Transient Activation of Hedgehog Pathway Rescued ...clincancerres.aacrjournals.org/content/clincanres/20/1/140.full.pdf · Transient Activation of Hedgehog Pathway Rescued Irradiation-Induced

Cancer Therapy: Preclinical

Transient Activation of Hedgehog Pathway RescuedIrradiation-Induced Hyposalivation by Preserving SalivaryStem/Progenitor Cells and Parasympathetic Innervation

Bo Hai1, Lizheng Qin1,4, Zhenhua Yang1,5, Qingguo Zhao1, Lei Shangguan1,6, Xinyu Ti1,6, Yanqiu Zhao1,3,Sangroh Kim2, Dharanipathy Rangaraj2, and Fei Liu1

AbstractPurpose: To examine the effects and mechanisms of transient activation of the Hedgehog pathway on

rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer.

ExperimentalDesign:Mouse salivary glands and cultured human salivary epithelial cells were irradiated

by a single 15-Gy dose. TheHedgehog pathwaywas transiently activated inmouse salivary glands, by briefly

overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human

salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was

examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary

flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), para-

sympathetic innervation, and expressionof related geneswere examinedbyflowcytometry, salisphere assay,

immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA.

Results: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh

overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary

function in male mice, which is related with preservation of functional SSPCs and parasympathetic

innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the

Bmi1 and Chrm1–HB-EGF pathways. The preservation of parasympathetic innervation was associated with

the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related

withmaintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human

salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation.

Conclusions: These findings suggest that transient activation of the Hedgehog pathway has the potential

to restore salivary gland function after irradiation-induced dysfunction. Clin Cancer Res; 20(1); 140–50.

�2013 AACR.

IntroductionHead and neck cancers account for approximately 3.2%

of all cancers in the United States with approximately342,550 survivors in 2012 (1, 2). Radiotherapy is the mostcommon form of treatment for head and neck cancers, and

nondiseased salivary glands are often exposed to radiother-apy. Due to the exquisite radiosensitivity of salivary glands,irreversible hyposalivation is common in long-term survi-vors of head and neck cancers treated with radiotherapyeven when various new techniques have been applied tominimize the irradiation (IR) damage to salivary glands (3).Hyposalivation exacerbates dental caries and periodontaldisease, and severely impairs the quality of life of patients.Irreversible hyposalivation has been attributed to the loss offunctional salivary stem/progenitor cells (SSPC) that con-tinuously replenish aged saliva-producing cells normally(4). Current treatments for IR-induced xerostomia canonly temporarily relieve these symptoms. Several newapproaches, including gene transfer and stem cell infusion,have shown promise in restoring salivary gland function byprotection or regeneration of saliva-producing cells (5).However, little is known about the molecular regulationof SSPCs and their functional restoration.

Hedgehog (Hh) signaling is required for salivary branch-ing morphogenesis (6), and is activated during the regen-erationof adult salivary glands (7). TheHh receptor Patched

Authors' Affiliations: 1Institute for Regenerative Medicine at Scott andWhite, Molecular and Cellular Medicine Department, Texas A&M HealthScience Center; 2Department of Radiation Oncology, Scott and WhiteHospital, Temple, Texas; 3Lutheran Medical Center, Brooklyn, New York;4Beijing Stomatological Hospital, Capital Medical University, Beijing;5Department of Orthodontics, School of Stomatology; and 6Xijing Hospital,Fourth Military Medical University, Xi'an, Shaanxi, China

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

B. Hai and L. Qin contributed equally to this work.

Corresponding Author: Fei Liu, 5701 Airport Rd., Module C, Temple, TX76502. Phone: 254 771 6813; Fax: þ001 254 771 6839; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-1434

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(1) January 1, 2014140

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(Ptch) acts as a negative regulator of Smoothened (Smo)that blocks production of repressor forms of the Gli tran-scription factors (GliR) and promotes production of theactivator forms (GliA). The binding of Hh ligands to Ptchunleashes Smo activity to tip the balance toward GliA thatconsequently activates the expressionofHh target genes (8).SonicHedgehog (Shh), onemajorHh ligand, is expressed inthe epitheliumof the embryonic salivary gland, andmay actwithin the epithelium in a juxtacrine manner to promoteproliferation and differentiation of epithelial cells (9). Insubmandibular glands (SMG) of the adult mouse, Hhsignaling is marginal, but is activated significantly duringfunctional regeneration after duct ligation (7).We report, inthis study, that IR inmousemodels does not activate theHhpathway in SMGs similar to occlusive damage, and transientactivation of the Hh pathway by Shh overexpression in thebasal epithelia of male mice rescued IR-induced hyposali-vation, likely through the preservation of SSPCs and para-sympathetic innervation. IR and transient Hh activation byother approaches similarly affected the expression of genesessential for the maintenance of SSPCs and parasympathet-ic innervation in the SMGs of female mice and culturedhuman salivary epithelial cells. These data indicated that thetransient activation ofHhpathway could potentially benefitsalivary gland function following radiotherapy of head andneck cancer.

Materials and MethodsMiceHh signaling activity was analyzed with B6;129-

Ptch1tm1Mps/J (Ptch1-lacZ) mice (The Jackson Laborato-ry). Mice carrying tetO-Shh (10) and Krt5-rtTA (11) trans-

genes were placed on doxycycline (Dox) chow (6 g/kg,Bio-serv) to induce Shh expression. Irradiation of mouseand measurement of stimulated saliva flow rate was aspreviously reported (11). All animal procedures wereapproved by the Texas A&M Health Science Center(TAMHSC) and the Institutional Animal Care and UseCommittee of the Scott and White Hospital.

Quantitative reverse transcription PCR analysisQuantitative reverse transcription PCR (qRT-PCR) was

done as reported previously (7). A qRT-PCR analysis formiRNAs was performed with TaqMan miRNA assays(AppliedBiosystems)usingU6 snRNAas the referenceRNA.

ELISA and Western blotFresh SMG samples were homogenizedwith 40-mL T-PRE

reagent containing protease inhibitors (Pierce) per milli-gram followed by centrifugation at 10,000g for 5minutes tocollect the supernatant for ELISA and Western blot. Theconcentration of Bdnf and Nrtn in saliva and SMG sampleswas examined with ELISA kits (Insight Genomics andMyBioSource). Western blot analysis was conducted asreported previously (7) with antibodies for Aqp5 (Abcam1:5,000), p21Waf1 (Millipore, 1:500), and GFRa2 (Abcam1:1,000).

Flow cytometryTheantibodiesusedwereagainst c-kit or Sca-1 (BioLegend,

1:50), Bmi1 (Abcam, 1:100), Gli1 (Thermo, 1:100), orChrm1 (ABBIOTEC, 1:100). For Bmi1 and Gli1 staining,cells were permeabilized with FIX & PERM reagents (LifeTechnologies). The stained cellswere analyzedonaCytomicsFC500 flow cytometer (Beckman Coulter), and data wereprocessed using the manufacturer’s software (CXP).

Histology and immunofluorescence stainingFrozen SMG sections were stained with an acetylcholin-

esterase (AChE) rapid staining kit (MBL) or an antibodyagainst Chrm1 (1:2,000; R&D Abs). The AChE stain wasquantified with NIS-Elements AR software (Nikon).

SAG treatmentThe small-molecule Hh agonist smoothened agonist

(SAG) (EMD) or vehicle were administered to femalePtch1-lacZ mice through SMG cannulation (2 mg/g), thenfollowed by daily intraperitoneal (i.p.) injection of 5 mg/gfor 3 days. SMGs samples were collected 1 day after the lastinjection for X-gal staining and qRT-PCR analysis.

Isolation and treatment of human salivary epithelialcells

Healthy human salivary gland samples from patients (2males and 2 female)with an age range of 25 to 61 years wereprovided by the Cooperative Human Tissue Network(CHTN), Southern and Mid-Western Divisions, a NationalCancer Institute-supported resource. Human SMGepithelial cells were isolated as reported previously (12)and cultured in mammary epithelial medium CnT-27

Translational RelevanceIrreversible hyposalivation is common in survivors of

head and neck cancer who were treated with radiother-apy even when various new techniques were applied tominimize the irradiation damage to the salivary glands.Current treatments for irradiation-induced hyposaliva-tion can only temporarily relieve symptoms of xerosto-mia. The morbidity is caused by the loss of functionalepithelial stem/progenitor cells. Recent studies indicatedthat parasympathetic innervation is also essential for theregeneration of salivary glands and is impaired by radio-therapy. This article reports on a novel finding that thetransient activation of Hedgehog pathway after irradia-tion rescued salivary gland dysfunction by preservingboth salivary SSPCs and parasympathetic innervation ina mouse model. Moreover, in cultured human salivarygland epithelial cells, transient Hedgehog activationafter irradiation rescued expression of genes essentialfor the maintenance of SSPCs and parasympatheticinnervation similarly as in mice. These findings sug-gested that targeting the Hedgehog pathway is a prom-ising option to treat irradiation-induced hyposalivation.

Transient Hh Activation Rescued IR-Induced Hyposalivation

www.aacrjournals.org Clin Cancer Res; 20(1) January 1, 2014 141




(Zen-Bio). Passage-4 cells were treated with a 15-Gy single-dose IR; then, some cells were infected with adenovirusesencoding humanGli1 or GFP (Applied Biological MaterialsInc.; MOI ¼ 10) 3 days later. All cells were collected 7 daysafter IR for analysis.

Effects of transient Hh activation in SMGs on SCC VIItumors

The mouse SCC VII tumor model was established, andsome tumors were irradiated as previously reported (13).Adenoviral vectors encoding GFP or rat Shh (AdGFP orAdShh, Applied Biological Materials Inc.) were delivered at109 particles per SMG by retrograde ductal instillation onday 0 (inoculation of 3 � 104 SCC VII cells) or day 9 (3 �105 cells). Mice were euthanized when the tumor diameterwas approximately 15 mm.

Statistical analysesAll quantified data were analyzed using one-way ANOVA

followed by Tukey multiple-comparison test. Statisticalanalysis and graphical generation of data were done withthe GraphPad Prism software.

ResultsHh activity in SMGs after irradiation or transient Shhoverexpression

We reported previously that the Hh signaling is activatedduring functional regeneration of the salivary gland afterligation of the main excretory ducts of SMGs (7). UsingPtch1-lacZ Hh reporter transgenic mice, we confirmed thatthe Hh activity is marginal in SMGs of adult mice, but isremarkably elevated in ductal epithelia after duct ligation(Supplementary Fig. S1). To examine the effect of IR on Hhsignaling in the salivary gland, Ptch1-lacZmice were treatedwith 15-Gy single-dose IR in the head and neck region, andthe SMGswere collected3, 7, or 14days after IR for qRT-PCRanalysis and X-gal staining for lacZ activity. IR did notsignificantly affect the expression of reporter Ptch1-lacZand endogenous Ptch1 gene in the SMGs of both male andfemale mice (Fig. 1A and Supplementary Fig. S1, P > 0.05,n ¼ 3). The expression of another Hh target gene Gli1 wasnot significantly affected by IR in male SMGs, but wassignificantly decreased in female SMGs on days 7 and 14(Fig. 1A,P <0.05, n¼3). These results indicated that IR doesnot activate the Hh pathway in SMGs.

Figure 1. Effects of IR and Shh onHh activity in SMGs. A, SMGsamples from Ptch1-lacZ Hhreporter transgenic mice werecollected before (D0) or 3, 7, or 14days after 15-Gy single-dose IR,and analyzed with qRT-PCR forrelative mRNA expression (n ¼ 3).B, Krt5-rtTA/tetO-Shh mice werenot treated (NT) or induced withDox for 7 days, then sacrificed tocollect SMGs for qRT-PCRanalysis of transgenes and genesrelated with Hh pathway (n ¼ 3).C and D, SMGs of male Krt5-rtTA/tetO-Shh/Ptch1-lacZ mice werecollected for X-gal staining onsections (C) or salispheres (D)without or with Dox inductionfor 7 days.

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Clin Cancer Res; 20(1) January 1, 2014 Clinical Cancer Research142




To modulate the Hh pathway, we generated Krt5-rtTA/tetO-Shh mice that overexpress Shh in basal epithelial cellsincluding those in the ducts of salivary glands upon Doxinduction. Seven days of Dox induction significantly upre-gulated the expression of the Hh target genes Gli1, Ptch1,Hhip, Bmi1, and Jag2 in SMGs ofmale, but not female, mice(Fig. 1B). The much higher expression level of Ptch1, com-pared with Ptch2I, indicated that Ptch1 is the dominant Hhreceptor in SMGsof both gender (Fig. 1B),whereas thePtch1expression in SMGs from females ismuch lower than that inmales before or after Dox induction (Fig. 1B). Similarly,although the expression of Gli2 and Smo in SMGs beforeinduction (Supplementary Fig. S6) was comparablebetween males and females, the expression of Gli1 withdominant activator activity (14) was much lower, whereasthe expression ofGli3with dominant repressor activity (14)was much higher in females (Fig. 1B). In addition, theinduction of Shh transgene in SMGs from females is muchless efficient, likely due to the lower expression of Krt5 andrtTA (Fig. 1B). All these factors likely contribute to theinsufficient Hh activation by Shh in SMGs of females. Inthe SMGs of male Krt5-rtTA/tetO-Shh/Ptch1-lacZ mice, theexpression of lacZ Hh reporter was marginal before Doxinduction and was significantly elevated in ductal epitheliaafter 7 days of Dox induction (Fig. 1C). The upregulation ofthe Ptch1-lacZ reporter and Hh target genes after transientShh overexpression appeared comparable with that afterduct ligation (Fig. 3C,�2-fold for Ptch1 in both cases and12vs. 7-fold forGli1, Supplementary Fig. S1; ref. 7), indicatingthat the overall Hh activation by transient Shh overexpres-sion is comparable with that during functional SMG regen-eration. SSPCs can be enriched by spherical culture to formsalispheres (15). In salisphere cells from male Krt5-rtTA/tetO-Shh/Ptch1-lacZ mice, expression of lacZ Hh reporterwas very weak without Dox induction, but was significantlyincreased in a subset of salisphere cells after 7 days of Doxinduction (Fig. 1D). These data indicated that Hh pathway

was efficiently activated in ductal epithelia and SSPCs inSMGsofmaleKrt5-rtTA/tetO-ShhmiceuponDox induction.

Rescue of IR-induced hyposalivation by transient Hhactivation

To evaluate the potential of transient activation of the Hhpathway in the restoration of salivary gland function afterIR, adult male Krt-rtTA/tetO-Shhmice were treated with 15-Gy single-dose IR in the head and neck region; then, Shhexpression was induced from days 0, 3, or 90 after IR for 7days. As expected, IR significantly reduced the saliva flowrate in mice throughout the observation period from days30 to 120 (Fig. 2A, P < 0.05, n¼ 5). Hh activation both 3 or90 days after IR significantly improved the saliva flow ratecomparedwith IR group throughout the observation periodafter Dox induction, whereas concurrent Hh activationimproved the saliva flow rate only from day 60 after IR toa much less extent than Hh activation from day 3 (Fig. 2A,n ¼ 5). Consistently, the expression of acinar markersAquaporin5 (Aqp5) and cholinergic receptor muscarinic 3(Chrm3) in SMGs was significantly downregulated by IRon day 120, but improved by transientHh activation 0, 3, or90 days after IR as indicated by qRT-PCR and Western blot,with the highest improvement in the day-3 group (Fig. 2B–D, n ¼ 3). Seven days of Dox treatment in female Krt-rtTA/tetO-Shhmice or male wild-type C57BL/6mice 3 or 90 daysafter IR had no significant effects on IR-induced decrease ofsaliva flow rate and expression of Aqp5 and Chrm3 (Sup-plementary Fig. S2, P > 0.05, n ¼ 5), indicating that therescue effects of Dox induction in male Krt-rtTA/tetO-Shhmice was mediated by efficient Hh activation.

Preservation of salivary stem/progenitor cells andBmi1 signaling pathway after IR by transient Hhactivation

SSPCs can be identified by the expression of surfacemarkers c-Kit and Sca-1 (16). Flow cytometric analysis

Figure 2. Transient Hh activationrescued radiation-inducedhyposalivation and expression ofacinar markers. A, the stimulatedwhole saliva flow rate in male Krt5-rtTA/tetO-Shh mice irradiated (IR)or IR and induced with Dox for 7days starting from day 0, 3, or 90after IR (IR þ Dox D0, D3, or D90)were standardized with the bodyweight and normalized to that oftheNTgroup (n¼5). B–D, 120daysafter IR, SMG samples werecollected and analyzed with qRT-PCR (B and C) andWestern blot (D)for the expression of the acinarmarkers Aqp5 and Chrm3normalized to Gapdh (n ¼ 3).�, P < 0.05; ��, P < 0.01.






found that the population of c-Kitþ/Sca-1þ cells was veryrare in nontreated SMGs, but was significantly expandedafter 7 days of Hh activation (Fig. 3A, 0.85% � 0.33% vs.0.06%� 0.02% in theNT group,mean� SEM, P < 0.05, n¼3). However, 60 days after Hh activation, the percentage ofc-Kitþ/Sca-1þ cell population was not significantly affectedcompared with control mice (Supplementary Fig. S3), indi-cating that the effect ofHhactivation on expansionof SSPCsis transient. The rarity of c-Kitþ/Sca-1þ cells in NT SMGsmade it difficult to evaluate the effects of IR and transientHhactivation after IR on this cell population. Therefore, weexamined the number of salisphere-forming cells per gland(Fig. 3B, n¼ 3) that reflects the number of functional SSPCs,and it was significantly reduced several days after IR (15).Shh overexpression for 7 days in male mice increased thenumbers of salisphere-forming cells per SMG significantly(P < 0.05), consistent with the expansion of the c-Kitþ/Sca-1þ cell population. IR decreased salisphere numbers by day

10 as expected, whereas Hh activation 3 days after IRsignificantly ameliorated IR-induced decrease of salispherenumbers (P < 0.05). Consistently, as indicated by qRT-PCRanalysis of SMG tissues (Fig. 3C, n ¼ 3), the expression ofc-Kit mRNA was significantly upregulated by transient Hhactivation, decreased by IR on both days 10 and 120,recovered on days 10 and 120 by transient Hh activation3 days after IR, and upregulated on day 120 by transient Hhactivation 90 days after IR (P < 0.05). In contrast, theexpression of the salivary progenitor cell marker Ascl3(17) was not significantly affected by Hh activation or byIR on day 10, but was significantly decreased on day 120after IR, and recovered by transient Hh activation 3 or 90days after IR (P < 0.05). Consistent with the reportedimpairment of proliferation by IR (18), the expression ofproliferation-related genes Prc1 and Top2a (19) was signif-icantly decreased by IR (P < 0.05). Transient Hh activationdid not significantly affect the expression of proliferation

Figure 3. Transient Hh activationpreserved functional SSPCs andthe activity of Bmi1 pathwayimpaired by IR. A, flow cytometricanalysis of c-Kitþ/Sca-1þ cells inSMGs. B, salisphere numbers fromeachwhole SMGcollected 10 daysafter IR or 7 days after Doxtreatment. C, expression of genesassociated with SSPCs andproliferation examined by qRT-PCR. D, flow cytometric analysis ofBmi1þ cells in SMGs. E, Westernblot and relative quantification ofp21Waf1. n ¼ 3 for all studies. ns,not significant.

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markers Prc1, Top2a, and the proliferative cell nuclearantigen (PCNA) in nonirradiated SMGs (Fig. 3C and Sup-plementary Fig. S4A, P > 0.05), but significantly inducedrecovery of the expression of Prc1 and Top2a decreased by IR(Fig. 3C, P < 0.05).IR-induced apoptosis in salivary glands peaked on days 2

to 3 after IR and decreased significantly thereafter (11, 20),which may have contributed to SSPCs loss. Hh signaling isantiapoptotic in most circumstances (21). However, theTunel assay (Supplementary Fig. S4B) indicated that, ondays 7 and 11 after IR, Hh activation from day 3 did notsignificantly affect the IR-induced apoptosis, suggesting thatthebeneficial effects of post-IR transientHhactivation is notlikely associated with the inhibition of apoptosis. Interest-ingly, although concurrent transient Hh activation (days 0–7) significantly inhibited apoptosis on day 3, it significantlyincreased apoptosis ondays 7 and11,whichmay contributeto the much less improvement of salivary function in thisgroup. Thedelayed increase of apoptosis ondays 7 and11 inthe concurrent Hh activation group might be associatedwith increased Ptch1 expression that is proapoptotic inthe absence of Hh ligands (22) and decreased expressionof the antiapoptosis protein p21Waf1 (23) after Hh activa-tion as shown further.The Hh target gene Bmi1 is associated with the mainte-

nance of various adult epithelial stem cells throughrepression of cyclin-dependent kinase (CDK) inhibitorsp16Ink4a/p19Arf and p21Waf1 (24–26). As indicated byqRT-PCR analysis (Fig. 3C, n ¼ 3), the expression of Bmi1mRNA in SMGs was significantly upregulated by transientHh activation, decreased by IR on day 10 but not on day120, and recovered by transientHh activation 3days after IR(P < 0.05). Meanwhile, the expression of p16Ink4a andp21Waf1 mRNA was regulated conversely to that of Bmi1 byHh activation and IR, and remained at a high level on day120 after IR (P < 0.05). Flow cytometric analysis (Fig. 3D, n¼ 3) confirmed that the Bmi1þ cell population in SMGswassignificantly increased after 7 days of Hh activation (1.95%� 0.23% vs. 1.02% � 0.07% in the NT group, P < 0.05),decreased on day 10 after IR (0.22% � 0.06%, P < 0.05 vs.NT), and was preserved by Hh activation 3 days after IR(0.74% � 0.22%, P > 0.05 vs. NT). Western blot analysisconfirmed that the expression of p21Waf1 in SMGs wassignificantly increased by IR on day 120 but reversed bytransient Hh activation 3 or 90 days after IR (Fig. 3E, P <0.05, n ¼ 3). In addition, flow cytometry indicated thatBmi1 is expressed in a subpopulation of c-Kitþ SSPCs that isexpanded by Hh activation (Supplementary Fig. S5). Thesedata indicated that the rescue of SSPCmaintenance after IRby Hh activation is related to the Bmi1 pathway.

Preservation of parasympathetic innervation, Chrm1–HB-EGF signaling and expression of neurotrophicfactors after IR by transient Hh activationParasympathetic innervation is essential for both organ-

ogenesis and regeneration of salivary glands after ductligation (27, 28), whereas IR reduced parasympatheticinnervation in the salivary glands of both human adults

and mouse embryos (29). By examining the activity orexpression of two markers of parasympathetic nerve, AChEand glial cell line-derived neurotrophic factor family recep-tor a2 (GFRa2; Fig. 4A and B, n¼ 3), we confirmed that IRimpaired parasympathetic innervation similarly in adultmouse SMGs on day 120 (P < 0.01). The impairment ofAChE activity by IR was significantly ameliorated by tran-sient Hh activation 3 or 90 days after IR (P < 0.05), whereasGFRa2 expression was remarkably increased by transientHh activation alone (P < 0.01) and was not significantlydecreased in SMGs that underwent transient Hh activation3or 90days after IR comparedwith that in theNTgroup (P>0.05). These data indicated that the parasympathetic inner-vation is preserved by transient Hh activation after IR.

Hh signaling is known to promote innervation or regen-eration of various peripheral nerves via maintenance of

Figure 4. Transient Hh activation preserved the parasympatheticinnervation and the expression of neurotrophic factors impaired by IR.A, AChE staining on SMG sections and the quantification of stainingintensity. B, Western blot and relative quantification of GFRa2. C,expression of neurotrophic factors andmiR-206 examined by qRT-PCR.D, concentrations of Bdnf in whole saliva or SMG homogenates and thatof SMG Nrtn detected by ELISA. n ¼ 3 for all studies.






neural structure (30), induction of angiogenesis (31), orinduction of neurotrophic factors such as brain-derivedneurotrophic factor (Bdnf) via repression of miR-206 orother unknown mechanisms (32, 33). Bdnf, Nerve growthfactor (Ngf), and Neurturin (Nrtn) are expressed in salivaryglands (34), and are essential for parasympathetic innerva-tion in embryonic mouse SMGs (29) or other tissues (35,36). As indicated by qRT-PCR (Fig. 4C, n¼ 3), transient Hhactivation significantly increased miR-206 expressionand decreased Bdnf expression, whereas IR significantlyincreased miR-206 expression and decreased Bdnf expres-sion both on days 10 and 120, and transient Hh activationafter IR significantly reversed the effects of IR on expressionof miR-206 and Bdnf on day 7 or 30 after Hh activation (P <0.05). The expression ofNgf andNrtnwas only significantlydecreased by IR on day 10 or 120, respectively (P < 0.05).Similar to Bdnf, the expression of Ngf and Nrtn was signif-icantly upregulated by transient Hh activation, and restoredor elevated by transient Hh activation after IR on day 10 or120, respectively (P < 0.05). ELISA confirmed that the Bdnflevel in SMG homogenates was significantly increased bytransient Hh activation, whereas the levels of Bdnf andNrtnin SMG homogenates and whole saliva Bdnf were signifi-cantly decreased on day 120 after IR and restored by

transient Hh activation either 3 or 90 days after IR (Fig.4D, P < 0.05, n ¼ 3).

During salivary organogenesis, parasympathetic innerva-tion signals via a cholinergic receptormuscarinic 1 (Chrm1)to promotes epithelial morphogenesis and proliferation ofprogenitor cells by transactivating theHeparin-Binding EGF(HB-EGF) pathway (27), which activates the expression ofgenes associated with cell-cycle progression such as cyclinA2 (Ccna2; ref. 37). Chrm1 expression is repressed by miR-107 (38), which is upregulated by IR (39) but decreased incells expressing high level of Shh (40). With qRT-PCRanalysis (Fig. 5A, n ¼ 3), we found that, in mouse SMGs,the expression of miR-107 was significantly decreased bytransient Hh activation and increased by IR as expected andrecovered by transient Hh activation after IR within 7 or 30days (P < 0.05); consistently, the expression of Chrm1 andCcna2 was significantly increased by transient Hh activa-tion, decreased by IR, and recovered by transient Hhactivation after IR (P < 0.05). Flow cytometry assay (Fig.5B, n¼ 3) indicated that the proportion of Chrm1þ cells inSMGs was significantly increased after transient Hh activa-tion (6.79% � 1.55% vs. 1.63% � 0.24% in the NT group,P < 0.05), decreased on day 10 after IR (0.07%� 0.03%, P <0.05 vs.NT), andwaspreservedbyHhactivation3days after

Figure 5. Transient Hh activationpreserved the activity of the Chrm1pathway impaired by IR. A,expression of miR-107, Chrm1,and Ccna2 examined by qRT-PCR(n ¼ 3). B, flow cytometric analysisof Chrm1þ cells in SMGs. C,immunofluorescence staining ofChrm1.

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IR (1.88% � 0.21%, P > 0.05 vs. NT). Immunofluorescentstaining for Chrm1 (Fig. 5C) indicated that, in nontreatedSMGs, Chrm1 is expressed weakly in acinar structures andstrongly in ductal structures; IR significantly downregulatedChrm1 expression particularly in ductal structures, whereastransient Hh activation after IR restored Chrm1 expression.These data suggested thatChrm1–HB-EGF signaling activitywaspreservedby transientHhactivation after IR,whichmaycontribute to the preservation of functional SSPCs in com-bination with preserved parasympathetic innervation.

Effects of IR and transient Hh activation on SMGs offemale mice and human salivary epithelial cellsSexual dimorphism is well recognized in the salivary

glands of rodents, and the SMGs of male rodents containa unique structure—granular convoluted tubule (GCT)—that is not present in SMGs of human or female rodents(41). To determine whether the data obtained earlier frommalemice are gender-specific, we examined the effects of IRand transient Hh activation with a small-molecule Hhagonist SAG (SmoothenedAgonist) in SMGsof femalemiceon the basis of the comparable expression of Smoothened inSMGs betweenmales and females (Supplementary Fig. S6).As indicated by qRT-PCR (Fig. 6A, n ¼ 3), in female SMGs,

IR affected the expression of genes related to SSPC main-tenance (c-Kit,Bmi1, p16,Chrm1 andCcna2) ondays 10 and120 and the gene essential for parasympathetic innervation(Bdnf) on day 10 after IR in amanner similar to that inmaleSMGs (P < 0.05). Local delivery of SAG by cannulationefficiently activated the Hh pathway in ductal SMG epithe-lial cells of female mice as indicated by induction of thePtch1-lacZ Hh reporter (Supplementary Fig. S6). Similar tothat inmale SMGs transiently overexpressing Shh, qRT-PCRindicated that SAG treatment significantly upregulated theexpression of c-kit, Bmi1, Bdnf, Chrm1, and Ccna2, anddecreased p16 expression (P < 0.05).

To examine the effects of IR and transient Hh activationon human salivary glands, we isolated human salivaryepithelial cells from biopsy samples. These cells showed atypical cobblestone morphology of epithelial cells in cul-ture, and most cells expressed the epithelial marker Krt14and/or the salivary glandular cell marker Aqp5 (Supple-mentary Fig. S7). Similar to that in mouse SMGs, qRT-PCR(Fig. 6B, n¼ 4) indicated that, in human salivary epithelialcells collected on day 10 after IR, the mRNA expression ofBMI1, BDNF, CHRM1, and CCNA2 was significantlydecreased, whereas that of P16 and P21 was significantlyincreased (P < 0.05). These IR-induced changes of geneexpression were not significantly affected by infection withcontrol adenovirus encoding GFP (GFP-Ad; P > 0.05), butwas significantly reversed by infection with adenovirusencoding Gli1 (Gli1-Ad) 3 days after IR (P < 0.05). Westernblot assay confirmed that the expression of P21Waf1 proteinwas upregulated by IR, and this upregulation was notaffected by GFP-Ad infection, but was repressed by Gli1-Adinfection (Fig. 6C, P < 0.05, n ¼ 4).

Taken together, these data indicated that the effects of IRand transient Hh activation on SMGs of female mice andcultured human salivary epithelial cells are similar to thatin male mice, and suggested that the transient activation ofthe Hh pathway might restore the expression of genesrelated to SSPC maintenance and parasympathetic inner-vation impaired by IR to rescue IR-induced hyposalivationin humans.

Effects of Shh gene transfer within SMGs on solidtumor

Because the activation of the Hh pathway in head andneck cancer is linked to poorer outcomes (42), it is impor-tant to determine whether transient Hh activation withinthe salivary gland can affect Hh activity and the growth ofsuch preexisting cancers. Squamous cell carcinoma is themost commonhead and neck cancer; therefore, we used themouse SCC VII tumor model to test this possibility. Infect-ing SCC VII cells in vitro with adenovirus-encoding rat Shh(AdShh) significantly increased the expression of Hh targetgenes Gli1 and Ptch1 in 7 days (Supplementary Fig. S8A),indicating that these cells are Hh-responsive. Retrogradedelivery of AdShh into SMGs ofmice carrying subcutaneousSCC VII tumors significantly increased the expression of therShh transgene and the Hh target genes Gli1 and Ptch1 inSMGs in 7 days (n ¼ 4, P < 0.05), but had no significant

Figure 6. Effects of IR and transient Hh activation on gene expression inSMGs of female mice and human salivary epithelial cells. A and B, geneexpression examined by qRT-PCR in SMGs of female C57BL/6 mice 10or 120 days after IR or in SMGs of female Ptch1-lacZ mice treated withSAG for 3 days (A, n ¼ 3), and in human salivary epithelial cells 10 daysafter IRwith or without infection of Gli1 or GFP adenoviruses (B, n¼ 4). C,Western blot and relative quantification of human P21 (n ¼ 4).






effect on the expression of these genes in tumors at this timepoint or on tumor growth with or without IR of tumorthroughout the observation period (Supplementary Fig.S8B–D, P > 0.05). Similar results were found when AdShhwas delivered into SMGs right after subcutaneous inocula-tion of 10 times fewer SCC VII cells (SupplementaryFig. S8E). These data indicated that local Hh activationcould be achieved in SMGs without promoting the growthof preexisting tumors outside the salivary glands.

DiscussionThe lossof functional SSPCs is believed tobeamajor cause

of IR-induced hyposalivation (4). We reported here thattransient Hh activation promotes SSPC expansion withoutIR and rescues SSPC maintenance after IR. Hh signalingregulates adult stem/progenitor cells upstream of the Bmi1pathway in other epithelial tissues (24, 43). Bmi1 is atranscriptional repressor that regulates stem cell self-renewalthrough the repression of important cell–cycle-regulatorygenes including p16INK4A, p19ARF, and p21Waf1 (24–26).Our data indicated that, in the salivary glands, the expressionof Bmi1 and its target genes is highly associatedwith changesof the functional SSPCpopulation causedby IRand transientHh activation, suggesting that the Bmi1 pathway may medi-ate the effect of Hh activation on SSPC maintenance.

In human salivary glands (29). Hh signaling can protectas well as promote the recovery of various peripheral nervesfrom injuries (30–32).We showedhere that, in adultmouseSMGs, the expression of multiple neurotrophic factors, theparasympathetic innervation, and the Chrm1–HBEGF sig-naling pathway connecting the parasympathetic innerva-tion to the maintenance of stem/progenitor cells weresignificantly impaired by IR and rescued by transient Hhactivation after IR, whichmay also contribute to IR-inducedhyposalivation and the rescue by Hh activation. Themechanisms of regulation by IR and Hh activation on theexpression of Bdnf and Chrm1 were related with miR-206and -107 respectively, but other mechanisms and those onthe expression of Ngf and Nrtn remain to be explored.

Although it was reported that aberrant regulation of Hhsignaling is associated with tumorigenesis of head and neckcancer (44), 7-day induction of Shh overexpression in basalepithelia in the present study led to Hh activation compa-rable with that observed after duct ligation and did notresult in any detectable tumor in mice for up to 4 monthsafter induction. Prolonged Hh activation for 15 weeks insalivary glands by Gli1 transgene resulted in hyperplasia,but these lesions regressed after withdrawal of transgeneexpression and became histologically normalized (45).These data indicated that the probability of inducing cancerin adult salivary glands by transient Hh activation at near-physiologic levels is likely very rare. For potential applica-tion in survivors of head and neck cancer, great cautionshould be exercised to only activate the Hh pathway in thesalivary gland to eliminate the risk of prompting growth,relapse, or metastasis of preexisting cancers. Advantageous-ly, salivary glands are well encapsulated, and local gene/

protein/drug delivery can be easily achieved by cannulationvia the orifices in the mouth. Previous reports and ourpreliminary data have indicated that transgenes deliveredby thismethodwould neither significantly increase levels oftransgenes and/or protein products systemically nor thegrowth of preexisting tumors (13).

Interestingly, in SMGs of female mice, transient Shhoverexpression failed to activate the Hh pathway efficientlyand rescue the IR-induced hyposalivation, which is relatedto the lower expression of both endogenous components ofHhpathway andShh transgene. For future studywith femalemice and, probably, other animal models, such genderdifferences should be considered and a higher dose or analternative agonist may be needed to achieve efficient Hhactivation in female salivary glands. In SMGs of femalemiceand human salivary epithelial cells from female patients,transient Hh activationwas achievedwith other approachesand similarly regulated expression of genes associated withSSPCs andparasympathetic innervation, suggesting that thetransient Hh activation may also rescue IR-induced hypo-function in females. Even in the worst scenario that thisstrategy only works in males, it will still be of significantimportance because approximately two-thirds of thepatients with head and neck cancer are male (1, 2).

In summary, we have been able to rescue salivary glandfunction by transient activation of Hh signaling in a mousemodel. The mechanism is associated with the preservationof functional stem/progenitor cells and parasympatheticinnervation. We observed similar rescue effects on theexpression of genes related to both aspects in culturedhuman salivary epithelial cells, suggesting the potential oftransient Hh activation in treating IR-induced hyposaliva-tion in human patients.

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

Authors' ContributionsConception and design: B. Hai, F. LiuDevelopment of methodology: B. Hai, L. Qin, F. LiuAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B. Hai, L. Qin, Z. Yang, Y. Zhao, S. KimAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): B. Hai, Z. Yang, X. Ti, F. LiuWriting, review, and/or revision of themanuscript: B. Hai, S. Kim, F. LiuAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Shangguan, D. Rangaraj, F. LiuStudy supervision: D. Rangaraj, F. LiuOther: Q. Zhao

AcknowledgmentsThe authors sincerely thank Drs. Adam Glick of Pennsylvania State

University, Jeff Whitsett of Cincinnati Children’s Hospital Medical Center,and Changyu Zheng of the National Institutes of Health for providing Krt5-rtTA mice, tetO-Shh mice, and SCC VII cells respectively.

Grant SupportThis study was financially supported by the NIH/NIDCR

1RC1DE020595-01 (FL) and 1R01DE022975-01 (FL).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMay 23, 2013; revised September 24, 2013; acceptedOctober 11,2013; published OnlineFirst October 22, 2013.

Hai et al.





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2014;20:140-150. Published OnlineFirst October 22, 2013.Clin Cancer Res Bo Hai, Lizheng Qin, Zhenhua Yang, et al. Stem/Progenitor Cells and Parasympathetic InnervationIrradiation-Induced Hyposalivation by Preserving Salivary Transient Activation of Hedgehog Pathway Rescued

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