cxcr4 activation deﬁnes a new subgroup of sonic …molecular and cellular pathobiology cxcr4...

Molecular and Cellular Pathobiology

CXCR4 Activation Defines a New Subgroup of SonicHedgehog–Driven Medulloblastoma

Rajarshi Sengupta1, Adrian Dubuc6, Stacey Ward1, Lihua Yang2, Paul Northcott6, B. Mark Woerner1,Kirsten Kroll3, Jingqin Luo5, Michael D. Taylor6, Robert J. Wechsler-Reya7, and Joshua B. Rubin1,4

AbstractMedulloblastoma prognosis tends to be poor, despite aggressive therapy, but defining molecular subgroups

may identify patients who could benefit from targeted therapies. This study used human gene array andassociated clinical data to identify a new molecular subgroup of medulloblastoma characterized by coactivationof the Sonic hedgehog (SHH) and CXCR4 pathways. SHH–CXCR4 tumors were more common in the youngestpatients where they were associated with desmoplastic histology. In contrast to tumors activating SHH but notCXCR4, coactivated tumors exhibited greater expression of Math1 and cyclin D1. Treatment with the CXCR4antagonist AMD3100 inhibited cyclin D1 expression and maximal tumor growth in vivo. Mechanistic investiga-tions revealed that SHH activation stimulated CXCR4 cell surface localization and effector signaling activity,whereas SHHabsence causedCXCR4 to assume an intracellular localization. Taken together, ourfindings define anew medulloblastoma subgroup characterized by a functional interaction between the SHH and CXCR4pathways, and they provide a rationale to clinically evaluate combined inhibition of SHH and CXCR4 formedulloblastoma treatment. Cancer Res; 72(1); 122–32. �2011 AACR.

Introduction

Medulloblastoma is the most common malignant braintumor of childhood. Despite aggressive multimodal therapy,5-year event-free survival and overall survival rates remainless than 70% (1). Furthermore, survivors often suffer severeneurologic, cognitive, and endocrine side effects. Thus, thereis a need for new and improved therapy. The development ofnovel therapies will be facilitated by definition of molecularsubgroups of disease in which the primary drivers of malig-nant growth are distinguished and the cell(s) of originidentified.

Multiple prognostically significant histologic subtypes ofmedulloblastoma are recognized (2, 3). Recent gene expressionprofiling has also revealed medulloblastoma to comprise dis-tinct molecular subgroups characterized by dysregulations in(i) WNT, (ii) Sonic hedgehog (SHH), (iii) Myc, or (iv) as yet

unidentified signaling pathways (4–6). Moreover, genetic anal-yses and lineage tracings have determined that at least 2distinct progenitor populations give rise to medulloblastoma,cerebellar granule neuron precursors (GNP), and cells of thelower rhombic lip (7–9).

Medulloblastomas derived from GNPs frequently exhibitconstitutive activation of the SHH pathway as a resultof mutations in patched (Ptc), suppressor of fused (Sufu; refs.10–13), or smoothened (Smo; ref. 14). Sonic subgroup tumorscan exhibit desmoplastic, classic, or anaplastic large cellhistologies. These differing histologies are associated withdistinct outcomeswith desmoplastic tumors possessing super-ior survival and anaplastic large cell tumors possessing inferiorsurvival, comparedwith classic tumors (1). Thus, SHHpathwayactivation alone does not determinemedulloblastoma biology,even within the SHH subgroup tumors.

A role for coacting pathways during tumorigenesis is alsoindicated by studies of a mouse model of medulloblastomasecondary to heterozygous loss of Ptc. Ptc nullizygous preneo-plastic lesions develop in the cerebellum of greater than 50% ofthesemice. However, only 14% to 20% of these animals developmedulloblastoma, indicating that events in addition to muta-tional activation of the SHH pathway are necessary for tumor-igenesis (15, 16).

A potentially important coacting factor is the chemokineCXCL12. During GNP development CXCL12 and its receptorCXCR4 are required for maximal SHH-induced proliferation(17). Moreover, CXCL12 and CXCR4 are expressed in severalbrain tumors including medulloblastoma, and the level ofCXCR4 expression has prognostic significance (18–20). Inhi-bition of CXCR4 with the specific antagonists AMD3100 or

Authors' Affiliations: Departments of 1Pediatrics, 2Pathology, 3Develop-mental Biology, and 4Anatomy andNeurobiology, 5Division ofBiostatistics,Washington University School of Medicine, St Louis, Missouri; 6Division ofNeurosurgery and Labatt Brain Tumor Research Center, Hospital for SickChildren, Toronto, Ontario, Canada; and 7Sanford-Burnham MedicalResearch Institute, La Jolla, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Joshua B. Rubin, Department of Pediatrics, Divi-sion of Hematology/Oncology, Washington University School of Medicine,CampusBox 8208, 660SouthEuclid Ave, St Louis,MO63110. Phone: 314-286-2790; Fax: 314-286-2892; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1701

�2011 American Association for Cancer Research.

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Published OnlineFirst November 3, 2011; DOI: 10.1158/0008-5472.CAN-11-1701

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AMD3465 blocked the intracranial growth of xenografts of thedesmoplastic medulloblastoma-derived cell line Daoy, sug-gesting that CXCR4 activation may also be important formaximal SHH-driven medulloblastoma growth (21).Hereinwe report that overexpression of CXCR4 occurs in the

SHH subgroup of medulloblastoma. Moreover, in vitro as wellas in vivo data indicate that the SHH pathway enhances CXCR4surface localization and signaling, resulting in a progrowthtranscriptional response, maximal tumor growth, and a sen-sitivity to the antitumor effects of AMD3100. On the basis ofthese findings, we propose an additional molecular subgroupof medulloblastoma in which the SHH and CXCR4 pathwayssynergize to promote tumor growth, and for which combinedSHH and CXCR4 antagonism may provide a therapeuticbenefit.

Materials and Methods

Animals were used in accordance with an established Ani-mal Studies Protocol approved by The Washington UniversitySchool of Medicine Animal Studies Committee. Chemicalswere obtained from Sigma unless otherwise noted.

Cell culturePrimary ND2:SmoA1 medulloblastoma isolates. Tumors

were isolated from SmoA1mice by surgical dissection.Meningeswere removed and the tissue was dissociated with Accutase-enzyme cell detachmentmedium (eBiosciences) containing 125units/mL DNase (3� for 15 minutes). Cells were strainedthrough a 70-mm nylon mesh (Falcon) and plated onto poly-D-lysine–coated (20 mg/mL) plates (2 � 106/mL) in Dulbecco'sModified Eagle's Medium/F12 (Life Technologies) containingN2 (Life Technologies), 20mmol/L KCl, and 36mmol/L glucose.Human medulloblastoma (Daoy) cells were obtained from

American Tissue Culture Collection and used at low passage(<10) as previously described (21).GNP cells. Primary cultures of purified GNPs were pre-

pared from postnatal day 6 wild-type C57/BL6 mice as previ-ously described (21).

Growth factor and drug treatmentsIn vitro treatments. SmoA1 tumor, GNPs, Daoy, 293T, or

HOSX4 cells were serum starved (24 hours) and treated with 1mg/mL CXCL12 (PeproTech). Pretreatments were with/with-out 1 mg/mL SHH (R&D) or 10 mmol/L cyclopamine [in 2%sodium phosphate/citrate buffer (pH¼ 3) containing 2-hydro-propyl-b-cyclodextrin (HPBCD)] for 12 hours prior to CXCL12treatment.AMD3100 treatment of ND2:SmoA1 flank xenografts.

Subcutaneous flank implants of 4 � 106 SmoA1þ/� medul-loblastoma cells in Matrigel were made in nude mice(Jackson). Tumor growth was monitored using serial cali-per-based volumetric measures. Mice harboring tumorsthat exhibited steady growth over a 4-week period andwere approximately 10 mm � 10 mm (length � width)underwent subcutaneous insertion of Alzet minipumpsfilled with AMD3100 (30 mg/mL; n ¼ 7) or PBS (n ¼ 5).Serial tumor volume measurements continued until mice

were euthanized 11 days after initiating treatment. Tumorswere isolated and fixed in Prefer for immunohistochemistryor lysed in TRIzol for RNA analysis.

cAMP measurementIntracellular cAMP concentrations in SmoA1 medulloblas-

toma or Daoy cells pretreated with/without cyclopamine (10mmol/L, 12 hours) then CXCL12 (1 mg/mL) for varying times(0–20 minutes) were determined as described (22).

Western blot analysesWhole-cell lysates. SDS-PAGE of cell lysates (40 mg/lane)

was carried out as described (21).Gai activation assays. Activated Gai pull-down assays

(NewEast Biosciences) followed the manufacturer's protocol.In brief, serum-starved cells (48 hours) were stimulated withCXCL12 (1 mg/mL, 10 minutes). Cell lysates were incubated for1 hour with an anti-active Gai antibody and Protein A-agarosebeads. Activated Gai was quantified by Western blot analysisusing an anti-Gai antibody.

Cell surface protein purification. Daoy cells (10 � 106)were pretreated with cyclopamine or vehicle for 12 hoursfollowed by CXCL12 (1 mg/mL, 30 minutes) or vehicle. TheCell Surface Protein Isolation Kit (Pierce) was used asdescribed (23).

The following antibodies were used in Western blot analy-ses: anti-CXCR4 [Leinco Tech Inc. (1:1,000)], anti-Akt, anti-phospho-Akt (Ser473), anti-ERK, and anti-phospho-ERK (CellSignaling; 1:1,000); anti-b-actin (Sigma; 1:10,000). Odyssey V3.0(LI-COR) image acquisition/analysis system was used fordetection and quantification of fluorescent bands. Normaliza-tion was to Actin or total content of specific proteins asindicated. Data are expressed as percentage of control (mean� SEM). Statistical analysis was carried out by one-wayANOVA. P values are reported in legends.

Measurement of changes in intracellular calciumPostnatal day 6 GNPs were cultured on poly-D-lysine–

coated glass bottom culture dishes (MatTak Corporation)and pretreated with/without SHH (1 /g/mL) for 12 hours.Daoy cells were pretreated with cyclopamine (10 mmol/L) orvehicle for 12 hours. Calcium flux was measured as described(17). Changes in cytosolic-free calcium were determined bymonitoring excitation fluorescence using a dual-wavelengthexcitation source fluorimeter (Fura-2 filter, TILL micro-scope, TILL photonics GmbH Lochhamer Schlag 19Germany) in response to sequential excitation at 340 nmand 380 nm. Data are presented as the relative ratio offluorescence at 340 and 380 nm.

Cell proliferation assayA total of 1 � 105 SmoA1 tumor cells cultured in poly-D-

lysine–coated 96-well flat-bottom plates were treated withAMD3100 (2.5 ng/mL) or CXCL12 (1 mg/mL) as indicated.Proliferation was determined per manufacturer's instructionsby the CellTiter 96 AQueous One Cell Proliferation AssaySystem (Promega) and a mQuant microplate Reader (Bio-TekInstruments).

SHH and CXCR4 Signaling in Medulloblastoma

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Reverse transcription PCRComplementary DNA and quantitative PCR was carried out

with RNA isolated from flank tumors as described (22). Reac-tion conditions included: 95�C (10 minutes), followed by 40cycles of 95�C (30 seconds) and various annealing tempera-tures (1 minute), a final extension at 72�C (1 minute), followedby a melting curve from 55 to 95�C. No-template controls werenegative. All reactions were normalized to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the same plate.

Fold change was calculated by the formula 2 �DDCtð Þ.

ImmunohistochemistryHuman medulloblastoma specimens were obtained from

the Department of Pathology at Washington University (St.Louis, MO) per an Institutional Review Board–approved pro-tocol for the use of archived pathologic specimens. Stainingwas accomplished as described (24) with the addition ofTyramide Signal Amplification (Perkin Elmer). Antibodiesused and dilutions; CXCR4 clone 12G5 at 1:6,500 (R&D Sys-tems), CXCR7 clone 11G8 at 1:1,000 (kind gift from MarkPenfold, Chemocentryx), CXCL12 at 1:500 (PeproTech), andMouse IgG 1 mg/mL (Jackson ImmunoResearch).

Human tissue samples, RNA preparationPrimary medulloblastoma (n ¼ 112) and normal cerebella

(fetal n ¼ 9, adult n ¼ 5) were obtained in accordance withthe Hospital for Sick Children (Toronto, Canada) ResearchEthics Board and profiled on Affymetrix GeneChip HumanExon 1.0ST arrays by the Toronto Centre for Applied Geno-mics (TCAG) as published (4). Molecular subgroups wereestablished as previously described for 103 of 112 samples(4).

CXCR4-high versus -low medulloblastoma classrationale

Samples were divided into CXCR4-high or -low expresserson the basis of their expression patterns relative to averagefetal cerebellar levels in a relaxed and stringent analysis.Tumors with more than 1.5-fold (relaxed) and more than 2-fold (stringent) expression levels were classified as CXCR4-highexpressers and those with less than �1.5-fold (relaxed) or lessthan �2-fold (stringent) as CXCR4-low expressers.

Molecular analysis of CXCR4-high versus -low tumorsTo detect gene expression changes associated with CXCR4-

high versus -low expression, Gene Set Enrichment Analysis(GSEA; ref. 25) was used. As previously discussed (4), a com-parative enrichment score for ranked genes in CXCR4-highversus -low expressers was calculated using 1,000 permuta-tions permitting the detection of statistically significant genesand pathways associating with each phenotype.

Results

CXCR4 is overexpressed in the SHH subgroup ofmedulloblastoma

Gene expression profiling has established 4 molecular sub-groups of medulloblastoma: WNT, SHH, group C, and group D,

with distinct demographics, clinical presentation, transcrip-tional profiles, genetic abnormalities, and outcome (4). Thetranscriptional profile of the SHH subgroup resembles an earlydevelopmental stage of cerebellar GNPs when their prolifera-tion is driven by SHH (26). During early development, SHH-induced GNP proliferation is modulated by multiple factorsincluding CXCR4, a G-protein–coupled receptor (GPCR), alsoimplicated in medulloblastoma growth (18, 21). We evaluatedCXCR4 expression in normal human fetal and adult cerebel-lum, and acrossmedulloblastoma subtypes. CXCR4 expressionwas high in the fetal cerebellum and declined significantly withage. A broad range of CXCR4 expression levels were evident inmedulloblastoma with Group C and D tumors exhibiting lowlevel, adult cerebellum-like CXCR4 expression, and WNT andSHH subtype tumors exhibiting high level, more fetal cerebel-lum-like levels of expression (Fig. 1A). While CXCR4 waspresent in both the WNT and SHH subtype tumors, only theSHH subtype tumors exhibited CXCR4 overexpression relativeto the normally high levels found in the fetal cerebellum.

The SHH medulloblastomas could be further categorizedinto CXCR4-high and -low expressing subgroups (Fig. 1B). Themajority of SHH medulloblastomas belonged to the CXCR4-high subgroup (�80%). The level of CXCR4 expression wascorrelated with other prognostically important features ofdisease such as histology and age. Desmoplastic tumors wereexclusively in the CXCR4-high subgroup. Anaplastic large celltumors occurred at a rate of only 4% in the CXCR4-high,compared with 25% in the CXCR4-low subgroup. In addition,nearly half of the CXCR4-high tumors occurred in infants,whereas only 13% of CXCR-low tumors occurred in this agegroup.

While rare mutations in CXCR4 have been described inmedulloblastoma (27), CXCR4 activation appears to be liganddependent. The CXCR4 ligand, CXCL12 was detected in geno-mic profiling of the SHH subgroup, and as previously described(18, 21), was primarily limited to the endothelium of tumor-associated blood vessels (Fig. 1C). Recently, a second CXCL12receptor, CXCR7,was identified and shown to be expressed andfunctional in certain brain tumors such as glioblastoma (28).To determine whether CXCR7 might also mediate CXCL12effects in the SHH subgroup of medulloblastoma we analyzedgenomic data as well as tissue sections for evidence of CXCR7expression. Little to no CXCR7mRNA expression was detectedin microarray analyses. Immunohistochemical staining oftumor sections from 3 separate human desmoplastic medul-loblastoma specimens revealed CXCR7 expression primarilylimited to endothelial cells (Fig. 1C). This was in contrast toCXCR4 expression, which as previously described, was evidentat high levels throughout the specimens (18, 21). Thus CXCR4appears to be the sole mediator of CXCL12 effects onmedulloblastoma.

SHH pathway activation is required for CXCR4 signalingin medulloblastoma

Understanding how the SHH and CXCR4 pathways inter-act could elucidate new mechanisms of medulloblastoma-genesis and identify novel therapeutic targets. A comparisonof CXCR4 activation between postnatal day 6 cerebellar

Sengupta et al.

Cancer Res; 72(1) January 1, 2012 Cancer Research124




GNPs and Daoy cells revealed that Daoy cells, which possessconstitutive activation of the SHH pathway exhibit signifi-cantly stronger responses to CXCL12 than GNPs (21). Wepostulated that among the differences between GNPs andDaoy cells were the degree of SHH pathway activation andthat SHH would potentiate CXCR4 function. To test thishypothesis we evaluated whether blockade of SHH signalingin medulloblastoma cells abrogated the effects of CXCL12.For these studies we used primary tumor cells derived fromspontaneous medulloblastoma that arise in ND2:SmoA1mice which possess an activating mutation in smo, consti-tutive SHH pathway signaling, and a high incidence ofmedulloblastoma (29). We treated dissociated SmoA1 tumorcells in vitro for 12 hours with the SHH pathway antagonistcyclopamine or vehicle and measured the effects onCXCL12-induced cAMP suppression. CXCL12 stimulationresulted in a significant reduction of intracellular cAMPlevels, which was completely blocked by pretreatment withcyclopamine, but not its inactive stereo-isomer, tomatidine(Fig. 2A). The effect of SHH had specificity for CXCR4 ascyclopamine pretreatment had no effect on cAMP elevationin response to PACAP, the ligand for the Gas-coupled GPCR,PAC1 (Fig. 2B; ref. 30).The earliest stages of CXCR4 signaling involve Gai activa-

tion. Thus, we examined the effect of cyclopamine on CXCR4-Gai coupling. Intracellular Gai activation was measured by

immunoprecipitation of the GTP-bound (activated) form ofGai. Acute stimulation of SmoA1 tumor cells with CXCL12(1mg/mL, 10 minutes) resulted in a significant increase in Gaiactivation, which was blocked by cyclopamine pretreatment(Fig. 2C and D). These data indicated that CXCL12-inducedcAMP suppression in SmoA1 cells was a direct consequence ofCXCL12-induced Gai activation and that the SHH pathway isrequired for this action. Together, these data support thehypothesis that mutational activation of SHH pathway sensi-tizes CXCR4 function.

We usedDaoy cells to further explore themolecular basis forSHH effects on CXCR4 signaling. Similar to SmoA1 cells,CXCL12-induced activation of Gai and its downstream effec-tors in Daoy cells was dependent upon activation of the SHHpathway (Supplementary Fig. S1A and S1B). CXCL12-inducedcAMP suppression (Supplementary Fig. S1C), F-actin polymer-ization (Supplementary Fig. S1D), calcium mobilization (Sup-plementary Fig. S1E and S1F), and extracellular signal-regu-lated kinase (ERK) 1/2 and Akt activation (Supplementary Fig.S1G and S1H) were all blocked by cyclopamine pretreatment.Consistent with Gai-dependant signaling, CXCL12-inducedcalcium flux and F-actin polymerization was completely abol-ished by pertussis toxin (data not shown). Inhibition of CXCR4function was not the result of changes in total CXCR4 expres-sion which was unaffected by cyclopamine (SupplementaryFig. S1G).

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Figure 1. CXCR4 is overexpressed in the SHH subgroup of medulloblastoma. A, CXCR4 expression in normal cerebella (fetal n ¼ 9, adult n ¼ 5) andmedulloblastoma (MB; n ¼ 103) samples profiled on Affymetrix Exon arrays. Statistically significant upregulation of CXCR4 is evident in SHH subgrouptumors relative to normal fetal cerebellar (P < 3.25E-2) and non-SHHmedulloblastoma (P < 3.41E-5) levels. B, histologic and age associations for CXCR4-highand -low SHHmedulloblastomas. Tumors were classified as high or low on the basis of 2-fold differences compared with the average normal fetal cerebellarlevels of CXCR4. C, representative sections from a human desmoplastic medulloblastoma specimen stained for CXCL12, CXCR4, and CXCR7. Brown,positive staining. Mouse IgG controls were negative. Scale bar, 50 mm.






To address the possibility that cyclopamine might havedirect off-target effects on CXCR4, we treated HOSX4 cellswith CXCL12 in the presence and absence of cyclopamine.HOSX4 cells are an osteosarcoma cell line that overexpressesCXCR4 but does not express components of the SHH pathwaysuch as Ptc and Smo. In these cells, CXCL12-induced ERK1/2phosphorylation was unaffected by cyclopamine (Supplemen-tary Fig. S2A and S2B). Thus, CXCR4 signaling is not alwaysdependent upon SHH action and cyclopamine has no directeffects on CXCR4.

CXCR4 function is SHH dependent in GNPsTo determine whether regulation of CXCR4 by the SHH

pathway might be limited to transformed cells, we culturednormal GNPs in the presence or absence of SHH for 12 hoursandmeasured CXCL12-stimulated calcium flux. In the absenceof continuous SHH treatment, SHH pathway activation isdownregulated in GNPs by 12 hours postisolation. This isevident in mRNA abundance of the SHH transcriptional targetGli1 (Supplementary Fig. S3A). CXCL12 treatment of GNPs inthe absence of SHH had little to no effect on calcium flux (Fig.3A). In contrast, pretreatment of GNPs with SHH for 12 hourssensitized them to CXCL12 treatment, resulting in substantialCXCL12-induced calcium flux (Fig. 3A and B). Thus, SHHpathway activation can promote CXCR4 pathway responsive-ness even in normal, nonneoplastic cells.

It is possible that SHH effects on GNPs were not the resultof direct effects of SHH on CXCR4 but rather a consequence

of SHH's mitogenic effects and expansion of a CXCL12-responsive subpopulation of cells. To exclude this possibil-ity, we carried out parallel studies in 293T cells. 293T cellsexhibited basal and SHH-induced activation of the SHHpathway as evidenced by both SHH-stimulated and cyclo-pamine-inhibited Gli1 expression (Supplementary Fig. S3B–S3D). Neither SHH nor cyclopamine had any effect on 293Tcell number (Supplementary Fig. S3E). Basal responsivenessof the CXCR4 pathway was also evident as CXCL12 treat-ment resulted in Gai activation, cAMP suppression andphosphorylation of ERK1/2 and Akt (Supplementary Fig.S4A–S4F). Consistent with SHH pathway–dependent activa-tion of CXCR4 signaling, each of these CXCL12 effects wasblocked by pretreatment with cyclopamine. Therefore, weconclude that the SHH pathway directly modulates CXCR4signaling rather than merely expanding the relative abun-dance of a CXCL12-responsive subpopulation.

Together, these in vitro studies clearly indicate that crosstalkbetween the SHH and CXCR4 pathways, in both normal andneoplastic cells, can be an important regulator of CXCR4signaling.

SHH pathway regulates cell surface expression of CXCR4A potential mechanism for regulating CXCR4 coupling to

Gai activation is through control of CXCR4 cell surface local-ization. To quantitate SHH pathway effects on CXCR4 surfacelocalization we carried out surface biotinylation and surfaceprotein isolation on Daoy cells treated with CXCL12 and

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Figure 2. SHH pathway activation isrequired for CXCR4 signaling inmedulloblastoma. A, CXCL12-induced changes in cAMP levelsweremeasuredbyELISA inSmoA1tumor cells pretreated (12 hours)with cyclopamine (10 mmol/L, graytriangles), its inactive isomertomatidine (10 mmol/L, lighter graycircles), or vehicle control (HPBCD,black squares) and normalizedto t ¼ 0 values (�, P < 0.001for the difference between thecyclopamine and vehicle/tomatidine conditions by 2-wayANOVA, n ¼ 3). B, cyclopaminepretreatment did not affectPACAP-activated cAMP induction(n ¼ 3). C, CXCL12(1 mg/mL, 10 minutes) inducedsignificant Gai activation (GTPloading) without changing total Gaior CXCR4 levels in whole-celllysates. Cyclopamine (10 mmol/L)pretreatment blocked CXCL12effects. D, the mean � SEM (n¼ 3)for CXCL12 activation of Gai(�, P < 0.001) and cyclopamineblockade of this effect (#, P <0.001). IB, immunoblotting; IP,immunoprecipitation.

Sengupta et al.





cyclopamine. CXCL12 stimulation for 30 minutes led tosignificant loss of surface CXCR4 through internalization(Fig. 4A and B). Interestingly, exposure to cyclopamine for12 hours also significantly reduced cell-surface CXCR4, whichwas further reduced upon subsequent CXCL12 treatment.Cell surface CXCR4 levels are determined by the balance

between receptor internalization and insertion in the plasmamembrane. These data suggest that cyclopamine might blockthe membrane insertion of CXCR4, leading to an overallreduction in surface receptor levels. Neither CXCL12 norcyclopamine had any effect on the surface levels of PAC1,again suggesting that SHH effects have specificity for CXCR4rather than for GPCRs in general. Taken together, these datasuggest that SHHmodulates CXCR4 signaling by regulating thecell surface fraction of CXCR4.

CXCR4 is necessary for maximal growth of SHH-drivenmedulloblastoma

CXCL12 has a positive effect on SHH-induced GNP prolif-eration (17). To determine whether crosstalk between the SHHand CXCR4 pathways similarly affected the growth of medul-loblastoma cells, we measured proliferation of SmoA1 cells inresponse to CXCL12. Addition of CXCL12 to primary tumorcultures for 12 hours had a small but significant effect on cellproliferation. This effect was completely blocked by the CXCR4antagonist AMD3100 suggesting that maximal tumor cellproliferation may require both the SHH and CXCR4 pathways(Fig. 5A).

To test whether CXCR4 enhanced medulloblastomagrowth in vivo, we examined the effect of AMD3100 treat-ment on the growth of SmoA1 xenografts. Primary SmoA1tumor cells were isolated and injected into the flanks ofnude mice. Tumor volume was monitored over a 4-week

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Figure 3. SHH enhances CXCL12-induced CXCR4 signaling in GNP cells.A, CXCL12-induced (1 mg/mL) calcium flux was measured in P6 GNPs inthe absence (PBS, black tracings) or presence (preSHH, gray tracings) of1 mg/mL SHH pretreatment. Arrows indicate the start of CXCL12treatment. Representative fluorescence ratios (340/380) from 2 individualcells from each treatment group are presented. B, mean and SEM fordifferences between peak calcium flux ratios without SHH pretreatment(white bar) or with SHH pretreatment (black bar) was determined from 3separate experiments with 5 groups of cells analyzed per experiment.��, P < 0.001 as determined by 2 tailed t-test.

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Figure 4. SHH regulates cell surface CXCR4 levels. A, biotinylated plasmamembraneproteins fromDaoy cells treatedwith cyclopamine (10mmol/L,12 hours) or vehicle, followed by CXCL12 stimulation (1 mg/mL for 30minutes) were isolated. Surface and total CXCR4 as well as surfacePAC1 content were determined by Western blot analysis. B, means �SEM for cell surface CXCR4 (dark gray bars) and PAC1 receptor (lightgray bars) band densities normalized to control (vehicle). CXCL12 andcyclopamine pretreatment significantly lowered CXCR4 levels(�, P < 0.001, n ¼ 3).






period and animals that exhibited steady tumor growthunderwent implantation of subcutaneous osmotic infusionpumps filled with either PBS or AMD3100. Control animals(PBS group) all exhibited an increase in tumor volume overthe 2-week experimental period. AMD3100 treatment led tosignificant inhibition of tumor growth indicating thatCXCR4 potentiates SHH-driven medulloblastoma growthin vivo (Fig. 5B).

To further examine the importance of CXCR4 to medullo-blastoma growth, we treated Ptcþ/�, math1GFP mice withAMD3100. Medulloblastoma develops in about 15% of thesemice with a peak incidence between 16 and 24 weeks(10, 15, 16, 31, 32). Six tumors developed in the PBS (n ¼ 56)and 8 in the AMD3100 (n ¼ 57) group. While not statisticallydifferent, the median time to tumor presentation was delayed

to 14.9weeks in the AMD3100 group comparedwith 10.6 weeksin the PBS group (Supplementary Fig. S5B).

Coactivation of SHH and CXCR4 pathways results in aunique expression profile in medulloblastoma

The correlation between CXCR4 expression and histologicsubtype of medulloblastoma, together with the impact ofCXCR4 activation on model medulloblastoma growth, sug-gested that coactivation of the SHH and CXCR4 pathwaysmight result in a unique progrowth phenotype. We deter-mined that CXCR4-high and -low medulloblastoma pos-sessed unique transcriptional profiles and could be distin-guished by gene array analysis (Fig. 6). Prominent amongthose genes with differential expression was Atoh1 (Math1),a HLH transcription factor known to regulate GNP

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Figure 5. CXCR4 activation isnecessary for maximal growth ofSHH-driven medulloblastomas. A,proliferation of SmoA1 tumor cellswas measured as described inMaterials and Methods aftertreatment with media alone(vehicle), CXCL12 (1 mg/mL),AMD3100 (2.5 ng/mL), or acombination of the 2. Meanabsorbances� SEM normalized tovehicle are shown (n ¼ 3). CXCL12induced modest but significantproliferation comparedwith vehicle(�, P < 0.001); this was blocked byAMD (#, P < 0.001). B, AMD3100blocked SmoA1 tumor growth inmice. Presented are tumor volumemeasurements for each mouse inthe control (PBS) and AMD3100-treated (AMD) groups obtainedover the 11-day treatment period.Each bar represents the volumemeasurement for a single mouseon a given day. Each day (0, 2, 4, 7,9, and11) is represented by a shadebetween white to black asindicated. Treatment was initiatedon day 0, 4 weeks after tumorimplantation. Fold growth for day11 compared with day 1 isindicated beneath eachmouse andmean fold tumor growth over theentire treatment period for PBS(n ¼ 7, dark box) and AMD3100-treated (n ¼ 5, dark circle) animalsis shown in the inset. AMDtreatment significantly reducedtumor volume (�,P < 0.05, by 2-wayANOVA).

Sengupta et al.





proliferation and be required for medulloblastomagenesis.In addition, multiple SHH target genes (Hhip, Neurog1) weremore highly expressed in CXCR4-high tumors. To distin-guish between genes regulated by CXCR4 and genes merely

coregulated along with CXCR4, we isolated SmoA1 tumorcells from flank tumors treated with AMD3100 or PBS andmeasured mRNA abundance by quantitative PCR (Supple-mentary Table S1). Four genes appeared to be regulated byCXCR4 (Table 1). Remarkably, CXCR4 significantly sup-pressed the expression of PPP2R2C (�60%), the regulatorysubunit of the phosphatase PP2A and elevated the expres-sion of cyclin D1 (�20%). These changes in gene expressioncould enhance SHH-driven growth by promoting cell-cycleprogression (cyclin D1) and blocking differentiation viaPP2A-mediated dephosphorylation of S6 kinase (PPP2R2C;ref. 33). Together, these data suggest a model in whichfunctional interaction between the SHH and CXCR4pathways results in a unique progrowth phenotype inmedulloblastoma.

Discussion

Medulloblastoma, the most common malignant pediatricbrain tumor, arises from multiple cerebellar cell lineages andcan be driven by several genetic abnormalities. Recent geneexpression profiling and immunohistochemical studies haveelucidated 4 distinct molecular subgroups with unique expres-sion profiles (3, 4, 6). Three subgroups are characterized byabnormal activation of the Wnt, SHH, or Myc pathways. Thefourth subgroup includes tumors whose molecular founda-tions are not yet specified.

Clinically, Wnt tumors carry the best prognosis and thosemedulloblastoma with MYC alterations, the worst (34). SHHtumors exhibit an intermediate outcome (1). Identifying thepathways altered in each of these tumor subgroups isnecessary for the development of novel and molecularlytargeted therapies and in addition, promises to improveoutcome through molecular stratification of patients forclinical trials. In this regard, identification of a medulloblas-toma as belonging to the SHH subgroup may not providecomplete characterization. SHH pathway activation alonedoes not determine medulloblastoma biology. SHH medul-loblastoma can include low-risk desmoplastic tumors ormore aggressive anaplastic disease. Hence, secondary

CXCR4-high CXCR4-low

CXCR4OR51L1NEUROG1NS3ST5C4ORF18ISLR2HHIPAPOB48RATP6V0A1MAL2FLRT3PSMALSLC17A7SULF1QPCTKLRF1C10RF129IRAK3ITIH2FREM1SPAG6KLHDC6C180RF12PNPLA4RPL39LBNC2FLRT2OR7G2OR52E4FAM123AHCN1ASAMATOH1TBR1AKR1C2NOGCD200ASPNIF144PROKR2LCE3CRPS4Y2TMEM100C60RF32OPN4CYP39A1OTORMYBPHCOLEC12CLGNOPCMLPRAMEF1FLJ46210BHLHB3KCNQ5RNF175WBSCR19TRHDEPLCL1RASD2SGCDPXT1OR9A2AGTRL1MYOCDMCTP1PDE5AAQP9NDST3GALBBOX1BEST3TMEM144REG1BHS6ST2C20ORF42GFAPCRABP1DDX43PPP2R2CPCDHB2AQP4KCNJ10PMCHHOXA2STS-1TCERG1LNETO1PDE10AHSPB8FLJ21272MTMR8LGLV6-57PTPRKTEX15PTTG1ATAD4RP6-213H15SLC4A11SLC4A8

Figure 6. CXCR4-high versus -low SHH tumors are molecularly distinctGene Set Enrichment Analysis (GSEA) heatmap of the most differentiallyexpressed genes in CXCR4-high versus -low SHH tumors identified byphenotypic comparison using signal-to-noise metrics.

Table 1. Genes that are significantly alteredafter CXCR4 inhibition of SmoA1 flank tumors:CXCR4-regulated genes

Sample mRNA Ct (primer) �Ct (GAPDH)

Fold change[2 �DDCtð Þ]

P(t test)

PBS Cyclin D1 4.43 1.00AMD Cyclin D1 4.76 0.79 0.04PBS PPP2R2C 4.17 1.00AMD PPP2R2C 3.52 1.58 <0.01PBS PLCb4 7.67 1.00AMD PLCb4 6.58 2.13 0.04PBS PLCL1 8.15 1.00AMD PLCL1 7.01 2.21 0.02






pathways, acting in concert with SHH must also determinebiology within the SHH subtype. In the current study, weshow that the level of CXCR4 expression further divides theSHH group of medulloblastoma into relevant subsets. Weidentified 2 medulloblastoma populations with either highor low CXCR4 expression. These 2 subsets of SHH-drivenmedulloblastoma possess distinct molecular, epidemiologic,and histologic profiles. CXCR4-high tumors are predomi-nantly either desmoplastic or classic histology and occurmost frequently in the youngest patients. In contrast,CXCR4-low tumors more commonly occur in older patientsand exhibit classic or large cell anaplastic histology. Thus,CXCR4 expression may distinguish populations of SHHmedulloblastoma with different prognoses.

The impact of CXCR4 in the SHH subgroup of medullo-blastomas appears to be a consequence of unique molecularinteractions that occur between these pathways. Our datasuggest that SHH regulates CXCR4 activity at one of theearliest stages of its signaling. While SHH did not affectoverall CXCR4 levels, prolonged inhibition of the SHH path-way reduced the cell surface fraction of CXCR4 and abrogatedcellular responses to CXCL12. Cell surface localization ofCXCR4, like other GPCRs, is regulated through desensitiza-tion, involving receptor phosphorylation and arrestin-medi-ated internalization, followed by either degradation or recy-cling to the cell surface (35–37). SHH could potentially affectany of these processes. Recent studies have highlighted thecomplexity of such heterologous regulation of GPCR inter-nalization/recycling. Yu and colleagues showed that ligand-induced activation of Neurokinin 1 receptors (NK1R) pro-duced a nonreciprocal inhibition of ligand-induced mu-opioidreceptor endocytosis as a consequence of NK1R-dependentsequestration of arrestins on endosome membranes (38). Inaddition, nerve growth factor (NGF) has been shown tomodulate the recycling of delta-opioid receptors to the surfacemembrane of central synaptic terminals (39).

We previously described alterations in CXCR4 signaling as aconsequence of reduced desensitization in astrocytes null forthe tumor suppressor neurofibromin (Nf1; ref. 22). Loss of Nf1resulted in hyperactivation of the RAS-MAP kinase pathwayand resultant ERK-dependent inhibition of GRK2, a kinaseinvolved in CXCR4 desensitization. GRK2 inhibition was cor-related with reduced phosphorylation of CXCR4 and sustainedCXCL12-induced suppression of cAMP. Interestingly, GRK2and b-arrestin 2 also directly interact with Smo and regulateSHH-signaling (40, 41). Thus, activation of the SHH pathwaymight modulate CXCR4 signaling through regulation of thedesensitization machinery, including GRK2 and b-arrestin 2.

The biologic significance of interactions between the SHHand CXCR4 pathways may lie in the unique transcriptionalprofile of the CXCR4-high tumors. PPP2R2C is the regulatorysubunit of the phosphatase PP2A, which enhances SHH-drivenproliferation by stabilizing N-myc (42). In addition, PP2Ablocks differentiation within the granule lineage by inactivat-ing S6 kinase (33). SHH directly induces expression of thecatalytic subunit of PP2A and here we show that CXCR4suppresses expression of the regulatory subunit of PP2A. Botheffects could increase PP2A activity and promote proliferation.

Cyclin D1 is a regulator of cyclin-dependent kinases such asCDK4/CDK6, which are critical for G1 to S transition and cell-cycle progression. Induction of cyclinD1 expression is essentialfor SHH-induced proliferation (43, 44). CXCR4 enhanced cyclinD1 expression in the SHH-medulloblastomas and this is likelyto be a critical component of CXCR4's positive effects on tumorgrowth. Furthermore, CXCR4's effect on cyclin D1 expressionsuggests that high CXCR4 expression and/or activation mayfeed back and augment SHH signaling.

Significantly, CXCR4 activation appeared to be necessaryfor maximal proliferation of SHH-driven tumors. In SmoA1tumor models, the CXCR4 antagonist AMD3100 significantlyinhibited tumor growth. These data indicate that dualinhibition of SHH and the CXCR4 pathways may provideadditional benefit in treating those SHH subtype medullo-blastomas that also express CXCR4. A recent clinical reportdescribed a single patient with medulloblastoma whosetumor was initially sensitive to SHH antagonist therapy, butwho subsequently experienced an aggressive recurrencebecause of mutation in Smo (45–47). The ability of AMD3100to inhibit the growth of SmoA1-driven tumors suggests thatperhaps CXCR4 antagonism might address this mechanismof resistance.

The interplay between the SHH and CXCR4 pathwayscould function in normal development and cancer biologyin any circumstances where the elements of both pathwaysare coexpressed. In addition to medulloblastoma, compo-nents of the CXCR4 and SHH pathways are expressed inprimary specimens of breast, pancreatic, and prostate can-cers as well as in gliomas and rhabdomyosarcomas (24, 48–53). It will be important to determine whether coexpressionof these pathways provides additional prognostic value andwhether combined antagonism of these pathways may haveadded benefit in the treatment of these cancers. Defininghow SHH and CXCR4 modulate each other's signaling islikely to open up new avenues of inquiry in each of thesefields and new approaches to treating any cancer in whichboth pathways are operative.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Nicole Warrington for technical assistance and criticalreading of the manuscript. Calcium flux measurements were made through theRegional Center for Biodefense and Emerging Infectious Diseases Research LiveCell Microscopy Core Facility at the Washington University School of Medicine

Grant Support

This work was supported by RO1NS052323-04S1 (RW-Reya) andRO1CA118389 (J.B. Rubin). M.D. Taylor holds a CIHR Clinician-Scientist PhaseII Award and is supported by grants from the NIH (R01CA148699), and thePediatric Brain Tumor foundation.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 17, 2011; revised October 29, 2011; accepted October 31, 2011;published OnlineFirst November 3, 2011.

Sengupta et al.





References1. Hatten ME, Roussel MF. Development and cancer of the cerebellum.

Trends Neurosci 2011;34:134–42.2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A,

et al. The 2007 WHO classification of tumours of the central nervoussystem. Acta Neuropathol 2007;114:97–109.

3. Gilbertson RJ, Ellison DW. The origins of medulloblastoma subtypes.Annu Rev Pathol 2008;3:341–65.

4. Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S,et al. Medulloblastoma comprises four distinct molecular variants. JClin Oncol 2011;29:1408–14.

5. Thompson MC, Fuller C, Hogg TL, Dalton J, Finkelstein D, Lau CC,et al. Genomics identifies medulloblastoma subgroups that areenriched for specific genetic alterations. J Clin Oncol 2006;24:1924–31.

6. Ellison DW, Dalton J, Kocak M, Nicholson SL, Fraga C, Neale G, et al.Medulloblastoma: clinicopathological correlates of SHH, WNT, andnon-SHH/WNT molecular subgroups. Acta Neuropathol 2011;121:381–96.

7. Gibson P, Tong Y, Robinson G, Thompson MC, Currle DS, Eden C,et al. Subtypes of medulloblastoma have distinct developmentalorigins. Nature 2010;468:1095–9.

8. Yang ZJ, Ellis T,Markant SL, Read TA, Kessler JD, BourboulasM, et al.Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells. Cancer Cell 2008;14:135–45.

9. Schuller U, Heine VM, Mao J, Kho AT, Dillon AK, Han YG, et al.Acquisition of granule neuron precursor identity is a critical determi-nant of progenitor cell competence to form SHH-induced medullo-blastoma. Cancer Cell 2008;14:123–34.

10. Corcoran RB, Scott MP. A mouse model for medulloblastoma andbasal cell nevus syndrome. J Neuro Oncol 2001;53:307–18.

11. Ellison D. Classifying the medulloblastoma: insights from morphologyandmolecular genetics. Neuropathol Appl Neurobiol 2002;28:257–82.

12. Pomeroy SL, Tamayo P, Gaasenbeek M, Sturla LM, Angelo M,McLaughlin ME, et al. Prediction of central nervous system embryonaltumour outcome based on gene expression. Nature 2002;415:436–42.

13. Taylor MD, Liu L, Raffel C, Hui CC, Mainprize TG, Zhang X, et al.Mutations in SUFU predispose to medulloblastoma. Nat Genet2002;31:306–10.

14. Reifenberger J,WolterM,WeberRG,MegahedM,RuzickaT, Lichter P,et al. Missense mutations in SMOH in sporadic basal cell carcinomasof the skin and primitive neuroectodermal tumors of the centralnervous system. Cancer Res 1998;58:1798–803.

15. Goodrich LV, Milenkovic L, Higgins KM, Scott MP. Altered neural cellfates and medulloblastoma in mouse patched mutants. Science1997;277:1109–13.

16. Oliver TG, Read TA, Kessler JD, Mehmeti A, Wells JF, Huynh TT, et al.Loss of patched and disruption of granule cell development in apreneoplastic stage of medulloblastoma. Development 2005;132:2425–39.

17. Klein RS, Rubin JB, Gibson HD, DeHaan EN, Alvarez-Hernandez X,Segal RA, et al. SDF-1 alpha induces chemotaxis and enhances Sonichedgehog-induced proliferation of cerebellar granule cells. Develop-ment 2001;128:1971–81.

18. Rubin JB, KungAL, KleinRS,Chan JA,SunY, Schmidt K, et al. A small-molecule antagonist of CXCR4 inhibits intracranial growth of primarybrain tumors. Proc Natl Acad Sci U S A 2003;100:13513–8.

19. Bian XW, Yang SX, Chen JH, Ping YF, Zhou XD, Wang QL, et al.Preferential expressionof chemokine receptor CXCR4by highlymalig-nant human gliomas and its association with poor patient survival.Neurosurgery 2007;61:570–8; discussion 578–9.

20. Calatozzolo C, Maderna E, Pollo B, Gelati M, Marras C, Silvani A,et al. Prognostic value of CXCL12 expression in 40 low-gradeoligodendrogliomas and oligoastrocytomas. Cancer Biol Ther2006;5:827–32.

21. Yang L, JacksonE,Woerner BM,Perry A, Piwnica-WormsD, Rubin JB.Blocking CXCR4-mediated cyclic AMP suppression inhibits braintumor growth in vivo. Cancer Res 2007;67:651–8.

22. Warrington NM, Woerner BM, Daginakatte GC, Dasgupta B, Perry A,Gutmann DH, et al. Spatiotemporal differences in CXCL12 expression

and cyclic AMP underlie the unique pattern of optic glioma growth inneurofibromatosis type 1. Cancer Res 2007;67:8588–95.

23. Sengupta R, Burbassi S, Shimizu S, Cappello S, Vallee RB, Rubin JB,et al. Morphine increases brain levels of ferritin heavy chain leading toinhibition ofCXCR4-mediated survival signaling in neurons. JNeurosci2009;29:2534–44.

24. Woerner BM,WarringtonNM,KungAL, Perry A, Rubin JB.WidespreadCXCR4 activation in astrocytomas revealed by phospho-CXCR4-spe-cific antibodies. Cancer Res 2005;65:11392–9.

25. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA, et al. Gene set enrichment analysis: a knowledge-based approachfor interpreting genome-wide expression profiles. Proc Natl Acad SciU S A 2005;102:15545–50.

26. Lee Y, Miller HL, Jensen P, Hernan R, Connelly M, Wetmore C, et al. Amolecular fingerprint for medulloblastoma. Cancer Res 2003;63:5428–37.

27. Schuller U, Koch A, Hartmann W, Garre ML, Goodyer CG, Cama A,et al. Subtype-specific expression and genetic alterations of thechemokinereceptor gene CXCR4 in medulloblastomas. Int J Cancer2005;117:82–9.

28. Hattermann K, Held-Feindt J, Lucius R, Muerkoster SS, Penfold ME,Schall TJ, et al. The chemokine receptor CXCR7 is highly expressed inhuman glioma cells and mediates antiapoptotic effects. Cancer Res2010;70:3299–308.

29. Hallahan AR, Pritchard JI, Hansen S, Benson M, Stoeck J, Hatton BA,et al. The SmoA1 mouse model reveals that notch signaling is criticalfor the growth and survival of Sonic hedgehog-induced medulloblas-tomas. Cancer Res 2004;64:7794–800.

30. Nicot A, Lelievre V, Tam J, Waschek JA, DiCicco-Bloom E. Pituitaryadenylate cyclase-activating polypeptide and Sonic hedgehog inter-act to control cerebellar granule precursor cell proliferation. J Neurosci2002;22:9244–54.

31. Kim JY, Koralnik IJ, LeFave M, Segal RA, Pfister LA, Pomeroy SL.Medulloblastomas and primitive neuroectodermal tumors rarelycontain polyomavirus DNA sequences. Neuro Oncol 2002;4:165–70.

32. Wetmore C, Eberhart DE, Curran T. The normal patched allele isexpressed in medulloblastomas from mice with heterozygous germ-line mutation of patched. Cancer Res 2000;60:2239–46.

33. Mainwaring LA, Kenney AM. Divergent functions for eIF4E and S6kinase by Sonic hedgehog mitogenic signaling in the developingcerebellum. Oncogene 2011;30:1784–97.

34. ChoYJ, TsherniakA, TamayoP,SantagataS, LigonA,GreulichH, et al.Integrative genomic analysis of medulloblastoma identifies a molec-ular subgroup that drives poor clinical outcome. J Clin Oncol2011;29:1424–30.

35. Busillo JM, ArmandoS, Sengupta R,Meucci O, BouvierM, Benovic JL.Site-specific phosphorylation of CXCR4 is dynamically regulated bymultiple kinases and results in differential modulation of CXCR4signaling. J Biol Chem 2010;285:7805–17.

36. Lefkowitz RJ. G protein-coupled receptors. III. New roles for receptorkinases and beta-arrestins in receptor signaling and desensitization. JBiol Chem 1998;273:18677–80.

37. Freedman NJ, Lefkowitz RJ. Desensitization of G protein-coupledreceptors. Recent Prog HormRes 1996;51:319–51; discussion 352–3.

38. Yu YJ, Arttamangkul S, Evans CJ, Williams JT, von Zastrow M.Neurokinin 1 receptors regulate morphine-induced endocytosis anddesensitization of mu-opioid receptors in CNS neurons. J Neurosci2009;29:222–33.

39. Bie B, Zhang Z, Cai YQ, Zhu W, Zhang Y, Dai J, et al. Nerve growthfactor-regulated emergence of functional delta-opioid receptors. JNeurosci 2010;30:5617–28.

40. Chen W, Ren XR, Nelson CD, Barak LS, Chen JK, Beachy PA, et al.Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science 2004;306:2257–60.

41. Atkinson PJ, Dellovade T, Albers D, Von Schack D, Saraf K, Needle E,et al. Sonic hedgehog signaling in astrocytes is dependent on p38mitogen-activated protein kinase and G-protein receptor kinase 2. JNeurochem 2009;108:1539–49.






42. Sjostrom SK, Finn G, Hahn WC, Rowitch DH, Kenney AM. The Cdk1complex plays a prime role in regulating N-myc phosphorylation andturnover in neural precursors. Dev Cell 2005;9:327–38.

43. Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, Lin SM,et al. Transcriptional profiling of the Sonic hedgehog response: acritical role for N-myc in proliferation of neuronal precursors. ProcNatl Acad Sci U S A 2003;100:7331–6.

44. Kenney AM, Cole MD, Rowitch DH. Nmyc upregulation by Sonichedgehog signaling promotes proliferation in developing cerebellargranule neuron precursors. Development 2003;130:15–28.

45. Romer J, Curran T. Targeting medulloblastoma: small-molecule inhi-bitors of the Sonic hedgehog pathway as potential cancer therapeu-tics. Cancer Res 2005;65:4975–8.

46. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, et al.Treatment ofmedulloblastomawith hedgehog pathway inhibitorGDC-0449. N Engl J Med 2009;361:1173–8.

47. YauchRL, DijkgraafGJ, Alicke B, Januario T, AhnCP,Holcomb T, et al.Smoothened mutation confers resistance to a Hedgehog pathwayinhibitor in medulloblastoma. Science 2009;326:572–4.

48. Holm NT, Abreo F, Johnson LW, Li BD, Chu QD. Elevated chemokinereceptor CXCR4 expression in primary tumors following neoadjuvant

chemotherapy predicts poor outcomes for patients with locallyadvanced breast cancer (LABC). Breast Cancer Res Treat 2009;113:293–9.

49. Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H,Nomura M, et al. Hedgehog signaling pathway is a new therapeutictarget for patients with breast cancer. Cancer Res 2004;64:6071–4.

50. Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ,LauwersGY, et al.Hedgehog is anearly and latemediator of pancreaticcancer tumorigenesis. Nature 2003;425:851–6.

51. Azoulay S, Terry S, Chimingqi M, Sirab N, Faucon H, Gil Diez deMedina S, et al. Comparative expression of Hedgehog ligands atdifferent stages of prostate carcinoma progression. J Pathol 2008;216:460–70.

52. Ehtesham M, Sarangi A, Valadez JG, Chanthaphaychith S, BecherMW, Abel TW, et al. Ligand-dependent activation of the hedgehogpathway in glioma progenitor cells. Oncogene 2007;26:5752–61.

53. Diomedi-Camassei F,McDowell HP, De IorisMA, Uccini S, Altavista P,Raschella G, et al. Clinical significance of CXC chemokine receptor-4and c-Met in childhood rhabdomyosarcoma.ClinCancerRes 2008;14:4119–27.

Sengupta et al.





2012;72:122-132. Published OnlineFirst November 3, 2011.Cancer Res Rajarshi Sengupta, Adrian Dubuc, Stacey Ward, et al. Driven Medulloblastoma

−CXCR4 Activation Defines a New Subgroup of Sonic Hedgehog

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