Chemokine receptor 7 (CCR7) upregulation, which mediates immune cell survival and migration to lymph nodes, has recently been associated with nodal metastasis of squamous cell carcinoma of the head and neck (SCCHN). However, the mechanism of CCR7 in tumor progression, its downstream signaling mediators, and interactions with other pathways contributing to metastasis of SCCHN have not been determined. We hypothesized that inflammatory chemokine-mediated signals could also promote tumor proliferation and mitogenic effects. Functional assays showed that chemotaxis and invasion of metastatic SCCHN cells were dependent on phosphoinositide-3 kinase (PI3K) and its substrate, activated phospholipase Cγ-1. In addition, treatment of CCR7+ metastatic SCCHN cells with CCL19 (MIP-3β) showed rapid activation of the prosurvival, PI3K/Akt pathway. Transactivation of EGFR-mediated and mitogen-activated protein kinase signaling pathways, which can promote migration and survival in parallel, did not appear to contribute to the functional or biochemical effects of CCR7 stimulation. Thus, proinflammatory chemokine signals that mediate activation, trafficking and survival of tumor-infiltrating immune cells in the tumor microenvironment actually appear to induce signals for progression of cancer cells. The CCR7-mediated pathway in metastatic SCCHN cells functions independently of EGFR signal transduction and therefore may represent an additional target for therapeutic intervention to prevent tumor progression and metastasis.
Survival for patients with squamous cell carcinoma of the head and neck (SCCHN) is only 30–40%, mainly due to the frequent presence of metastasis at diagnosis (Greenlee et al., 2001). Thus, an urgent goal in head and neck oncology is to develop improved systemic therapeutic agents for clinical use. A better understanding of molecular pathways that mediate a unique pattern of tumor invasion and metastasis is necessary to enable the development of therapies designed to prevent tumor dissemination.
Chemokines are small, secreted molecules that signal through G-protein-linked receptors. Chemokines can be made and secreted by many different cell types, including tumor cells and tumor-infiltrating immune cells. Recently, metastatic SCCHN cells have been shown to express chemokine receptor 7 (CCR7), which may enable their access to the lymphatic system and facilitate spread to regional lymph nodes (Wang et al., 2004). Immune cells utilize CCR7 to transmit a prosurvival signal, mediated by the phosphoinositide-3 kinase (PI3K)/protein kinase B (PKB/Akt) cascade (Pilkington et al., 2004; Sánchez-Sánchez et al., 2004), which may be analogous in epithelial cancer cells. In order to determine the mechanism(s) by which chemokines promote tumor growth and spread, we have studied the downstream events mediating CCR7-induced migration, invasion, and survival in metastatic SCCHN cells.
In immune cells, CCR7 signal transduction is activated by proinflammatory cytokines (Mailliard et al., 2004), and appears to be mediated by PI3Ks, a family of lipid kinases that play a crucial role in cellular processes associated with malignant behavior, including cell growth, migration, and survival (Benistant et al., 2000; Mills et al., 2001; Luo et al., 2003). Once activated, Akt/PKB inhibits cell death pathways by directly phosphorylating and inactivating proapoptotic proteins, including Bad and procaspase 9 (Datta et al., 1999; Amornphimoltham et al., 2004; Wells and Lillien, 2004). Also, Akt/PKB has been implicated as a signaling intermediate upstream of survival genes, dependent on the transcription factor NF-κB, which induces cellular inhibitors of apoptosis (Beraud et al., 1999; Kane et al., 2001). Furthermore, activation of Akt/PKB has been shown to be important in progression of SCCHN (Tosi et al., 2005).
CCR signaling has been studied recently in melanoma (Murakami et al., 2003; Li et al., 2004), but similar data in epithelial cancer have not been widely reported. This is crucial since many epithelial cancers, such as SCCHN, are being treated clinically by inhibitors targeting the erb-B family of receptors. A recent publication indicated the importance of HER2/neu (erb-B2) and CXCR4 expression. Thus, signaling interactions between these different receptor families in SCCHN must be evaluated in vitro.
In SCCHN, the potential for cross-talk between CCR7 and EGFR (erb-B1)-mediated signaling pathways is predicated on the observations that other G-protein-coupled receptors (GPCRs), such as for bradykinin and prostaglandin E2, can promote SCCHN invasion and proliferation (Lango et al., 2002; Thomas et al., 2003), through activation of EGFR (Lui et al., 2003). Thus, determining CCR7-dependent interactions with other associated pathways is critical to developing the most effective therapeutic targets. Also, because small-molecule inhibitors, as monotherapy against EGFR-induced signals, have not provided sufficient activity in vivo, studies identifying and targeting novel EGFR-independent pathways are needed. Our study of three separate metastatic cell lines demonstrates intact CCR7-mediated survival and invasion under conditions completely blocking EGFR, which support the investigation of CCR7 pathway as a novel target for therapeutic intervention and blockade against SCCHN.
CCR7-mediated invasion of metastatic SCCHN cell lines is regulated by PI3K
Since our primary SCCHN cell lines (PCI-4A, -15A, -37A) express CCR6, while autologous metastatic tumor cell lines (PCI-4B, -15B, -37B) express CCR7 (Wang et al., 2004), we analysed their capability to migrate in vitro in response to the respective chemokine ligand. Transwell migration assays were performed after pretreatment with the PI3K-specific inhibitor (LY294002) at 10 μ M and 37°C for 4 h. These experiments showed that PI3K inhibition significantly blocked CCR7-mediated cell migration as compared with background control levels established with media alone or CCL20 (100 ng/ml), the ligand for CCR6. Chemotaxis assays in response to CCL19 were performed for PCI-4B, PCI-15B, and for PCI-37B, as shown in Figure 1a. SCCHN cell lines derived from primary tumors (PCI-4A, -15A, or -37A) showed no activation in response to CCR7 ligands (data not shown).
In addition to chemotactic ability, we evaluated the invasive capacity mediated by CCR7 in metastatic SCCHN cell lines, PCI-4B and -37B (Figure 1b). In vitro invasion through MATRIGEL was assessed after exposure of these cells to the CCR7 ligand, CCL19, in the presence or absence of LY294002, or pretreatment with a CCR7-specific blocking mAb. Figure 1b shows that treatment of cells with LY294002 (10 μ M, at 37°C for 4 h) significantly abolished this effect (P<0.01), indicating the dependence of invasion on PI3K activity.
CCR7-dependent phosphorylation of PLCγ-1 and Akt / PKB occurs in response to CCL19
We next studied CCL19-induced downstream signaling molecules activated by PI3K in different metastatic SCCHN cell lines PCI-4B (Figure 2a) and PCI-37B (Figure 2b) to identify the signal transduction pathways responsible for chemotaxis and invasion observed above. CCL19-induced CCR7-mediated signaling targets were determined by immunoblot in the presence or absence of PI3K inhibition, using LY294002. As shown, CCL19 induced phospho-PLCγ-1 and phospho-Akt/PKB activation to levels of 2–3-fold above the baseline, which was determined by incubation with media alone. Phosphorylation of these molecules was blocked selectively by the PI3K inhibitor, LY294002, linking their activation to the PI3K-dependent functional effects described above. These experiments were repeated at least three times with similar results.
Inhibition of PLCγ-1, but not Akt / PKB, activation blocks CCL19-induced invasion
To determine whether PLC blockade abrogated SCCHN cell invasion in vitro, we performed MATRIGEL invasion assays, using two SCCHN cell lines, derived from metastatic tumors. As shown in Figure 4, because U73122 inhibits all of the cellular PLCs, we used PLCγ-1 antisense oligonucleotides (Thomas et al., 2003) to test the consequence of specific abrogation of phospho-PLCγ-1 after CCR7 stimulation. The antisense oligonucleotide was directed against the ATG start site region. SCCHN cells (PCI-4B and -37B) were plated in invasion chambers at a high density and treated with CCL19 (100 ng/ml), CCL19+U73122 (3 μ M), or CCL19+antisense oligonucleotides against p-PLCγ-1 (12.5 μ M). Both the PLC inhibitor as well as PLCγ-1 antisense oligonucleotides abrogated in vitro invasion of a representative SCCHN cell line (Figure 3a). Immunoblotting in PCI-15B cells demonstrated that the PLCγ-1 antisense oligonucleotides decreased levels of phosphorylated PLCγ-1 by approximately 60% (Figure 3b). However, under conditions that block activation of Akt/PKB, we did not observe any reduction in CCL19-induced cell motility using a chemotaxis assay (not shown).
CCR7-mediated MATRIGEL invasion is EGFR-independent
Cross-talk with EGFR-mediated signaling could occur either through convergence of intracellular second messenger pathway(s) or by enhanced extracellular ligand secretion and subsequent receptor activation. To rule out either of these possibilities, we show that CCL19-induced, CCR7-mediated phosphorylation of PLCγ-1 in PCI-4B cells was blocked by the PI3K inhibitor (LY294002), but not by the EGFR inhibitor (AG1478) or by pre-incubation of cells with an extracellular EGFR-blocking mAb (C225). Note that blockade of phospho-EGFR levels was measured by either phospho-EGFR-specific mAb (Tyr 1173) or by sequential immunoprecipitation of total EGFR, then blotting for phospho-tyrosine-containing molecules (Figure 4a). Similar results were seen in experiments with two other metastatic SCCHN cell lines, PCI-37B (Figure 4b). In addition, to ensure that results were not confounded by growth-factor-containing media, these experiments were performed with serum-free conditions and growth factor starvation for 48–72 h.
As SCCHN cell invasion can be mediated by GPCRs via cross-talk with EGFR, which can also activate PLCγ-1 (Thomas et al., 2003), we performed in vitro MATRIGEL invasion assays, similar to those described above, in the presence or absence of the EGFR specific tyrosine kinase inhibitor, AG1478 (250 nM, pretreated at 37°C for 4 h). These experiments showed that, under serum-free, growth factor starvation conditions leading to >90% inhibition of EGFR activation in metastatic cells, PCI-4B, -15B, and -37B, little or none of this invasive effect was reduced (Figure 4c, P>0.05). EGFR-expressing, metastatic PCI-4B, -15B, and -37B cells retained significant capacity to invade through MATRIGEL, in response to CCL19 treatment.
CCR7-mediated signal transduction is independent of EGFR activation
Under these conditions (serum starvation for 48 h), immunoblot experiments showed that CCL19 did not induce additional phosphorylation of EGFR or its downstream target pMAPK (not shown). A second technique, immunoprecipitation of EGFR and subsequent blotting using anti-phospho-tyrosine Ab, also showed the same result (Figure 5a). Differences between intensity of EGFR bands using either technique are seen. This may reflect the relative lack of specificity associated with p-EGFR-specific mAb (Figure 5, lanes 1–4), whereas the sequential precipitation of EGFR followed by blotting for the p-Tyr-containing fraction (Figure 5a) may show a less prominent band at baseline. Further experiments in PCI-15B cells showed that neither anti-EGFR antibody nor EGFR inhibitor could block MIP-3β-induced phospho-Akt/PKB and phospho-PLCγ-1 activation (Figure 5b). The same results were seen in the other metastatic cell line, PCI-4B and -37B (data not shown). Thus, CCR7-mediated signal transduction identifies an EGFR-independent signaling pathway in these cells, leading to invasion and critical downstream mediators, phospho-Akt/PKB and phospho-PLCγ-1.
CCRs can couple distinct signaling pathways in lymphocytes and mediate migration, cell growth, and transcriptional activation (Ganju et al., 1998; Sotsios and Ward, 2000). One particular signaling pathway controlled by PI3K has been the focus of much attention in immune cells, with respect to its activation by CCRs and the role it plays in regulating cell migration (Vanhaesebroeck and Waterfield, 1999; Curnock and Ward, 2003; Uchida et al., 2003). Pharmacological and genetic studies have now convincingly shown that both CC and CXC chemokines stimulate PI3K-dependent chemotaxis of inflammatory cells (Curnock et al., 2002; Biswas and Sodhi, 2002). In this study, we present evidence that CCR7 mediates survival and invasion of metastatic SCCHN cells via activation of PI3K, indicating that this oncogene may also be critically involved in tumor progression and metastasis. Upon specific ligand stimulation with CCL19, CCR7 activated important downstream signaling mediators, Akt/PKB and PLCγ-1, into their active, phosphorylated forms. These findings indicate an important role for CCR7-mediated signaling in SCCHN invasion and metastasis, via PI3K-dependent activation.
Others have shown the antiapoptotic effects of CCL19-induced, CCR7-mediated signaling in mature dentritic cells (Sánchez-Sánchez et al., 2004). Thus, in addition to chemotaxis and invasion, CCR7 activation of Akt/PKB suggests a role for survival of metastatic tumor cells in vivo. Further studies in vitro and in animal models will be needed to clarify the potential clinical role for blockade of this effect in conjunction with interruption of EGFR signal transduction.
Several important pathways critical for cellular growth and survival were not activated by CCR7-mediated effects. The mitogen-activated protein kinase (MAPK/ERK) pathway has been reported to be important for proliferation and invasion of SCCHN cell lines mediated in part by EGFR activation (Thomas et al., 2003). Despite our clear evidence that CCR7-mediated signal transduction is PI3K-dependent, we observed no induction of the MAP/MEK/ERK pathway in response to MIP-3β (data not shown). We found that CCL19 enhanced phospho-PLCγ-1 and phospho-Akt/PKB generation and our preliminary experiments suggest that activation of NF-κB is involved (data not shown) (Li et al., 2000). These results indicate that the MAPK/ERK pathway is not involved in ligand-induced CCR7 signal transduction.
The correlation between EGFR-mediated PLCγ-1 signaling in SCCHN and its effects on cellular behavior has recently been reported (Thomas et al., 2003), raising the question as to the potential collaboration between these otherwise distinct signaling pathways. As a number of other GPCRs expressed by SCCHN cells appear to act in association with EGFR-dependent pathways (Ellis et al., 1999; Wang et al., 2002), often through trans-activation, we investigated whether these actions and CCR7-dependent signals are mediated through an EGFR-dependent mechanism. We found that CCR7 signal transduction is intact in the presence of nearly complete EGFR inhibition. Neither MATRIGEL invasion nor downstream CCR7 signal transduction appeared to be reduced in the presence of EGFR tyrosine kinase inhibitors or extracellular EGFR-blocking mAb. This finding indicates CCR7-mediated signals act through a separate, EGFR-independent pathway, which may represent a novel target for therapeutic intervention against SCCHN survival, invasion, and metastasis. Also, because the clinical results of EGFR inhibition as monotherapy have been disappointing, our data provide preliminary evidence for the potential therapeutic value of the combined CCR7 and EGFR pathway blockade.
While CCR7 ligands, CCL19 and SLC, are produced by a number of cellular sources, the precise intercellular mechanism mediating inflammatory signaling and CCR7 expression in the tumor microenvironment has not been identified. Inflammatory cytokines, hypoxia, and other stimuli may act proximally in the cascade to induce CCR7 expression in tumor cells and subsequent signal transduction, leading to invasion and metastasis. Further work will be necessary to understand the sequence of events leading to the CCR7-mediated metastatic phenotype, to enable the testing of therapeutic strategies aimed at blocking these carcinogenic and metastatic effects. In addition, our data support the preclinical evaluation of combination small-molecule inhibitors of both CCR7-mediated and EGFR-induced signaling pathways to enhance the anticancer effect in animal model systems.
Materials and methods
SCCHN cell lines PCI-4A/B, PCI-15A/B, and PCI-37A/B (A: derived from primary tumor, B, derived from lymph node metastasis from the same SCCHN patient) were characterized at the University of Pittsburgh (Heo et al., 1989). Cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA), which contained 8% (v/v) heat-inactivated fetal bovine serum (Equitech-Bio, Ingram, TX, USA), 100 U/ml penicillin G, and 100 μg/ml streptomycin (Invitrogen). These cells do not secrete EGF, but secretion of other EGFR ligands, TGF-α and amphiregulin, has been reported (Grandis et al., 1998).
Reagents and antibodies
The CCR7 chemokine ligand, CCL19 (MIP-3β), and the CCR6 ligand CCL20 (MIP-3α) were purchased from R&D Systems (Minneapolis, MN, USA). The PI3K inhibitor, LY294002, and EGFR inhibitor, AG1478, were purchased from Calbiochem (San Diego, CA, USA). The Akt/PKB inhibitor (124005) was purchased from EMD Biosciences. Antibodies used included anti-hCCR7 mAb (2H4) from BD Biosciences (San Jose, CA, USA), rabbit anti-phospholipase Cγ-1 (PLCγ-1), anti-phospho-PLCγ-1, anti-Akt/PKB and anti-phospho-Akt/PKB (Cell Signaling Technologies, Beverly, MA, USA), rabbit anti-ERK2 (C-14) and mouse anti-phospho-ERK (E-4) (Santa Cruz Biotechnology, CA, USA), and β-actin (Calbiochem). Anti-EGFR (sc-03), Anti-phospho-EGFR (Tyr 1173, sc-1173), and Anti-PY99 (sc-7020) were bought from Santa Cruz Biotechnology, CA, USA. U73122 (BioMol, Plymouth Meeting, PA, USA) was used to block PLC activity. Antisense PLCγ-1 oligonucleotides were obtained from Pharmacon, Inc. (Thomas et al., 2003). The Annexin-V apoptosis detection kit was used as per the manufacturer (BD Biosciences).
Cell migration studies were performed as described previously (Wang et al., 2004). Briefly, disposable 96-well chemotaxis chambers (ChemoTx Neuroprobe, Gaithersburg, MD, USA) with an 8 μm pore size and 6 mm width/well were run in triplicate, in AIM-V medium. Aliquots of the chemokine (29 μl) were added to the wells at a concentration of 500 ng/ml. The cell suspensions (5 × 104 cells/50 μl), which were pretreated with PI3K inhibitor, LY294002 (EMD Biosciences, San Diego, CA, USA), at 10 μ M and 37°C for 4 h were placed in the top chamber of the filter. Cells were then removed with a cell harvester. A 50 μl aliquot of Trypsin-EDTA was added for 3 min at 37°C, 5% CO2 before centrifuging at 1500 r.p.m. for 5 min. Cells in each lower well were counted under a light microscope in at least five different fields (original magnification, × 200). Mean±s.d. was recorded for each condition, and index calculated based on the control, random migration.
MATRIGEL invasion assay
Cell invasion was quantified in vitro using MATRIGEL-coated semipermeable, modified Boyden inserts with a pore size of 8 μm (Becton-Dickinson Biosciences Discovery Labware, Bedford, MA, USA). Briefly, 2.5 × 104 cells, which were pretreated with/without AG1478 (250 nM) or LY294002 (10 μ M) for 4 h, or anti-hCCR7 antibody (3 μg/ml) for 4 h and resuspended in 0.5 ml of serum-free AIM-V media, were seeded in triplicate in the inserts containing the MATRIGEL membrane (27.2 μg per chamber). CCL19 (0.5 ml at 500 ng/ml) in AIM-V was loaded into each lower chamber as a chemoattractant, for 36 h at 37°C in a 5% CO2 atmosphere. Cells were fixed and stained with Baxter Diff-Quik Stain Set (Dade Behring, Newark, DE, USA), counted under a microscope ( × 200) as a sum of five high-power fields distributed randomly on the central membrane, and reported as mean±s.d. Invasion was reported as a ratio of nonspecific cell invasion (from media pulsed wells).
Briefly, 70–80% confluent cells were serum-starved for 72 h. Cells were then treated with/without 10 μ M LY294002 (PI3K inhibitor) at 37°C for 4 h, or with/without 10 μ M LY294002 and 250 nM AG1478 (EGFR inhibitor), at 37°C for 4 h, followed by stimulation with 100 ng/ml CCL19 (CCR7 ligand), 37°C for 10 min. After treatment, cells were harvested in lysis buffer (10 mM Tris HCl, pH 7.6, 50 mM Na4P2O7, 50 mM NaF, 1 mM NaV3O4, 1% Triton X-100 and 1 × protease inhibitor of protein tyrosine phosphatases), sonicated for 3 s and centrifuged at 4°C, 14 000 r.p.m. for 30 min. The supernatant protein was normalized and 50 μg of protein was size-fractionated through a 10% SDS–PAGE gel, transferred to nitrocellulose and immunoblotted with the indicated mAbs. A second experimental set was performed. Cells were treated with/without U73122 (PLC inhibitor, 3 μ M, at 37°C for 48 h), PLCγ-1 antisense phosphothiolated oligonucleotides (12.5 μ M, at 37°C for 48 h), Akt/PKB inhibitor (10 μ M, at 37°C for 4 h). Then, using immunoblotting, we determined p-Akt/PKB and p-PLCγ-1 protein expression.
Immmunoprecipitation of EGFR
Briefly, 60–70% confluent cells were serum-starved for 72 h, then treated with or without 250 nM AG1478 (EGFR inhibitor), at 37°C for 4 h, followed by stimulation with 100 ng/ml CCL19 (CCR7 ligand) or 10 ng/ml EGF (EGFR ligand), 37°C for 10 min. Cells were harvested in lysis buffer and 3 μl of neutralizing anti-EGFR mAb (Upstate Cell Signaling, Charlottesville, VA, USA) was used for precipitation. After the addition of anti-EGFR Ab, 50 μl Protein G agarose beads (Invitrogen, Carlsbad, CA, USA) were added to each sample and the mixture was rotated overnight at 4°C. After centrifugation, the beads were washed with Western lysis buffer three times, followed by addition of 30 μl of 2 × sample buffer containing β-mercaptoethanol. Beads were boiled for 5 min, incubated on ice for 1 min, then centrifuged to remove agarose beads prior to electrophoresis.
Data were expressed as mean±standard deviation (s.d.) of repeated assays. Statistical differences between the two groups were evaluated using the unpaired Student's t-test. The calculations were performed with the software Statview™ Abacus Concepts, Berkley, CA, USA). Values of P<0.05 were considered significant.
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This work was funded by the American Head and Neck Society/American Academy of Otolaryngology (to RLF), and by the University of Pittsburgh Cancer Institute and the Eye and Ear Foundation, through the Stout Family Fund for Head and Neck Cancer Research.
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