Defects in the Fanconi Anemia Pathway in Head and Neck ... · Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and

Biology of Human Tumors

Defects in the Fanconi Anemia Pathway in Headand Neck Cancer Cells Stimulate Tumor CellInvasion through DNA-PK and Rac1 SignalingLindsey E. Romick-Rosendale1, Elizabeth E. Hoskins1, Lisa M. Privette Vinnedge1,Grant D. Foglesong1, Marion G. Brusadelli1, S. Steven Potter2, Kakajan Komurov1,Samantha A. Brugmann2, Paul F. Lambert3, Randall J. Kimple4, Elizabeth L. Virts5,Helmut Hanenberg5,6,7, Maura L. Gillison8, and Susanne I.Wells1

Abstract

Purpose: Head and neck squamous cell carcinoma (HNSCC)remains a devastating disease, and Fanconi anemia (FA) genemutations and transcriptional repression are common. Invasivetumor behavior is associated with poor outcome, but relevantpathways triggering invasion are poorly understood. There is asignificant need to improve our understanding of geneticpathways and molecular mechanisms driving advanced tumorphenotypes, to develop tailored therapies. Here we sought toinvestigate the phenotypic and molecular consequences of FApathway loss in HNSCC cells.

Experimental Design: Using sporadic HNSCC cell lines withand without FA gene knockdown, we sought to characterize thephenotypic and molecular consequences of FA deficiency. FApathway inactivation was confirmed by the detection of classic

hallmarks of FA following exposure to DNA cross-linkers. Cellswere subjected to RNA sequencing with qRT-PCR validation,followed by cellular adhesion and invasion assays in the presenceand absence of DNA-dependent protein kinase (DNA-PK) andRac1 inhibitors.

Results: We demonstrate that FA loss in HNSCC cells leads tocytoskeletal reorganization and invasive tumor cell behavior in theabsence of proliferative gains. We further demonstrate that cellularinvasion following FA loss is mediated, at least in part, throughNHEJ-associated DNA-PK and downstream Rac1 GTPase activity.

Conclusions: These findings demonstrate that FA loss stimu-lates HNSCC cell motility and invasion, and implicate a target-able DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.Clin Cancer Res; 22(8); 2062–73. �2015 AACR.

IntroductionIn the general population, HNSCC is associated with smoking,

alcohol consumption, andHPV infection, and represents the sixthleading type of cancer worldwide, with an annual incidence of500,000. More than half of sporadic HNSCCs are diagnosed atlocally advanced or metastatic stages, and approximately 50% oftreated patients relapse with local or distant metastasis, both

bearing poor rates of remission (1, 2). Unfortunately, decadesof research have not improved HNSCC outcomes significantly,and the classic therapeutic option, a combination of surgery,radiation, and chemotherapy, leaves patients permanently dis-figured. Thus, there is a need to improve our understanding of thebiologic processes driving local invasiveness and develop novelapproaches for the treatment of late-stage tumors. Previous exomesequencing data demonstrated that 11% of HPVþ and HPV�

HNSCCs harbor nonsynonymous mutations in FA DNA repairgenes (3). In addition to classic loss-of-function mutations,transcriptional repression of FANCB, FANCC, FANCF, FANCJ,and FANCM (e.g., by promoter methylation) has been noted indysplastic HN tissue and HNSCC (4, 5). This suggests that loss ofFA pathway function can provide a selective advantage to HNSCCcells. However, the underlying biologic mechanisms, aside fromgenome instability, remain poorly understood.

Major advances in understanding how defective DNA repairpathways contribute to tumorigenesis have been achieved bystudying DNA repair deficiency syndromes that often manifestduring the first years of life (6). Fanconi anemia (FA) is a rareinherited disorder where every cell of an affected individual isexquisitely sensitive to DNA cross-linking agents including mito-mycin C or cisplatin (for reviews on mechanisms and diseasemanifestations see (refs. 7 and 8 and references within). Clini-cally, patients with FA are characterized by frequent congenitalabnormalities, early progressive bone marrow failure, and a highpropensity for developingmalignancies, especially acutemyeloidleukemia, and squamous cell carcinoma of the head and neck,

1Cancer and Blood Diseases Institute, Cincinnati Children's HospitalMedical Center, Cincinnati, Ohio. 2Division of Developmental Biology,Cincinnati Children's Hospital Medical Center,Cincinnati,Ohio. 3McAr-dle Laboratory for CancerResearch,UniversityofWisconsin School ofMedicine and Public Health, Madison, Wisconsin. 4Department ofHuman Oncology, University of Wisconsin School of Medicine andPublic Health, Madison,Wisconsin. 5Department of Pediatrics, IndianaUniversity School of Medicine, Indianapolis, Indiana. 6Department ofOtorhinolaryngology, Heinrich Heine University, Duesseldorf, Ger-many. 7Department of Pediatrics III, University Children's HospitalEssen, University of Duisburg-Essen, Essen, Germany. 8Internal Med-icine—Hematology & Oncology, Comprehensive Cancer Center, TheOhio State, University College of Medicine, Columbus, Ohio.

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

Corresponding Author: Susanne Wells, Cancer and Blood Diseases Institute,Cincinnati Children's Hospital, 3333 Burnet Ave., MLC 7013, S7-206, Cincinnati,OH 45229. Phone: 513-636-5986; Fax: 513-636-2880; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-2209

�2015 American Association for Cancer Research.

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esophageal, and anogenital regions. In fact, FA patients withHNSCCs are usually diagnosed at a young age with advancedtumor stages that have a poor prognosis (9, 10). FA has beenassociated with recessive mutations in one of 19 FA genes (11),which play a crucial role in triggering and coordinating funda-mental mechanisms of DNA repair for the maintenance ofgenome instability. When intact, the FA core complex, composedof the protein products of eight FA genes including FANCA, isassembled at the site of DNA damage and triggers monoubiqui-tination of the central and evolutionarily conserved pathwaymembers FANCD2 and FANCI by the E3 ligase FANCL. Theactivated FANCD2/FANCI dimer then stabilizes the stalled rep-lication fork at the crosslink and coordinates the activity ofnucleases, TLS polymerases, and homologous recombinationfactors. Genes of the homologous recombination (HR) pathway,where biallelic mutations clinically cause FA-like syndromes, areBRIP1/FANCJ, BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN,and RAD51C/FANCO. Cells with biallelic mutations in FA genescan compensate for their HR defects by overactivating the error-prone nonhomologous end joining (NHEJ) pathway (12, 13),thus triggering inappropriate DNA repair. Individuals with het-erozygous loss-of-function germline mutations in DNA repairgenes also are at an increased risk for tumors, due to the loss of thewild-type allele in the malignant cells (6). Here, heterozygousgermline defects in the "late" genes of the FApathway (FANCD1/J/N/O/Q/S) are predominantly associated with the developmentof hereditary breast/ovarian and pancreatic cancer (14, 15).Furthermore, even sporadic tumors in patients with no familyhistory of cancer frequently harbormutations inDNA repair genes(3, 16–18).

Several lines of evidence suggest that FA pathway loss in theepidermis, in contrast to the hematopoietic system, promotesgrowth in the basal stem and progenitor cell compartment. First,in murine models, genetic loss of Fancd2 cooperated with trans-genic HPV16 E7 expression targeted to basal epithelial cells topromote the development of HNSCC (19). Importantly, Fancd2loss alonewas already sufficient for a subtle yet consistent increasein basal cell proliferation in E7-negative control mice, thushighlighting a pro-proliferative role for FA pathway defects inthe normal epidermis and in an HPV-negative environment.

Second, using HPV-immortalized human keratinocytes, wehave previously reported that FANCA or FANCD2 knockdowndrive proliferation and HPV E7–dependent hyperplasia in three-dimensional (3D) organotypic epithelial raft but not in two-dimensional keratinocyte culture systems (20). Third, we haverecently reported that defects in the FA pathway stimulate HPVgenome amplification and accumulation of the HPV E7 onco-protein with concomitant cellular proliferation (21, 22). Fourth,FA patient–derivedHNSCC cell lines were shown to harbor eithersimilar or increased stem cell populations when compared withsporadic HNSCC lines, using tumor sphere formation, CD44positivity, or ALDH1 status as experimental end points (23, 24).

To define the functional effects of acquired FA deficiency inHNSCC cells, we generated isogenic FA HNSCC models usingshRNA-mediated stable knockdown and rescue strategies in HPV-positive and -negative tumor cell lines. While depletion of the keyFA pathway components FANCA, FANCD2, and FANCJ inducedclassical FA phenotypes in these cells when exposed to DNAcrosslinkers, minor to no effects on tumor cell growth wereobserved under standard culture conditions. Surprisingly, how-ever, under these same conditions, FA loss caused cytoskeletalreorganization and dramatic increases in tumor cell invasivenessin the absence of proliferative gains in HPV-positive and negativeHNSCC cells. Invasive properties were associated, at least in part,with increased activities of the NHEJ-associated DNA-dependentprotein kinase (DNA-PK) and the Rac1 GTPase, thus linking FA-associated DNA damage responses with cytoskeletal machineries.In summary, these data highlight new and unexpected signalingconnections between the FA DNA repair pathway and invasivetumor phenotypes in HNSCC. These might be targeted for thedevelopment of novel treatment strategies to lower mortality andimprove the prognosis of patients with advanced HNSCCs.

Materials and MethodsCell culture

The UM-SCC1, UM-SCC6, and UM-SCC47 cell lines werederived and maintained as previously described (25). All celllines were authenticated regularly by their morphologic charac-teristics and analysis of corresponding genetic and molecularmarkers. Nontargeting, FANCD2-, FANCA-, and FANCJ(TRCN49915) specific short hairpin RNA (shRNA)-expressinglentiviral vectors were obtained through the Sigma MISSIONshRNA program (Sigma Aldrich) as previously described (20).The MIEG-HPV-16-E7 (GenScript) construct was developed fromthe pMIEG3 retroviral vector, a kind gift fromDr. DavidWilliams(Boston Children's Hospital, Boston, MA), and has beendescribed earlier. The EcoRI-E7-His(6)-FLAG-XhoI sequence, GA-ATTCGGCGGCCGCGCCAC CATGCATGGAGATACACCTACAT-TGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCT-CTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGA-TGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAG-CCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACG-CTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTT-TGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCA-TCTGTTCAGAAACCAGACTACAAGGACGACGATGACAAGCAT-CACCATCACCATCACTAACTCGAG, was created by flanking theLXSN-HPV16-E7 gene with primers to introduce EcoRI and in-frame His(6)-FLAG-Stop-XhoI sequence. The PCR product wasproduced, cut, and inserted into the EcoRI and XhoI sites in thepMIEG vector by GenScript. RD114 pseudotype retroviruses were

Translational Relevance

Head and neck squamous cell carcinoma (HNSCC) is adevastating cancer type with poor outcomes particularly whendiagnosed at advanced, invasive stages. Targetable genes andpathways that stimulate HNSCC progression remain poorlyunderstood. A significant proportion of HNSCCs harbormutations in FA DNA repair genes. Furthermore, individualswith germline loss-of-function mutations in FA genes areuniquely predisposed to aggressive HNSCC development.Herein, we show that loss of the FA pathway in HNSCC cellsstimulate plasma membrane reorganization and tumor cellinvasiveness in vitro and in vivo, that is reliant upon NHEJ-associated DNA-dependent protein kinase (DNA-PK) andRac1 GTPase. These findings implicate this important DNArepair pathway in the suppression of advanced tumor pheno-types, and identify new treatment options tailored to FA-deficient HNSCCs.

FA Loss Drives Tumor Cell Invasion

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produced in 293T cells using an established protocol in theHanenberg laboratory. Cells were transduced at 30% to 50%confluence for a total of 4 hour for retroviruses or 8 hours forlentiviruses in a final concentration of 8 mg/mL Polybrene. ForMIEG-based vectors, cells were sorted on GFP. Following sortingand replating, cells were then transduced with either nontarget-ing or FANCD2-specific shRNA. For the lentiviral vectors, cellswere selected and carried in 1.25 mg/mL puromycin. A fusionconstruct between EGFP and FANCD2 cDNA was generated andverified by direct sequencing (H. Hanenberg, Unpublished obser-vations). The Rac1 inhibitor NSC23766 was a generous gift byDr. Yi Zheng (Cincinnati Children's Hospital Medical Center,Cincinnati, Ohio).

Cell-cycle measurementsCells were seeded for 24 hours and then either left untreated or

treated for 24 hours with 0.25 mg/mL or 0.5 mg/mL melphalan(Sigma-Aldrich). Cells were trypsinized and 5 � 105 cells werewashed and prepared to assess BrdUrd incorporation according tothe manufacturer's instructions (APC BrdU Flow Kit, BD Phar-mingen). The cells were pulsed with 10 mmol/L BrdUrd for 45minutes. Cell-cycle profiles were detected using 7AAD, withsamples acquired on a BD FACSCanto instrument (BD Bios-ciences), and the results were analyzed using FlowJo (Treestar).

RNA sequencingIndividual samples were aligned to the Hg19 genome using

TopHat v1.4.1. Gene quantificationwas performedwithCufflinksv2.0.0 with the "-G -u -b" parameters and the Ensembl genemodel. Gene-level quantifications were used throughout. Datawere analyzed with GeneSpring 12.6.1 NGS, filtering to removeduplicates, and filtering on post alignment readmetrics to removereads with mapping quality below 40, or with more than onematch to genome, or failing vendor QC. Quantification wascarried out withDESeq as normalization algorithm and thresholdnormalized counts to 1, baseline to median of all samples. Wefiltered to remove genes with fewer than 3 RPKM in at least onesample. Differential expression was determined with the AudicClaverie test (P < 0.05, FC > 1.5). Functional enrichment analysiswas carried out with ToppGene and cytoscape figures were madeusing ToppCluster.

Western blot analysisWhole-cell protein extracts were harvested and lysed using 1X

Laemmli buffer. Blotting was performed as previously described(21). Antibodies used were rabbit polyclonal FANCA (CascadeBioscience), rabbit polyclonal FANCD2 (Novus), rabbit poly-clonal FANCJ (Novus), mouse monoclonal DNA-PKcs (Abcam),rabbit polyclonal phospho-DNA-PKcs (S2056; Abcam), and actin(Seven Hills Bioresearch). For detection of HPV16 E7, a primaryantibody mix of mouse monoclonal anti-16E7 antibody [1:150,8C9 (Invitrogen) and a 1:200 dilution of ED17 (Invitrogen)] wasused.

Three-dimensional epithelial raft culturesOrganotypic rafts were generated as described previously

(20). Briefly, a total of 1 � 106 UM-SCC1 cells were platedon a collagen matrix with embedded feeder fibroblasts. Expo-sure to the liquid–air interface resulted in the generation ofstratified epithelium with differentiation properties that reflect

its natural human counterpart. The tissue was fixed in 2%paraformaldehyde after 16 days of growth, embedded inparaffin, sectioned, and morphologically examined by hema-toxylin and eosin staining.

Immunofluorescence and DIC imagingImmunofluorescence was performed as previously described

(20) using the Leica DM 5000B. Cells were plated onto coverslipsinto 6-well plates at equal cell number and collected after 24hoursof incubation. Cells were fixed in 4% paraformaldehyde andrinsed in PBS prior to staining with Rhodamine phalloidin(1:200; Invitrogen) and DAPI to stain all nuclei. Differentialinterference contrast (DIC) images were acquired at the time ofPhalloidin imaging. Cells on coverslips were treated overnightwith 20 mmol/L NSC23766 prior to fixation.

Invasion assaysBioCoat Matrigel transwell invasion assays were performed

as per manufacturer's instructions (BD Biosciences). Cells wereresuspended in serum-free media in the top chamber andallowed to invade through Matrigel to serum-containing mediain the bottom chamber. For UMSCC-1, 1.5 � 105 cells wereseeded per transwell while for UMSCC-6 and UMSCC-47, 2.5�105 cells were seeded. Invasion was allowed to proceed for 22hours prior to fixation in methanol and staining with Giemsa.Total numbers of invaded cells were quantified for each trans-well using ImageJ.

Migration assaysCostar transwell migration assay were performed as per man-

ufacturer's instructions (Corning Incorporated). Briefly, cells wereresuspended in serum-freemedia in the top chamber and allowedto migrate through the polycarbonate membrane to the serum-containingmedia located in the bottom chamber. A total of 1.5�105 UM-SCC1 NTsh and D2sh cells were seeded per transwell.Migration was allowed to proceed for 16 hours prior to fixation inmethanol and staining with Giemsa. The total number of migrat-ing cells was quantified for each transwell using ImageJ. Sixindependent migration assay experiments were performed.Graphs were generated using GraphPad Prism 6, and a pairedstudent t test was applied to determine significance.

Chicken chorioallantoic membrane assaysFertilized White Leghorn chicken eggs were incubated while

rotating at 37�C in a humidified atmosphere (>60% relativehumidity). After 48 hours, 0.5 mL of albumin was removed fromeach egg, and the eggs were placed into a nonrotating incubationchamber at 37�C in a humidified atmosphere. After 48 hours, theeggs were windowed to expose the chorioallantoic membrane(CAM), vasculature, and viable embryo. To evaluate tumor cellinvasion, a thinly sliced pipette ring was placed on top of thechorion layer of the membrane and a 25-mL suspension of500,000 UM-SCC1 cells that were either FA-deficient or -profi-cient and embedded in Matrigel were pipetted into the ring ontothe membrane. Egg shell windows were covered with scotch tapeand the eggs were returned to the nonrotating incubator. After anadditional 72 hours of incubation, CAMs with tumor cells wereharvested, fixed in 4% paraformaldehyde, processed, embeddedin paraffin blocks, and sectioned. Five-mm sections were utilizedfor standard hematoxylin and eosin staining.

Romick-Rosendale et al.

Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research2064




Rac1 activity assayThe Active Rac1 Pull-Down and Detection Kit (Thermo Scien-

tific) was used to detect active Rac1 in the absence and presence ofDNA-PKcs inhibitor NU7026 (Tocris Bioscience). Briefly, cellswere plated at equal cell number and allowed to adhere. Cellswere treated with either vehicle or 10 mmol/L NU7026 for 24hours. Prior to harvesting, cells were exposed to a 30-minute pulseof bleomycin (10 mg/mL). Cells were harvested and lysed asdirected by the kit and protein concentrations were determinedusing thePierce BCAProteinAssayKit according tomanufacturinginstructions (Thermo Scientific). Equivalent amounts of proteinwere loaded onto the column and the protocol outlined withinthe assay kit was followed. Following elution of activated Rac1from the column, Western blot analysis for Rac1-GTP was per-formed using the anti-Rac1mousemAb (1:1,000) provided in theassay kit. GTPgS (positive control) and GDP (negative control)were used as controls in the pull-down assays.

Statistical analysisStatistical significance was determined using two-way ANOVA

with Sidak post hoc tests using ana value of 0.05 for all calculationsusing phalloidin projection data. All other significance was deter-mined using a Student t test. Statistical analyses were performedusing GraphPad Prism 6 software.

ResultsClassical FA phenotypes result from FA knockdown in HPV-positive and negative HNSCC cancer cells

We have recently analyzed whole-exome sequencing data oftherapy-na€�ve HNSCCs (26) and found that a significant propor-tion of sporadicHNSCCs harbor somaticmutations in FA and FA-related genes (3). To confirm thiswith sequencing data fromotherpatient cohorts, we queried The Cancer Genome Atlas (TCGA;ref. 27; n¼ 306), as well as whole sequencing data from a set of 34primary human HNSCCs. A total of 11.1% and 17.6%, respec-tively, of such tumors harbored non-synonymous (N.S.) muta-tions in the 15 FA genes that had been identified at that time:FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE,FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN,FANCP, and FANCO (Fig. 1A). To explore possible biologic effectsof FA loss in systems mimicking HPVþ and HPV� HNSCC, wecreated the following knockdown models using previously pub-lished lentiviral shRNA vectors (28). First, an HPV-negative UM-SCC1 cell line was transduced with HPV16 E7, and subsequentlyknocked down for FANCD2. Second, the same cell line wasknocked down for FANCA, FANCD2, and FANCJ in the absenceof E7. Third, a secondHPV� cell line, UM-SCC6, and anHPVþ cellline, UM-SCC47, were similarly knocked down for FANCA andFANCD2. Western blot analyses verified efficient FA proteindepletion for all HNSCC cell lines transduced with either FANCA,-D2, and -J shRNA expression vectors (Fig. 1B and SupplementaryFig. S1). Cells transduced with the non-targeting (NTsh) controlshRNAdid not result in FA depletion (as expected). To ensure thatFANCD2 depletion induced classical FA phenotypes, DNA cross-linker sensitivity of knockdown versus control cells was quanti-fied. FA lymphoblasts and fibroblasts predictably respond tomelphalan exposure with a G2–M cell-cycle arrest (28, 29). Asexpected, FANCD2-deficient UM-SCC1 cancer cells treated withmelphalan also displayed an increase in the proportion of cells inG2–Mwhen compared with control NTsh cells (Fig. 1C). FANCJ-

deficientUM-SCC1 cells responded to themelphalan treatment ina similar manner when compared with the FANCD2-deficientcancer cells (data not shown). UM-SCC6 andUM-SCC47HNSCCcell lines depleted for FA proteins were similarly sensitive tomelphalan (data not shown); thus, FA knockdown induces char-acteristic hallmarks of FA in HPV-positive and negative HNSCCcells. To determine the consequences of FA loss onUM-SCC1 cellsgrown under standard culture conditions, we quantified prolif-eration rates based on BrdUrd incorporation (Fig. 1D). FANCD2and FANCJ loss did not increase the proliferation of UM-SCC1cells, but either decreased or did not change proliferation (Fig. 4Dand Supplementary Fig. S1B). InHPV16E7–expressingUM-SCC1cells, FANCD2 loss did not affect cellular proliferation (Supple-mentary Fig. S1C). Similar decreases or no effectswere observed inUM-SCC6 and UM-SCC47 cell lines (data not shown). Takentogether, FA knockdown in HNSCC cell lines did not alter andsometimes reduced cellular growth under standard two-dimen-sional culture conditions, and conferred classical FA phenotypesin the presence of DNA crosslinkers.

FA loss deregulates epithelial HNSCC morphology andcytoskeletal organization

To assess global expression patterns induced by perturbing theFA pathway, we performed RNA sequencing (RNASeq) of theabove HPV16 E7–positive UM-SCC1 cells, either depleted forFANCD2 or control transduced (Fig. 1B). A number of genes weredifferentially expressed in the FANCD2sh compared with theNTsh HNSCC cells (Supplementary Table S1). Following identi-fication of genes with significantly altered expression, ToppGenewas used to perform gene ontology analysis and redundancieswere eliminated. A large number of gene ontology hits wereidentified (Supplementary Table S2). Figure 2A lists top candidatebiologic processes which, based on gene expression alterations,may be regulated by FANCD2 loss. In line with our previouslypublished data wherein FA pathway loss impaired keratinocytedifferentiation in organotypic epithelial raft models, the expres-sion of a number of differentiation-associated genes was reducedby FANCD2 knockdown (Supplementary Tables S1 and S2).Importantly, this was not accompanied by the induction ofproliferative gene signatures, in agreement with a lack of prolif-erative gains in the above FA HNSCC cell populations understandard conditions and with previously published data usingpatient-derived cell lines (Fig. 1D and data not shown; ref. 20).Among the top biologic processes were cellular motility andinvasion. Increased expression of the intermediate filamentvimentin was noted which has already been linked to a numberof cancers as a mesenchymal marker for invasive potential (30).Vimentin induction in FAHNSCC cells was validated by qRT-PCR(Fig. 2B). In contrast, the expression of other genes involved inclassical epithelial-to-mesenchymal transition (EMT), such as E-cadherin, Snail1, or Twist1 was not altered. Perhaps related to theobserved regulation of genes involved in cellular motility, weobserved a marked alteration in the expression of genes involvedin cellular adhesion and locomotion (Fig. 2A). Further morpho-logic examination of FA-depleted UM-SCC1 cells by DIC micros-copy revealed a clear difference in cellular shape and spatialarrangement. FANCD2 and FANCJ knockdown in the UM-SCC1cell line impaired SCC epithelial morphology: cells physicallyseparated from each other but remained connected by intercel-lular projections that were largely absent in the control HNSCCcells (Fig. 2C). To further investigate the intercellular projections,






the F-actinmarker phalloidin was used to stain actin filaments. Asshown inFig. 2D, FANCD2andFANCJ knockdown cells tended toseparate butmaintained long intercellular projections, in contrastto control cells which harbored tight epithelial cell–cell contacts.Taken together, FA pathway loss in HNSCC cells deregulatedtranscriptomes associated with cellular motility and adhesion,and this was accompanied by morphologic and cytoskeletal

responses including the formation of intercellular protrusions.To assess the ability of FA-deficient versus proficient tumor cellsto grow as 3D tissues, we engineered 3D organotypic epithelialtumor rafts from NTsh or FANCD2sh UM-SCC1 cells (Fig. 2E).Interestingly, while FA-deficient cells were able to grow andassemble into 3D tissue, we noted their occasional presence inthe underlying collagenmatrix, a feature which was not shared by

Figure 1.Generation of FA-deficient, HPV-positive and negative, HNSCC cell models. A, the somatic mutations table (MAF file) for head and neck squamous carcinoma(HNSC) samples was obtained from TCGA data portal and from an independent cohort of sporadic HNSCC tumors from Ohio State University (OSU).Analysis ofmutational data from 306 (TCGA) and 34 (OSU) sporadic HNSCC tumors determined that 11.1% and 17.6%, respectively, harbored non-synonymous (N.S.)mutations in one of 16 FA genes, respectively. B, UM-SCC1 HNSCC (HPV16 E7-positive and –negative) cells were knocked down for FANCA, FANCD2,and FANCJ, followed by Western blot analysis for verification of protein depletion. C, FA knockdown by shRNA transduction leads to classical FA phenotypes.UM-SCC1 cells were treated with melphalan, and subjected to flow cytometry–based cell-cycle analysis. The numbers listed indicate percentages of cellsin G2–M following melphalan exposure. FANCD2-deficient cells were increased for the proportion of cells in G2–M when compared with the NTsh control cellpopulation. D, BrdUrd incorporation in FANCD2- and FANCJ-deficient compared with control UM-SCC1 cells reveals a slight decrease in proliferation.






the FA-proficient counterparts. We therefore tested the possibilitythat FA-deficient cells harbored increased invasive properties.

FA pathway loss in HNSCC cells promotes cellular invasionTumor cell invasiveness was determined directly using Matri-

gel transwell assays. Both HPV16 E7þ and E7� UM-SCC1 cellswere significantly more invasive when knocked down forFANCD2 compared with empty vector controls (Fig. 3A andB). Invasion was not an indirect consequence of increasedproliferation (Fig. 1D, data not shown) or increased cellularadhesion (Supplementary Fig. S2A). Similarly, FANCA andFANCJ knockdown also stimulated invasion in UM-SCC1 cells(Fig. 3C). To rule out any off-target effects for the lentiviralknockdown approach, we next expressed a shRNA-resistantFANCD2 construct in FANCD2sh UM-SCC1 cells (Fig. 3D). Aspredicted, the introduction of EGFP-FANCD2 was sufficient to

rescue UM-SCC1 FANCD2 shRNA cells from invasion (Fig.3D). Expression of the EGFP-FANCD2 fusion protein wasconfirmed by Western blot analysis on the right. Finally,FANCA and FANCD2 knockdown in UM-SCC6 and UM-SCC47cells also stimulated tumor cell invasion (Fig. 3E and F). Asexpected, the increased invasiveness of the cancer cells corre-lated with increased motility seen by standard migration assays(Supplementary Fig. S2B). To further assess the invasive prop-erties of FA-deficient versus -proficient tumor cells, we utilizedCAM assays (Supplementary Fig. S3). Interestingly, FA-deficientcells were occasionally able to invade into the chorion mem-branes of living chick embryos, and more specifically appearedto invade as cell clusters; however, invasion was never observedwith their FA-proficient counterparts. Taken together, FA lossleads to a dramatic increase of invasive capacity and motility inHPV-positive and negative HNSCC cells.

Figure 2.FA loss in HNSCC cells induces cytoskeletal andmorphological alterations. A, RNASeq and subsequent ToppGene analysis of FANCD2-deficient versus control cellsreveals cellular processes with significantly altered gene expression. A list of the top candidate biologic processes is shown. B, verification of RNASeq geneexpression results was performed on a select number of genes using qRT-PCR. qRT-PCR also revealed that some classical EMTmarkers were not uniformly elevatedin FA-deficient HNSCCs, with the exception of vimentin. C, morphological examination of FA-depleted HNSCC cells compared with FA-proficient controlsby DIC microscopy shows differences in spatial arrangement and cell shape as well as increased intercellular protrusions in FANCD2- and FANCJ-deficient cells.D, intercellular projectionswere examined further by stainingwith the F-actinmarker phalloidin and subsequent immunofluorescence experiments. Quantification ofintercellular projections was performed on three independent experiments and the errors bars represent SD. Asterisks indicate significance at � , P < 0.05;�� , P < 0.001; and �� , P < 0.0001 (ANOVA). E, UM-SCC1 epithelial raft culture shows UM-SCC1 FANCD2sh knockdown cells in the underlying collagenmatrix as compared with UM-SCC1 NTsh control cells.






Invasion in response to FA loss requires DNA-PK activityFA cells exhibit characteristic sensitivity to DNA crosslinkers,

and defects in error-free DNA repair by homologous recombina-tion (HR). These defects are accompanied, under some circum-stances, by a corresponding increase in the activity of error-proneNHEJ pathway components (12, 13). NHEJ requires the activa-

tion of the catalytic subunit of DNA-PKcs and subsequent autop-hosphorylation on serine 2056 (31). To probe a possible func-tional involvement for DNA-PKcs signaling in FA-associatedinvasion, we first determined whether FANCD2- and FANCJ-deficient UM-SCC1 cells harbored activated DNA-PKcs. We thenverified the functionality of a DNA-PK inhibitor NU7026 in this

Figure 3.FA loss in a panel of HPV-negative and positive HNSCC cells promotes invasion. A, UM-SCC1 cells expressing HPV16 E7 and knocked down for FANCD2, comparedwith control NTsh cells were plated to invade through Matrigel-coated transwell for 22 hours. Invasive cells were quantified and representative images areshown. B and C, UM-SCC1 cells knocked down for FANCD2, or for FANCA and FANCJ compared with control NTsh cells were plated to invade througha Matrigel-coated transwell for 24 hours. Invasive cells were quantified and representative images are shown. D, invasion mediated by FANCD2 depletioncould be repressed by the expression of a knockdown-resistant eGFP-FANCD2 fusion gene. UM-SCC1 cells were transduced with an eGFP-FANCD2 retroviralexpression vector, and sorted for GFP-negative cells as a control population, or sorted for cells that express eGFP-FANCD2. Western blot analysis confirmsexpression of the fusion protein in GFP positive, but not in GFP-negative cells. Transwell assays demonstrate EGFP-FANCD2 mediated rescue from invasionthat is driven by FANCD2 loss. E and F, UM-SCC6 and UM-SCC47 knocked down for FANCA and FANCD2 were similarly used for transwell invasion assayscompared with controls.






system to probe the importance of DNA-PK activity for invasion(Fig. 4A). Control cells and their FANCD2 and FANCJ-depletedcounterparts were subjected to a pulse of bleomycin to stimulateDNA damage signaling, and the cells were then treated withvehicle or with the DNA-PKcs inhibitor NU7026. Cells wereanalyzed for activated DNA-PKcs phosphorylated on serine2056. FANCD2 and particularly FANCJ loss stimulated DNA-PKcs phosphorylation compared with control (Fig. 4A, lanes1–3), and autophosphorylation was completely eliminated byNU7026. Next, we determined whether DNA-PKcs activationwas functionally important for FA-deficient cancer cell invasion.DNA-PKcs inhibition by NU7026 suppressed the invasive phe-notype in FANCD2shUM-SCC1 cells (Fig. 4B), but did not impaircellular viability (Fig. 4C). Importantly, DNA-PKcs inhibitionalso reduced control cell invasion under these conditions, thusindicating that DNA-PK activation contributes to HNSCCcell invasion. The ability of NU7026 to suppress invasion inFA-deficient cells was also observed in HPVþ FANCD2sh (Sup-plementary Fig. S4A) and FANCAsh (Supplementary Fig. S4B)UM-SCC47 cells.

Invasion in response to FA loss requires Rac1 GTPaseactivity

Ras-related small GTPase Rho/Rac/CDC42 signaling pathwaysare key players in cellularmorphology and invasion (32). Becausethe activation of Rac1 was reported to produce intercellularprojections similar to the ones noted for FA-deficient cells (Fig.2; refs. 33, 34), we carried out Rac1-GTP pull downs to firstdetermine whether Rac1 is activated in FA HNSCC cells, and ifso, to probe a possible role for DNA-PKcs in its regulation.FANCJsh knockdown cells were chosen based on the observationthat DNA-PK activation was maximal in FANCJ when comparedwith FANCD2knockdown cells in Fig. 4A. Interestingly, FANCJsh-transduced UM-SCC1 cells showed increased Rac1 activity whencompared with their NTsh control–transduced counterparts (Fig.5A, compare lanes 1 and 3). Treatment with DNA-PKcs inhibitordid not affect Rac1 activity in the control NTsh cells (Fig. 5A,compare lanes 1 and 2), but significantly lowered Rac1 activity inFANCD2sh and FANCJsh cells (Fig. 5A, compare lanes 3 and 4).Taken together, these results suggest that Rac1 activation down-stream from FA loss is, at least in part, dependent uponDNA-PKcsactivity. We next sought to examine the requirement for Rac1GTPase activity in the characteristic cytoskeletal reorganizationand invasion of FA-deficient HNSCC cells. We utilizedNSC23766, a small molecule that has been shown to specificallyinhibit Rac1 structural and functional activity but does not affectthe activity of other Rho-related small GTPases (35, 36). Inter-estingly, NSC23766 reduced the number of intercellular

Figure 4.DNA-PK activity promotes cellular invasiveness. A, FA loss stimulated DNA-PKcs phosphorylation in UM-SCC1 cells. Western blot analysis showsactivation of DNA-PKcs by phosphorylation at Ser2056 following FANCD2and FANCJ loss. The functionality of the DNA-PKcs inhibitor, NU7026, wasverified in the same cells. Cells were treated with either DMSO or NU7026 for24 hours and exposed to a short 30-minute burst of bleomycin. B, DNA-PKcsactivation contributes to invasion. Cells were control transduced or knockeddown for FANCD2, and the number of invasive cells was quantified followingovernight incubation. Experiments were carried out in duplicate and SD wascalculated. C, DNA-PKcs inhibition does not affect cell viability as is shown byvisual examination of the cells.











protrusions and stimulated cell–cell adhesion in FANCD2- andFANCJ-deficient cells compared with control UMSCC1 cells asassessed by DIC (Fig. 5B, top) and phalloidin staining (Fig. 5B,bottom panel and quantification below). To determine whetherNSC23766 was also capable of suppressing cellular invasion inresponse to FANCD2 loss, FA-proficient and -deficient HNSCCcells were treated withNSC23766 or vehicle over the course of thetranswell assay. Indeed, cellular invasion following FANCD2 losswas repressed by NSC23766 which did not significantly affectcontrol cell invasion (Fig. 5C) or cellular growth (Fig. 5D).NSC23766 could also suppress invasion in FA-deficient HPVþ

UM-SCC47 cells (Supplementary Fig. S4C). Together, these datademonstrate that FA loss and subsequent DNA-PK activationpromote Rac1 activity to induce cytoskeletal aberrations andinvasive tumor phenotypes in HNSCC cells (see Fig. 5E for aworking model).

DiscussionEach year, approximately 40,000 new patients are diagnosed

with head and neck cancer, predominantly HNSCC, in the UnitedStates, and this number continues to rise. Two main causativefactors in the majority of oral, oropharyngeal, and laryngealcarcinomas are smoking and alcohol use; however, a growingpercentage of these head and neck cancers, approximately 25% atpresent, have been attributed to HPV infection (37). Cellular andmolecular characteristics ofHPV–positive and –negativeHNSCCsare still being explored, although these are distinct biologic andclinical entities (38). Herein, we link the loss of the FADNA repairpathway in HNSCC cells with stimulated invasive potentialregardless of HPV status, and uncover novel roles for DNA-PK(unrelated to DNA repair) in promoting Rac1 activation andinvasive behavior.

Locoregional dissemination of malignant tumor cells is asso-ciated with poor outcome, and is crucially dependent on themigratory and invasive properties of the cancer cell. Tumor cellmigration requires the formation of protrusions, leading edgeattachment to the surrounding extracellularmatrix, contraction ofthe cell to pull the cell body towards the leading edge, anddetachment of the trailing edge. FA-deficient HNSCC cells dis-played expression changes for genes known toplay key roles in cellmotility, cell–cell adhesion and locomotion. Furthermore, weobserved the presence of pronounced protrusions in FA-deficientcells compared with their FA-proficient counterparts and notedaltered cell morphology. Together with these morphologicphenotypes, we showed a significant increase in motility andinvasion of FA-deficient cells compared with their FA-proficientcounterparts. Invasion was induced by the loss of either upstream

(FANCA), central (FANCD2), and downstream (FANCJ) com-ponents of the FA pathway, and was reversible by the reintro-duction of a knockdown-resistant construct. Together, thesedata support a scenario whereby the intact FA DNA repairmachinery suppresses the transition of transformed to invasiveHNSCC phenotypes.

DNA crosslink processing utilizes multiple repair pathwayswhose coordination appears to be a major function of the FApathway. Recently, two reports have demonstrated that FA lossengages the DNA damage sensor kinase DNA-PK, a process that islikely followed by aberrant DNA repair by NHEJ and resultingcharacteristic FA chromosome pathologies. As such, FA proteinsmight govern the decision to channel double strand breaks (DSB)into homologous recombination (HR) in favor of the competingDNA-PK–associated error-prone NHEJ pathway (12, 13). Weshow that FA loss in HNSCC cells increases the levels of active,autophosphorylated DNA-PKcs, and functionally implicate thisDNA damage sensor in advanced HNSCC for the first time. Whilethe nuclear role of DNA-PK in DNA repair is well characterized,noncanonical activities have emerged as well. DNA-PK has beenimplicated in inflammation through the phosphorylation of NF-kB1 (39), and in metabolic gene regulation through interactionwith and phosphorylation of USF-1 (40). Furthermore, a numberof novel cytoplasmic DNA-PK substrates were published recentlythat participate in cytoskeletal regulation (41). These includemembers of the 14-3-3 protein family, vimentin, and desmopla-kin. The authors showed that DNA-PK activation decreasedmotility in melanoma cell lines, in contrast to increased invasionin our HNSCC cells. Thus, whereas advanced cancer phenotypesare regulated by DNA-PK in both systems, the direction of theobserved regulation may be cell type specific. Such differencesmight reflect significant complexity in the regulation of cytoskel-etal components by DNA-PK. Further analysis identified a novellink between DNA-PKcs and Rac1 signaling in FA HNSCC cellswherein a specific DNA-PK inhibitor decreased active Rac1-GTPprotein levels. One possible mechanismmight be supported by aprevious finding that DNA-PKcs can physically interact with theCDC42/Rac1 guanine exchange factor, ARHGEF6, in ovariancancer cells (42). In addition, growing evidence supports correla-tions between DNA damage signaling and Rac1 activity (43–45),although the mechanisms and functional relevance of pathwaycross-talk are largely unclear. Whether DNA-PKcs activates Rac1directly through guanine nucleotide exchange factors (GEF) orindirectly through signal transduction cascades is currently underinvestigation.

Rac1 is a well-known regulator of the cellular actin cytoskele-ton, adhesion, barrier function, and migration. Like other mem-bers of the Rho family, Rac1 cycles between GDP-bound inactive

Figure 5.Morphologic aberrations and invasive tumor phenotypes triggered by FA loss are dependent upon Rac1 activation. A, Rac1-GTP pulldown assays show thatDNA-PK inhibition attenuates Rac1 activation in FA-deficient cells. All lanes are from the same Western blot analysis. B, the small-molecule Rac1 inhibitorNSC23766 was utilized at 20 mmol/L concentrations that did not affect cellular growth. To assess the effects of Rac1 inhibition on aberrant morphology and tumorcell invasion triggered by FA loss, UM-SCC1 cells were plated at equal densities and imaged by DIC microscopy prior to and following an 18-hour exposure to20 mmol/L NSC23766 (left). NSC23766 treated and untreated UM-SCC1 cells were stained for phalloidin and imaged (right). The reduction in intercellularmembrane projections following treatment with the Rac1 inhibitor is quantified and included below the images. C, invasion of NSC23766 or vehicletreated FA-deficient versus -proficient UM-SCC1 cells was evaluated by transwell assays. Experiments were carried out in duplicate and SD was calculated.D, UM-SCC1 exposure to NSC23766 at 15 mmol/L and 20 mmol/L concentrations did not affect cellular fitness and growth for the duration of the invasionassays, 22 hours. E, a working model depicts the consequences of FA deficiency in HNSCC cells. Loss of FA pathway function results in increased DNAstress and activation of the DNA-PK sensor kinase through autophosphorylation on S2056. DNA-PK activity is directly or indirectly required for downstreamRac1 activation, and subsequent Rac1-dependent SCC cell invasion.






and GTP-bound active states (46). These GTPases are controlledby two classes of regulatory molecules: activating GEFs, andrepressive GTPase-activating proteins (GAP). Previously pub-lished work found that Rac1 was required for Kras-mediatedtumorigenesis in skin epithelium and the lung (47, 48). Moreimportantly, Rac1 activity strongly is associated with cell motilityand tumor metastasis (49, 50). We found that both cell mor-phology (protrusion formation) and cellular invasion weredependent upon Rac1 activation, and show a reversion of theFA-deficient HNSCC cell phenotype to a more epithelial-likemorphology following inhibition of Rac1. We also observed asignificant decrease in cancer cell invasion of FA-deficient cellsfollowing treatmentwith theRac1 inhibitor. Taken together, thesefindings link the loss of the FA pathway with increased Rac1activation and downstream cytoskeletal aberrations and aggres-sive invasive potential. Of importance, Rac1 inhibitors are apotential novel therapeutic option for sporadic HNSCC carryingFA mutations, as well as an alternative, non-genotoxic treatmentfor HNSCC in patients with FA.

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

Authors' ContributionsConception and design: L.E. Romick-Rosendale, E.E. Hoskins, L.M. PrivetteVinnedge, S.I. WellsDevelopment of methodology: L.E. Romick-Rosendale, E.E. Hoskins, G.D.Foglesong, S.A. BrugmannAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.E. Romick-Rosendale, E.E. Hoskins, L.M. Privette

Vinnedge, G.D. Foglesong, M.G. Brusadelli, P. Lambert, R.J. Kimple, E.L. Virts,M.L. GillisonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.E. Romick-Rosendale, E.E. Hoskins, G.D. Fogle-song, M.G. Brusadelli, S.S. Potter, K. Komurov, S.I. WellsWriting, review, and/or revision of the manuscript: L.E. Romick-Rosendale,E.E. Hoskins, L.M. Privette Vinnedge, G.D. Foglesong, P. Lambert, R.J. Kimple,S.I. WellsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.E. Romick-Rosendale, E.E. HoskinsStudy supervision: E.E. Hoskins, S.I. WellsOther (provided new innovative reagents): H. Hanenberg

AcknowledgmentsThe authors thank Dr. James Lessard of Cincinnati Children's Hospital

Medical Center (CCHMC) and Seven Hills Bioresearch (Cincinnati, OH) forhis gift of the C4 pan-actin mAb used in this work. The authors also thank Drs.Stella Davies, Parinda Mehta, and Kasiani Myers of CCHMC and the CincinnatiChildren's Fanconi Anemia Comprehensive Care Center for thoughtful exper-imental guidance and discussion.

Grant SupportThis work was supported in part by NIH award RO1 CA102357 (to S.I.

Wells). H. Hanenberg is supported by the Lilly Foundation Physician/Scientistinitiative.

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 September 12, 2015; revised November 9, 2015; accepted Novem-ber 10, 2015; published OnlineFirst November 24, 2015.

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2016;22:2062-2073. Published OnlineFirst November 24, 2015.Clin Cancer Res Lindsey E. Romick-Rosendale, Elizabeth E. Hoskins, Lisa M. Privette Vinnedge, et al. SignalingCells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Defects in the Fanconi Anemia Pathway in Head and Neck Cancer

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