Chemokine Receptor Expression in Oral Squamous Cell Carcinoma: Correlation with Growth Factor- and Cytokine- Mediated Cell Migration in Vitro

Chemokine Receptor Expression in Oral Squamous Cell Carcinoma: Correlation with Growth Factor- and Cytokine- mediated Cell Migration in vitro

Katsumi Muraoka1, Kazuhiko Okumura2, Hiroyuki Kitajo3, Hidetaka Kato1 and Makoto Arisue1

1Division of Oral and Maxillofacial Surgery, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido (Chief: Professor Makoto Arisue) 2Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido (Chief: Professor Takanori Shibata) 3Institutu of personalized medical Science Health Sciences University of Hokkaido (Chief: Professor Tomofumi Kawakami)

Abstract: Metastasis is the chief cause of mortality in cancer patients. Recently, chemokines and chemokine receptors were shown to play an important role in the metastasis of various cancers. We examined the role of chemokine receptor-mediated signaling in the invasion potential of human oral squamous cell carcinoma (OSCC) cell lines that were derived from 5 primary tumors and 6 cervical lymph node metastases. Compre- hensive analysis of the mRNAs for human chemokine receptors showed that the OSCC cell lines had uniform expression patterns of chemokine receptors. Overall, there were no consistent differences in the expression of chemokine receptors between primary site- and lymph node metastasis-derived cell lines. However, a highly invasive OSCC cell line (SAS-H1) expressed up-regulation of CCR5, CCR6, CCR7, CXCR1, CXCR6 and CX3CR1 compared to a poorly invasive OSCC cell line (SAS-L1). Then we examined whether factors in the tumor microenvironment regulated chemokine receptor expression in SAS-H1 cells. Speciﬁcally, transforming growth factor (TGF) -β1 enhanced the expression of CCR5, CCR6, CCR7 and CX3CR1. Pretreatment of SAS-H1 cells with transforming growth factor (TGF) -β1 increased the expression of CCR7 and CX3CR1, and then enhanced CCL21- and CX3CL1-induced directional migration (1.5-fold enhancement as compared with untreated control). In addition, CX3CL1 increased the adhesion of SAS-H1 cells on uncoated tissue culture plates. Neither chemokine stimulated cell proliferation. Treatment of SAS-H1 cells with CX3CL1 activated the phosphotidylinositol-3-kinase (PI3K) and MEK signal transduction pathways. Our results suggest that chemokine receptor-mediated signaling is involved in the local invasion and metastasis of human OSCC.

Key words: Chemokine receptors, TGF-β1, TNF-α, INF-γ, IL-1β

Abbreviations

TGF-β1: transforming growth factor-β1 TNF-α: tumor necrosis factor-α INF-γ: interferon-γ IL-1β: interleukine-1β PI3K: phosphatidylinositol 3 kinase MEK: mitogen-activated protein kinase/extracellular signal-regulated kinase

Received 6/13/07; revised 8/15/07; accepted 8/21/07. Grant support: This work is supported in part by the grant from Scientiﬁc Research on Priority Areas (C)(2), #17592098 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Requests for reprints: Dr. Kazuhiko Okumura, Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Hu- man Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa Ishikari-Tobetsu Hokkaido, 061–0293, Japan, Phone: +81–133–23–1429, Fax: +81–133–23–1429, E-mail: [email protected] 74 Oral Science International Vol. 4, No. 2

Introduction OSCC cells via the activation of PI3K and MEK-mediat- Oral cancer is the most common malignant tumor of ed signaling pathways. the head and neck. Squamous cell carcinoma accounts for more than 90% of cancers of the oral cavity1. Pa- Materials and Methods tients diagnosed with early stage OSCC have a better prognosis, but failure of treatment of neck cancers is a 1. Cells and culture significant problem, and metastasis to the cervical Human OSCC cell lines were derived from primary lymph nodes occurs more frequently in OSCC2. tumors (SAS, SAS-H1, SAS-L1, IT-GA, SCC9) or from The development of metastatic tumors is accompa- lymph node metastases (HSC-2, HSC-3, HSC-4, IS- nied by loss of attachment and subsequent emigration FOM, OSC-19, OSC-20), as previously described6–10. In of cancer cells to tissues via lymphatic and/or blood ves- particular, highly invasive clone SAS-H1 cells and poor- sels. The chemokine superfamily consists of small, se- ly invasive clone, SAS-L1 cells were derived from hu- creted proteins (8-12 kDa) that are involved in lympho- man tongue poorly differentiated squamous cell carcino- cyte migration, and trafficking and homing throughout ma, and SAS cells were picked out by rat lung the lymphatic system. Chemokines are classified into endothelial cell monolayer assay7. Cells were cultured four highly conserved groups: CXC, CC, C and CX3C, in Dulbecco's Modified Eagle's Medium / Ham F-12 me- based on the relative positions of the first two cysteine dium (DMEM/F-12: Sigma), supplemented with 10% residues in the amino terminus of the protein. These heat-inactive fetal bovine serum (FBS), 100 unit/ml chemokine molecules constitute a superfamily of induc- penicillin G, and 100 μg/ml streptomycin (Gibco BRL). ible, secreted, proinflammatory proteins that are in- The immortalized human keratinocyte cell line HaCaT11 volved in a variety of immune responses, and act pri- as normal keratinocytes and the human monocytic marily as chemoattractants and activators of specific U937 cells as positive control of the expression of che- types of leukocytes. The specific effects of chemokines mokine receptors were cultured in DMEM/F-12 and Ro- on the target cell types are mediated by a family of sin- swell Park Memorial Institute 1640 medium (RPMI gle-chain, seven-transmembrane-spanning, G-protein- 1640: Sigma), respectively, supplemented as described coupled receptors. To date, 18 human chemokine recep- for OSCC cell lines. tors have been identified, which are part of a much bigger superfamily of G-protein-coupled receptors3. Im- 2. Cell Stimulation portant roles of chemokines and their receptors have Cells were either unstimulated, or stimulated with been demonstrated in inflammation and infection, as various concentrations (1-20 ng/ml) of growth factors well as in malignant tumors4. Recent studies have (TGF-β1, EGF, and HGF) or proinflammatory cytokines demonstrated that tumor cells express functionally ac- (IL-1β, TNF-α, and IFN-γ) for 24 hours (h). All growth tive chemokine receptors and expression of these recep- factors and cytokines were purchased from R&D Sys- tors appears to regulate cellular functions associated tems. Prior to stimulation, cells were washed twice with the process of metastasis5. with cold phosphate buffered saline (PBS). In the current study, we examined the expression of chemokine receptors in human OSCC cells derived from 3. Isolation of RNA and Reverse Transcription primary tumors and lymph node metastases. The re- PCR (RT-PCR) Analysis ceptors CCR3, CCR4, CCR7, CXCR2, CXCR6 and Total RNA was isolated from cells using the RNeasy CXCR1 were abundantly expressed in cells derived from Mini kit (Qiagen Inc.), according to the manufacturer's both primary tumors and lymph node metastases. In instructions. Reverse transcription was performed us- the highly invasive SAS-H1 cell line, TGF-β1 enhanced ing Superscript II (Invitrogen Corp.), and Oligo (dT)12–18 the expression of several chemokine receptors, as well primers (Gibco BRL). The products of reverse tran- as the ligand-dependent migratory response. We also scription were amplified using a Taq PCR Master Mix demonstrated that the interaction of CX3CR1 with its kit (Qiagen Inc.) and a thermocycler (GeneAmp PCR cognate ligand, CX3CL1/Fractalkine, may play a role in System 2400, PE Applied Biosystems). Primers for the the local invasion and lymph node metastasis of human human chemokine receptors were designed using prim- November, 2007 Chemokine Receptor Expression in Oral Squamous Cell Carcinoma 75 er selection (Web-based Sequence Analysis, SeqWeb 16 h (migration assay) or 48 h (invasion assay) at 37℃. Version 2.02, Accelrys Inc.), based on sequence data Cells on the lower surface of the membrane were from the National Center for Biotechnology Information stained, and counted under a light microscope (a total of databases. The sequences of the primers and annealing five different fields were counted at 200 × magnifica- temperatures for each chemokine receptor and β-actin tion). Where indicated, cells were preincubated with the (as a control) are presented in Table 1. Amplification MEK inhibitor PD98059, or the PI3K inhibitor LY294002 was carried out using 35 PCR cycles consisting of dena- for 6 h before being added to the upper chamber. turation for 30 seconds (sec) at 95℃, annealing for 30 sec at 55-63℃ (depending on the primer set, Table 1) 6. Matrigel invasion assay and extension for 2 minutes (min) at 72℃. Amplified As described above, the invasion assay upper chamber products were separated on 1.5% agarose gels and with an 8-μm porosity cell-permeable polycarbonate fil- stained with ethidium bromide. ter covered with Matrigel was placed on a cell culture Densitometry was performed using Image J analysis plate. Results were evaluated as described for cell mi- software (version 1.37, NIH). The mRNA levels of the gration. chemokine receptors were normalized to β-actin, and are presented as a fold-induction. 7. Cell adhesion assay Six-well tissue culture plates were either uncoated, or 4. Measurement of invasiveness precoated with 300 μg/ml Matrigel overnight at 4℃. The measurement of invasiveness in various human The plates were washed with PBS and then incubated OSCC cell lines was performed in 24-well cell culture in 0.5% bovine serum albumin (BSA) in DMEM/F-12 for chambers containing polycarbonate filter inserts (8-μm 1 h at 37℃. The plates were again rinsed with PBS and pore size; 353097, Cell Culture Insert, Falcon) and a re- dried. Cells were seeded (1 × 104/well) in 2 ml serum- constituted extracellular matrix membrane (Matrigel; free medium, and allowed to attach for 3 h at 37℃, in Becton Dickinson), as previously described12. Cell inva- the presence of varying concentrations of CCL21 or sion chambers were prepared by carefully placing CX3CL1. Nonadherent cells were removed by gentle Matrigel (30 μg/filter) onto the polycarbonate filter, fol- washing with PBS, then adherent cells were harvested lowed by incubation at room temperature overnight. by trypsinization and counted by light microscopy using Cells were resuspended in the appropriate medium sup- a hemocytometer. plemented with 1% FBS (2 × 104/100 μl), and seeded in the upper chamber. Conditioned medium, obtained by 8. Proliferation assay incubating NIH3T3 fibroblasts for 24 h in serum-free To evaluate cell proliferation, cells (5 × 104 cells) medium, was placed in the lower chamber as chemoat- were seeded on 35 mm plastic tissue culture dishes and tractant. The chambers were incubated for 48 h at allowed to adhere overnight. Cells were then cultured 37℃. Cells on the lower surface of the membrane were for 24 h before replacing the complete media with stained, and counted under a light microscope (a total of DMEM/F-12 supplemented with 1% BSA with different five different fields were counted at 200 × magnifica- concentrations of CCL21 or CX3CL1. At the indicated tion). times, the cells were subjected to trypsinization, and the number of cells in each dish was counted by trypan 5. Migration assay blue dye-exclusion using a light microscope and a hemo- Cell migration was evaluated using 24-well cell cul- cytometer. ture chambers containing polycarbonate filter inserts (8-μm pore size; 353097, Cell Culture Insert, Falcon). 9. Statistical analysis Cells were resuspended in the appropriate medium sup- Statistical data represents the mean ± standard de- plemented with 1% FBS (2 × 104/100 μl), and seeded in viation (SD) of three experiments performed in tripli- the upper chamber. Recombinant CCL21 and CX3CL1 cate. Statistical significance was determined using the (R&D Systems) were used as chemoattractants in the unpaired Student's t-test. Values of P ＜ 0.05 were con- lower compartment. The chambers were incubated for sidered significant. 76 Oral Science International Vol. 4, No. 2

Table 1 Primer sequences for RT-PCR analysis of chemokine receptors, with corresponding Genebank accession numbers and predicted sizes of the ampliﬁed products.

Genebank Name Sequence product size (bp) Tm (℃) accession No. Forward 5'-CTCTTCCTGTTCACGCTTC-3' NM001295 CCR1 595 63 Reverse 5'-CCAAATGTCTGCTCTGCTC-3' Forward 5'-GCTGTGTTTGCTTCTGTCC-3' BC095540 CCR2 530 60 Reverse 5'-GTCACTCCATCCACTGTCTC-3' Forward 5'-ACCAACATCTACCTGCTCAAC-3' BC130320 CCR3 624 61 Reverse 5'-GCTCCGCTCACAGTCATTTC-3' Forward 5'-GCACCAAAGAAGGCATCAAG-3' NM005508 CCR4 676 60 Reverse 5'-AACCCAAGGAAGAGGACCAC-3' Forward 5'-ACTGACATCTACCTGCTCAAC-3' NM00579 CCR5 725 59 Reverse 5'-TTTCTGAACTTCTCCCCGAC-3' Forward 5'-CTATGCCATCAACTTTAACTGC-3' BC037960 CCR6 650 59 Reverse 5'-ACTTCCTTCTCACACACCAC-3' Forward 5'-TACATCGGAGACAACACCAC-3' NM001838 CCR7 732 59 Reverse 5'-GAAGCTATGAAGACCACGAC-3' Forward 5'-CTTTTTGTCTTCTCCTTCCCC-3' NM005201 CCR8 485 59 Reverse 5'-ACCACAATGAGCACCAACC-3' Forward 5'-AGCGATGAGAGCACCAAAC-3' BC069678 CCR9 479 60 Reverse 5'-TCCAGCAACATAGACGACAG-3' Forward 5'-TGCTGGATACTGCCGATCTACTG-3' NM016602 CCR10 312 61 Reverse 5'-TCTAGATTCGCAGCCCTAGTTGTC-3' Forward 5'-GCAGATGAAGATTACAGCCC-3' BC028221 CXCR1 678 60 Reverse 5'-GACGACAGCAAAGATGACC-3' Forward 5'-ATCTATGCCCTGGTATTCCTG-3' NM001557 CXCR2 277 61 Reverse 5'-GGTAACGGTCCACACTGATG-3' Forward 5'-CAACGCCACCCACTGCCAATACAA-3' NM001504 CXCR3 447 63 Reverse 5'-CAGGCGCAAGAGCAGCATCCACA-3' Forward 5'-CCCACCATCTACTCCATCATC-3' BC020968 CXCR4 719 59.5 Reverse 5'-TGCACAGTGTTCTCAAACTC-3' Forward 5'-AACTACCCGCTAACGCTGGAAATGGAC-3' BC110352 CXCR5 694 63 Reverse 5'-CACGGCAAAGGGCAAGATGAAGACC-3' Forward 5'-TCAATGACAGCAGCCAGGAG-3' NM006564 CXCR6 679 63 Reverse 5'-AGGAACACAGCCATCACCAG-3' Forward 5'-CAACAGCAAGAAGCCCAAG-3' NM001337 CX3CR1 548 60 Reverse 5'-ACACGATGACCACCAGAAG-3' Forward 5'-GCACCACCTTTTTTTACTATGACC-3' BC095533 XCR1 328 62 Reverse 5'-AAGATGCTGCTGTAGAGGCTG-3' Forward 5'-AGGCCAACCGCGAGAAGATGACC-3' AB004047 β-actin 343 58 Reverse 5'-GAAGTCCAGGGCGACGTAGCAC-3'

Results tors in a panel of human OSCC cell lines derived from either primary tumors (n ＝ 5) or cervical lymph node 1. Chemokine receptor mRNA proﬁles in human metastases (n ＝ 6). Receptor mRNA was measured by OSCC cells semi-quantitative RT-PCR. The mRNA expression pat- We analyzed the expression of 18 chemokine recep- terns of chemokine receptors in human OSCC cells, Ha- November, 2007 Chemokine Receptor Expression in Oral Squamous Cell Carcinoma 77

Fig. 1 Chemokine receptor mRNA proﬁles in human OSCC cells. Semi-quantitative RT-PCR analysis of 18 chemokine receptors in OSCC cell lines, HaCaT epithelial keratinocytes, and U937 monocytes.

CaT epithelial keratinocytes, and U937 monocytic cells chemokine receptors. CCR2, CCR9, CCR10, CXCR1 are illustrated in Fig. 1. HaCaT cells and U937 cells and CXCR3 were expressed in the majority of OSCC uniformly expressed chemokine receptors, but U937 cell lines and in HaCaT cells. CCR3, CCR4, CCR5, cells up-regulated receptor mRNA more than HaCaT CXCR2 and CXCR6 were consistently up-regulated in cells. Overall, human OSCC cells expressed a variety of almost all OSCC cells (1.4-3.5, 1.8-2.2, 1.3-5.3, 1.3-1.9 78 Oral Science International Vol. 4, No. 2

Fig. 2 Invasiveness of human OSCC cell lines. OSCC cells were plated onto Matrigel-coated ﬁlters, and NHI-3T3 ﬁbroblast-conditioned medium was placed into the lower chamber, as described in Materials and Methods.

and 1.3-2.7 fold increase, respectively) as compared 132.5 ± 18.4 and 138.2 ± 24.7 invaded cells/field, re- with HaCaT cells. In contrast, XCR1 was highly ex- spectively). The invasion potential of these OSCC cells pressed in only four OSCC cell lines (IT-GA: 2.4 fold, IS- correlated with the up-regulation of expression of a FOM: 2 fold, OSC-19: 1.3 fold and SCC9: 1.7 fold), than great variety of chemokine receptors (6-17 different re- the other OSCC cell lines. Furthermore, CXCR4 was ceptors). In contrast, four cell lines (SAS-L1, HSC-2, highly expressed in U937 cells (3 fold) and only five HSC-3 and HSC-4) exhibited low invasiveness (40.2 ± OSCC cells (IT-GA: 2 fold, IS-FOM: 2 fold, OSC-19: 1.9 13.9, 36.7 ± 10.8, 82 ± 19.2 and 85.3 ± 20.5 invaded fold, OSC-20: 1.6 fold and SCC9: 1.9 fold) as compared cells/field, respectively) that had a very restricted profile with HaCaT cells. Thus, no consistent differences in the of chemokine receptors, with mRNA expression for 1-9 expression of any of the chemokine receptors were ob- different receptors. These results indicated that highly served in OSCC cells derived from cervical lymph node invasive human OSCC cells express a wider array of metastases compared to those derived from primary tu- chemokine receptors than poorly invasive OSCC cells. mors. To further investigate the correlation of invasion activity and chemokine receptor expression, we compared 2. Human OSCC cells that expressed a variety of the constitutive expression of chemokine receptors in chemokine receptors exhibited high invasion poorly invasive (SAS-L1) and highly invasive (SAS-H1) activity cells using RT-PCR. In SAS-H1 cells, CCR5, CCR6, Next we measured the invasiveness of 11 human CCR7, CXCR1, CXCR6 and CX3CR1 were consistently OSCC cell lines through an 8-μm porosity cell-perme- up-regulated compared to SAS-L1 cells. These recep- able polycarbonate filter covered with Matrigel. As a tors were either undetectable or expressed at markedly chemoattractant, NIH3T3 fibroblast conditioned medi- lower levels in poorly invasive SAS-L1 cells (Fig. 3). um was used. As shown in Fig. 2, seven OSCC cell lines These results suggested that highly invasive OSCC cells (SAS, SAS-H1, IT-GA, IS-FOM, OSC-19, OSC-20 and express a higher level and a wider array of chemokine SCC9) exhibited a high invasiveness (135.2 ± 22.2, receptors than poorly invasive OSCC cells. 192.7 ± 35.6, 162.5 ± 28.8. 183.0 ± 32.2, 106 ± 20.2, November, 2007 Chemokine Receptor Expression in Oral Squamous Cell Carcinoma 79

Fig. 3 Correlation between invasion potential and chemokine receptor mRNA expression. RT-PCR analysis of highly invasive SAS-H1 and poorly invasive SAS-L1 cells. SAS-H1 cells exhibited up-regulation of various chemokine receptors compared to SAS-L1 cells. 100bp, molecular marker. The results of RT-PCR for β-actin demonstrate the loading of equal amounts of DNA on the gel.

3. Growth factors or proinflammatory cytokines regulated CXCR1 mRNA expression (data not shown). modified chemokine receptor expression in These results indicated that factors in the tumor micro- highly invasive SAS-H1 cells environment regulate the expression of chemokine re- To assess whether factors in the tumor microenviron- ceptors in highly invasive OSCC cells. ment regulated chemokine receptor expression, we analyzed receptor expression in SAS-H1 cells treated with 4. TGF-β1 up-regulates CCL21-stimulated migra- growth factors or pro-inflammatory cytokines using tion of OSCC cells, but not growth semi-quantitative RT-PCR. TGF-β1 up-regulated the The cognate ligand of CCR7, which was highly ex- expression of CCR5, CCR6, CCR7 and CX3CR1 in a pressed in SAS-H1 cells, is CCL21. The previous result dose-dependent manner. In contrast, epidermal growth showed that treatment of SAS-H1 cells with TGF-β1 in- factor (EGF) up-regulated the expression of CXCR6 creased the expression of CCR7 compared to untreated mRNA only (Fig. 4). Tumor necrosis factor (TNF)-α in- SAS-H1 cells in a dose-dependent manner. We next ex- creased the expression of both CXCR6 and CX3CR1. amined the effect of TGF-β1 on CCL21-mediated Interferon (IFN)-γ was the only pro-inflammatory cyto- SAS-H1 cell invasion. TGF-β1 was added to SAS-H1 kine that induced the up-regulation of CCR6 mRNA. cells for 24 h prior to treatment with CCL21, and then Interleukin (IL)-1β up-regulated CX3CR1 mRNA only removed 16 h post-stimulation with CCL21. The inva- (Fig. 5). Hepatocyte growth factor (HGF) had no effect sion activity of SAS-H1 cells increased in response to on chemokine receptor expression in SAS-H1 cells (data CCL21 in a dose-dependent manner, and TGF-β1 en- not shown). Furthermore, none of the growth factors or hanced CCL21-stimulated invasion (Fig. 6A). Further- proinflammatory cytokines examined in this study up- more, CCL21 also induced the migration of SAS-H1 80 Oral Science International Vol. 4, No. 2

Fig. 4 Effect of growth factor stimulation on chemokine receptor mRNA expression in highly invasive SAS-H1 cells. SAS-H1 cells were incubat- Fig. 5 Effect of proinﬂammatory cytokine stimulation ed with or without the indicated growth factors on chemokine receptor mRNA expression in at various concentrations. After 24 h, chemo- highly invasive SAS-H1 cells. SAS-H1 cells were kine receptor and β-actin mRNA levels were incubated with or without the indicated proin- analyzed. Chemokine receptor mRNA levels ﬂammatory cytokines at various concentrations. were normalized to those of β-actin and are After 24 h, chemokine receptor and β-actin shown as fold-activation. mRNA levels were analyzed. Chemokine receptor mRNA levels were normalized to those of β-actin and are shown as fold-activation. cells in a dose-dependent manner, and TGF-β1 enhanced CCL21-stimulated migration (Fig. 6B). Howev- CX3CL1 on SAS-H1 cell invasion, and found that inva- er, CCL21 did not increase the adhesion of SAS-H1 cells sion and cell migration were induced by CX3CL1 in a on Matrigel-coated plates (Fig. 6C), or cell proliferation dose-dependent manner (Fig. 7A and 7B). CX3CL1 also (Fig. 6D). These results suggested that CCL21 is a induced the adhesion of SAS-H1 cells to uncoated cul- functionally active ligand of CCR7, and induces OSCC ture dishes (Fig. 7C), but had no effect on cell prolifera- cell invasion. Furthermore, TGF-β1 enhanced the tion (Fig. 7D). To determine the potential role of PI3K CCL21-stimulated invasion and migration of OSCC and/or MEK-mediated signaling pathways in the effects cells. of CX3CL1 on SAS-H1 cells, we examined CX3CL1-induced migration in the presence of the PI3K inhibitors 5. CX3CL1 induced adhesion and migration are (LY294002) or MEK inhibitor (PD98059). Both inhibi- modulated by PI3K and MEK tors inhibited the migration of SAS-H1 cells in a dose- CX3CL1, also known as Fractalkine, is a membrane- dependent manner compared to untreated controls, bound chemokine that functions as a chemoattractant which indicated that CX3CL1-induced migration is me- and an adhesion molecule. We examined the effect of diated by PI3K and MEK (Fig. 7E and 7F, respectively). November, 2007 Chemokine Receptor Expression in Oral Squamous Cell Carcinoma 81

At all concentrations tested (0-50 μM), LY294002 or PD98059 alone did not produce cytotoxic effects.

Discussion Tumor cell migration is an important factor in the in- tricate processes of local invasion and metastasis. It has been shown that highly invasive tumor cells have a higher migratory response than poorly invasive tumor cells. However, the molecular basis of local invasion and metastasis is not fully understood. Recent evidence points to similarities in the formation of metastases and leukocyte trafﬁcking, a process that is tightly regulated by chemokines and chemokine receptors5,13. In the current study, we carried out a comprehensive in vitro analysis of the expression of 18 chemokine receptors in a panel of human OSCC-derived cell lines. OSCC cells expressed an array of chemokine receptors. The majority of OSCC cell lines, similar to HaCaT cells, expressed all 18 chemokine receptors, with the exception of CXCR5. Interestingly, the majority of OSCC cell lines exhibited an up-regulation of CCR3, CCR4, CCR7, CXCR2, CXCR6 and CX3CR1 compared to HaCaT cells. CCR3 and CCR4 are expressed in adult T-cell leukemia, and CCR3 has been implicated in the recruitment of tumor cells in the skin14. The ligand for CCR4 has been found on the cutaneous endothelium15. Thus, these receptors may play a role in promoting local invasion. CCR7 is involved in the migration of T and B lymphocytes and dendritic cells to draining lymph nodes4, and has been associated with lymph node metastasis in breast cancer, gastric cancer, nasopharyngeal carcinoma, esophageal carcinoma and head and neck carcino- ma16–20. CCR7 expression correlates with metastatic potential and poor prognosis17,21–23 and its cognate ligand, Fig. 6 The effect of TGF-β1 on CCL21-induced invasion, CCL21, is present at high levels in the draining lymph migration and adhesion in SAS-H1 cells. (A) Cells nodes of many cancers17,23. CXCR2 and its cognate li- were stimulated with or without TGF-β1 (10 ng/ ml) for 24 h, then subjected to a Matrigel invasion gand CXCL8 (IL-8) have been linked to cell proliferation assay in the presence of the indicated concentra- of colon carcinoma24. CXCL8 is produced by OSCC tions of CCL21, as described in Materials and 25 Methods. (B) Migratory response of SAS-H1 cells cells , and may promote the proliferation of OSCC cells following stimulation with the indicated concen- that express CXCR2. The cognate ligand of CXCR6, trations of CCL21. (C) Cells were treated with or CXCL16, is expressed in lung, liver and lymph nodes, without 100 ng/ml of CCL21 and adhesion was monitored on plates coated with 300 μg/ml of which suggests that CXCR6 expression in tumor cells Matrigel. (D) Cell proliferation of SAS-H1 cells af- facilitates lung, liver and lymph node metastasis26–28. ter stimulation with CCL21. SAS-H1 cells were grown in serum-free medium with different con- CX3CR1 has been implicated in the development of centrations of CCL21. The number of cells was skeletal metastases in prostate cancer29. The CXCR1 li- counted after 5 and 7 days of culture. Data repre- gand CX3CL1, or Fractalkine, is markedly induced in sents the mean ± S.D. of three independent experiments performed in triplicate. ＊P ＜ 0.05 endothelial cells by proinﬂammatory cytokines, such 82 Oral Science International Vol. 4, No. 2

Fig. 7 Effect of TGF-β1 on CX3CL1-induced invasion, migration and adhesion in SAS-H1 cells. (A) Cells were stimulated with or without TGF-β1 (10 ng/ml) for 24 h, then subjected to a Matrigel invasion assay in the presence of the indicated concentrations of CX3CL1, as described in Materials and Methods. (B) The migratory response of SAS-H1 cells was analyzed after stimulation with the indicated concentrations of CX3CL1. (C) Cells were treated with or without 100 ng/ml of CX3CL1, and adhesion was monitored on uncoated plates. (D) Cell proliferation of SAS-H1 cells after stimulation with CX3CL1. SAS-H1 cells were grown in serum-free medium with different concentrations of CX3CL1. The number of cells was counted after 5 and 7 days of culture. Inhibition of the PI3K (E) and MEK (F) pathways suppresses the cell migration to CX3CL1 in SAS-H1 cells. Cells were pretreated with the indicated concentrations of LY29002 or PD98059 for 12 h, stimulated with CX3CL1 (10 ng/ml), and subjected to a migration assay, as described in Materials and Methods. Data represents the mean ± S.D. of three independent experiments performed in triplicate. ＊P ＜ 0.05 November, 2007 Chemokine Receptor Expression in Oral Squamous Cell Carcinoma 83 as TNF-α, IL-1 and IFN-γ30. In the current study, amined whether factors in the tumor microenvironment CX3CR1-expressing OSCC cells migrated towards regulated chemokine receptor expression in SAS-H1 CX3CL1. Thus, CX3CR1/Fractalkine production by in- cells. Both growth factors and proinflammatory cyto- flamed endothelial cells may promote the local invasion kines induced the up-regulation of chemokine receptors of OSCC cells. On the other hand, we observed that in a dose-dependent manner. TGF-β1, in particular, CXCR4 and XCR1 expression was increased in highly- enhanced the expression of CCR5, CCR6, CCR7 and invasive OSCC cells. CXCR4 expression is low or ab- CX3CR1 in SAS-H1 cells. TGF-β1 is a multifunctional sent in normal epithelial cells16,31. Therefore, CXCR4 growth factor that elicits numerous cellular effects of expression is generally a characteristic of carcinoma the metastatic process35. Our previous study suggested cells5. It has been reported that CXCR4 is up-regulation that TGF-β1 might enhance the invasiveness of OSCC in lymph node metastatic cells, in comparison with non- cells36. The stimulation of invasion by TGF-β1 is di- metastatic OSCC and normal epithelial cells31. Howev- rectly associated with its ability to enhance the expres- er, we presented that CXCR4 expression was up-regu- sion of matrix metalloproteinases37. Therefore, we sug- lated in both primary OSCC cells and lymph node gest that TGF-β1 stimulates directly invasion and the metastatic OSCC cells. Furthermore, XCR1 is the "C" expression of chemokine receptors-mediated invasion. sub-family of chemokine receptors and contains only Additionally, IFN-γ and TNF-α increased the expres- one member32. The cognate ligand of XCR1, lymphotac- sion of CCR6 and CXCR6, respectively, in a dose-depen- tin, has been reported to induce migration of T, B lym- dent manner, and CX3CR1 was up-regulated by IL-1β. phocytes, NK cells or neutrophils, and XCR1 expresses CX3CR1 was also up-regulated by TGF-β1 and TNF-α. in these cells. XCR1/lymphotactin regulates T and B In addition, IFN-γ did not induce CX3CR1 mRNA ex- lymphocytes and neutrophil trafficking, and thereby pression. These results indicate that factors in the tu- also their roles in inflammatory and immunological mor microenvironment regulate the expression of cer- responses33. Interestingly, only four highly-invasive tain chemokine receptors in highly invasive OSCC cells, OSCC cell lines had up-regulation of XCR1. However, it and that this may play a role in increased migration of is unknown whether XCR1/lymphotactin is implicated tumor cells into surrounding tissues. In support of this, in promoting the migratory phenotype of a variety of tu- we showed that treatment of SAS-H1 cells with TGF-β1 mor cells. Additional studies are needed to understand potentiated CCL21-and CX3CL1/Fractalkine-induced the relationship between invasion activity and chemo- cell migration. In addition, CX3CL1/Fractalkine not kine receptor expression in OSCC cells. In the current only induced migration, but also increased cell adhe- study, we demonstrated that CCR5, CCR6, CCR7, sion. CXCR1, CXCR6 and CX3CR1 were consistently up-reg- We demonstrated that PI3K and/or MEK, which are ulated in highly invasive SAS-H1 cells, but were unde- integral components of the intracellular signaling path- tectable or expressed at markedly lower levels in poorly ways that regulate cell survival and proliferation38,39, invasive SAS-L1 cells. These results suggest that high- are involved in CX3CL1-induced cell migration activity. ly invasive OSCC cells express a wider array and higher Our results are in agreement with a recent report that levels of chemokine receptors than poorly invasive CX3CL1 induces cell adhesion- and migration-associat- OSCC cells. ed signaling in prostate cancer cells29. Thus, our results In OSCC, there is often chronic inflammation at the indicate that the signaling events that are triggered fol- primary tumor site. In fact, many human cancers pres- lowing stimulation of the CX3CR1 receptor by its cog- ent with leukocyte infiltration into the tumor tissue. nate ligand involve PI3K and MEK. Infiltrated leukocytes are regulated by a complex che- In summary, we have shown that highly and poorly mokine network that influences not only the extent and invasive OSCC cells are associated with the expression phenotype of the infiltrate, but also tumor cell growth, of different repertoires of chemokine receptors. The dis- survival, and invasion, and angiogenesis. Leukocytes tinct chemokine receptor profile of highly invasive produce a variety of growth factors and proinflammato- OSCC cells correlated with the activation of chemokine/ ry cytokines that induce the expression of chemokine chemokine receptor signal pathways that may be in- receptors in tumor cells34. In the current study, we ex- volved in local invasion and lymph node metastasis. 84 Oral Science International Vol. 4, No. 2

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