Chemokine receptors in head and neck cancer: Association with metastatic spread and regulation during chemotherapy

Authors

  • Anja Muller,

    Corresponding author
    1. Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany
    • Departments of Radiation Oncology, Dermatology or Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany
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    • Fax: +49-211-811-7316.

    • The first 3 authors contributed equally to this work.

  • Eniko Sonkoly,

    1. Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany
    2. Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany
    3. Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany
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    • The first 3 authors contributed equally to this work.

  • Christine Eulert,

    1. Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany
    2. Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany
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    • The first 3 authors contributed equally to this work.

  • Peter Arne Gerber,

    1. Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Robert Kubitza,

    1. Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Kerstin Schirlau,

    1. Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Petra Franken-Kunkel,

    1. Department of Radiation Oncology, Heinrich-Heine-University, Duesseldorf, Germany
    2. Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Christopher Poremba,

    1. Department of Pathology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Carl Snyderman,

    1. Department of Otorhinolaryngology, University of Pittsburgh, PA, USA
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  • Lars-Oliver Klotz,

    1. Institute of Molecular Biology and Biochemistry, Heinrich-Heine-University, Duesseldorf, Germany
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  • Thomas Ruzicka,

    1. Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Henning Bier,

    1. Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany
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  • Albert Zlotnik,

    1. Neurocrine Biosciences Inc., San Diego, CA, USA
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  • Theresa L. Whiteside,

    1. Hillman Cancer Center, University of Pittsburgh, PA, USA
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  • Bernhard Homey,

    Corresponding author
    1. Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany
    • Departments of Radiation Oncology, Dermatology or Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany
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    • The last 2 authors contributed equally to this work.

  • Thomas K. Hoffmann

    Corresponding author
    1. Department of Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Germany
    • Departments of Radiation Oncology, Dermatology or Otorhinolaryngology, Heinrich-Heine-University, Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany
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    • The last 2 authors contributed equally to this work.


Abstract

Head and neck carcinomas are histologically and clinically heterogeneous. While squamous cell carcinomas (SCC) are characterized by lymphogenous spread, adenoid cystic carcinomas (ACC) disseminate preferentially hematogenously. To study cellular and molecular mechanisms of organ-specific metastasis, we used SCC and ACC cell lines and tumor tissues, obtained from patients with primary or metastatic disease. Comprehensive analysis at the mRNA and protein level of human chemokine receptors showed that SCC and ACC cells exhibited distinct and nonrandom expression profiles for these receptors. SCC predominantly expressed receptors for chemokines homeostatically expressed in lymph nodes, including CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR)5. No difference in expression of chemokine receptors was seen in primary SCC and corresponding lymph node metastases. In contrast to SCC, ACC cells primarily expressed CXCR4. In chemotaxis assays, ACC cells were responsive to CXCL12, the ligand for CXCR4. Exposure of ACC cells to cisplatin resulted in upregulation of CXCR4 on the cell surface, which was repressed by the transcriptional inhibitor, α-amanitin. Treatment of ACC cells with CXCL12 resulted in the activation of Akt and ERK1/2 pathways. Furthermore, CXCL12 suppressed the rate of apoptosis induced by cisplatin in ACC cells, suggesting that signaling via CXCR4 may be part of a tumor cell survival program. Discrimination of the chemokine receptor profile in SCC and ACC in vitro and in tissues provided insights into their distinct biologic and clinical characteristics as well as indications that chemokine receptors might serve as future therapeutic targets in these malignancies. © 2005 Wiley-Liss, Inc.

Head and neck carcinomas (HNC) include tumors with different histological phenotypes and distinct clinical characteristics. Squamous cell carcinomas (SCC) of the upper aerodigestive tract mucosa represent the most common histological subtype.1, 2 Adenoid cystic carcinoma (ACC) is a rare malignant epithelial tumor, arising from salivary glands.3 Both tumor types are characterized by distinct metastatic patterns. While SCC frequently metastasizes to regional lymph nodes, ACC is more aggressive, disseminating to distant sites, particularly, the lung and the liver.2, 4, 5, 6 In both these tumor types, current treatment regimens result in fairly good locoregional control, but little progress has been 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 therapeutic options for the control of disseminated tumor spread. Owing to their distinct metastatic patterns, ACC and SCC provide an interesting model to investigate the so far poorly understood molecular mechanisms of organ-specific metastasis.

Recent studies have demonstrated that tumor cells express functionally active chemokine receptors and that expression of these receptors appears to regulate cellular functions associated with the process of metastasis.8, 9, 10 Chemokines are a superfamily of small, cytokine-like proteins that selectively attract and activate different cell types.11, 12 The CXC chemokine receptor (CXCR)4 is known to be expressed in several cancer types, including breast cancer,8 melanoma13 and lung cancer.14 These cancer types may use CXCR4 to metastasize to target organs. Furthermore, CXCR4 expression has been implicated in metastatic spread in vivo, in animal 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 chemokine receptors, influencing their metastatic behavior. CC chemokine receptor (CCR)7 is abundantly expressed in primary tumors and lymph node metastases of SCC in vivo. ACC cells express high levels of CXCR4 both in vitro and in vivo, whereas CXCR4 expression is undetectable or low in SCC. Furthermore, the exposure of ACC cells to the antineoplastic agent, cisplatin, results in the upregulation of CXCR4 on the cell surface, providing survival signals to tumor cells.

Abbreviations:

ACC, adenoid cystic carcinoma; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; ERK, extracellular signal-regulated kinase; HNC, head and neck carcinoma; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3′-kinase; PKB, protein kinase B; RT-PCR, reverse transcription polymerase chain reaction; SCC, squamous cell carcinoma.

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 provided by Dr. W.L. Qiu (Shanghai, China). SCC cell lines were derived from 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/SCC T#17, UT-SCC-24A/SCC T#24) or from lymph node metastases (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 were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine and antibiotic/antimycotic solution (all from Gibco, Eggenstein, Germany); as the only exception, cell line ACC-2 (ACC T#2) was cultured in RPMI medium (Gibco), with identical supplementation. For control purposes, primary mucosal keratinocytes were separated from healthy oral mucosa, with the patients' informed consent. Tissue samples were cut into small strips and incubated in Dispase I solution (Roche Diagnostics, Mannheim, Germany) overnight at 4°C. On the following day, the mucosa was peeled off the lamina propria and incubated in trypsin–EDTA solution at 37°C for 30 min. A suspension of primary mucosal cells was prepared in keratinocyte serum-free medium (keratinocyte-SFM), supplemented with antibiotic/antimycotic solution (all from Gibco). Cells were seeded into 75-cm2 tissue-culture flasks and propagated in keratinocyte-SFM. To harvest or passage the cell 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 cells were incubated for 72 hr, and on day 3, fresh culture medium, supplemented with different concentrations of cisplatin (1, 3 or 9 μg/ml, Bristol-Myers Squibb, Munich, Germany), α-amanitin (5 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) or combinations of cisplatin and α-amanitin, was added, and controls received medium alone. The antitumor effects were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) test, as described previously.18

Quantitative real-time RT-PCR (TaqMan®)

Total RNA of tumor cells and keratinocytes was extracted (TRIzol reagent, Invitrogen, Carlsbad, CA, USA), DNase-treated and reverse-transcribed, as described previously.8, 19 Complementary DNA was quantitatively analyzed for the expression of human chemokines and chemokine receptors by the fluorogenic 5′-nuclease PCR assay, as reported.8, 19 Specific primers and probes were obtained from Applied Biosystems (Foster City, CA, USA). Gene-specific PCR products were continuously measured during 40 cycles, with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Quantitative real-time RT-PCR was performed with SYBR green as reporter, for reactions using chemokine receptor-specific primers, and FAM as reporter, for reactions using chemokine receptor-specific primer/probe combinations. Probes for the internal positive control (ribosomal 18s RNA) were associated with the VIC reporter. Target gene expression was normalized between different samples, based on the values of ribosomal 18s RNA expression. Plasmid cDNA for chemokine receptors were used for quantification of target gene-specific mRNA expression.

Flow cytometry

To analyze chemokine receptor expression, cells were stained with 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 (BD PharMingen), 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-conjugated streptavidin (BD PharMingen) were used. CCR8 was stained using unlabeled anti-CCR8 (210-762-R100, goat IgG, Alexis Biochemicals, Lausanne, Switzerland) and PE-conjugated swine anti-goat IgG (Caltag, Burlingame, CA, USA). Flow-cytometric analysis was performed using a FACScan flow cytometer, equipped with a single, 488-nm argon-ion laser and CELLQuest software (Becton Dickinson, San Jose, CA, USA). All tumor cell lines as well as primary human mucosal keratinocytes were stained 2 times for CCR1–10 and CXCR1–6. For the detection of intracellular CCR7 and CXCR5, we additionally applied the IntraStain kit (DAKO, Glostrup, Denmark).

Tissue samples and immunohistochemistry

Tissue blocks of primary ACC (n = 57) were obtained from the University of Pittsburgh Medical Center, USA. Paraffin blocks of primary tumors (n = 29) and lymph node metastases (n = 12 matched pairs) from patients with SCC were obtained from surgical specimens at the Department of Otorhinolaryngology, University Hospital Düsseldorf, Germany. The study was approved by the local Ethic Committees, and informed consent was secured from each patient prior to sample acquisition. Histopathological diagnosis was confirmed in all tissue samples. Tumors were routinely formalin-fixed, paraffin-embedded and sectioned at 3–5-μm slices, which were stained with anti-human CXCR4 antibody, as previously described.8 For immunohistochemical analyses of CCR7, tumor sections were fixed in acetone and preprocessed with H2O2, followed by an avidin and biotin blocking step (VECTOR Blocking Kit, Vector Laboratories, Burlingame, CA, USA). Sections were stained with monoclonal antibody against human CCR7 (R&D Systems), or isotype control antibody (BD PharMingen). Development of the staining was performed with a DAKO AEC-Kit (DAKO). Sections were counterstained with hematoxylin. Histopathological evaluation was performed by 2 independent investigators on a scale of 0–2+, depending on the intensity of the immunoreactivity (0, negative immunostaining; 1+, weakly positive immunostaining; 2+, moderately/strongly positive immunostaining). For immunocytochemical analysis of chemokine receptors in cell lines, cells were grown on slides for 72 hr, washed with PBS, fixed in methanol at −20°C for 5 min, treated with acetone for 30 sec, air-dried and stained for chemokine receptors, as described earlier.

Chemotaxis assays

Migration was assayed in 24-well, cell-culture chambers, using inserts with 8-μm pore membranes, as previously described.8 Membranes were precoated with fibronectin (5 μg/ml, Becton Dickinson). ACC (T#3) and SCC (T#1) cells were resuspended in chemotaxis buffer (DMEM; 0.1% BSA, Sigma-Aldrich; 12 mM HEPES, Invitrogen) at 0.5–1 × 105 cells/ml. Either the medium (0.6 ml) alone or the media supplemented with different concentrations of the ligands CXCR4 and CXCL12 (human recombinant CXCL12, R&D Systems) was added to the lower chamber. After incubation for 9 (ACC) or 15 hr (SCC), cells which have migrated through the pores were stained and counted under a light microscope in at least 5 high-power fields. Different time intervals were chosen, because of the observed difference in the basic migratory potential of ACC and SCC (control/blank). FCS (5%) was used as a positive control to assure the general capacity of tumor cells to perform migratory responses. All chemotaxis assays were performed 3 times in triplicate wells.

Western blotting and immunodetection

For Western blotting, ACC (T#3) cells were treated with human recombinant CXCL12 (500 ng/ml; R&D Systems) for the indicated times, lysed in 2× 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. Samples were heated at 95°C for 5 min and applied to SDS-polyacrylamide gels of 10% (w/v) acrylamide, followed by electrophoresis and tank-blotting onto polyvinylidene difluoride membranes. Immunodetections were performed using polyclonal antibodies, at dilutions recommended by the manufacturer. Anti-phospho-ERK1/2, anti-total ERK1/2, anti-phospho-Akt (Ser473) and anti-total Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Detection of apoptosis

For morphological analysis of apoptosis, ACC cells were treated with cisplatin or the combination of cisplatin and human recombinant CXCL12 for 24 hr, and stained with 5 μg/ml Hoechst 33342 (Sigma) to reveal nuclear condensation and fragmentation by fluorescence microscopy. To quantify apoptosis, at least 5 fields with ˜200 cells per field were examined in each well.

Results

Cell lines of ACC and SCC exhibit distinct chemokine receptor expression profiles

To determine the differences in chemokine receptor profiles of ACC and SCC, 2 histologically and clinically different subtypes of HNC, we performed a comprehensive analysis of the expression of all known chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, CX3CR1 and CXCR1) in human ACC (n = 2) and SCC cell lines derived from both primary tumors (n = 10) and lymph node metastases (n = 3) of HNC patients. Absolute quantities of receptor mRNA were measured by quantitative real-time RT-PCR (Fig. 1), and their cell-surface expression (for CCR1–CCR10 and CXCR1–CXCR6) was determined by flow cytometric analysis (Fig. 2 and Table I).

Figure 1.

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

Figure 2.

Distinct chemokine receptor expression of ACC and SCC cell lines. (a, b) Flow cytometric analysis of CXCR4 expression on ACC (T#3) (a) and SCC (T#1) (b) cells. (c, d) Flow cytometric analysis of surface (c) and intracellular (d) CCR7 expression in SCC (T#1) cells. (e, f) Immunocytochemical analysis of SCC (T#1) cells using isotype antibody (e) or specific antibody for CCR7 (f). Magnification: ×200. (gi) Flow cytometric analysis of surface (g) or intracellular (h) CXCR5 expression in SCC (T#6) cells as well as surface CXCR5 expression after 24 hr of serum starvation in SCC (T#6) cells (i). Filled histogram, isotype; black line, CXCR4, CCR7 and CXCR5, respectively. Representative data for 1 of 2 ACC and 1 of 13 (b, c, e, f) or 3 (d, g–i) SCC cell lines evaluated.

Table I. Surface Chemokine Receptor Expression in SCC and ACC Cell Lines
 CCR1CCR2CCR3CCR4CCR5CCR6CCR7CCR8CCR9CCR10CXCR1CXCR2CXCR3CXCR4CXCR5CXCR6
  • 1

    KC, keratinocytes.

  • 2

    Percentage of positive cells. Values are means of two measurements.

  • 3

    T, cell line derived from primary tumor.

  • 4

    M, cell line derived from metastatic lymph node.

Mucosal KC1222
SCC T#132
SCC T#224
SCC T#323
SCC T#42432
SCC T#64524
SCC T#723
SCC T#8253
SCC T#1024225623
SCC M#10444212726
SCC T#17262
SCC M#17242
SCC T#242
SCC M#243
ACC T#26425
ACC T#395

Figure 1 illustrates chemokine receptor mRNA expression in ACC and SCC cells, as well as primary mucosal keratinocytes. The latter exhibited a very limited repertoire of chemokine receptors, with CXCR1 being the predominant family member expressed at a significant level (Fig. 1m); epidermal keratinocytes from 3 different donors showed an almost identical expression pattern (data not shown). ACC cells had a very restricted profile of chemokine receptors, with mRNA expression for several receptors, and CXCR4 was the only chemokine receptor expressed at a high level compared to SCC cells (Fig. 1p). In contrast to ACC, SCC cells expressed a considerable variety of chemokine receptors. CXCR1 and CXCR2 genes, encoding chemokine receptors reported to be associated with CXCL8-mediated proliferation and invasion of cancer cells,20 were expressed in the majority of SCC cells, but were absent in ACC cells (Figs. 1m and 1n). Compared to mucosal keratinocytes, a consistent upregulation of receptor mRNA 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 were consistently although variably expressed in different SCC cell lines, but were not detectable or expressed at markedly lower levels in ACC cells (Figs. 1g and 1q). Interestingly, CCR7, CXCR3 and CCR3 were reported to be associated with lymph node metastasis in other cancer types.21, 22, 23, 24, 25 While CXCR4 was the only receptor with high expression in ACC cells, SCC cells displayed negligible levels of CXCR4 mRNA (Fig. 1p).

At the protein level, flow cytometric analysis confirmed high cell surface expression of CXCR4 (25–95%) in ACC cells (Fig. 2a and Table I), while SCC cell lines or primary mucosal keratinocytes were found to express little or no CXCR4 (Fig. 2b and Table I). Notably, some SCC cell lines showed low but detectable surface CXCR4 expression (≤4%; Table I). For CCR7, despite significant expression at the mRNA level, no surface protein could be 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 representative SCC cell lines expressing high levels of CCR7 mRNA and one ACC cell line. All 3 SCC cell lines showed high intracellular CCR7 expression (Fig. 2d). Intracellular CCR7 expression in SCC cell lines was further confirmed by immunocytochemical analysis (Figs. 2e and 2f). However, ACC cells demonstrated negligible amounts of intracellular CCR7 (data not shown). These data suggest that, in culture, CCR7 protein is either not secreted or is rapidly shed from the cell surface. To assess whether microenvironmental factors regulate CCR7 expression, surface CCR7 was analyzed by flow cytometry in SCC cells after serum starvation, or treatment with inflammatory cytokines or growth factors, including TNF-α, TGF-β and EGF, or supernatant of endothelial cells or fibroblasts. However, none of these factors induced surface CCR7 expression (data not shown). Similarly to CCR7, CXCR5 was not detected on the surface of SCC cells by flow cytometric analysis (Table I and Fig. 2g). However, intracellular staining of 3 representative SCC cell lines demonstrated high levels of intracellular CXCR5 (Fig. 2h), in accordance with their abundant CXCR5 mRNA expression. Moreover, after 24 hr of serum starvation, CXCR5 was detected on the cell surface (15% positive cells) of SCC cells (Fig. 2i).

Flow cytometric analyses showed weak surface protein expression for CCR3 in all SCC, and for CCR6 in one ACC and all SCC cell lines. All other receptors were undetectable or expressed at a low level on the surface of cultured ACC and SCC cells or mucosal keratinocytes (Table I). Interestingly, SCC cell lines derived from lymph node metastases showed no consistent difference in the expression of any of the chemokine receptors compared to those derived from primary tumors (Fig. 1 and Table I).

Tissue immunohistochemistry confirms differences in the chemokine receptor expression profile in ACC versus SCC

To assess whether the difference in CXCR4 and CCR7 expression between ACC and SCC cells is relevant in vivo, immunohistochemical analyses of 57 primary ACC and 29 primary SCC were performed. Although CXCR4 was not detectable in normal salivary gland (Fig. 3a), 56/57 primary ACC exhibited strong (2+) CXCR4 expression (Fig. 3a). Only one ACC patient demonstrated weak (1+) expression of CXCR4. In contrast to ACC, primary SCC was predominantly negative for CXCR4, (Fig. 3b) but abundantly expressed CCR7 (Figs. 3g and 3h). Notably, a minority of primary SCC expressed low levels of CXCR4 (1+, 9/29, Fig. 3c), and only 1/29 SCC showed high CXCR4 expression (2+). In 2 CXCR4-positive SCC samples, in particular, malignant cells at the leading edge of the tumor exhibited strong CXCR4 expression (Fig. 3d), suggesting that CXCR4 might have a role in tumor invasion.

Figure 3.

CXCR4 expression in tissues of primary ACC and SCC and CCR7 expression in tissues of primary tumors as well as matching lymph node metastases of SCC. Representative immunohistochemical data on the chemokine receptors CXCR4 (af) and CCR7 (g, h). (a) Primary tumors of ACC patients demonstrate strong and homogenous CXCR4 expression (a; right) while adjacent normal salivary gland expresses no CXCR4 protein (a; left). (bd) Variable CXCR4 protein expression in primary tumors of SCC patients. (b) Absence of CXCR4 protein in a primary SCC. The arrow indicates CXCR4-expressing endothelial cells. (c) Weak CXCR4 expression in another primary SCC (arrows) and no CXCR4 immunoreactivity in adjacent normal mucosa (left). (d) Enhancement of CXCR4 protein expression at the invasive front (arrows) of a primary 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: ×250 (a, b), ×100 (ch).

In accordance with our in vitro findings, CCR7 was expressed in 23 out of 26 (88%) primary tumors of SCC. In 7 out of 26 (27%) SCC, a strong CCR7 expression (2+) could be observed.

To investigate possible differences in chemokine receptor expression in primary SCC and matching lymph node metastases, we performed immunohistochemical staining of matched pairs from 12 patients with nodal-positive SCC. CXCR4 was absent in 5/12 primary tumors and 7/12 corresponding lymph node metastases of SCC (Figs. 3e and 3f; Table II). Six of 12 primary tumors and 5/12 lymph node metastases expressed CXCR4 at low levels (1+); however, strong expression (2+) was only detected in one of the primary tumors (Table II). We could not observe a consistent difference in the expression of CXCR4 between primary tumors and their corresponding lymph node metastases.

Table II. CXCR4 and CCR7 Expression in Primary Tumors of SCC and Their Corresponding Lymph Node Metastases
 Chemokine receptor expression
01+2+
  • 1

    T, primary tumors.

  • 2

    M, lymph node metastases.

CXCR4T1(n = 12)561
M2(n = 12)750
CCR7T (n = 12)084
M (n = 12)273

In contrast to CXCR4, CCR7 was expressed in all primary tumors and 10/12 corresponding lymph node metastases (Table II and Figs. 3g and 3h). Strong CCR7 expression (2+) was observed in 8/12 primary SCC and 5/12 lymph node metastases (Table II and Figs. 3g and 3h). The expression of CCR7 in both primary tumors and metastases suggests that this receptor could play a role in lymphogenic metastasis of SCC. However, no consistent up- or downregulation was found in lymph node metastases compared to the corresponding primary tumors.

CXCR4 is functionally active in ACC cells and mediates directional migration

CXCL12, the corresponding ligand of CXCR4, is highly expressed in the lung and liver, organs that represent major destinations of ACC metastasis.4, 8 Consequently, we asked whether CXCR4 expressed in ACC cells is functionally active upon ligand binding. When the highly CXCR4-expressing ACC cells (T#2) (Table I) were incubated with various concentrations of recombinant human CXCL12, binding of the ligand and time-dependent internalization of the chemokine receptor was seen by flow cytometry (data not shown). Furthermore, a transwell migration assay was performed to examine the effect of CXCL12 on migration of ACC and SCC cells (ACC T#3 and SCC T#1). ACC cells were able 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 show a chemotactic response to CXCL12 gradients (Fig. 4 and data not shown).

Figure 4.

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

The antineoplastic agent cisplatin upregulates CXCR4 on tumor cells

Patients with HNC frequently undergo chemotherapy with cisplatin.26, 27 Hence, we next sought to investigate the regulation of chemokine receptors on tumor cells, during the exposure to this antineoplastic agent. CXCR4-expressing ACC cells were exposed to sublethal doses of cisplatin at the concentrations of 1, 3 and 9 μg/ml. These concentrations are comparable to those observed in vivo in sera of HNC patients,28 and caused 24–78% inhibition of cell growth, as determined by MTT assays (data not shown). Flow cytometric analysis of ACC (T#3) cells showed that cisplatin dose-dependently induced the expression of CXCR4 on the cell surface (Figs. 5a and 5b). After 24-hr incubation with cisplatin, a 1.5- to 2-fold increase in mean fluorescence for CXCR4 was observed (Fig. 5b). To investigate the underlying mechanism of cisplatin-induced CXCR4 expression, ACC cells were co-incubated with cisplatin and α-amanitin, an inhibitor of RNA polymerase II-mediated transcription. In the presence of α-amanitin, the induction of CXCR4 was decreased by up to 85% (Fig. 5c), suggesting that induction of CXCR4 by cisplatin requires gene transcription. In contrast to ACC cells, no significant upregulation of surface CXCR4 was detected on SCC (T#6) cells after cisplatin-treatment (data not shown).

Figure 5.

ACC cells express elevated levels of surface CXCR4 after cisplatin-treatment (ac) and show enhanced survival in the presence of CXCL12 (d, e). (a) Flow cytometric analysis of CXCR4 protein expression in untreated ACC (T#3) cells (black line) or cells treated with 1 μg/ml (dotted line), 3 μg/ml (broken line), or 9 μg/ml (grey line) cisplatin. Filled histogram shows isotype control. (b) Relative mean fluorescence 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 μg/ml cisplatin (broken line) or 3 μg/ml cisplatin and 5 μg/ml α-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 Western blotting, using phosphospecific antibodies. Examination for the total expression of Akt/PKB and ERK1/2 ensured equal loading of proteins in 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. Results are 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 μg/ml), or a combination of cisplatin (9 μg/ml) and CXCL12 (500 ng/ml). Results are expressed as mean + SD of 4 independent experiments. (***p < 0.001, Student's t-test).

CXCL12/CXCR4 interaction activates survival pathways in tumor cells

To assess whether signaling via CXCR4 activates pathways involved in cell survival and proliferation in ACC cells, we focused 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 phosphatidylinositol 3′-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 involved in cell proliferation, differentiation and survival.30 ACC cells were incubated in serum-free medium, containing 500 ng/ml CXCL12, and total cell lysates were collected at several time points. Lysates were examined for phosphorylation of Akt/PKB and ERK1/2, using Western blot analysis. Our results indicate that CXCL12 significantly 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 ERK1/2 was sustained for up to 30 min, and decreased back to control levels after 60 min. Examination for the total expression of MAP kinases ensured equal loading of proteins in each lane. These data suggest that CXCL12/CXCR4 interaction in ACC cells activates intracellular signaling pathways involved in cell survival and proliferation.

To examine whether the activation of Akt/PKB and ERK1/2 by CXCL12 is indeed associated with increased cell survival, ACC cells were incubated with cisplatin or the combination of cisplatin and CXCL12, or left untreated, stained with Hoechst 33342, and the percentage of apoptotic cells was quantified, based on their characteristic morphology. In our experiments, the baseline apoptotic rate of ACC cells was 2.5% on average. Cisplatin (9 μg/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 condensed and fragmented cell nuclei. After 24 hr, the rate of apoptosis induced by cisplatin was significantly decreased by co-incubation with CXCL12 (Fig. 5e, p < 0.001). The rate of cisplatin-induced apoptosis was still reduced by CXCL12 after 48 hr (data not shown). These results suggest that CXCL12 indeed suppresses apoptosis induced by cisplatin, thus promoting survival of ACC cells.

Discussion

The presence of local and distant metastases is a key criterion of the malignant phenotype and represents an important prognostic as well as survival-limiting factor for most cancer types. In general, cancer cells metastasize to distinct organs in a nonrandom manner. To investigate cellular characteristics that may be important in the process of organ-specific metastasis, we selected 2 different types of HNC: SCC, characterized by frequent presence of lymph node metastases, and ACC, a rare cancer type characterized by hematogenous dissemination. These 2 tumor entities serve as a model to study the organ-specificity of the metastatic process.

The formation of metastases is thought to be the result of several sequential steps that share many similarities with leukocyte trafficking, a process critically regulated by chemokines and their receptors.31 In this study, we provide evidence that SCC and ACC cells display a distinct chemokine receptor expression profile. SCC cells express a broad variety of chemokine receptors. The majority 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 cells themselves,33 may promote the proliferation and invasion of SCC cells expressing CXCR1 and CXCR2. In SCC cell lines, we consistently observed upregulation of CCR7 and CXCR5 mRNA, while mRNA for these receptors was absent or expressed at a low level in normal mucosal keratinocytes and ACC cells. Interestingly, the corresponding ligands of these receptors (CCL19, CCL21 and CXCL13, respectively) are homeostatically expressed in lymph nodes.34, 35, 36 CCR7 expression is critical for the migration of naïve lymphocytes and mature dendritic cells to draining lymph nodes.31 Furthermore, CCR7 expression has been associated with lymph node metastasis in several cancer types, such as melanoma, lung cancer, gastric cancer and esophageal cancer.21, 22, 23, 24, 25

In our panel of SCC cell lines, CCR7 protein was only detectable intracellularly, whereas in tissues, surface expression was evident in primary tumors examined by immunohistochemistry, suggesting that the transport of CCR7 protein to the cell surface may require microenvironmental factors present only in vivo. Indeed, our previous observations show that IL-1β, TNF-α or EGF induce relevant chemokine receptors on the surface of melanoma cells (unpublished data). However, no surface CCR7 expression was detected in SCC cells either after serum starvation, or after treatment with inflammatory cytokines or growth factors, suggesting that either a combination of factors or yet unidentified compounds are required for the recruitment of CCR7 to the surface of SCC cells. The majority of primary tumors and lymph node metastases of SCC expressed CCR7. Thus, CCR7 might play a role in directing SCC cells to the draining lymph nodes and contribute to the frequent presence of lymph node metastases in SCC patients, as also suggested by Wang et al.37 It has been proposed that SCC cells of the head and neck region mimick the differentiation and migration pattern of dendritic cells, displaying a CCR6+CCR7 phenotype within the skin and a CCR6CCR7+ phenotype on their way to local draining lymph nodes.37 However, in our study, no differences in CCR7 expression could be observed neither in matched primary SCC cell lines at the mRNA level, nor in tissue sections of primary SCC tumors and matching lymph node metastases at the protein level. Our results are in accordance with recent results of Ding and coworkers,22 demonstrating CCR7 expression in both primary tumors and lymph node metastases of esophageal SCC. These observations are in line with recent studies, demonstrating that primary tumors are similar to the corresponding metastatic tumors in their gene expression signature.38, 39 Thus, regulation of chemokine receptors may be a relatively early event during tumorigenesis.

CXCR5 participates in the trafficking of B and T cells to secondary lymphoid organs.34, 35 Recently, it has been suggested that, in leukemic B cell lymphomas, CXCR5 may be associated with the spread of malignant B cells to lymphoid tissues.40, 41 Here, we show the abundant expression of CXCR5 transcripts and the presence of surface CXCR5 on SCC cells. Our observation is consistent with the concept that metastasis and leukocyte trafficking share underlying mechanisms and suggests that CXCL13/CXCR5 interactions may provide a novel complementary pathway, mediating lymphogenic spread.

The only chemokine receptor found to be highly expressed in ACC but not in SCC was CXCR4. In ACC cells, CXCR4 signaling resulted in the induction of directional tumor cell migration, supporting its role in tumor invasion and metastasis. Recent studies uncovered the fundamental role of CXCL12/CXCR4 interactions in physiological processes, such as organogenesis, hematopoiesis and homing of progenitor cells to liver and bone marrow.42 In addition to its complex physiological functions, CXCR4 has been shown to be pivotal during the metastatic spread of tumor cells to distant organs.8, 14, 43, 44, 45 ACC most frequently metastasizes to the lung and liver,4, 6 and CXCL12, the only known CXCR4 ligand, is abundantly expressed in these organs.8 The abundant expression of CXCR4 in the predominantly hematogenously metastasizing ACC suggests that CXCR4 may play a role in directing ACC cells to their metastatic sites. In contrast to the frequent hematogenous dissemination, lymphogenous metastases in ACC are extremely rare,4, 46, 47 despite the fact that the lymph nodes are also an abundant source of CXCL12. Although several studies have suggested a role for CXCR4 in lymph node metastasis,48, 49, 50 accumulating evidence suggests that CXCR4 is not a major participant in lymphatic tumor spread, including large-scale gene expression data in metastatic head and neck SCC.51 In lymph nodes, CXCL12 is expressed by stromal cells in close vicinity to high endothelial venules52 (unpublished observations) and contributes to the homing of memory T cells to lymph nodes via the bloodstream but not the lymphatics.53 Hence, the microanatomical distribution of CXCL12 in lymph nodes may partially explain why lymphogenous metastasis is not observed in ACC with abundant CXCR4 expression. Moreover, extravasation requires a complex interplay of chemokine receptors, adhesion molecules and other factors, and ACC cells may be deficient in some factors that are essential for extravasation or other steps in the process of lymphogenous metastasis.

In addition to ACC, also a subset of SCC tumors demonstrated CXCR4 expression in vivo, which is in accordance with recent studies.48, 54, 55 Notably, 2 of the CXCR4-expressing SCC tumors showed enhanced CXCR4 expression at the leading edge of the tumor, suggesting a possible role for this receptor in primary tumor invasion, as it also has been suggested in prostate and ovarian cancer.56, 57

The induction of apoptosis in tumor cells is a major goal of cancer chemotherapy. Cisplatin, a chemotherapeutic agent used in the therapy of HNC, exerts its cytotoxic effect by forming DNA adducts, which in turn activate a complex network of pathways that finally culminate in apoptosis.58 However, following cisplatin-exposure, prosurvival pathways are also activated.58, 59 The fate of a cell is determined by the balance between proapoptotic and prosurvival pathways. In the present study, we demonstrate that sublethal doses of cisplatin induced CXCR4 on the surface of malignant cells. In the presence of the transcriptional inhibitor α-amanitin, the upregulation of CXCR4 by cisplatin was markedly repressed, suggesting that gene transcription is required for this induction. Moreover, we show that in ACC cells CXCL12 stimulation resulted in the activation of Akt and ERK1/2, and MAP kinases are involved in signal transduction pathways that are generally associated with cell survival and proliferation.29, 30 Our findings are supported by recent observations, showing that CXCL12 can induce survival and proliferation signals in several cell types, such as CD4+ T cells,60 embryonic neural cells,61 as well as cancer cells, including breast cancer, pancreatic cancer and glioblastoma.45, 62, 63, 64 In addition to the activation of survival pathways, we also provide evidence that CXCL12 reduces the rate of apoptosis induced by cisplatin in ACC cells. We propose that suppression of apoptosis via CXCR4 signaling may lead to increased tumor cell viability and might contribute to cisplatin resistance and the failure of antineoplastic treatment of metastatic ACC.

In the present study, we have shown that differences in the metastastic patterns of 2 histologically distinct tumors of the head and neck, ACC and SCC, are associated with a different repertoire of chemokine receptors. The distinct chemokine receptor profile of ACC is also associated with signaling pathways involved in cell survival and proliferation.

Currently, intense efforts are underway to identify small molecule antagonists for chemokine receptors that could be useful for treating disseminated cancer. It appears that the efficacy of conventional chemotherapeutic treatment might be enhanced by combination with chemokine receptor neutralization to repress prosurvival pathways and enhance tumor cell apoptosis. These and other strategies, based on emerging mechanistic data, represent some of the promising new directions in the therapy of metastatic disease.

Ancillary