Akt signaling. In addition, the total of 2,009 genes with significantly altered expression in heat map of the WNT pathway showed increase in WNT3, þ CD133 cells compared with CD133 cells. There are 167 stem DVL2, and CTNNB1 and decrease in expression of the repres- þ genes from the literature after subtraction of 80 genes (green sors such as AXIN2 and GSK3B, in CD133 SCC cells (Sup- þ circle). Our data indicate that the sorted CD133 cells are plementary Fig. S2D). enriched for a stem cell gene expression signature. Moreover, a We hypothesized that the NOTCH, NF-kB, and WNT path- þ heat map of 80 signature genes shows that the genes are mainly ways could form interactive networks in cSCC CD133 CSC distributed in two clusters (Fig. 2A). Cluster I includes 32 upre- populations and used Ingenuity Pathway Analysis (IPA) to þ gulated stem-related genes in CD133 cells, including several reveal the potential regulatory relationships annotated among NOTCH signaling pathway genes NOTCH1, JAG2, and MAML1; these three pathways. The results showed experimental evi- WNT pathway genes WNT3, FZD1, and CTNNB1; and Sonic dence for altered expression and annotated links between NF- Hedgehog pathway genes GLI2, SUFU, PTCH1, and GCNF (Germ kB with the NOTCH (Fig. 2D) or WNT pathway (Fig. 2E). The Cell Nuclear Factor). Cluster II shows 48 downregulated genes network map suggests the hypothesis that noncanonical þ in the CD133 group, including NOTCH signaling-related NOTCH1 signaling could contribute to the activation of REL genes JAG1, NOTCH4, and downstream components RBPJ, and and RELB in cSCC (Fig. 2D). The interaction of NF-kBwiththe NUMB; Wnt signaling negative regulator gene GSK3B and hedge- WNT–b-catenin pathway is more indirectly through other hog signaling regulator HHIP. Unexpectedly, we also detected nodes and pathways (Fig. 2E). increased expression of hematopoietic cell determinant CD8A in þ þ several CD133 samples. CD8a has been associated with CD133 Independent validation of stem and pathway signature lymphomyeloid stem cells and cytotoxic T lymphocytes, which gene expression using Nanostring in a different set of cSCC could potentially infiltrate cSCC and be detected among a subset tissue samples of samples (16–19). To independently validate the microarray results, we verified þ GeneGo analysis of this 80-stem gene signature revealed the gene expression levels with sorted CD133 and CD133 cells NOTCH pathway, NOTCH1-mediated NF-kB pathway, WNT from seven additional SCC tumors, using the Nanostring nCoun- pathway, Hedgehog, and NOTCH-mediated EMT signaling ter Gene Expression assay, for 149 selected genes from the stem- pathways are the top five most significantly ranked pathways in and top-ranked cancer-related pathway gene signatures. Notch þ CD133 cSCC cells (Supplementary Fig. S2B; Supplementary signaling pathway components NOTCH1, NOTCH2, and MAML1 Table S3). To further investigate what kinds of signaling pathways genes were confirmed to have higher expression levels, while JAG1 þ contributed in the CD133 population, we analyzed pathways and EP300 showed decreased expression (Fig. 3A). The genes ranking for all 2,089 significantly different expressed genes based involved in the NF-kB pathway also showed upregulated expres- on the ANOVA paired test results (Supplementary Fig. S2C). The sion, including IL1R1, IRAK2/4, CHUK, and RELB, as well as broader analysis showed that molecular mechanisms of cancer, FAS, TLR6, PRKACA, MMP8, and ICAM4. In the WNT pathway, regulation of EMT pathways, PI3K/AKT, mTOR, NF-kB, ERK/ increased WNT3 and DVL2 expression and decreased GSK3B and þ MAPK signaling, and RAR activation are within the top 20 path- DVL3 expression in CD133 cells were independently verified þ ways in the CD133 cell subset. using Nanostring. Because NOTCH, NOTCH1-mediated NF-kB, and WNT sig- Furthermore, genes in several other signaling pathways dis- naling appeared in top-ranked pathways in both stem cell playing genomic or expression alterations and implicated in supervised and unsupervised analyses, we generated heat maps pathogenesis of SCC (8–10) were confirmed by Nanostring þ for genes included in the top-ranked signaling/stem gene sig- validation (Fig. 3B and C). In CD133 cells, increased gene natures from the 2,089 differentially expressed genes in cSCC expression was observed in EGFR/ERBB, IGF1R, AKT, and mTOR (Fig. 2B and C; Supplementary Fig. S2D). The heat map of the and STAT family genes, as well as those implicated in FGF, MAPK, NOTCH pathway showed two distinct clusters (Fig. 2B): cluster and TGF-b signaling pathways (Fig. 3B). Among other validated I displayed relative increase in gene expression, of ligand JAG2, genes, cell cycle–related genes FBXW11 and PPP2R5C are over- receptors NOTCH1, NOTCH2, NOTCH3, and downstream expressed, while YAP1 and CTNNA1 related to cell growth and transcription factor MAML1; cluster II and III revealed down- differentiation are decreased (Fig. 3C). Additionally, chromatin regulated gene expression, including ligands JAG1, DLL1, recep- regulatory factors including DNMT1 and EZH2 genes were tors NOTCH4, glycosylation modification factor POFUT2, and decreased, while HDAC6 is overexpressed. The differential expres- þ þ downstream transcription factor component RBPJ. CD133 sion of mRNA for several of these genes in CD133 versus cSCC did not exhibit significant differential expression of CD133 cells is not always congruent with the predicted effects canonical NOTCH target genes HES and HEY1,consistentwith of gene mutations (NOTCH1), copy-number alterations (YAP1), a recent study that reported defective transcription of HES and or overall expression (EZH2) observed in SCC subsets versus HEY1 in subsets of human HNSCC with wild-type NOTCH nonmalignant epithelia (7–9, 19, 20), underscoring the impor- genes (15). 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 altered expression of 80 upregulated or downregulated stem gene signature in sorted CD133þ cells compared with CD133 cells after gating for the CD61 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 2,089 signiﬁcantly different expressed genes in CD133þ CSCs compared with CD133 tumor cells. IPA analysis of the network of NF-kB and NOTCH pathway 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 indicates upregulated genes, and the blue color indicates downregulated genes. The different shapes indicate different function category of the molecules as indicated.

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Figure 3. Validation of differentially expressed genes in the CD133þ CSC population using Nanostring expression assay. Totally 149 signiﬁcantly altered genes involved in stem cell gene signature or signaling pathways were selected and validated using Nanostring nCounter gene expression assay. The consistently altered genes 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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þ Inhibiting NOTCH reduces the CD133 cell population 77% respectively (P < 0.001) compared with the control siRNA As NOTCH signaling has been implicated in suppressing dif- group (Fig. 4E, left). NOTCH2 siRNA showed similar but weaker ferentiation or promoting growth in different SCC (7, 15, 21), we inhibitory effects on nuclear DNA-binding activity of these three first examined the functional effects of two different g-secretase transcription factor components, and combination of NOTCH2 inhibitors (DAPT and RO4929097) that inhibit NOTCH, upon with NOTCH1 siRNA did not further inhibit NF-kB nuclear þ the CD133 subset in the human cSCC cell line SCC13 (23). We binding activity (Fig. 4E, middle and right). These results provide confirmed that SCC13 exhibits a similar small percentage of evidence for NOTCH-MODULATED NF-kB pathway activity, þ þ CD133 cells varying between 0.2% and 6%, depending on which could potentially promote the CD133 population in culture conditions, and displays clonogenic and sphere-forming cSCC cells. potential, as observed by primary cSCC tumors. Three concentra- þ tions of DAPT (1, 5, 10 mmol/L) and RO4929097 (1, 3, 5 mmol/L) IKK–NF-kB pathway signaling promotes the CD133 cell were selected based on ranges found to include IC50 for other population in cSCC þ tumor cell lines (24). FACS analysis shows that the CD133 cell As IKKa (CHUK) and other NF-kB pathway components were þ population is significantly inhibited by 5 to 10 mmol/L DAPT differentially overexpressed in CD133 SCC tumor cells, we and 1 mmol/L RO4929097, supporting a functional role of screened SCC13 cells for the effects of wedelolactone, an inhibitor þ NOTCH1 in the maintenance of CD133 cells (Fig. 4A). We next shown to inhibit IKKa and b kinases, and IKKa-dependent NF-kB þ tested if these two NOTCH inhibitors could affect CD133 cell activation in SCC cells (25, 26). Wedelolactone inhibits TNF-a colony formation of SCC13, as well as UM-SCC-46, an HNSCC inducible NF-kB reporter activity assayed 24 and 48 hours after line verified to express wtNOTCH1 and pathway genes by whole treatment in SCC13 cells (Fig. 5A). To determine the effects of þ þ exome and RNA sequencing (Hui Cheng, unpublished observa- NF-kB inhibition on SCC CD133 cells, we analyzed the CD133 tions). Colony formation of both SCC13 and UM-SCC46 cells cell population with or without wedelolactone treatment. The was significantly decreased by DAPT or RO4929097 (Fig. 4B; blue, FACS results showed lower concentrations of 1 to 5 mmol/L þ red bars). wedelolactone reduce CD133 cells, while 10 mmol/L wedelo- þ To independently examine the specificity of these findings for lactone significantly decreases the CD133 population (Fig. 5B, þ þ NOTCH1 or 2 differentially expressed in tumor CD133 cells P < 0.001). The effect of wedelolactone on the CD133 popula- in Fig. 3A, we examined the expression of NOTCH1 and 2 protein, tion is not due to general cytotoxicity. The above results suggest þ and tested the effects of small interfering (si)RNAs silencing the NF-kB pathway contributes to maintaining the cSCC CD133 þ NOTCH1 and NOTCH2 on the CD133 cell population in two CSC population in vitro. cell lines. We confirmed that NOTCH1 protein of expected size is þ expressed and knocked down efficiently by siRNA in both SCC13 Expression and activation of IKKa promote the CD133 cell and UMSCC46 cells, while weak expression of NOTCH2 protein population in cSCC is detected only with prolonged exposure and weakly inhibited As both our microarray and Nanostring verification data (Fig. 4C, top). Consistent with the differential expression and showed there is increased CHUK (IKKa) gene expression in þ þ knockdown of NOTCH proteins, the relative CD133 cell num- CD133 cells, and IKK inhibitor wedelolactone could decrease þ ber decreased 40% of control with NOTCH1 knockdown alone, the CD133 population in human SCC13 cells, we independently while no effect was attributable to the limited expression and investigated the specific role and function of IKKa for the main- þ knockdown of NOTCH2 alone or when combined with NOTCH1 tenance of CD133 CSC in cSCC. To achieve this, we first tested siRNA (Fig. 4C, bottom). Together, the functional effects observed the effect of siRNA knockdown of endogenous IKKa on the þ with pharmacologic and genetic approaches suggest NOTCH1 CD133 cells. Knockdown by siRNA efficiently inhibited cyto- þ þ signaling contributes to the CD133 population and colony- plasmic and nuclear of IKKa (Fig. 5C) and reduced the CD133 forming capability in a cSCC as well as an HNSCC cell line population by 50% compared with the control group in SCC13 expressing wild-type NOTCH1. cells (Fig. 5D). Similar effects were also observed in the HNSCC cell line UMSCC46. Next, we examined the effects of introducing Inhibiting NOTCH reduces NF-kB transcriptional and nuclear IKKa wild-type (WT) or activated or inactivated genetic IKKa þ DNA-binding activity in cSCC cells mutants upon the CD133 population. These include IKKa with Because NOTCH1-mediated NF-kB activation is the second serine phospho-acceptor sites substituted with constitutively acti- þ top-ranked GeneGo analysis signature for the CD133 cell pop- vating glutamate residues (EE); inactivating alanines (AA); or ulation, and aberrant activation of NF-kB has been observed in Lysine44 substituted with alanine, which produces a catalytically SCC (8, 25), we explored the possibility that NOTCH signaling inactive form (KA; ref. 25). Cells expressing IKKa WT or EE protein þ enhances NF-kB pathway signaling, by assay for NF-kB reporter significantly increased the CD133 population 2.1- (P < 0.01) and gene activity after NOTCH inhibition. As shown in Fig. 4D, 3.5-fold (P < 0.001) in SCC13 cells, whereas the inactive IKKa þ inhibitors of g-secretase and Notch activation, DAPT and forms AA or KA exhibited no increase in the CD133 subset (Fig. þ RO4929097, decrease TNF-a–induced NF-kB reporter activity in 5E). Furthermore, we confirmed that the increase in the CD133 human SCC13 cells in a dose-dependent manner. To further population by overexpression of wild-type IKKa (WT) or its active confirm the above results, siRNA was used to specifically knock form (EE) can be significantly inhibited by IKKa siRNA knockdown NOTCH1 and/or NOTCH2, and nuclear DNA binding of down (Fig. 5F). five NF-kB nuclear transcription factor subunits was assayed and To further examine the potential relationship between IKKa þ quantified. The results showed that after transfection with and CD133 population in cSCC in vivo, we performed immu- NOTCH1 siRNA, the DNA-binding activity of canonical NF-kB nofluorescent (IF) staining of cSCC specimens. Figure 5G shows þ pathway subunit p65 (RelA), and noncanonical pathway subunits colocalization of IKKa and CD133 immunostaining in cell p52, and RELB, were significantly decreased to 64%, 80%, and subpopulations in two different primary cSCC tissues. The tumor

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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 cell population 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 siRNA were 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-kB activity was stimulated with TNF-a (10 ng/mL) and normalized with b-gal. E, Silencing NOTCH 1, NOTCH2, or combined NOTCH1, and 2 siRNA partially decreases p65, p52, and RelB nuclear activation by binding assay. All results are shown as mean þ SD; , P < 0.05; , P < 0.01; , P < 0.001. The experiments were done in triplicate.

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Figure 5. Modulation of CD133þ CSCs by IKK inhibitor and IKKa expression, which colocalized with CD133 expression in cSCC tissues. A, IKK inhibitor wedelolactone inhibits NF-kB activity in SCC13 cells. Cells were transfected with NF-kB luciferase reporter and b-Gal plasmids and treated with wedelolactone and TNF-a alone or in combination for 24 and 48 hours. Red, no TNF-a; blue, TNF-a treatment. , P < 0.05, compared with TNF-a-treated, non- wedelolactone group; #, P < 0.05, compared with non–TNF-a-treated group. B, Wedelolactone reduces CD133þ CSC population in SCC13 cells. Cells were treated with wedelolactone in different doses for 24 hours, stained with CD133-PE antibody, and the CD133þ population was analyzed with FACS. C, IKKa (CHUK) siRNA exhibited high knockdown efficiency in both cytoplasmic and nuclear compartment of SCC13 cells. D, IKKa (CHUK) knockdown significantly decreased CD133þ CSC population both in SCC13 and UMSCC46 cells. E, Overexpression IKKa (CHUK) active forms dramatically increases the CD133þ CSC population in SCC13 cells. F, Decreased CD133þ population caused by IKKa (CHUK) siRNA knockdown could be reversed by introducing WT IKKa or IKKa active forms (EE). Cells were cotransfected with different active forms of IKKa (WT, EE, AA, and KA) plus IKKa siRNA with control siRNA. CD133þ population was analyzed after 48 hours transfection. , P < 0.05, compared with WT or EE control siRNA groups; #, P < 0.05, compared with vector control siRNA group. G, The frozen sections of cSCC tissues were coimmunostained with anti-IKKa and CD133 primary antibodies followed by Alexa- Fluor-488 (green) and Alexa-Fluor-555 (red) secondary antibodies. DAPI (blue) was used for visualizing nuclei, and H&E staining is showing morphology of tumor specimens. Magnification, 200. All data represent mean and SD of one representative of three independent experiments, and each conducted in triplicate.

specimens from 2 patients in Fig. 5G and Supplementary Fig. S3C lation of cells is seen along the interface of malignant epithelia and S3D show merged staining of CD133 and IKKa, as well as with intervening inﬂammatory (Supplementary Fig. S3C and 3D) with DAPI, at the basal layer and leading edge of epithelial tumor or ﬁbrovascular stroma (Fig. 5G). In addition, Supplementary cell nests shown in adjacent H&E sections. The costained popu- Fig. S3A and 3B shows merged staining of CD133 and IKKa for

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cells in two additional SCC tumors, over a wider region at 100 ing studies of cSCC and HNSCC provide evidence for gene copy magnification. These data are consistent with the differential alterations or mutations in components or coregulators of the þ coexpression of these detected in CD133 cells from multiple NOTCH (NOTCH 1,2), WNT (AJUBA), cancer-related MAPK and tumors, and functional effects of IKKa genomic mutants on PI3K growth (HRAS, RASA1, BRAF, PIK3CA), NF-kB prosurvival þ CD133 cells in cell lines. Together, these observations support and death pathways (FADD, BIRC2/3, CASP8, RIPK4). Lastly, our þ a role for increased IKKa expression in the CD133 population in findings revealed a link between the NOTCH and NF-kB signal cSCC cells and tumors. pathways and expression of several of their key components, NOTCH1, IKKa, RELA, and RELB, contributing to the mainte- þ þ Knockdown of RELA and/or RELB inhibits the cSCC CD133 nance of the CD133 CSC population and its stem-like pheno- population and its colony/spheroid formation phenotype typic features (Fig. 6E). IKKa can mediate downstream canonical and alternative We uncovered a NOTCH cSCC CSC gene signature and þ NF-kB pathway activation through RELA and RELB, which are showed that NOTCH plays a role in maintaining the CD133 þ found to be overexpressed in CD133 cells. Next, we examined cell population. Blocking NOTCH by either of two different þ the knockdown effects of RELA and/or RELB on the CD133 g-secretase inhibitors, or NOTCH1 by siRNA, reduced the per- þ population in cSCC SCC13 and HNSCC UMSCC46 cells centage of CD133 SCC cells, and their capacity for clonogenic þ (Fig. 6A). The CD133 cell populations were clearly reduced by sphere formation, which are established surface and phenotypic 54% with RELA siRNA, 43% with RELB siRNA, and 43% with markers of CSC/TIC (3). The NOTCH pathway plays a key role combined knockdown in SCC13 cells (Fig. 6B, blue bars). Similar in cell–cell communication via interaction of membrane-bound results were observed in UMSCC46 cells (Fig. 6B, green bars). JAG ligands and NOTCH receptors (27). The NOTCH receptor Next, we quantified the colony/spheroid formation ability in family is composed of 4 family members (27). In HNSCC and SCC13 and UMSCC46 cells after knockdown of RELA and/or cSCC, inactivating and missense mutations in NOTCH1 and þ RELB. Clonogenic colonies from CD133 cells were reduced to 2 have been observed in a subset of tumors, suggesting that 78%, 63%, and 55% by RELA or RELB siRNA knockdown alone, the NOTCH may function as a tumor suppressor in those or in combination in SCC13 cells (Fig. 6C, pink bars). Spheroid contexts (8, 9, 28, 29). Complexes of NOTCH receptor intra- formation in UMSCC46 cells also decreased to 76%, 69%, and cellular domains (NICD) with canonical NOTCH transcription- 68%, respectively (Fig. 6C, purple bars; Fig. 6D). Together, the al cofactors can activate HES1/HEY1 repressors that trigger above data indicate both IKKa and NF-kB components RELA and epithelial differentiation. Intriguingly, differential expression þ þ RELB play an important role in maintaining the CD133 popu- of HES1/HEY1 was not detected among CD133 cSCC over- lation and its cancer stem–like features in human SCC. expressing NOTCH1 and other signal components, and there was decreased expression of key canonical transcription factor fi Discussion RBPJ. These ndings are consistent with recent studies in human HNSCC, where tumors expressing WT NOTCH1 included sub- To our knowledge, this is the first study to demonstrate the sets without evidence of HES1/HEY1 activation, and where þ CD133 gene expression signature and its top-ranked signaling NOTCH inhibitors reduced proliferation, through an unknown pathways in a purified cancer stem cell–like subset from primary mechanism (15). cSCC tumors. There are four important findings from our In this regard, we provide evidence that NOTCH can promote research. First, our optimized isolation and separation further noncanonical cross-activation of nuclear factor-kB (NF-kB/REL), þ þ enriched for high purity CD133 cytokeratin cells, overcoming a a family of signal activated transcription factors that are often significant technical obstacle and enabling whole gene expression aberrantly activated in cSCC, HNSCC, and cervical SCC (25, profiling detection and validation of stem and cancer genes in the 30–33). The activation of canonical NF-kB1/RELA and alternate þ small CD133 population from clinical tumor samples. Second, NF-kB2/RELB transcription factors is mediated by inhibitor- our study provides a global overview of the gene signatures of kappaB kinase (IKK)a/b/g or IKKa complexes, respectively þ þ CD133 CSC in the human cSCC. Using microarray, Nanostring (25). Our profiling of CD133 cSCC cells unveiled increased validation and bioinformatics analyses, these data provide a expression of multiple components of the NOTCH and NF-kB valuable resource for hypothesis generation and testing for func- signaling pathways, including key components NOTCH1, IKKa tional and clinical role of different signaling components and (CHUK), RELA, and RELB (Fig. 6E). We showed that inhibition of þ pathways in cSCC CD133 CSC. Our stem gene expression NOTCH by g-secretase or NOTCH1 by siRNA functionally par- profiling data identified 2,000 candidate genes differentially tially inhibits NF-kB reporter and nuclear DNA binding of NF-kB þ þ expressed in CD133 cSCC cells, including an 80-gene set of p52, RELA and RELB along with the CD133 phenotype, and that known stem cell signatures. Third, we demonstrated upregulated knocking down overexpressed IKKa, RELA, and RELB similarly þ expression of genes of key signaling pathways and networks from inhibited the CD133 phenotype. The partial reduction of NF-kB the CSC population by analyzing the top-ranked pathways activity by inhibition of NOTCH signaling is expected, because þ involved with GeneGO and IPA platform for the CD133 tran- NOTCH are not the only molecules that directly modulate NF-kB þ script signature. The CD133 transcript signatures represent path- activity in SCC (8, 25). ways implicated in self-renewal and epithelial differentiation, Interestingly, our study also establishes that IKKa is an impor- þ such as NOTCH, WNT, SHH, and NF-kB, and cancer-related tant regulator for maintaining the CD133 CSC population in pathways PTEN, PI3K/AKT/mTOR, NGF, MAPK, EGFR/ERBB, cSCC cells. Notably, NOTCH1 and IKKa have previously been FGF, JAK/STAT, TP53, and TGF-b. Genomics and experimental shown to physically and functionally interact in normal kerati- studies provide evidence supporting the biologic and clinical nocytes and cervical SCC cells (33, 34). NOTCH1 knockdown significance of several of these pathways to SCC biology (7, 8). modulated TNFa-induced NF-kB activity and promoter binding Complementing our observations, recent whole exome sequenc- in 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 pathway activation. A, RELA and RELB siRNA exhibited high efﬁciency of siRNA knockdown. B, Knockdown of RELA and/or RELB inhibited CD133þ population. After 48 hours of transfection, the CD133þ population was analyzed by FACS. C, Knockdown of RELA or RELB inhibited colony and spheroid formation abilities. After transfection for 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 siRNA þ knockdown after 14 days of culture (40). E, Schematic illustration of activated Notch enhances NF-kB pathway activation in cSCC CSC population. The CD133 cSCC CSC gene signature is demonstrated with activated NOTCH signaling, which includes NOTCH1, NOTCH2, and JAG2 increased expressing. The activated NOTCH pathway then enhances CHUK(IKKa), one of the key upstream components of NF-kB signaling pathway. The active CHUK(IKKa) forms complexes with 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.

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g-secretase pharmacologic inhibition of TNF-induced NF-kB always overlap, they may contain distinct stem cell subpopula- activity in cSCC cells. In cervical SCC, NOTCH1 and IKKa also tions. Our prior and current study of skin SCC tumors established þ promoted chemotherapeutic resistance, a feature often that the relatively rare CD133 population is a biologically attributed to CSC. Our data indicate that WT or activating phos- and therapeutically relevant subset enriched for TIC and pho-acceptor site mutants for TNF-mediated IKKa activation sphere-forming stem phenotypes. While our studies did not þ increase CD133 cells, whereas inactivated IKKa mutants did preclude that there are CSC-bearing overlapping or distinct mar- þ not, implicating the role of IKKa in the expansion of CD133 kers, this study explores and experimentally validates a role for þ þ CSC. Further supporting this, IKKa colocalized in CD133 cells in related NOTCH and IKK–NF-kB signaling in the CD133 pop- malignant epithelia of cSCC tumors. Besides its role in NF-kB ulation CSC phenotype. This study provides information about activation, IKKa may also function as a cofactor to regulate other gene expression patterns and signaling pathways for this CSC key transcription factor genes that control cell proliferation and population in human primary cSCC, which could help differentiation, such as E2F1/BMI1 (35). Intriguingly, IKKa has identify potential drug targets for CSC to complement current also been reported to bind the promoter and reciprocally repress therapies for cSCC. Notch canonical target gene HES1 (36), potentially helping to explain the lack of HES1 induction among NOTCH-related genes Disclosure of Potential Conflicts of Interest we detected, and the paradoxical role of NOTCH1 and IKKa in No potential conflicts of interest were disclosed. promoting CSC expansion without triggering NOTCH-mediated terminal differentiation. Authors' Contributions Although the involvement of the IKK–NF-kB signaling path- Conception and design: X.X. Quan, Z. Chen, C. Van Waes way in promoting SCC has been demonstrated (25, 30–33), its Development of methodology: X.X. Quan, N.V. Hawk, Z. Chen þ role in the maintenance of a CD133 CSCpopulationincSCC Acquisition of data (provided animals, acquired and managed patients, has not previously been reported. Our gene expression profiling provided facilities, etc.): X.X. Quan, P.S. Meltzer, A. Montemarano, M. Braun, Z. Chen and functional analysis reveals the important role of increased Analysis and interpretation of data (e.g., statistical analysis, biostatistics, NF-kB signaling pathway and key transcription factors RELA computational analysis): X.X. Quan, W. Chen, S.K. Lee, P.S. Meltzer, Z. Chen, þ andRELBinpromotingCD133 CSCs in cSCC. Our finding C. Van Waes that elevated NF-kB signaling is important to maintain CSCs Writing, review, and/or revision of the manuscript: X.X. Quan, Z. Chen, is supported by microarray expression profiling in human C. Van Waes embryonic stem cell (ESC), which demonstrates that RELA is Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X.X. Quan, S.K. Lee, D.W. Petersen, Z. Chen essential to maintain ESC pluripotency (37). Study supervision: X.X. Quan, Z. Chen, C. Van Waes The evidence for altered expression of components of the Other (ran experiments, lab technician): J. Coupar PI3K/AKT/mTOR, MAPK, FGF, ERBB, and STAT signaling or activating pathways is consistent with evidence for frequent Acknowledgments genomic alterations affecting these pathways in HNSCC and This study has been supported by the Intramural Research Program of the cSCC (38). These pathways are also important for controlling National Cancer Institute and the National Institute on Deafness and Other the self-renewal and growth of CSCs (39–43). PI3K and MAPK Communication Disorders (X. Quan, J. Coupar, S.K. Lee, Z. Chen, and C. Van – Waes were supported by intramural projects ZIA-DC-000016, ZIA-DC- signaling is implicated in SCC growth and CSC (44 48). The 000073, and ZIA-DC-000074, which were awarded to C. Van Waes). We regulatory role of the STAT pathway on CSCs self-renewal was especially thank Dr. Jonathan C. Vogel, who is deceased, for his guidance in reported in HNSCC (49). Hence, our profiling data suggest that initiating this project, helpful discussionsandcommentsonthepurification þ multiple signaling pathways may contribute to the stemness of the CD133 cell subpopulation, and the design and setup of microarray signature in cSCC. Consistent with this, we observed only experiment. We thank Dr. Mark Udey in the Dermatology Branch, NCI, NIH, partial inhibition of CSC phenotypes when blocking NOTCH Bethesda, Maryland, for many comments and strong support for the project. – We thank Dr. Thomas Hornyak, in the Dermatology Department, University and IKK NF-KB signal. Thus, combination therapy inhibiting of Maryland, School of Medicine, Baltimore, Maryland, for his comments and CSCs may be more effective, because pharmacologic inhibition support. We thank Dr. William G. Telford, in the Experimental Transplanta- þ of one pathway may only partially reduce the CD133 cell tion and Immunology Branch, NCI, NIH, Bethesda, for his advice on cell flow population. cytometry experiments. This study utilized the high-performance computa- Several stemness makers have been studied in SCC and other tional capabilities of the Biowulf Linux cluster at the NIH, Bethesda, Maryland cancers, including CD44, ALDH1, and CD133 (3, 4, 5, 50). (https://hpc.nih.gov/systems/). Among these, CD44 and ALDH1 are expressed in relatively The costs of publication of this article were defrayed in part by the broader populations in tumor and cell lines, and additional payment of page charges. This article must therefore be hereby marked markers or criteria are often used to enrich and characterize the advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate CSC subpopulations. In our prior (3) and current study, the this fact. þ CD133 population showed relatively lower expression of CD44, and <1.5-fold difference in ALDH1 compared with the CD133 Received May 18, 2017; revised January 7, 2018; accepted June 20, 2018; population. As ALDH1, CD44, and CD133 populations do not published first June 29, 2018.

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2048 Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics

Targeting Notch1 and IKKα Enhanced NF-κB Activation in CD133+ Skin Cancer Stem Cells

Xin Xin Quan, Nga Voong Hawk, Weiping Chen, et al.

Mol Cancer Ther 2018;17:2034-2048. Published OnlineFirst June 29, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-0421

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