Chemokine receptors in head and neck cancer: association with metastatic

spread and regulation during chemotherapy

Anja Muller1*�, Eniko Sonkoly1,2,3�, Christine Eulert1,3�, Peter Arne Gerber1, Robert Kubitza2, Kerstin Schirlau3,Petra Franken-Kunkel1,2, Christopher Poremba4, Carl Snyderman5, Lars-Oliver Klotz6, Thomas Ruzicka2,Henning Bier

3, Albert Zlotnik

7, Theresa L. Whiteside

8, Bernhard Homey

2*� and Thomas K. Hoffmann3*�

1Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany2Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany3Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany4Department of Pathology, Heinrich-Heine-University, Duesseldorf, Germany5Department of Otorhinolaryngology, University of Pittsburgh, PA, USA6Institute of Molecular Biology and Biochemistry, Heinrich-Heine-University, Duesseldorf, Germany7Neurocrine Biosciences Inc., San Diego, CA, USA8Hillman Cancer Center, University of Pittsburgh, PA, USA

Head and neck carcinomas are histologically and clinically hetero-geneous. While squamous cell carcinomas (SCC) are character-ized by lymphogenous spread, adenoid cystic carcinomas (ACC)disseminate preferentially hematogenously. To study cellular andmolecular mechanisms of organ-specific metastasis, we used SCCand ACC cell lines and tumor tissues, obtained from patients withprimary or metastatic disease. Comprehensive analysis at themRNA and protein level of human chemokine receptors showedthat SCC and ACC cells exhibited distinct and nonrandom expres-sion profiles for these receptors. SCC predominantly expressedreceptors for chemokines homeostatically expressed in lymphnodes, including CC chemokine receptor (CCR) 7 and CXC chemo-kine receptor (CXCR)5. No difference in expression of chemokinereceptors was seen in primary SCC and corresponding lymphnode metastases. In contrast to SCC, ACC cells primarily ex-pressed CXCR4. In chemotaxis assays, ACC cells were responsiveto CXCL12, the ligand for CXCR4. Exposure of ACC cells to cis-platin resulted in upregulation of CXCR4 on the cell surface,which was repressed by the transcriptional inhibitor, a-amanitin.Treatment of ACC cells with CXCL12 resulted in the activation ofAkt and ERK1/2 pathways. Furthermore, CXCL12 suppressedthe rate of apoptosis induced by cisplatin in ACC cells, suggestingthat signaling via CXCR4 may be part of a tumor cell survivalprogram. Discrimination of the chemokine receptor profile inSCC and ACC in vitro and in tissues provided insights into theirdistinct biologic and clinical characteristics as well as indicationsthat chemokine receptors might serve as future therapeutic tar-gets in these malignancies.' 2005 Wiley-Liss, Inc.

Key words: head and neck cancer; squamous cell carcinoma;adenoid cystic carcinoma; chemokine receptor; metastasis

Head and neck carcinomas (HNC) include tumors with differenthistological phenotypes and distinct clinical characteristics. Squa-mous cell carcinomas (SCC) of the upper aerodigestive tractmucosa represent the most common histological subtype.1,2

Adenoid cystic carcinoma (ACC) is a rare malignant epithelialtumor, arising from salivary glands.3 Both tumor types are charac-terized by distinct metastatic patterns. While SCC frequentlymetastasizes to regional lymph nodes, ACC is more aggressive,disseminating to distant sites, particularly, the lung and theliver.2,4–6 In both these tumor types, current treatment regimensresult in fairly good locoregional control, but little progress hasbeen made in the treatment of disseminated diseases.6,7 Hence,metastatic spread represents an important survival-limiting factor.

A detailed characterization of mechanisms underlying organ-specific metastasis may lead to the development of novel thera-peutic options for the control of disseminated tumor spread.Owing to their distinct metastatic patterns, ACC and SCC providean interesting model to investigate the so far poorly understoodmolecular mechanisms of organ-specific metastasis.

Recent studies have demonstrated that tumor cells express func-tionally active chemokine receptors and that expression of thesereceptors appears to regulate cellular functions associated with theprocess of metastasis.8–10 Chemokines are a superfamily of small,cytokine-like proteins that selectively attract and activate differentcell types.11,12 The CXC chemokine receptor (CXCR)4 is knownto be expressed in several cancer types, including breast cancer,8

melanoma13 and lung cancer.14 These cancer types may useCXCR4 to metastasize to target organs. Furthermore, CXCR4expression has been implicated in metastatic spread in vivo, in ani-mal models of breast cancer and melanoma.8,13

Here, we report that 2 clinically important subgroups of HNC,ACC and SCC, express a distinct, nonrandom pattern of chemokinereceptors, influencing their metastatic behavior. CC chemokinereceptor (CCR)7 is abundantly expressed in primary tumors andlymph node metastases of SCC in vivo. ACC cells express high levelsof CXCR4 both in vitro and in vivo, whereas CXCR4 expression isundetectable or low in SCC. Furthermore, the exposure of ACC cellsto the antineoplastic agent, cisplatin, results in the upregulation ofCXCR4 on the cell surface, providing survival signals to tumor cells.

Material and methods

Cell lines and culture

Two ACC cell lines, established from salivary gland tumors(ACC-2/ACC T#2 and ACC-3/ACC T#3),15 were kindly providedby Dr. W.L. Qiu (Shanghai, China). SCC cell lines were derivedfrom primary tumors of the head and neck region (UD-SCC-1–4/SCC T#1–4, UD-SCC-6/SCC T#6, UD-SCC-7A/SCC T#7, UD-SCC-8/SCC T#8, UM-SCC-10A/SCC T#10, UM-SCC-17A/SCCT#17, UT-SCC-24A/SCC T#24) or from lymph node metastases

Grant sponsor: Research Commission/Committee, University ofD€usseldorf; Grant sponsor: German Cancer Foundation (Deutsche Kreb-shilfe/Mildred Scheel Stiftung); Grant sponsor: German Research Founda-tion; Grant number: SFB503; Grant sponsor: National Institute of Health,USA; Grant number: RO1-DE13918, PO1-DE12321.

�The first 3 authors contributed equally to this work.�The last 2 authors contributed equally to this work.*Correspondence to: Departments of Radiation Oncology, Dermatol-

ogy or Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf,Moorenstr. 5, D-40225 Duesseldorf, Germany. Fax: 149-211-811-7316.E-mail: tkhoffmann@uni-duesseldorf.de or a.mueller@uni-duesseldorf.deReceived 18 March 2005; Accepted after revision 5 August 2005DOI 10.1002/ijc.21514Published online 5 December 2005 in Wiley InterScience (www.

interscience.wiley.com).

Abbreviations: ACC, adenoid cystic carcinoma; CCR, CC chemokinereceptor; CXCR, CXC chemokine receptor; ERK, extracellular signal-regulated kinase; HNC, head and neck carcinoma; MAPK, mitogen-acti-vated protein kinase; PI3K, phosphatidylinositol 30-kinase; PKB, proteinkinase B; RT-PCR, reverse transcription polymerase chain reaction; SCC,squamous cell carcinoma.

Int. J. Cancer: 118, 2147–2157 (2006)' 2005 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

(UM-SCC-10B/SCC M#10, UM-SCC-17B/SCC M#17, UT-SCC-24B/SCC M#24), as previously described.16,17 Tumor cells werecultured in DMEM, supplemented with 10% FCS, 2 mM L-gluta-mine and antibiotic/antimycotic solution (all from Gibco, Eggen-stein, Germany); as the only exception, cell line ACC-2 (ACCT#2) was cultured in RPMI medium (Gibco), with identical sup-plementation. For control purposes, primary mucosal keratino-cytes were separated from healthy oral mucosa, with the patients’informed consent. Tissue samples were cut into small strips andincubated in Dispase I solution (Roche Diagnostics, Mannheim,Germany) overnight at 4�C. On the following day, the mucosawas peeled off the lamina propria and incubated in trypsin–EDTAsolution at 37�C for 30 min. A suspension of primary mucosalcells was prepared in keratinocyte serum-free medium (keratino-cyte-SFM), supplemented with antibiotic/antimycotic solution (allfrom Gibco). Cells were seeded into 75-cm2 tissue-culture flasksand propagated in keratinocyte-SFM. To harvest or passage thecell lines, almost confluent monolayers were detached with 0.05%trypsin/0.02% EDTA solution (Boehringer, Mannheim, Germany).

For treatment with the antineoplastic agent cisplatin, tumor cellswere incubated for 72 hr, and on day 3, fresh culture medium, sup-plemented with different concentrations of cisplatin (1, 3 or 9 lg/ml,Bristol-Myers Squibb, Munich, Germany), a-amanitin (5 lg/ml,Sigma-Aldrich, St. Louis, MO, USA) or combinations of cisplatinand a-amanitin, was added, and controls received medium alone.The antitumor effects were determined with the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) test, asdescribed previously.18

Quantitative real-time RT-PCR (TaqMan1)

Total RNA of tumor cells and keratinocytes was extracted(TRIzol reagent, Invitrogen, Carlsbad, CA, USA), DNase-treatedand reverse-transcribed, as described previously.8,19 Complemen-tary DNA was quantitatively analyzed for the expression of humanchemokines and chemokine receptors by the fluorogenic 50-nucle-ase PCR assay, as reported.8,19 Specific primers and probes wereobtained from Applied Biosystems (Foster City, CA, USA). Gene-specific PCR products were continuously measured during 40cycles, with the ABI PRISM 7000 Sequence Detection System(Applied Biosystems). Quantitative real-time RT-PCR was per-formed with SYBR green as reporter, for reactions using chemo-kine receptor-specific primers, and FAM as reporter, for reactionsusing chemokine receptor-specific primer/probe combinations.Probes for the internal positive control (ribosomal 18s RNA) wereassociated with the VIC reporter. Target gene expression was nor-malized between different samples, based on the values of riboso-mal 18s RNA expression. Plasmid cDNA for chemokine receptorswere used for quantification of target gene-specific mRNA expression.

Flow cytometry

To analyze chemokine receptor expression, cells were stainedwith the following reagents: PE-conjugated anti-human CCR1,anti-CCR2, anti-CCR3 (all from R&D Systems, Minneapolis,MO, USA), anti-CCR4 (BD PharMingen, San Diego, CA), anti-CCR5 (R&D Systems), anti-CCR6 (BD PharMingen), anti-CCR7(R&D Systems), anti-CCR9 (R&D Systems), anti-CXCR1 (BDPharMingen), anti-CXCR2 (BD PharMingen), anti-CXCR3, anti-CXCR4, anti-CXCR5 or anti-CXCR6 (all from R&D Systems).For CCR10 staining, biotinylated anti-human CCR10 (clone 1908;DNAX Research Institute, Palo Alto, CA, USA) and PE-conju-gated streptavidin (BD PharMingen) were used. CCR8 was stainedusing unlabeled anti-CCR8 (210-762-R100, goat IgG, Alexis Bio-chemicals, Lausanne, Switzerland) and PE-conjugated swine anti-goat IgG (Caltag, Burlingame, CA, USA). Flow-cytometric analy-sis was performed using a FACScan flow cytometer, equippedwith a single, 488-nm argon-ion laser and CELLQuest software(Becton Dickinson, San Jose, CA, USA). All tumor cell lines aswell as primary human mucosal keratinocytes were stained 2times for CCR1–10 and CXCR1–6. For the detection of intracellu-lar CCR7 and CXCR5, we additionally applied the IntraStain kit(DAKO, Glostrup, Denmark).

Tissue samples and immunohistochemistry

Tissue blocks of primary ACC (n 5 57) were obtained from theUniversity of Pittsburgh Medical Center, USA. Paraffin blocks ofprimary tumors (n 5 29) and lymph node metastases (n 5 12matched pairs) from patients with SCC were obtained from surgi-cal specimens at the Department of Otorhinolaryngology, Univer-sity Hospital D€usseldorf, Germany. The study was approved bythe local Ethic Committees, and informed consent was securedfrom each patient prior to sample acquisition. Histopathologicaldiagnosis was confirmed in all tissue samples. Tumors were rou-tinely formalin-fixed, paraffin-embedded and sectioned at 3–5-lmslices, which were stained with anti-human CXCR4 antibody, aspreviously described.8 For immunohistochemical analyses ofCCR7, tumor sections were fixed in acetone and preprocessedwith H2O2, followed by an avidin and biotin blocking step (VEC-TOR Blocking Kit, Vector Laboratories, Burlingame, CA, USA).Sections were stained with monoclonal antibody against humanCCR7 (R&D Systems), or isotype control antibody (BD PharMin-gen). Development of the staining was performed with a DAKOAEC-Kit (DAKO). Sections were counterstained with hematoxy-lin. Histopathological evaluation was performed by 2 independentinvestigators on a scale of 0–21, depending on the intensity of theimmunoreactivity (0, negative immunostaining; 11, weakly posi-tive immunostaining; 21, moderately/strongly positive immunos-taining). For immunocytochemical analysis of chemokine recep-tors in cell lines, cells were grown on slides for 72 hr, washed withPBS, fixed in methanol at 220�C for 5 min, treated with acetonefor 30 sec, air-dried and stained for chemokine receptors, asdescribed earlier.

Chemotaxis assays

Migration was assayed in 24-well, cell-culture chambers, usinginserts with 8-lm pore membranes, as previously described.8

Membranes were precoated with fibronectin (5 lg/ml, BectonDickinson). ACC (T#3) and SCC (T#1) cells were resuspended inchemotaxis buffer (DMEM; 0.1% BSA, Sigma-Aldrich; 12 mMHEPES, Invitrogen) at 0.5–1 3 105 cells/ml. Either the medium(0.6 ml) alone or the media supplemented with different concen-trations of the ligands CXCR4 and CXCL12 (human recombinantCXCL12, R&D Systems) was added to the lower chamber. Afterincubation for 9 (ACC) or 15 hr (SCC), cells which have migratedthrough the pores were stained and counted under a light micro-scope in at least 5 high-power fields. Different time intervals werechosen, because of the observed difference in the basic migratorypotential of ACC and SCC (control/blank). FCS (5%) was used asa positive control to assure the general capacity of tumor cells toperform migratory responses. All chemotaxis assays were per-formed 3 times in triplicate wells.

Western blotting and immunodetection

For Western blotting, ACC (T#3) cells were treated with humanrecombinant CXCL12 (500 ng/ml; R&D Systems) for the indi-cated times, lysed in 23 SDS-PAGE buffer [125 mM Tris-HCl,4% (w/v) SDS, 20% (w/v) glycerol, 100 mM DTT, 0.2% (w/v)bromophenol blue (pH 6.8)], followed by brief sonication. Sam-ples were heated at 95�C for 5 min and applied to SDS-polyacryla-mide gels of 10% (w/v) acrylamide, followed by electrophoresisand tank-blotting onto polyvinylidene difluoride membranes.Immunodetectionswereperformedusingpolyclonalantibodies,atdi-lutions recommended by the manufacturer. Anti-phospho-ERK1/2, anti-total ERK1/2, anti-phospho-Akt (Ser473) and anti-totalAkt antibodies were purchased from Cell Signaling Technology(Beverly, MA, USA).

Detection of apoptosis

For morphological analysis of apoptosis, ACC cells weretreated with cisplatin or the combination of cisplatin and humanrecombinant CXCL12 for 24 hr, and stained with 5 lg/ml Hoechst33342 (Sigma) to reveal nuclear condensation and fragmentation

2148 MULLER ET AL.

by fluorescence microscopy. To quantify apoptosis, at least 5fields with ~200 cells per field were examined in each well.

Results

Cell lines of ACC and SCC exhibit distinct chemokine receptorexpression profiles

To determine the differences in chemokine receptor profiles ofACC and SCC, 2 histologically and clinically different subtypesof HNC, we performed a comprehensive analysis of the expres-

sion of all known chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, CX3CR1 and CXCR1) in human ACC (n 5 2) and SCCcell lines derived from both primary tumors (n 5 10) and lymphnode metastases (n 5 3) of HNC patients. Absolute quantities ofreceptor mRNA were measured by quantitative real-time RT-PCR(Fig. 1), and their cell-surface expression (for CCR1–CCR10 andCXCR1–CXCR6) was determined by flow cytometric analysis(Fig. 2 and Table I).

Figure 1 illustrates chemokine receptor mRNA expression inACC and SCC cells, as well as primary mucosal keratinocytes.

FIGURE 1 – ACC and SCC exhibit distinct chemokine receptor mRNA profiles. Comprehensive quantitative real-time RT-PCR analyses of allknown chemokine receptors (CCR1–CCR10 (a–j), CXCR1–CXCR6 (m–r), CX3CR1 (k) and XCR1 (l)) in ACC (n 5 2) and SCC cell lines,derived from either primary tumors (T; n5 10) or matching lymph node metastases (M; n5 3; 3 matched pairs) or human primary mucosal ker-atinocytes (n5 1). Values are expressed as femtograms of target gene in 25 ng of total cDNA.

2149CHEMOKINE RECEPTORS IN HEAD AND NECK CANCER

FIGURE 2 – Distinct chemokine receptor ex-pression of ACC and SCC cell lines. (a, b) Flowcytometric analysis of CXCR4 expression onACC (T#3) (a) and SCC (T#1) (b) cells. (c, d)Flow cytometric analysis of surface (c) and intra-cellular (d) CCR7 expression in SCC (T#1) cells.(e, f) Immunocytochemical analysis of SCC(T#1) cells using isotype antibody (e) or specificantibody for CCR7 (f). Magnification: 3200. (g–i) Flow cytometric analysis of surface (g) orintracellular (h) CXCR5 expression in SCC(T#6) cells as well as surface CXCR5 expressionafter 24 hr of serum starvation in SCC (T#6)cells (i). Filled histogram, isotype; black line,CXCR4, CCR7 and CXCR5, respectively. Rep-resentative data for 1 of 2 ACC and 1 of 13 (b, c,e, f) or 3 (d, g–i) SCC cell lines evaluated.

2150 MULLER ET AL.

The latter exhibited a very limited repertoire of chemokine recep-tors, with CXCR1 being the predominant family memberexpressed at a significant level (Fig. 1m); epidermal keratinocytesfrom 3 different donors showed an almost identical expression pat-tern (data not shown). ACC cells had a very restricted profile ofchemokine receptors, with mRNA expression for several recep-tors, and CXCR4 was the only chemokine receptor expressed at ahigh level compared to SCC cells (Fig. 1p). In contrast to ACC,SCC cells expressed a considerable variety of chemokine recep-tors. CXCR1 and CXCR2 genes, encoding chemokine receptorsreported to be associated with CXCL8-mediated proliferation andinvasion of cancer cells,20 were expressed in the majority of SCCcells, but were absent in ACC cells (Figs. 1m and 1n). Comparedto mucosal keratinocytes, a consistent upregulation of receptormRNA was observed in the majority of SCC cell lines for CCR3,CCR7, CXCR3, CXCR5 and CXCR6 (Figs. 1c, 1g, 1o, 1q and 1r).Among this set of chemokine receptors, CCR7 and CXCR5 wereconsistently although variably expressed in different SCC celllines, but were not detectable or expressed at markedly lowerlevels in ACC cells (Figs. 1g and 1q). Interestingly, CCR7,CXCR3 and CCR3 were reported to be associated with lymphnode metastasis in other cancer types.21–25 While CXCR4 was theonly receptor with high expression in ACC cells, SCC cells dis-played negligible levels of CXCR4 mRNA (Fig. 1p).

At the protein level, flow cytometric analysis confirmed highcell surface expression of CXCR4 (25–95%) in ACC cells (Fig. 2aand Table I), while SCC cell lines or primary mucosal keratino-cytes were found to express little or no CXCR4 (Fig. 2b andTable I). Notably, some SCC cell lines showed low but detectablesurface CXCR4 expression (�4%; Table I). For CCR7, despitesignificant expression at the mRNA level, no surface protein couldbe detected in SCC cells in vitro, using flow cytometry (Fig. 2c).To determine whether CCR7 protein is stored in the cytoplasm,we performed intracellular staining for CCR7 in 3 representativeSCC cell lines expressing high levels of CCR7 mRNA and oneACC cell line. All 3 SCC cell lines showed high intracellularCCR7 expression (Fig. 2d). Intracellular CCR7 expression in SCCcell lines was further confirmed by immunocytochemical analysis(Figs. 2e and 2f). However, ACC cells demonstrated negligibleamounts of intracellular CCR7 (data not shown). These data sug-gest that, in culture, CCR7 protein is either not secreted or is rap-idly shed from the cell surface. To assess whether microenviron-mental factors regulate CCR7 expression, surface CCR7 was ana-lyzed by flow cytometry in SCC cells after serum starvation, ortreatment with inflammatory cytokines or growth factors, includ-ing TNF-a, TGF-b and EGF, or supernatant of endothelial cells orfibroblasts. However, none of these factors induced surface CCR7expression (data not shown). Similarly to CCR7, CXCR5 was notdetected on the surface of SCC cells by flow cytometric analysis

(Table I and Fig. 2g). However, intracellular staining of 3 repre-sentative SCC cell lines demonstrated high levels of intracellularCXCR5 (Fig. 2h), in accordance with their abundant CXCR5mRNA expression. Moreover, after 24 hr of serum starvation,CXCR5 was detected on the cell surface (15% positive cells) ofSCC cells (Fig. 2i).

Flow cytometric analyses showed weak surface protein expres-sion for CCR3 in all SCC, and for CCR6 in one ACC and all SCCcell lines. All other receptors were undetectable or expressed at alow level on the surface of cultured ACC and SCC cells ormucosal keratinocytes (Table I). Interestingly, SCC cell linesderived from lymph node metastases showed no consistent differ-ence in the expression of any of the chemokine receptors com-pared to those derived from primary tumors (Fig. 1 and Table I).

Tissue immunohistochemistry confirms differences in thechemokine receptor expression profile inACC versus SCC

To assess whether the difference in CXCR4 and CCR7 expressionbetween ACC and SCC cells is relevant in vivo, immunohistochemi-cal analyses of 57 primary ACC and 29 primary SCC were per-formed. Although CXCR4 was not detectable in normal salivarygland (Fig. 3a), 56/57 primary ACC exhibited strong (21) CXCR4expression (Fig. 3a). Only one ACC patient demonstrated weak(11) expression of CXCR4. In contrast to ACC, primary SCC waspredominantly negative for CXCR4, (Fig. 3b) but abundantlyexpressed CCR7 (Figs. 3g and 3h). Notably, a minority of primarySCC expressed low levels of CXCR4 (11, 9/29, Fig. 3c), and only1/29 SCC showed high CXCR4 expression (21). In 2 CXCR4-posi-tive SCC samples, in particular, malignant cells at the leading edgeof the tumor exhibited strong CXCR4 expression (Fig. 3d), suggest-ing that CXCR4 might have a role in tumor invasion.

In accordance with our in vitro findings, CCR7 was expressedin 23 out of 26 (88%) primary tumors of SCC. In 7 out of 26(27%) SCC, a strong CCR7 expression (21) could be observed.

To investigate possible differences in chemokine receptorexpression in primary SCC and matching lymph node metastases,we performed immunohistochemical staining of matched pairsfrom 12 patients with nodal-positive SCC. CXCR4 was absent in5/12 primary tumors and 7/12 corresponding lymph node metasta-ses of SCC (Figs. 3e and 3f; Table II). Six of 12 primary tumorsand 5/12 lymph node metastases expressed CXCR4 at low levels(11); however, strong expression (21) was only detected in oneof the primary tumors (Table II). We could not observe a consis-tent difference in the expression of CXCR4 between primarytumors and their corresponding lymph node metastases.

In contrast to CXCR4, CCR7 was expressed in all primarytumors and 10/12 corresponding lymph node metastases (Table II

TABLE I – SURFACE CHEMOKINE RECEPTOR EXPRESSION IN SCC AND ACC CELL LINES

CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10 CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6

Mucosal KC1 – – – – – 22 – – – 2 – – – – – –SCC T#13 – – – – – 2 – – – – – – – – – –SCC T#2 – – 2 – – 4 – – – – – – – – – –SCC T#3 – – 2 – – 3 – – – – – – – – – –SCC T#4 – – 2 – – 4 – – – – – 3 – 2 – –SCC T#6 – – – – – 4 5 – – – – 2 – 4 – –SCC T#7 – – 2 – – 3 – – – – – – – – – –SCC T#8 – – 2 – – 5 – – – – – – – 3 – –SCC T#10 – 2 4 2 – – 2 – 5 – 6 – – 2 3 –SCC M#104 – – 4 – – 4 – – 2 12 7 – – 2 6 –SCC T#17 – – 2 – – 6 – – – – – – 2 – – –SCC M#17 – – 2 – – 4 – – – – – 2 – – – –SCC T#24 – – 2 – – – – – – – – – – – – –SCC M#24 – – – – – 3 – – – – – – – – – –ACC T#2 – – – – – 6 – – 4 – – – – 25 – –ACC T#3 – – – – – – – – – – – – – 95 – –

1KC, keratinocytes.–2Percentage of positive cells. Values are means of two measurements.–3T, cell line derived from primary tumor.–4M, cellline derived from metastatic lymph node.

2151CHEMOKINE RECEPTORS IN HEAD AND NECK CANCER

FIGURE 3 – CXCR4 expression in tissues of primary ACC and SCC and CCR7 expression in tissues of primary tumors as well as matchinglymph node metastases of SCC. Representative immunohistochemical data on the chemokine receptors CXCR4 (a–f) and CCR7 (g, h). (a) Pri-mary tumors of ACC patients demonstrate strong and homogenous CXCR4 expression (a; right) while adjacent normal salivary gland expressesno CXCR4 protein (a; left). (b–d) Variable CXCR4 protein expression in primary tumors of SCC patients. (b) Absence of CXCR4 protein in aprimary SCC. The arrow indicates CXCR4-expressing endothelial cells. (c) Weak CXCR4 expression in another primary SCC (arrows) and noCXCR4 immunoreactivity in adjacent normal mucosa (left). (d) Enhancement of CXCR4 protein expression at the invasive front (arrows) of aprimary SCC (representative of 2/29 SCC). Absence of CXCR4 (e, f), however, abundant expression of CCR7 (g, h) showing membrane staining(g, inset) in a primary tumor and its corresponding lymph node metastasis of SCC. Magnifications: 3250 (a, b),3100 (c–h).

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and Figs. 3g and 3h). Strong CCR7 expression (21) was observedin 8/12 primary SCC and 5/12 lymph node metastases (Table IIand Figs. 3g and 3h). The expression of CCR7 in both primarytumors and metastases suggests that this receptor could play a rolein lymphogenic metastasis of SCC. However, no consistent up- ordownregulation was found in lymph node metastases compared tothe corresponding primary tumors.

CXCR4 is functionally active in ACC cells and mediatesdirectional migration

CXCL12, the corresponding ligand of CXCR4, is highlyexpressed in the lung and liver, organs that represent major desti-nations of ACC metastasis.4,8 Consequently, we asked whetherCXCR4 expressed in ACC cells is functionally active upon ligandbinding. When the highly CXCR4-expressing ACC cells (T#2)(Table I) were incubated with various concentrations of recombi-nant human CXCL12, binding of the ligand and time-dependentinternalization of the chemokine receptor was seen by flow cyto-metry (data not shown). Furthermore, a transwell migration assaywas performed to examine the effect of CXCL12 on migration ofACC and SCC cells (ACC T#3 and SCC T#1). ACC cells wereable to migrate toward CXCL12 in a dose-dependent manner(Fig. 4, p < 0.05). In contrast to ACC, SCC cells expressing no(T#1) or low amounts (T#6) of CXCR4 (see Table I) did not showa chemotactic response to CXCL12 gradients (Fig. 4 and data notshown).

The antineoplastic agent cisplatin upregulates CXCR4on tumor cells

Patients with HNC frequently undergo chemotherapy with cis-platin.26,27 Hence, we next sought to investigate the regulation ofchemokine receptors on tumor cells, during the exposure to thisantineoplastic agent. CXCR4-expressing ACC cells were exposedto sublethal doses of cisplatin at the concentrations of 1, 3 and9 lg/ml. These concentrations are comparable to those observedin vivo in sera of HNC patients,28 and caused 24–78% inhibitionof cell growth, as determined by MTT assays (data not shown).Flow cytometric analysis of ACC (T#3) cells showed that cisplatindose-dependently induced the expression of CXCR4 on the cellsurface (Figs. 5a and 5b). After 24-hr incubation with cisplatin, a1.5- to 2-fold increase in mean fluorescence for CXCR4 wasobserved (Fig. 5b). To investigate the underlying mechanism ofcisplatin-induced CXCR4 expression, ACC cells were co-incu-bated with cisplatin and a-amanitin, an inhibitor of RNA polymer-ase II-mediated transcription. In the presence of a-amanitin, theinduction of CXCR4 was decreased by up to 85% (Fig. 5c), sug-gesting that induction of CXCR4 by cisplatin requires gene tran-scription. In contrast to ACC cells, no significant upregulation ofsurface CXCR4 was detected on SCC (T#6) cells after cisplatin-treatment (data not shown).

CXCL12/CXCR4 interaction activates survival pathwaysin tumor cells

To assess whether signaling via CXCR4 activates pathwaysinvolved in cell survival and proliferation in ACC cells, wefocused our attention on the activation of Akt/protein kinase B(Akt/PKB) and the extracellular signal-regulated kinases 1 and 2(ERK1/2). Akt/PKB is a known downstream effector of phosphati-

dylinositol 30-kinase (PI3K), and has been implicated in signal-transduction pathways, promoting cell survival.29 The mitogen-activated protein kinase (MAPK) ERK1/2 is known to be involvedin cell proliferation, differentiation and survival.30 ACC cells wereincubated in serum-free medium, containing 500 ng/ml CXCL12,and total cell lysates were collected at several time points. Lysateswere examined for phosphorylation of Akt/PKB and ERK1/2,using Western blot analysis. Our results indicate that CXCL12 sig-nificantly induces activation of Akt/PKB and ERK1/2 (Fig. 5d).The activation was clearly evident for both kinases after 5 min,and showed its maximum after 15 min. Phosphorylation of Akt/PKB was still maintained at 60 min, while activation of ERK1/2was sustained for up to 30 min, and decreased back to control lev-els after 60 min. Examination for the total expression of MAP kin-ases ensured equal loading of proteins in each lane. These datasuggest that CXCL12/CXCR4 interaction in ACC cells activatesintracellular signaling pathways involved in cell survival and pro-liferation.

To examine whether the activation of Akt/PKB and ERK1/2 byCXCL12 is indeed associated with increased cell survival, ACCcells were incubated with cisplatin or the combination of cisplatinand CXCL12, or left untreated, stained with Hoechst 33342, andthe percentage of apoptotic cells was quantified, based on theircharacteristic morphology. In our experiments, the baseline apop-totic rate of ACC cells was 2.5% on average. Cisplatin (9 lg/ml)induced a low level of apoptosis after 6 hr (9.5%, data not shown)and significant amounts of apoptosis (44%) after 24 hr, with con-densed and fragmented cell nuclei. After 24 hr, the rate of apopto-sis induced by cisplatin was significantly decreased by co-incuba-tion with CXCL12 (Fig. 5e, p < 0.001). The rate of cisplatin-induced apoptosis was still reduced by CXCL12 after 48 hr (datanot shown). These results suggest that CXCL12 indeed suppressesapoptosis induced by cisplatin, thus promoting survival of ACCcells.

Discussion

The presence of local and distant metastases is a key criterionof the malignant phenotype and represents an important prognosticas well as survival-limiting factor for most cancer types. In gen-eral, cancer cells metastasize to distinct organs in a nonrandommanner. To investigate cellular characteristics that may be impor-

TABLE II – CXCR4 AND CCR7 EXPRESSION IN PRIMARY TUMORS OF SCCAND THEIR CORRESPONDING LYMPH NODE METASTASES

Chemokine receptor expression

0 11 21

CXCR4 T1(n5 12) 5 6 1M2(n5 12) 7 5 0

CCR7 T (n5 12) 0 8 4M (n5 12) 2 7 3

1T, primary tumors.–2M, lymph node metastases.

FIGURE 4 – ACC but not SCC cells demonstrate chemotacticresponses to CXCL12 gradients. Transwell chemotaxis assays withACC (T#3) or SCC (T#1) cells in response to different concentrationsof CXCL12 (1, 10, 100, 1,000 and 1,500 ng/ml). Results are expressedas mean number of migrated cells in 5 fields (black histogram, ACCT#3; hatched histogram, SCC T#1). Error bars indicate the SD for 1out of 3 representative experiments performed in triplicates. *p <0.05 compared to control by Student’s t-test.

2153CHEMOKINE RECEPTORS IN HEAD AND NECK CANCER

tant in the process of organ-specific metastasis, we selected 2 dif-ferent types of HNC: SCC, characterized by frequent presence oflymph node metastases, and ACC, a rare cancer type characterizedby hematogenous dissemination. These 2 tumor entities serve as amodel to study the organ-specificity of the metastatic process.

The formation of metastases is thought to be the result of sev-eral sequential steps that share many similarities with leukocytetrafficking, a process critically regulated by chemokines and theirreceptors.31 In this study, we provide evidence that SCC and ACCcells display a distinct chemokine receptor expression profile.SCC cells express a broad variety of chemokine receptors. Themajority of SCC cell lines, similarly to mucosal keratinocytes,express significant levels of CXCR1 and CXCR2 transcripts.These 2 chemokine receptors have been associated with CXCL8-mediated proliferation of melanoma and colon cancer cells.20,32

Hence, it is conceivable that CXCL8, also produced by SCC cellsthemselves,33 may promote the proliferation and invasion of SCC

cells expressing CXCR1 and CXCR2. In SCC cell lines, we con-sistently observed upregulation of CCR7 and CXCR5 mRNA,while mRNA for these receptors was absent or expressed at a lowlevel in normal mucosal keratinocytes and ACC cells. Interest-ingly, the corresponding ligands of these receptors (CCL19,CCL21 and CXCL13, respectively) are homeostatically expressedin lymph nodes.34–36 CCR7 expression is critical for the migrationof na€ıve lymphocytes and mature dendritic cells to draining lymphnodes.31 Furthermore, CCR7 expression has been associated withlymph node metastasis in several cancer types, such as melanoma,lung cancer, gastric cancer and esophageal cancer.21–25

In our panel of SCC cell lines, CCR7 protein was only detect-able intracellularly, whereas in tissues, surface expression was evi-dent in primary tumors examined by immunohistochemistry, sug-gesting that the transport of CCR7 protein to the cell surface mayrequire microenvironmental factors present only in vivo. Indeed,our previous observations show that IL-1b, TNF-a or EGF induce

FIGURE 5 – ACC cells express elevated levels of surface CXCR4 after cisplatin-treatment (a–c) and show enhanced survival in the presenceof CXCL12 (d, e). (a) Flow cytometric analysis of CXCR4 protein expression in untreated ACC (T#3) cells (black line) or cells treated with1 lg/ml (dotted line), 3 lg/ml (broken line), or 9 lg/ml (grey line) cisplatin. Filled histogram shows isotype control. (b) Relative mean fluores-cence values for CXCR4 in ACC (T#3) cells, treated with cisplatin in the indicated concentrations, as determined by flow cytometric analysis.Results are expressed as relative mean fluorescence as compared to untreated samples. Error bars indicate SD of 3 independent experiments. (c)Flow cytometric analysis of CXCR4 protein expression in untreated ACC (T#3) cells (black line) or cells treated with 3 lg/ml cisplatin (brokenline) or 3 lg/ml cisplatin and 5 lg/ml a-amanitin (grey line). (d) CXCL12 induces Akt/PKB and ERK1/2 phosphorylation in human ACC (T#3)cells. Cells were exposed to CXCL12 (500 ng/ml) for the given times, followed by analysis of Akt/PKB and ERK1/2 phosphorylation by West-ern blotting, using phosphospecific antibodies. Examination for the total expression of Akt/PKB and ERK1/2 ensured equal loading of proteinsin each lane. Human epidermal growth factor (EGF, 10 ng/ml), an activator of both Akt/PKB and ERK1/2 was used as a positive control. Resultsare representative of at least 3 independent experiments. (e) Percentage of apoptotic cells as determined by Hoechst 33342 staining in ACC(T#3) cells either left untreated, or treated with cisplatin (9 lg/ml), or a combination of cisplatin (9 lg/ml) and CXCL12 (500 ng/ml). Resultsare expressed as mean 1 SD of 4 independent experiments. (***p < 0.001, Student’s t-test).

2154 MULLER ET AL.

relevant chemokine receptors on the surface of melanoma cells(unpublished data). However, no surface CCR7 expression wasdetected in SCC cells either after serum starvation, or after treat-ment with inflammatory cytokines or growth factors, suggestingthat either a combination of factors or yet unidentified compoundsare required for the recruitment of CCR7 to the surface of SCCcells. The majority of primary tumors and lymph node metastasesof SCC expressed CCR7. Thus, CCR7 might play a role in direct-ing SCC cells to the draining lymph nodes and contribute to thefrequent presence of lymph node metastases in SCC patients, asalso suggested by Wang et al.37 It has been proposed that SCCcells of the head and neck region mimick the differentiation andmigration pattern of dendritic cells, displaying a CCR61CCR72

phenotype within the skin and a CCR62CCR71 phenotype ontheir way to local draining lymph nodes.37 However, in our study,no differences in CCR7 expression could be observed neither inmatched primary SCC cell lines at the mRNA level, nor in tissuesections of primary SCC tumors and matching lymph node meta-stases at the protein level. Our results are in accordance withrecent results of Ding and coworkers,22 demonstrating CCR7expression in both primary tumors and lymph node metastases ofesophageal SCC. These observations are in line with recent stud-ies, demonstrating that primary tumors are similar to the corre-sponding metastatic tumors in their gene expression signature.38,39

Thus, regulation of chemokine receptors may be a relatively earlyevent during tumorigenesis.

CXCR5 participates in the trafficking of B and T cells to secon-dary lymphoid organs.34,35 Recently, it has been suggested that, inleukemic B cell lymphomas, CXCR5 may be associated with thespread of malignant B cells to lymphoid tissues.40,41 Here, weshow the abundant expression of CXCR5 transcripts and the pres-ence of surface CXCR5 on SCC cells. Our observation is consis-tent with the concept that metastasis and leukocyte traffickingshare underlying mechanisms and suggests that CXCL13/CXCR5interactions may provide a novel complementary pathway, media-ting lymphogenic spread.

The only chemokine receptor found to be highly expressed inACC but not in SCC was CXCR4. In ACC cells, CXCR4 signal-ing resulted in the induction of directional tumor cell migration,supporting its role in tumor invasion and metastasis. Recent stud-ies uncovered the fundamental role of CXCL12/CXCR4 interac-tions in physiological processes, such as organogenesis, hemato-poiesis and homing of progenitor cells to liver and bone marrow.42

In addition to its complex physiological functions, CXCR4 hasbeen shown to be pivotal during the metastatic spread of tumorcells to distant organs.8,14,43–45 ACC most frequently metastasizesto the lung and liver,4,6 and CXCL12, the only known CXCR4ligand, is abundantly expressed in these organs.8 The abundantexpression of CXCR4 in the predominantly hematogenously meta-stasizing ACC suggests that CXCR4 may play a role in directingACC cells to their metastatic sites. In contrast to the frequent hem-atogenous dissemination, lymphogenous metastases in ACC areextremely rare,4,46,47 despite the fact that the lymph nodes are alsoan abundant source of CXCL12. Although several studies havesuggested a role for CXCR4 in lymph node metastasis,48–50 accu-mulating evidence suggests that CXCR4 is not a major participantin lymphatic tumor spread, including large-scale gene expressiondata in metastatic head and neck SCC.51 In lymph nodes, CXCL12

is expressed by stromal cells in close vicinity to high endothelialvenules52 (unpublished observations) and contributes to the hom-ing of memory T cells to lymph nodes via the bloodstream but notthe lymphatics.53 Hence, the microanatomical distribution ofCXCL12 in lymph nodes may partially explain why lymphoge-nous metastasis is not observed in ACC with abundant CXCR4expression. Moreover, extravasation requires a complex interplayof chemokine receptors, adhesion molecules and other factors, andACC cells may be deficient in some factors that are essential forextravasation or other steps in the process of lymphogenous meta-stasis.

In addition to ACC, also a subset of SCC tumors demonstratedCXCR4 expression in vivo, which is in accordance with recentstudies.48,54,55 Notably, 2 of the CXCR4-expressing SCC tumorsshowed enhanced CXCR4 expression at the leading edge of thetumor, suggesting a possible role for this receptor in primarytumor invasion, as it also has been suggested in prostate and ovar-ian cancer.56,57

The induction of apoptosis in tumor cells is a major goal of can-cer chemotherapy. Cisplatin, a chemotherapeutic agent used in thetherapy of HNC, exerts its cytotoxic effect by forming DNAadducts, which in turn activate a complex network of pathwaysthat finally culminate in apoptosis.58 However, following cispla-tin-exposure, prosurvival pathways are also activated.58,59 The fateof a cell is determined by the balance between proapoptotic andprosurvival pathways. In the present study, we demonstrate thatsublethal doses of cisplatin induced CXCR4 on the surface ofmalignant cells. In the presence of the transcriptional inhibitor a-amanitin, the upregulation of CXCR4 by cisplatin was markedlyrepressed, suggesting that gene transcription is required for thisinduction. Moreover, we show that in ACC cells CXCL12 stimu-lation resulted in the activation of Akt and ERK1/2, and MAP kin-ases are involved in signal transduction pathways that are gener-ally associated with cell survival and proliferation.29,30 Our find-ings are supported by recent observations, showing that CXCL12can induce survival and proliferation signals in several cell types,such as CD41 T cells,60 embryonic neural cells,61 as well as can-cer cells, including breast cancer, pancreatic cancer and glioblas-toma.45,62–64 In addition to the activation of survival pathways, wealso provide evidence that CXCL12 reduces the rate of apoptosisinduced by cisplatin in ACC cells. We propose that suppression ofapoptosis via CXCR4 signaling may lead to increased tumor cellviability and might contribute to cisplatin resistance and the fail-ure of antineoplastic treatment of metastatic ACC.

In the present study, we have shown that differences in themetastastic patterns of 2 histologically distinct tumors of the headand neck, ACC and SCC, are associated with a different repertoireof chemokine receptors. The distinct chemokine receptor profileof ACC is also associated with signaling pathways involved in cellsurvival and proliferation.

Currently, intense efforts are underway to identify small mole-cule antagonists for chemokine receptors that could be useful fortreating disseminated cancer. It appears that the efficacy of con-ventional chemotherapeutic treatment might be enhanced by com-bination with chemokine receptor neutralization to repress prosur-vival pathways and enhance tumor cell apoptosis. These and otherstrategies, based on emerging mechanistic data, represent some ofthe promising new directions in the therapy of metastatic disease.

References

1. Mao L, Hong WK, Papadimitrakopoulou VA. Focus on head and neckcancer. Cancer Cell 2004;5:311–16.

2. Bier H, Hoffmann T, Hauser U, Wink M, Ochler M, Kovar A,Muser M, Knecht R. Clinical trial with escalating doses of the antiepi-dermal growth factor receptor humanized monoclonal antibody EMD72 000 in patients with advanced squamous cell carcinoma of the lar-ynx and hypopharynx. Cancer Chemother Pharmacol 2001;47:519–24.

3. Sur RK, Donde B, Levin V, Pacella J, Kotzen J, Cooper K, Hale M.Adenoid cystic carcinoma of the salivary glands: a review of 10 years.Laryngoscope 1997;107:1276–80.

4. Hoffmann T. Metastatic adenoid cystic carcinomas of the salivaryglands. In: Lippert B, Werner J, eds. Metastasis in head and neck can-cer. Marburg: Tectum, 2001.405–12.

5. Mukherji SK, Armao D, Joshi VM. Cervical nodal metastases in squa-mous cell carcinoma of the head and neck: what to expect. Head Neck2001;23:995–1005.

6. Fordice J, Kershaw C, El-Naggar A, Goepfert H. Adenoidcystic carcinoma of the head and neck: predictors of morbidityand mortality. Arch Otolaryngol Head Neck Surg 1999;125:149–52.

2155CHEMOKINE RECEPTORS IN HEAD AND NECK CANCER

7. Genden EM, Ferlito A, Bradley PJ, Rinaldo A, Scully C. Neck diseaseand distant metastases. Oral Oncol 2003;39:207–12.

8. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McCla-nahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A,et al. Involvement of chemokine receptors in breast cancer metastasis.Nature 2001;410:50–6.

9. Liotta LA. An attractive force in metastasis. Nature 2001;410:24–5.10. Zlotnik A. Chemokines in neoplastic progression. Semin Cancer Biol

2004;14:181–5.11. Zlotnik A, Yoshie O. Chemokines: a new classification system and

their role in immunity. Immunity 2000;12:121–7.12. Campbell JJ, Butcher EC. Chemokines in tissue-specific and microen-

vironment-specific lymphocyte homing. Curr Opin Immunol 2000;12:336–41.

13. Murakami T, Maki W, Cardones AR, Fang H, Tun Kyi A, Nestle FO,Hwang ST. Expression of CXC chemokine receptor-4 enhances thepulmonary metastatic potential of murine B16 melanoma cells. Can-cer Res 2002;62:7328–34.

14. Spano JP, Andre F, Morat L, Sabatier L, Besse B, Combadiere C,Deterre P, Martin A, Azorin J, Valeyre D, Khayat D, Le Chevalier T,et al. Chemokine receptor CXCR4 and early-stage non-small cell lungcancer: pattern of expression and correlation with outcome. AnnOncol 2004;15:613–7.

15. He RG, Qiu WL, Zhou XJ. The establishment of Acc-2 and Acc-3and their morphological observation. J West China Stomatol 1986;6:1–3.

16. Ballo H, Koldovsky P, Hoffmann T, Balz V, Hildebrandt B,Gerharz CD, Bier H. Establishment and characterization of four celllines derived from human head and neck squamous cell carcinomasfor an autologous tumor-fibroblast in vitro model. Anticancer Res1999;19:3827–36.

17. Masters JR, Palsson B, eds.Human cell culture, vol. 2. London:Kluwer, 1999.

18. Hoffmann TK, Leenen K, Hafner D, Balz V, Gerharz CD, Grund A,Ballo H, Hauser U, Bier H. Antitumor activity of protein kinase Cinhibitors and cisplatin in human head and neck squamous cell carci-noma lines. Anticancer Drugs 2002;13:93–100.

19. Homey B, Wang W, Soto H, Buchanan ME, Wiesenborn A, Catron D,Muller A, McClanahan TK, Dieu-Nosjean M-C, Orozco R, RuzickaT, Lehmann P, et al. Cutting edge: the orphan chemokine receptor Gprotein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associatedchemokine CCL27 (CTACK/ALP/ILC). J Immunol 2000;164:3465–70.

20. Varney ML, Li A, Dave BJ, Bucana CD, Johansson SL, Singh RK.Expression of CXCR1 and CXCR2 receptors in malignant melanomawith different metastatic potential and their role in interleukin-8(CXCL-8)-mediated modulation of metastatic phenotype. Clin ExpMetastasis 2003;20:723–31.

21. Wiley HE, Gonzalez EB, Maki W, Wu M-t, Hwang ST. Expression ofCC chemokine receptor-7 and regional lymph node metastasis of B16murine melanoma. J Natl Cancer Inst 2001;93:1638–43.

22. Ding Y, Shimada Y, Maeda M, Kawabe A, Kaganoi J, Komoto I,Hashimoto Y, Miyake M, Hashida H, Imamura M. Association of CCchemokine receptor 7 with lymph node metastasis of esophageal squa-mous cell carcinoma. Clin Cancer Res 2003;9:3406–12.

23. Till KJ, Lin K, Zuzel M, Cawley JC. The chemokine receptor CCR7and a4 integrin are important for migration of chronic lymphocyticleukemia cells into lymph nodes. Blood 2002;99:2977–84.

24. Mashino K, Sadanaga N, Yamaguchi H, Tanaka F, Ohta M, Shibuta K,Inoue H, Mori M. Expression of chemokine receptor CCR7 is associ-ated with lymph node metastasis of gastric carcinoma. Cancer Res2002;62:2937–41.

25. Kyi AT, Dudli C, Burg G, Nestle FO. Functional expression of CCR3but not of CCR7 in lymphogenic melanoma metastasis 32nd ESDRAnnual Meeting, Geneva, Switzerland, 2002. p 13.

26. de Haan LD, De Mulder PH, Vermorken JB, Schornagel JH, Vermey A,Verweij J. Cisplatin-based chemotherapy in advanced adenoid cysticcarcinoma of the head and neck. Head Neck 1992;14:273–7.

27. Browman GP, Hodson DI, Mackenzie RJ, Bestic N, Zuraw L. Choos-ing a concomitant chemotherapy and radiotherapy regimen for squa-mous cell head and neck cancer: a systematic review of the publishedliterature with subgroup analysis. Head Neck 2001;23:579–89.

28. Johnsson A, Hoglund P, Grubb A, Cavallin-Stahl E. Cisplatin pharma-cokinetics and pharmacodynamics in patients with squamous-cell car-cinoma of the head/neck or esophagus. Cancer Chemother Pharmacol1996;39:25–33.

29. Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phos-phoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999;68:965–1014.

30. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: afamily of protein kinases with diverse biological functions. MicrobiolMol Biol Rev 2004;68:320–44.

31. Kunkel EJ, Butcher EC. Chemokines and the tissue-specific migrationof lymphocytes. Immunity 2002;16:1–4.

32. Li A, Varney ML, Singh RK. Expression of interleukin 8 and itsreceptors in human colon carcinoma cells with different metastaticpotentials. Clin Cancer Res 2001;7:3298–304.

33. Watanabe H, Iwase M, Ohashi M, Nagumo M. Role of interleukin-8secreted from human oral squamous cell carcinoma cell lines. OralOncol 2002;38:670–9.

34. Muller G, Hopken UE, Lipp M. The impact of CCR7 and CXCR5 onlymphoid organ development and systemic immunity. Immunol Rev2003;195:117–35.

35. Ohl L, Henning G, Krautwald S, Lipp M, Hardtke S, Bernhardt G,Pabst O, Forster R. Cooperating mechanisms of CXCR5 and CCR7 indevelopment and organization of secondary lymphoid organs. J ExpMed 2003;197:1199–204.

36. Matloubian M, David A, Engel S, Ryan JE, Cyster JG. A transmem-brane CXC chemokine is a ligand for HIV-coreceptor Bonzo. NatImmunol 2000;1:298–304.

37. Wang J, Xi L, Hunt JL, Gooding W, Whiteside TL, Chen Z, GodfreyTE, Ferris RL. Expression pattern of chemokine receptor 6 (CCR6) andCCR7 in squamous cell carcinoma of the head and neck identifies anovel metastatic phenotype. Cancer Res 2004;64: 1861–6.

38. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signa-ture of metastasis in primary solid tumors. Nat Genet 2003;33:49–54.

39. Weigelt B, Glas AM, Wessels LFA, Witteveen AT, Peterse JL, van’tVeer LJ. Gene expression profiles of primary breast tumors main-tained in distant metastases. Proc Natl Acad Sci USA 2003;100:15901–5.

40. Durig J, Schmucker U, Duhrsen U. Differential expression of chemo-kine receptors in B cell malignancies. Leukemia 2001;15:752–6.

41. Lopez-Giral S, Quintana NE, Cabrerizo M, Alfonso-Perez M, Sala-Valdes M, de Soria VGG, Fernandez-Ranada JM, Fernandez-Ruiz E,Munoz C. Chemokine receptors that mediate B cell homing to secon-dary lymphoid tissues are highly expressed in B cell chronic lympho-cytic leukemia and non-Hodgkin lymphomas with widespread nodulardissemination. J Leukoc Biol 2004;76:462–71.

42. Horuk R. Chemokines beyond inflammation. Nature 1998;393:524–5.43. Sun YX, Wang J, Shelburne CE, Lopatin DE, Chinnaiyan AM, Rubin

MA, Pienta KJ, Taichman RS. Expression of CXCR4 and CXCL12(SDF-1) in human prostate cancers (PCa) in vivo. J Cell Biochem2003;89:462–73.

44. Geminder H, Sagi-Assif O, Goldberg L, Meshel T, Rechavi G, Witz IP,Ben-Baruch A. A possible role for CXCR4 and its ligand, the CXC che-mokine stromal cell-derived factor-1, in the development of bone mar-row metastases in neuroblastoma. J Immunol 2001;167:4747–57.

45. Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima S,Kawaguchi M, Kobayashi H, Doi R, Hori T, Fujii N, Imamura M.Expression of stromal cell-derived factor 1 and CXCR4 ligand recep-tor system in pancreatic cancer: a possible role for tumor progression.Clin Cancer Res 2000;6:3530–5.

46. Allen MS, Jr, Marsh WL, Jr. Lymph node involvement by directextension in adenoid cystic carcinoma. Absence of classic emboliclymph node metastasis. Cancer 1976;38:2017–21.

47. van der Wal JE, Becking AG, Snow GB, van der Waal I. Distantmetastases of adenoid cystic carcinoma of the salivary glands and thevalue of diagnostic examinations during follow-up. Head Neck 2002;24:779–83.

48. Delilbasi CB, Okura M, Iida S, Kogo M. Investigation of CXCR4 insquamous cell carcinoma of the tongue. Oral Oncol 2004;40:154–7.

49. Kato M, Kitayama J, Kazama S, Nagawa H. Expression pattern ofCXC chemokine receptor-4 is correlated with lymph node metastasisin human invasive ductal carcinoma. Breast Cancer Res 2003;5:R144–50.

50. Uchida D, Begum NM, Tomizuka Y, Bando T, Almofti A, Yoshida H,Sato M. Acquisition of lymph node, but not distant metastatic poten-tials, by the overexpression of CXCR4 in human oral squamous cellcarcinoma. Lab Invest 2004;84:1538–46.

51. O’Donnell RK, Kupferman M, Wei SJ, Singhal S, Weber R,O’Malley B, Jr, Cheng Y, Putt M, Feldman M, Ziober B, MuschelRJ. Gene expression signature predicts lymphatic metastasis in squ-amous cell carcinoma of the oral cavity. Oncogene 2004;24:1244–51.

52. Okada T, Ngo VN, Ekland EH, Forster R, Lipp M, Littman DR, Cys-ter JG. Chemokine requirements for B cell entry to lymph nodes andPeyer’s patches. J Exp Med 2002;196:65–75.

53. Scimone ML, Felbinger TW, Mazo IB, Stein JV, von Andrian UH,Weninger W. CXCL12 mediates CCR7-independent homing of cen-tral memory cells, but not naive T cells, in peripheral lymph nodes.J Exp Med 2004;199:1113–20.

54. Uchida D, Begum NM, Almofti A, Nakashiro K, Kawamata H,Tateishi Y, Hamakawa H, Yoshida H, Sato M. Possible role of stro-mal-cell-derived factor-1/CXCR4 signaling on lymph node metastasisof oral squamous cell carcinoma. Exp Cell Res 2003;290:289–302.

55. Almofti A, Uchida D, Begum NM, Tomizuka Y, Iga H, Yoshida H,Sato M. The clinicopathological significance of the expression of

2156 MULLER ET AL.

CXCR4 protein in oral squamous cell carcinoma. Int J Oncol 2004;25:65–71.

56. Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman NS,McCauley LK. Use of the stromal cell-derived factor-1/CXCR4 path-way in prostate cancer metastasis to bone. Cancer Res 2002;62:1832–7.

57. Scotton CJ, Wilson JL, Scott K, Stamp G, Wilbanks GD, Fricker S,Bridger G, Balkwill FR. Multiple actions of the chemokine CXCL12on epithelial tumor cells in human ovarian cancer. Cancer Res2002;62:5930–8.

58. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis ofresistance. Oncogene 2003;22:7265–79.

59. Aoki K, Ogawa T, Ito Y, Nakashima S. Cisplatin activates survivalsignals in UM-SCC-23 squamous cell carcinoma and these signalpathways are amplified in cisplatin-resistant squamous cell carcinoma.Oncol Rep 2004;11:375–9.

60. Suzuki Y, Rahman M, Mitsuya H. Diverse transcriptional response ofCD41 T cells to stromal cell-derived factor (SDF)-1: cell survival

promotion and priming effects of SDF-1 on CD41 T cells. J Immunol2001;167:3064–73.

61. Chalasani SH, Baribaud F, Coughlan CM, Sunshine MJ, Lee VMY,Doms RW, Littman DR, Raper JA. The chemokine stromal cell-derived factor-1 promotes the survival of embryonic retinal ganglioncells. J Neurosci 2003;23:4601–12.

62. Smith MCP, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD. CXCR4 regulates growth of both primary andmetastatic breast cancer. Cancer Res 2004;64:8604–12.

63. Barbero S, Bonavia R, Bajetto A, Porcile C, Pirani P, Ravetti JL, ZonaGL, Spaziante R, Florio T, Schettini G. Stromal cell-derived factor 1astimulates human glioblastoma cell growth through the activation ofboth extracellular signal-regulated kinases 1/2 and akt. Cancer Res2003;63:1969–74.

64. Zhou Y, Larsen PH, Hao C, Yong VW. CXCR4 is a major chemokinereceptor on glioma cells and mediates their survival. J Biol Chem2002;277:49481–7.

2157CHEMOKINE RECEPTORS IN HEAD AND NECK CANCER