smad7 promotes healing of radiotherapy- induced oral ......translational cancer mechanisms and...

Translational Cancer Mechanisms and Therapy

Smad7 Promotes Healing of Radiotherapy-Induced Oral Mucositis without CompromisingOral Cancer Therapy in a Xenograft Mouse ModelJingjing Luo1,2, Li Bian2,3,4, Melanie A. Blevins5, Dongyan Wang2,4, Chao Liang2,DanfengDu2, FanglongWu1,2, Barry Holwerda6, Rui Zhao5, DavidRaben7, Hongmei Zhou1,Christian D. Young2,4, and Xiao-Jing Wang2,4

Abstract

Purpose: We previously reported preventive and therapeu-tic effects of Smad7, a multifunctional protein, on radiother-apy (RT)-induced mucositis in mice without promotinghuman oral cancer cell survival or migration in vitro. Thecurrent study aims to determine whether a Smad7-basedbiologic can treat existing oral mucositis during radiotherapyfor oral cancer and whether this treatment compromisesRT-induced cancer cell killing in neighboring oral cancer.

Experimental Design: We transplanted human oral cancercells into the tongues of mice and applied craniofacial irradi-ation to simultaneously kill tumor cells and induce oralmucositis, thus modeling RT and mucositis in oral cancerpatients. We topically applied a recombinant human Smad7protein fused with the cell-penetrating Tat tag (Tat-Smad7) tothe oral mucosa of tumor-bearing mice post RT when oralmucositis began to develop.

Results: Topically applied Tat-Smad7 penetrated cells inboth the oral mucosa and oral cancer, attenuatingTGFb and NF-kB signaling as well as inflammation at bothsites. Tat-Smad7 treatment alleviated oral mucositis withreductions in DNA damage and apoptosis in keratinocytes,but increased keratinocyte proliferation compared withvehicle-treated mucositis lesions. In contrast, adjacent oralcancer exposed to Tat-Smad7 did not show alterations inproliferation or direct DNA damage, but showed increasedoxidative stress damage and apoptosis compared with tumorstreated with vehicle.

Conclusions: Our results suggest that short-courseTat-Smad7 application to oral mucositis promotes its healingbut does not compromise the cytotoxic effect of RT on oralcancer and has context-specific effects on oral mucosa versusoral cancer.

IntroductionOral mucositis, painful ulceration of the oral mucosa, is a

common toxic effect of radiotherapy (RT) for bone marrowtransplant, craniofacial RT for head and neck cancer (HNC), andchemoradiotherapy for all cancer types (1). Approximately 70%of HNC patients develop oral mucositis during treatment, whichcan be severe enough to cause reduction in oral intake or prema-ture withdrawal from cancer treatment (1, 2). New RT technol-ogies such as intensity-modulated RT and stereotactic body RT(SBRT) more precisely target cancer lesions and spare morenormal tissue. However, these treatments do not reduce acute

toxicity in the oral mucosa of HNC patients (3), because neigh-boring mucosa is still exposed to high intensity RT, albeit at alower dose than the cancer. Further, certainpatients are at high riskfor oralmucositis regardless of treatment regimen (4). Palifermin,recombinant human keratinocyte growth factor (KGF), is the onlyFDA-approved targeted therapy for preventing oral mucositis inbone marrow transplant patients (4% of the at-risk population),but it has no effect on existing mucositis (5). Palifermin clinicaltrials in oral cancer patients showed modest prevention of severeoral mucositis (6, 7). A major challenge in treating oral mucositisis to repair ulceratedmucosawithout promoting cancer, as growth

1State Key Laboratory of Oral Diseases, Department of Oral Medicine, WestChina Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, P.R.China. 2Department of Pathology, University of Colorado Anschutz MedicalCampus, Aurora, Colorado. 3Department of Pathology, the First AffiliatedHospital of Kunming Medical University, Kunming, P.R. China. 4Allander Bio-technologies, LLC, Aurora, Colorado. 5Department of Biochemistry and Molec-ular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Color-ado. 6Mtibio, LasVegas, Nevada. 7Department of RadiationOncology, Universityof Colorado Anschutz Medical Campus, Aurora, Colorado.

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

J. Luo and L. Bian contributed equally to this article.

Current address for C. Liang: School of Basic Medical Sciences, ShanghaiUniversity of Traditional ChineseMedicine, Shanghai, P.R. China; Current addressfor D. Du: Henan Key Laboratory for Esophageal Cancer Research and

Department of Pathology of Basic Medical College, Zhengzhou University,Zhengzhou, Henan, P.R. China.

Corresponding Authors: Xiao-Jing Wang, University of Colorado AnschutzMedical Campus, 12800 East 19th Avenue, Mailstop 8104, Aurora, CO 80045.Phone 303-724-3001; Fax: 303-724-4730; E-mail: [email protected];Hongmei Zhou, State Key Laboratory of Oral Diseases, Department of OralMedicine,West ChinaHospital of Stomatology, SichuanUniversity, No. 14, Sec. 3,Renmin Road South, Chengdu, Sichuan 610041, P.R. China. Phone: 86-028-85503480; Fax: 86-028-85582167; E-mail: [email protected]; and ChristianD. Young, Allander Biotechnologies, LLC, 12635 E. Montview Blvd #100, Aurora,CO 80045. Phone: 720-266-7063; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-18-1081

�2018 American Association for Cancer Research.

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factors (e.g., KGF) and their receptors are often overexpressed incancer cells. To date, there is no FDA-approved drug to treat oralmucositis in cancer patients. Clinical trials for GC4419, a super-oxide dismutase mimetic, to treat oral mucositis in HNC patientsrevealed reductions in severe oral mucositis cases (8). GC4419requires i.v. infusions 1 hour prior to each of 14 doses of RT.Because it is difficult to predict which patients will develop severeoral mucositis, we sought to develop a therapeutic interventionthat is topically applied to existing oral mucositis and targetsmultiple pathogenic processes of oral mucositis.

Oral mucositis develops as a consequence of complex molec-ular and cellular pathobiology processes, leading to apoptosis ofbasal epithelia cells, loss of epithelial renewal, atrophic damage,excessive inflammation, and ulceration (9). Our previous studydiscovered that in addition to NF-kB activation, activated TGFbsignaling contributes greatly to RT-induced oral mucositis (10).TGFb is a potent growth inhibitor and apoptosis inducer forepithelial cells and a proinflammatory cytokine in oral mucosa(11). To dampen both TGFb and NF-kB pathways to treat oralmucositis, we developed a recombinant Smad7 protein thatcontains human Smad7 fused to the HIV-1 Tat protein transduc-tion domain. Tat-Smad7 protein rapidly penetrates cells uponcontact (10). Local delivery of Tat-Smad7 to mouse oral mucosashows prophylactic and therapeutic effects on RT-induced oralmucositis (10). A remaining question is how to effectively utilizethis therapeutic intervention without compromising RT-directedcancer cell killing. Smad7 can be either tumor suppressive ortumor promotive in different cancer types (12). For cancersoutside the oral cavity, the concern for potential systemictumor-promoting effects of Tat-Smad7 is relatively minorbecause orally administered Tat-Smad7 protein will be degradedas it passes through thedigestive tract.However, whenRT-inducedoral mucositis in oral cancer patients is treated with Tat-Smad7,the oral cancer will also be exposed to Tat-Smad7. Althoughincreasing epithelial cell proliferation and reducing apoptosis inoral mucositis promote wound healing, these effects could com-promise RT-directed cancer cell death. We have previously shownthat Tat-Smad7 increases survival of humanoral keratinocytes butnot head and neck squamous cell carcinoma (HNSCC) cells afterRT. Similarly, Tat-Smad7 increases migration of normal kerati-nocytes but not HNSCC cells (10). These in vitro data indicate thenecessity of in vivo testing of Tat-Smad7 using a model systemmimicking RT in human oral cancer with an RT dose sufficient to

induce oral mucositis. Although our previous study shows bothpreventive and therapeutic effect of Tat-Smad7 on oral mucositis(10), the current study focused on assessing the therapeuticeffect of Tat-Smad7 on promoting healing of existing oralmucositis in a model of oral cancer. We chose two HNSCCcell lines: FaDu, with a SMAD4 deletion representing alteredTGFb signaling found in tobacco-associated oral cancer (13),and UM-SCC-1, with wild-type SMAD4, representing intactTGFb signaling in tumor cells. We provide evidence that Tat-Smad7 application to RT-induced oral mucositis with neigh-boring oral cancer effectively promotes oral mucositis healingwhile paradoxically increasing tumor cell death in RT-treatedoral cancer.

Materials and MethodsCell lines

Two human oral cancer cell lines were used. FaDu was pur-chased from theATCC, andUM-SCC-1 (14)was provided byMTAthroughUniversity ofMichigan. Cells were authenticated by shorttandem repeat profiling (University of Colorado Cancer CenterProtein Production,Monoclonal Antibody, TissueCulture SharedResource) prior to our experiments. Cells were cultured in DMEMcontaining 10% FBS.

Generation and application of Tat-Smad7As previously described, we produced recombinant Tat-

tagged, human Smad7 protein as a GST fusion protein fromE. coli (10). Tat-Smad7 was cleaved from GST with PreScissionprotease followed by size exclusion chromatography to purifyTat-Smad7 protein as a monomer (Supplementary Fig. S1). Forin vivo treatment, 1 mg Tat-Smad7 (in 30 mL PBS containing 30%glycerol) was applied to mouse oral cavity; food/water waswithdrawn for 1 hour after treatment to minimize treatmentdisruption.

Orthotopic human oral cancer xenotransplantationAnimal experiments were performed in accordance to an Insti-

tutional Animal Care and Use Committee–approved protocol.We used female 8- to 10-week-old athymic nude mice (CharlesRiver Laboratories) as xenotransplantation recipients. Mice wereanesthetized with 80mg/kg ketamine and 12mg/kg xylazine (i.p.injection). Note that 105 oral cancer cells (FaDu or UM-SCC-1)were suspended in 20 mL of 50% PBS/50% Matrigel and injecteddirectly into the anterior/middle tongue using a syringe with30-gauge needle.

Craniofacial irradiation to oral tumor–bearing miceMice with tongue tumors (7 days after injection of cancer

cells) were cranially irradiated to induce oral mucositis using aRS2000 biological irradiator (Rad Source Technologies) aspreviously reported (10). Based on our previous observationthat a single 18 Gy dose has kinetics and severity of oralmucositis similar to 8 Gy x3 fractionated irradiation (10), wechose 18 Gy irradiation to reduce the death rate due to repeatedanesthesia for fractionated irradiation to oral tumor-bearingmice that had deteriorating health. Each mouse was anesthe-tized with 80 mg/kg ketamine and 12 mg/kg xylazine andplaced under a lead shield exposing only their head. The dayof irradiation was designated day 1. All animals were providedsoft food in addition to standard diet. On day 6, when micebegan to lose weight due to oral ulcer-associated reduced food

Translational Relevance

Oral mucositis, painful oral ulcerations, is one of the mostcommon toxic effects of radiotherapy (RT) and chemotherapyin cancer patients and can lead to therapy withdrawal or dosereduction. In head and neck cancer (HNC) patients, a majorchallenge in treating oral mucositis is to repair ulceratedmucosa without promoting neighboring cancer growth. Todate, there is no FDA-approved drug to treat oral mucositis inHNC patients. We studied a novel biological agent for treatingoral mucositis in a mouse model mimicking RT-inducedoral mucositis adjacent to oral cancer. Our preclinical datademonstrate the feasibility of a novel therapeutic approachfor treating existing oral mucositis without compromisingradiotherapy in neighboring cancer.

Smad7 Promotes Oral Mucositis Healing without Aiding Cancer

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intake, mice were divided into two groups (of equal weight andtumor size) and treated daily with Tat-Smad7 or vehicle. BrdU(125 mg/kg) was administered i.p. 2 hours before euthanasia.Mice were sacrificed and tongue samples collected on day 10 forpathologic evaluation and immunostaining.

Pathologic evaluation, immunostaining, and terminaldeoxynucleotidyl transferase dUTP nick end labeling assay

Tongue tissues were fixed in 10% formalin, embedded inparaffin, and cut in 5 mm sections. Tongue epithelium and tumorhistology were evaluated using hematoxylin and eosin (H&E)–stained slides. Open ulcer size, defined as complete loss ofepithelium, was measured using NIS (Nikon Intensilight) Ele-ments software by two independent investigators, and the resultsaveraged to determine ulcer size (mm). We performed immuno-histochemical and immunofluorescent staining as previouslydescribed (10). The primary antibodies used were guineapig anti-Keratin14 (1:200, Fitzgerald, 20R-CP200), chickenanti-Keratin5 (1:200, Biolegend, #905901), FITC-labeled anti-body to BrdUþ (BD Bioscience, 347583), rat anti-mouse CD45(1:50, BD Bioscience, 550539), rabbit anti-mouse F4/80 (1:400,Cell Signaling Technology, 70076), rat anti-mouse Ly6G (1A8,1:3,200, Biolegend #127602), rabbit anti–NF-kB subunit p50(1:100, Santa Cruz Biotechnology, SC-7178), rabbit anti-pSmad2(1:200, Cell Signaling Technology, 3101), rabbit anti-pSmad3(1:400, Abcam, ab52903), rabbit anti-pH2AX (1:100, CellSignaling Technology, 9718), and mouse anti–8-OHdG (1:100,Alpha Diagnostic, 8OHG11-M). Smad7 antibody was producedusing human Smad7 recombinant protein to immunize rabbits.Specificity of Smad7 antiserum was confirmed by Western blot(Supplementary Fig. S2). Secondary antibodies conjugated toAlexa Flour 594 (red) or 488 (green) were used (1:200 for all,Invitrogen). Terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) staining was performed with a TUNEL kit(Promega) according to the manufacturer's directions to detectapoptotic cells. Slides were mounted with coverslips using Fluor-omount-G or DAPI Fluoromount-G (SouthernBiotech).

Quantification of immunostainingIn oral mucosa, we quantified BrdUþ, pH2AX-, or 8-hydroxy-20-

deoxyguanosine (8-OHdG)–positive cells as cells per mm base-ment membrane length (including all epithelial cells), TUNEL-positive cells as cells per mm basement membrane length (includ-ing all epithelial layers and stroma above themuscle layer), CD45-positve cells as DAPI-positive cells per mm2 epithelial and stromaarea above muscle layer, nuclear pSmad2- or NF-kB p50–positivecells as the percentage of positive cells per total epithelial cell count(excluding sloughed epithelial cells induced by irradiation).Sequential 20x images along the basement membrane were quan-tified and averaged per sample. In oral cancer regions, K14- or K5-positive cells were defined as tumor cells. All tumor cells weremeasured together as the area of tumor (mm2), excluding stromalcells. We quantified percentage of nuclear pSmad2- or NF-kBp50–positive cells/total tumor (epithelial and stromal) cells. Wequantified BrdUþ, pH2AXþ, 8-OHdGþ, CD45þ, and TUNELþ cellsas cells permm2 tumor area. Ly6Gþ or F4/80þ cellswere quantifiedas cells per mm2 tumor area based upon staining intensity andmorphology todistinguish them fromnonspecific stainingofothercell types. For pH2AX staining, cells with more than three nuclearfoci were defined as pH2AX-positive cells. Five random 20x tumorimages were quantified and averaged per sample.

Statistical analysisStatistical analyses were performed using GraphPad Prism

software. Normality was determined by D'Agostino and Pearsonnormality test (or Shapiro–Wilk normality test for smaller sam-ples size, n < 8). Every dataset passed these normality tests;differences between two treatment groups were determined usingthe Student t test, and differences between more than two groupswere determined by one-way ANOVA with Tukey multiple com-parison test. Data are presented as the mean � SEM.

ResultsTat-Smad7 treatment reduced oral mucositis severity withoutprotecting adjacent oral cancer

To mimic RT-induced oral mucositis in oral cancer patients,we used a human oral cancer orthotopic tongue xenograftmodel in athymic nude mice. Bone marrow transplant patientsare immune suppressed and experience severe RT-induced oralmucositis (15), suggesting that T lymphocytes are not majorcontributors to mucositis. We transplanted two human oralcancer lines, FaDu or UM-SCC-1, into the tongues of athymicnude mice. The health of tumor-bearing mice rapidly deterio-rated due to cancer burden and compromised oral intake.Therefore, after tumors were established, we irradiated micewith 18 Gy cranial RT instead of fractionated RT to minimizedeath due to repeated doses of anesthesia required forfractionated RT. This irradiation dose induces oral mucositiswith the kinetics and severity similar to 3 � 8 Gy fractionatedRT in mice with obvious mucosal damage starting on day 5 afterRT (10). Six days after RT, when mice began to lose weight (10),we treated them with Tat-Smad7 produced as we previouslydescribed (ref. 10; Supplementary Fig. S1). We chose the doseand regimen of topical Tat-Smad7 application that effectivelytreated oral mucositis (1 mg/30 mL oral dose, daily). Vehicletreatment (30% glycerol in PBS) was used as control. Mice weresacrificed and tongues harvested 10 days after RT. We assessed ifTat-Smad7 protein was delivered to both oral mucosa and oralcancer with immunofluorescent staining using a human Smad7antibody that also cross-reacts with mouse Smad7 (Supple-mentary Fig. S2). Similar to our previous report (10), in the oralepithelium of Tat-Smad7–treated mice, Smad7 was present inboth the cytoplasm and the nucleus, whereas endogenousSmad7 levels in vehicle-treated mice were low (Fig. 1A). Intumors, Tat-Smad7 was detected in the cytoplasm of bothtumor epithelia and stroma (Fig. 1A), suggesting that TGFb inthese cells drives Smad7 cytoplasmic translocation to blockTGFb signaling (16). We measured tumor area microscopicallyand found that both FaDu and UM-SCC-1 tumor sizes werereduced in RT-treated mice and Tat-Smad7 treatment did notsignificantly affect tumor size compared with vehicle in thisshort-course treatment (Fig. 1B and C). Tongue mucosaadjacent to tumor showed RT damage and mucositis formationprimarily at the posterior dorsal surface which has fewercornified layers compared with tongue papillae at the tip ofthe tongue (Fig. 1F and G). Tat-Smad7–treated mice hadsignificantly smaller RT-induced tongue ulcers compared withvehicle-treated mice (Fig. 1D and E).

Histopathologic analysis revealed that irradiated epitheliumexhibited atrophy, flattened tongue papillae, thinning cuticle, andulceration compared with untreated epithelium (Fig. 1F and G).Irradiated mucosa treated with Tat-Smad7 showed less epithelial

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atrophy, smaller ulcerated area, and fewer immune cells com-pared with vehicle-treated mucosa (Fig. 1F and G; ref. 10). Com-pared with nonirradiated tumors, irradiated tumors near muco-sitis showed obvious cell damage with vacuolar degeneration andpyknotic nuclei or dysplastic cell shape with enlarged, smudgedchromatin. Apoptotic cells, inflammation, and interstitial fibrosiswere obvious in irradiated tumors. FaDu tumors were moresensitive to irradiation as demonstrated by more damaged tumorcells than UM-SCC-1 tumors (Fig. 1F and G).

Tat-Smad7 reduced NF-kB and TGFb signaling in both oralmucositis and oral cancer

To examine on-target effects of Tat-Smad7 on oral mucosaand cancer, we performed immunostaining for pSmad2and pSmad3, markers of TGFb pathway activation, andnuclear NF-kB p50, a marker of NF-kB pathway activation.

Oral mucosa after RT had more nuclear pSmad3þ cells com-pared with nonirradiated mucosa (Supplementary Fig. S3),similar to changes in pSmad2þ cells as previously reported(10). Fewer nuclear pSmad2þ and pSmad3þ cells were observedin irradiated oral mucosa and adjacent oral cancer in Tat-Smad7–treated mice compared with vehicle treatment (Fig. 2A, B, E, F, I,and J; Supplementary Fig. S3). In nonirradiated tongue tumors,both UM-SCC-1 and FaDu had more pSmad3þ cells than oralmucosa (Supplementary Fig. S3), indicating TGFb-dependentSmad3 activation (even in Smad4-deficient FaDu tumors) aspreviously reported (13). In addition, fewer nuclear NF-kBp50þ cells were observed in both irradiated oral mucosa andadjacent oral cancer in Tat-Smad7–treated mice compared withvehicle treatment (Fig. 2C, D, G, H, K, and L). These datademonstrate the on-target efficacy of Tat-Smad7 against knownSmad7 targets in tongue mucosa with oral tumors.

Figure 1.

Oral Tat-Smad7 treatment alleviated RT-induced oral mucositis but did not affect radiotherapy on neighboring oral cancer. A, Representativeimmunofluorescent images using Smad7 antibody to detect endogenous Smad7 or Tat-Smad7 in irradiated mouse tongue mucosa and neighboringxenografted FaDu tumor (no endogenous Smad7). Weak endogenous Smad7 was detected in mouse tongue mucosa and oral cancer stromal cells of mouse origin.K14 antibody was used to counterstain epithelial cells. B and C, Quantification of oral tumor size (mean � SEM) 10 days after 18 Gy RT in mice untreated(no RT), treated with RT and vehicle (RT þ Vehicle) or RT and Tat-Smad7 (RTþTat-Smad7). D and E, Quantification of oral ulcer diameter (mean � SEM)10 days after 18 Gy RT in mice treated as described in B and C. P values determined by the Student t test (two groups) or one-way ANOVA with Tukeymultiple comparison test (more than two groups). F and G, Representative H&E images of oral mucosa and oral cancer in tongue samples harvested from micebearing FaDu (F) or UM-SCC-1 (G) tumors 10 days after 18 Gy RT. Top plots present low power images of tongue epithelium and tumor. Yellow solid linesdefine the ulcer boundary. Black dotted lines delineate the boundary of tongue tumor. Scale bars, 200 mm. Mid plots present high power images of oralepithelium from nonirradiated posterior dorsal tongue mucosa and irradiated mucosa from the same region with ulcer. White dotted lines delineate epithelial cellsmigrated underneath the ulcer. Scale bars, 100 mm. Bottomplots present high power images of the oral cancer. Yellow arrows point to dying or apoptotic tumor cells.White arrows point to areas without obvious RT-induced damage. Scale bars, 100 mm.






Tat-Smad7 treatment alleviated inflammation and DNAdamage–associated cell death in oral mucositis lesions of oralcancer–bearing mice

We quantified leukocytes with CD45 staining and foundTat-Smad7–treated mucositis contained fewer CD45þ leukocytes(Fig. 3A and B). We next performed F4/80 and Ly6G staining todetermine relative levels of F4/80þmacrophages and Ly6Gþ cells[a marker of polymorphonuclear (PMN) neutrophils or PMN

myeloid-derived suppressor cells (MDSC)]. RT-induced oralmucositis harbors primarily neutrophils and macrophages (9)that were obvious in oralmucositis lesions of tumor-bearingmice(Fig. 3A). Tat-Smad7–treated mucositis had fewer PMNs andmacrophages than vehicle-treated lesions (Fig. 3A), consistentwith smaller ulcers and fewer inflammatory cells observedbyH&Eand CD45 staining. To examine epithelial slough due to DNAdamage–induced cell death and cessation of proliferation, we

Figure 2.

Oral Tat-Smad7 treatment attenuated nuclear pSmad2 and NF-kB p50 in RT-induced oral mucositis and adjacent cancer. A–D, Representativeimmunofluorescent staining of pSmad2 (A, B) and NF-kB subunit p50 (C, D) in RTþVehicle or RTþTat-Smad7–treated oral mucosa and oral cancer (FaDu:A, C; UM-SCC-1: B, D) with K14 or K5 epithelial cell counterstain. Scale bars, 100 mm for all sections. E–L, Quantifications of nuclear staining for pSmad2 or NF-kBp50 (mean� SEM). Nuclear pSmad2 andNF-kB p50–positive cells were quantified as a percentage of total epithelial cells in oral mucosa (FaDu: E,G; UM-SCC-1: I,K)or in tumor cells (FaDu: F, H; UM-SCC-1: J, L). P values determined by the Student t test.

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first performed pH2AX staining as a marker of DNA-damagedcells. Irradiated oral mucosa had numerous pH2AXþ cells, andTat-Smad7–treated oral mucosa had fewer pH2AXþ cells com-pared with vehicle-treated mucosa (Fig. 3A and C). Next, stainingof nuclear 8-OHdG was performed as a marker of oxidativeDNA damage (17). There were fewer 8-OHdGþ cells in oralmucosa of Tat-Smad7–treated mice compared with vehicle-trea-tedmice (Fig. 3A andD).We performed TUNEL assays to quantifyapoptosis and noted numerous apoptotic cells in the oral mucosaof irradiated mice. Tat-Smad7–treated mucosa contained fewerapoptotic cells than vehicle control (Fig. 3A and E). Together,these data demonstrate that Tat-Smad7 treatment reduces inflam-mation, DNA damage, and apoptosis associated with RT-inducedoral mucositis in tumor-bearing mice.

Tat-Smad7 reduced inflammation in irradiated oral cancerUnlike nonirradiated oral mucosa that had no obvious inflam-

mation (Fig. 1), leukocyte infiltration was apparent in nonirra-diated tumors that contained numerous F4/80þ macrophages

and Ly6Gþ cells (Supplementary Fig. S4). Very few infiltrated Bcells were detected (data not shown). In irradiated FaDu tumors,F4/80þ cells were still prominent, but Ly6Gþ cells were largelydiminished (Fig. 4A andCvs. Supplementary Fig. S4) even thoughLy6Gþ cells were numerous in adjacent mucositis (Fig. 3). BothF4/80þ cells and Ly6Gþ cells were still pronounced in UM-SCC-1–irradiated tumors (Fig. 4D, F, and G). Intriguingly, Tat-Smad7reduced the number of CD45þ leukocytes and F4/80þ macro-phages in both types of irradiated oral tumors and reduced Ly6Gþ

cells in irradiated UM-SCC-1 tumors (Fig. 4).

Effects of Tat-Smad7 on irradiated oral cancer diverge from itseffects on oral mucositis

Similar to oral mucositis, irradiated tumors had numerouspH2AXþ cells that were significantly more abundant than innonirradiated tumors (Fig. 5A; Supplementary Fig. S5). FaDutumors harbored more pH2AXþ cells than UM-SCC-1 tumors(Fig. 5D and E; Supplementary Fig. S5). However, unlike oralmucositis, there was no difference in the number of pH2AXþ cells

Figure 3.

Oral Tat-Smad7 treatment mitigated inflammation, DNA damage, and apoptosis in RT-induced oral mucositis. A, Representative immunostaining of markersfor immune cells (CD45, Ly6G, F4/80), DNA damage markers (pH2AX, 8-OHdG), and apoptosis (TUNEL) in RTþVehicle or RTþTat-Smad7–treated oralmucosa adjacent to irradiated FaDu tongue tumors. Similar staining patterns were also seen in irradiated mucosa adjacent to UM-SCC-1 tumors (not shown)and quantified in B to E. K5 or K14 antibody was used to counterstain epithelial cells. DAPI was used to counterstain nuclei for CD45 staining, and propidiumiodide (PI) was used to counterstain nuclei for TUNEL. Scale bars, 100 mm for all sections. B–E, Quantification (mean � SEM) of immunostaining markers inoral mucosa adjacent to irradiated FaDu and UM-SCC-1 shown in A. CD45þ cells (B) were quantified based on mm2 epithelial and stromal areas above themuscle layer. Quantifications for pH2AXþ cells (C), 8-OHdGþ cells (D), and TUNELþ cells (E) in keratinocytes per mm basement membrane length. P valueswere determined by the Student t test.






in oral cancers treated with Tat-Smad7 versus vehicle in bothtumor types (Fig. 5D and E). 8-OHdGþ cells were hardly detect-able in nonirradiated tumors (Supplementary Fig. S5) but wereinduced significantly by RT (Fig. 5B). In contrast to Tat-Smad7effects on oral mucositis, 8-OHdGþ cells in oral cancer weresignificantly increased in Tat-Smad7–treatedmice compared withvehicle (Fig. 5F and G). Basal levels of TUNELþ apoptotic cellswere present in nonirradiated tumors (Supplementary Fig. S5)and RT induced numerous apoptotic cells in both tumor types(Fig. 5C). Apoptotic cells in oral cancer were increased inTat-Smad7–treated mice compared with vehicle-treated(Fig. 5H and I), opposite of what was observed in Tat-Smad7–treated mucositis. We performed BrdU staining to quantitateproliferating cells. Adjacent to ulceration, Tat-Smad7–treated oralmucosa showed more proliferative BrdUþ cells along the epithe-lial basement membrane than vehicle-treated oral mucosa (Fig.6A–D). In contrast, Tat-Smad7–treated oral cancer had no differ-ence in proliferation compared with vehicle-treated oral cancer(Fig. 6A, B, E, and F). In summary, Tat-Smad7–treated mucositis

lesions had reduced RT-induced DNA damage, inflammation,and apoptosis and increased keratinocyte proliferation, whereasadjacent irradiated tumors treated with Tat-Smad7 demonstratedno resolution to DNA damage, decreased inflammation, andunaltered proliferation.

DiscussionOneof themajor challenges for treating oralmucositis in cancer

patients is that potential therapies reviving normal mucosalregeneration could also pose risk by protecting cancer cells. Weshow here that Tat-Smad7 topical application reduced activationof NF-kB and TGFb signaling and reduced inflammation in bothoralmucosa and cancer, but differentially affectednormalmucosaand oral cancer with respect to DNA damage and associated celldeath, and cell proliferation.

Our current study confirms that in oral mucositis-inducedacute inflammation associated with NF-kB and TGFb activation(10, 18), macrophages and neutrophils are predominant

Figure 4.

Oral Tat-Smad7 treatment reduced leukocyte infiltrate in irradiated tongue tumors. A and D, Representative images of CD45, F4/80 and Ly6G staining inFaDu tumors (A) and UM-SCC-1 tumors (D). K5 antibody was used to counterstain epithelial cells, and DAPI was used to counterstain nuclei for CD45 staining.Note that tumor cells after irradiation had more nonspecific staining in F4/80 than in nonirradiated tumors (Supplementary Fig. S4). Quantification of F4/80þ

macrophages in C and F is based upon staining intensity and morphology (indicated by yellow arrows, A and D). Black arrows in Ly6G vehicle panel inA point to a few remaining Ly6Gþ cells which were absent in Tat-Smad7–treated tumor. Scale bars, 100 mm for all sections. B and C, Quantifications of CD45þ

and F4/80þ cells in FaDu tumors. E–G,Quantifications of CD45þ, F4/80þ, and Ly6Gþ cells in UM-SCC-1 tumors. Immune cellswere quantified (mean� SEM) permm2tumor area. P values were determined by the Student t test.

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leukocyte types (9). Fewer macrophages and neutrophils inTat-Smad7–treated mucositis could reflect less damage (smallerulcers and fewer dead cells) or a quicker resolution of inflamma-tion. Oral cancer without RT showed chronic inflammation in thetumor microenvironment that harbored numerous F4/80þ puta-tive M2 macrophages and Ly6Gþ putative tumor-associated neu-trophils or PMN-MDSCs; both are known to be tumor-promotingleukocytes attracted by TGFb1 and also a major source ofTGFb1 (19). With RT, F4/80þ and Ly6Gþ cells remained plentifulin UM-SCC-1 tumors, and Tat-Smad7 reduced numbers of thesecells similar to TGFb inhibitors and NF-kB inhibitors used inclinical trials of metastatic cancer, which has an inflammatorytumor microenvironment (20, 21). The subtypes of theseleukocytes in irradiated tumors require further study, e.g., if theyhave shifted from PMN-MDSCs to neutrophils as part of acute

inflammation induced by RT. Interestingly, Ly6Gþ leukocyteswere largely diminished in irradiated FaDu tumors despitetheir presence in the adjacent mucosa. Because FaDu cells lackSmad4 but retain nuclear pSmad2þ and pSmad3þ, it is unclearif this altered TGFb signaling affected RT response–associatedchanges in Ly6Gþ cell infiltration or depletion in tumors. Incontrast, Tat-Smad7 still reduced F4/80þ macrophages in FaDutumors, possibly by blocking RT-induced TGFb signalingthrough Smad2/Smad3 and by direct blocking of non–Smad-mediated NF-kB activation by TGFb (22). These datahighlight the complex biology and heterogeneity of tumors inresponse to RT. The anti-TGFb/NF-kB effects of Smad7 couldhave a cascade effect on cytokine production directly or indi-rectly due to reduced leukocyte numbers. Therefore, futurein-depth studies are needed to dissect subtypes of immune

Figure 5.

Tat-Smad7 did not reduce RT-induced DNA damage but increased oxidative stress and apoptosis in irradiated tongue cancers. A–C, Representativestaining of pH2AX, 8-OHdG, and TUNEL in RT þ vehicle and RT þ Tat-Smad7–treated tongue cancer. K14 antibody was used to counterstain epithelialcells or propidium iodide (PI) to counterstain nuclei. Scale bars, 100 mm for all sections. D–I, Quantification (mean � SEM) of pH2AXþ, 8-OHdGþ, and TUNELþtumor epithelial cells in cancer per mm2 tumor epithelial area (FaDu: D, F, H; UM-SCC 1: E, G, I). P values were determined by the Student t test.






cells influenced by Smad7 as well as the production of cyto-kines by these cells.

Because reducing inflammation is insufficient to alleviate oralmucositis, other pathogenic processes must also be targeted foreffective treatment. Among these processes, RT-induced DNAdamage kills tumor cells but also initiates oral mucositis(1, 23). Therefore, reducing DNA damage or accelerating repairis key for oral mucositis healing, but such an effect is undesirablefor eradicating tumor cells. The selective reduction of RT-inducedDNA damage by Smad7 in oral mucosa (Fig. 3) might beexplained by its ability to facilitate DNA damage repair throughprotein–protein interactions with the DNA repair proteincomplex containing ATM and Mre11-Rad50-Nbs1 proteins(24). However, Tat-Smad7 did not reduce RT-induced DNAdamage in tumor cells (pH2AXþ cells). This may be part of thereason that intrinsic DNA repair defects in tumor cells (evidentin nonirradiated tumors) cannot be reversed by Tat-Smad7. Inaddition, TGFb-mediated DNA repair is an important mech-

anism of radioresistance in cancer treatment (25–29). In thiscontext, blocking TGFb signaling by Tat-Smad7 wouldenhance DNA damage in cancer cells. Further, RT-inducedoxidative stress is required to amplify DNA damage, causingcollateral damage leading to cancer cell death (30, 31) and oralmucositis (1, 23). Reduced oxidative stress marker 8-OHdGwith Tat-Smad7 treatment in oral mucositis is consistentwith previous reports that both TGFb and NF-kB pathwayscan induce oxidative stress (32–35). Paradoxically, TGFb isreported to reduce oxidative stress in a skin SCC model (36).This could explain why Tat-Smad7 treatment increased8-OHdGþ cells in oral cancer. Given these effects, it is notsurprising that DNA damage–associated apoptosis wasreduced by Tat-Smad7 in oral mucositis but increased inadjacent oral cancer. The effect of Tat-Smad7 on apoptosis isalso consistent with previous reports showing Smad7 reducedapoptosis in normal epithelia (10, 37) but enhanced apoptosisin cancer (38).

Figure 6.

Tat-Smad7 increased proliferation in irradiated oral mucosa but not neighboring oral cancer and summary of Tat-Smad7 actions.A andB,Representative staining ofBrdU-positive cells in irradiated mucosa and neighboring oral cancer (FaDu: A, UM-SCC-1: B). K14 antibody was used as counterstain epithelial cells. Scale bars,100 mm. C–F, Quantification (mean � SEM) of BrdUþ cells in oral mucosal keratinocytes per mm basement membrane length (adjacent to FaDu: C; adjacent toUM-SCC-1: D) and in cancer epithelial cells per mm2 tumor area (FaDu: E; UM-SCC-1: F). P values were determined by the Student t test. G, Smad7 functionalmechanisms in oralmucositis with neighboring irradiated oral cancer. RT directly activates TGFb andNF-kB, and induces DNAdamage. Smad7 alleviates RT-inducedDNAdamage (includingDNA strand breaks and oxidative damage), attenuates TGFb-induced growth arrest, attenuates apoptosis, and blocks inflammation inducedby TGFb and NF-kB, further promoting oral mucositis healing. H, Smad7 functional mechanisms in irradiated oral cancer. RT directly activates TGFb andNF-kB. Tumor cells harbor intrinsically damaged DNA and are thus more sensitive to RT-induced apoptosis. Smad7 enhances RT-induced oxidative damage thatconsequently increases apoptosis. Smad7 also attenuates TGFb and NF-kB activation–induced inflammation.

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In a subset of HNSCCs, endogenous Smad7 is activated byTGFb and NF-kB, which transcriptionally suppress Smad targetgenes that could either be tumor suppressive or promotive(22). Therefore, pharmacologic dosage of a Smad7-based bio-logic needs to be carefully assessed. Our study revealed thatHNSCCs derived from SMAD4 mutant (FaDu) and SMAD4wild-type (UM-SCC-1) cells responded to Tat-Smad7 similarly.This can be explained by the fact that the majority of humancancers escape Smad-dependent TGFb growth inhibition,which is also supported by Tat-Smad7 not affecting cancercell proliferation in our model. In addition, we have shownSMAD4-mutant epithelial cells rely primarily on Smad3-depen-dent signaling to mediate TGFb1-induced inflammation (13).Therefore, the use of Tat-Smad7 in SMAD4-mutant cancercould follow the same principle as TGFb inhibitor in treatingSMAD4-mutant metastatic cancer including pancreatic cancerthat has a high rate of SMAD4 mutation (39).

In summary, we provide evidence that local short-termTat-Smad7 protein delivery alleviated RT-induced oral muco-sitis without compromising RT-induced killing of neighboringoral cancer. Potential mechanisms for the context-specificeffects of Smad7 are as follows: In oral mucositis, Tat-Smad7attenuates TGFb-mediated growth arrest and apoptosis as wellas TGFb/NF-kB–mediated inflammation. Smad7 could alsodirectly reduce DNA damage or accelerate repair (Fig. 6G). Inneighboring oral cancer, Smad7 still attenuates TGFb/NF-kB–mediated inflammation, but is insufficient to reduce DNAdamage (Fig. 6H). In addition, Smad7 could block TGFb-mediated suppression of oxidative damage and consequentlyinduce apoptosis in tumor cells (Fig. 6H). Because high inten-sity/hypofractionated SBRT is increasingly used to treat oralcancers resistant to traditional low-dose fractionated RT, futurestudies that more closely mimic SBRT will be needed to assesstherapeutic efficacy of Tat-Smad7 in oral mucositis treatment.In addition, HNSCC mouse models with an intact immunesystem (13, 40) will further determine if effects of Tat-Smad7are influenced by T-cell–mediated tumor immunity. Futurestudies should carefully assess Tat-Smad7 dose-dependentefficacy to effectively treat oral mucositis but avoid potentialoncogenic effects on neighboring oral cancer, and assess the

effect of long-term Tat-Smad7 use on oral cancer progressionwith an intact immune microenvironment.

Disclosure of Potential Conflicts of InterestD.Raben is a consultant/advisory boardmember for EMDSerono. X.-J.Wang

is an employee of Veterans Affairs, reports receiving commercial research grantsfrom NIH to Allander, and holds ownership interest (including patents) inAllander Biotechnologies. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: J. Luo, D. Raben, X.-J. WangDevelopment of methodology: J. Luo, L. Bian, C. Liang, D. Du, H. Zhou,C.D. Young, X.-J. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Luo, L. Bian, M.A. Blevins, D. Wang, C. Liang, D.Du, F. Wu, B. Holwerda, R. ZhaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Luo, L. Bian, C. Liang, D. Du, F. Wu, H. Zhou,C.D. Young, X.-J. WangWriting, review, and/or revision of the manuscript: J. Luo, L. Bian,M.A. Blevins, D. Raben, H. Zhou, C.D. Young, X.-J. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. WuStudy supervision: R. Zhao, H. Zhou, X.-J. Wang

AcknowledgmentsWe thankDrs. Yosef Refaeli, Brian Turner, Steve Sonis, and Thomas Carey for

advice and an anonymous donation for support of this work. D. Raben issupported by the Marsico Endowment fund for research.

This work was supported by NIH grant DE024659 to C.D. Young andX.-J. Wang, and DE015953 to X.-J. Wang. X.-J. Wang is also a Research Biologistin Department of Veterans Affairs. J. Luo and F. Wu were supported by theNational Nature Science Foundation of China No. 81772898, and theState Scholarship Fund of China Scholarship Council No. 201506240122 andNo. 201606240201. M.A. Blevins was supported by T32 CA174648.

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 April 7, 2018; revised August 2, 2018; accepted August 31, 2018;published first September 5, 2018.

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2019;25:808-818. Published OnlineFirst September 5, 2018.Clin Cancer Res Jingjing Luo, Li Bian, Melanie A. Blevins, et al. Modelwithout Compromising Oral Cancer Therapy in a Xenograft Mouse Smad7 Promotes Healing of Radiotherapy-Induced Oral Mucositis

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