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[Cancer Research 61, 5911-5918, August 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Hepatocyte Growth Factor/Scatter Factor-induced Activation of MEK and PI3K Signal Pathways Contributes to Expression of Proangiogenic Cytokines Interleukin-8 and Vascular Endothelial Growth Factor in Head and Neck Squamous Cell Carcinoma1

Gang Dong2, Zhong Chen, Zhi-Yu Li, Ning T. Yeh, Caren C. Bancroft and Carter Van Waes

Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proangiogenic activity of hepatocyte growth factor (HGF)/scatter factor has been closely associated with its ability to stimulate endothelial cell chemotaxis, migration, proliferation, and capillary formation. However, the potential of HGF as a paracrine factor in regulating the expression of angiogenesis factors by tumor cells is not widely appreciated. We observed that increased HGF was correlated with higher levels of angiogenesis factors interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in serum of patients with head and neck squamous cell carcinoma (HNSCC) as compared with that in normal volunteers and hypothesized that HGF may regulate angiogenesis factor production by tumor cells through the activation of its receptor c-Met, which is expressed by HNSCC cells. To test this hypothesis, we examined the effect of HGF treatment on IL-8 and VEGF expression by a panel of primary keratinocytes and HNSCC lines. HGF induced a significant dose-dependent increase in IL-8 and/or VEGF cytokine production in eight HNSCC lines tested, which is not observed in normal keratinocytes. In addition, HGF increased mRNA expression of IL-8 in 3 of 6 and VEGF in 5 of 6 HNSCC lines. The increase in induction of these factors by HGF corresponded to an increase in phosphorylation of c-Met in HNSCC. HGF-induced phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) pathway substrate p42/p44erk and phosphatidylinositol 3'-kinase (PI3K) pathway substrate Akt provided evidence for downstream activation of MEK and PI3K pathways in HNSCC. Inhibitors of MEK (U0126) and PI3K (LY294002) blocked p42/p44erk and Akt, respectively, and partially blocked HGF-induced production of IL-8 and VEGF, whereas the combination of U0126 and LY294002 completely inhibited expression of IL-8 and VEGF by UMSCC-11A. Our results demonstrate that HGF can promote expression of angiogenesis factors in tumor cells through both MEK- and PI3K-dependent pathways. Understanding HGF/Met paracrine regulatory mechanisms between tumor and host cells may provide critical information for targeting of therapies against angiogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HGF3 and its tyrosine kinase receptor, c-Met, have been demonstrated to promote tumor progression and metastasis in experimental models and transgenic models (1 , 2) . By acting directly on tumor cells through binding to its high affinity receptor c-Met, HGF activates multiple signal pathways through receptor tyrosine kinase-mediated phosphorylation, leading to increased expression of transcription factors and proteases necessary for tumor proliferation and metastasis (3, 4, 5, 6) . Consistent with this notion, many epithelial tumors have been reported to exhibit elevated levels of c-Met expression through either genomic amplification or up-regulated gene expression (7, 8, 9, 10) . In HNSCC, two recent reports (11 , 12) found evidence for stepwise increases in c-Met protein levels in primary tumors and lymph node metastasis by immunohistochemistry, which suggested a correlation may exist between c-Met expression and degree of metastasis.

HGF is also a known angiogenesis factor by its ability to promote endothelial cell growth, survival, and migration both in vitro and in vivo (13 , 14) . Oncogenic activities of HGF have been proposed through its role in promoting angiogenesis in tumors (15 , 16) . Data supporting this hypothesis came from examining the correlation of vessel density and HGF production in tumor samples and xenograft models (15 , 17 , 18) . HGF is produced by stromal cells (such as fibroblasts and smooth muscle cells) in tumors (19 , 20) . However, IL-8 and VEGF, the prototype angiogenic factors reported to promote tumor growth and metastasis in numerous studies (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) , are often produced by tumor cells. A relationship, if any, between HGF and production of IL-8 and VEGF by tumor cells remains to be elucidated.

In the current study, we present an alternative hypothesis for HGF-mediated angiogenesis, which could occur through HGF induction of angiogenesis factor expression by tumor cells. We noted that serum levels of HGF are increased in parallel with angiogenesis factors such as IL-8 and VEGF in HNSCC patients. In vitro, HGF was found to induce VEGF and IL-8 production in HNSCC lines but had a minimal effect on keratinocytes. HGF-induced IL-8 and VEGF production correlated with the phosphorylation of c-Met and activation of MEK and PI3K pathways. Inhibition of these two pathways by small molecule inhibitors of MEK and PI3K completely blocked HGF-inducible IL-8 and VEGF production by HNSCC cells. Our data suggest that HGF-induced angiogenic factor production in tumor cells may be an important component in the HGF/Met paracrine regulatory loop in the tumor environment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Human recombinant HGF was either purchased from R&D Systems (Minneapolis, MN) or kindly provided by Dr. George F. Vande Woude from NCI. The plasmids encoding full-length human IL-8 and VEGF cDNA were kindly provided by Dr. Joost Oppenheim of NCI and Dr. Jian Tang of Beijing Medical University, respectively. The MEK inhibitor U0126 was purchased from Promega (Madison, WI). PI3K kinase inhibitor LY294002 was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). The bicinchoninic acid Protein Assay and Super Signal West Pico Chemiluminescent Detection kits were obtained from Pierce (Rockford, IL). Anti-c-Met polyclonal antibody Met (sc-161) and antibody-protein A-agarose conjugate [h-Met (C-28) AC, sc-161AC] were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine mouse monoclonal antibody 4G10 (05-321) is from Upstate Biotechnology (Lake Placid, NY). Phospho- and non-phospho-specific antibodies and control proteins for Erk1/2, JNK, p38, and Akt were purchased from New England Biolabs (Beverly, MA).

Collection of Human Serum.
All of the serum for cytokine studies was obtained with informed consent under an institutional review board-approved protocol at NIH (97-DC-0044). Serum from 15 patients with HNSCC and 12 unaffected age- and gender-matched control subjects was obtained for a prospective comparison of cytokine concentration (21) . No subjects had a history of prior malignancy, immunodeficiency, autoimmune disorders, and hepatitis or HIV infection. Blood was drawn from control subjects and patients before treatment. Sera were collected by centrifuging whole blood at 3000 rpm for 15 min at 10°C in a Sorvall RT6000D centrifuge (DuPont, Wilmington, DE), and the aliquots were stored at -80°C until used in ELISA assay.

Human Keratinocytes and HNSCC Lines.
Human UMSCC cell lines were provided by Dr. Thomas Carey and derived from patients with SCC arising from sites in the upper aerodigestive tract at the University of Michigan, Ann Arbor, MI, after informed consent, as described previously (22 , 37) . The cell lines established from each patient specimen are designated by a numeric designation, and where isolates from two different time points or anatomical sites were obtained from the same patient, the designation includes an alphabetical suffix (i.e., "A" or "B"). NA and CA922 are oral SCC lines described previously (38) and kindly provided by Frank Ondrey of the National Institute on Deafness and Other Communication Disorders with permission by Dr. Toshio Kuroki of the Institute of Medical Science, Tokyo, Japan. The cell lines used were maintained in Eagle’s MEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. Normal human adult skin keratinocytes were purchased from Cascade Biologicals, Inc., (Portland, OR) and cultured in keratinocyte serum-free medium supplemented with bovine pituitary extract, epidermal growth factor, and 0.08 mM calcium supplied from the same company. Primary keratinocytes were used during passage 2-4. Rhek-1A and Rhek-1A ras cell lines were kindly provided by Dr. Johng S. Rhim at Uniformed Service University at Rockville, MD. Rhek-1A was a human primary keratinocyte line immortalized by SV40 T antigen, and Rhek-1A ras was obtained by subsequent transfection of Rhek-1A with Harvey ras oncogene (39) .

Cell Culture Supernatants for ELISA.
UMSCC-9 and -11A were grown in complete media, and human keratinocytes were cultured overnight in KGM with 0.08 mM calcium at 5 x 104 cells in 1.0 ml/well in sterile 24-well culture plates. The next day, HNSCC cells were switched to Eagle’s MEM plus 1% BSA and treated with different doses of human recombinant HGF for 24 h. Culture supernatants were collected, centrifuged at 14,000 rpm for 5 min at 4°C to remove debris, and stored at -20°C for ELISA. In the experiments for testing inhibitor activity, cultured cells were pretreated with or without MEK inhibitor U0126 or PI3K inhibitor LY294002 for 60 min at 37°C. Forty ng/ml HGF was then added to the cell culture for 24 h, and the conditioned medium was collected and tested.

Cytokine Quantitation by ELISA.
Cytokines HGF, IL-8, and VEGF in human serum or cell culture supernatants were measured by ELISA according to the manufacturer’s suggestions (R&D Systems). The serum or culture supernatants were pretested by ELISA to determine the correct dilution allowing cytokine measurement to fall into the linear range of the standard curves. Concentrations of cytokines were interpolated from a standard curve performed by using recombinant cytokines. The absorbance of the colorimetric reaction was read by a microplate autoreader set at 450 nm (Biotek, Winooski, VT). Each sample was tested in duplicate by ELISA, and repeated experiments were performed for two or three times. The threshold sensitivity of ELISA detecting HGF, IL-8, and VEGF is 40, 10, and 9 pg/ml, respectively.

RNA Isolation and Northern Analysis of Cytokine Expression.
Total RNA was isolated from cultured tumor cells when 80–90% confluent in a 75-cm2 or 150-cm2 tissue culture flask using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Twenty µg of total RNA from each cell line was run on a 1.2% formaldehyde/agarose gel. Transferred nylon membranes were hybridized with DNA probes for IL-8 and VEGF cytokine genes using QuikHyb hybridization solution (Stratagene, La Jolla, CA). After hybridization and a wash under high-stringency conditions, the membrane was exposed to an X-OMAT AR film with an intensifying screen at -80°C. The films were developed by a M35 X-OMAT film processor (Kodak, Rochester, NY).

Western Blot and Immunoprecipitation Analyses.
Cells were plated in 100-mm tissue culture dishes and allowed to grow until 80–90% confluent. The day before the experiments, HNSCC cells were switched into Eagle’s MEM plus 1% BSA. Different concentrations of HGF were added to the cells in culture for 10 to 60 min. In experiments where inhibitors were used, cells were preincubated with inhibitors for 1 h before the addition of HGF. Whole cell extracts were made following a procedure described previously by Brown et al. (40) . Briefly, cells were rinsed twice with cold PBS and lysed in lysis buffer [50 mM Tris (pH 7.4), 400 mM NaCl, 50 mM NaF, 30 mM sodium PPi, 1 mM sodium pyrovendidate, 1% SDS, and 0.5% NP40]. A protease inhibitor tablet (Complete; Mini; catalogue 1836153; Boehringer Mannheim, Mannheim, Germany) was added/10 ml of lysis buffer before use. Cell lysate was collected from tissue culture dishes by scraping followed by passing through a 23-gauge needle for three times. Samples were then spun at 18,000 x g for 20 min in 4°C. Supernatants were aliquoted, snap frozen, and stored at -80°C. Protein concentrations were determined using a BCA protein assay kit following the manufacturer’s instructions (Pierce).

For Western blot analysis, 40 µg of cell extracts were resolved on an 8 x 8-cm denaturing SDS-polyacrylamide gel (Novex, San Diego, CA). After the transfer of protein to a nitrocellulose membrane, the blot was incubated sequentially with 3% dry milk in PBS for 1 h, polyclonal rabbit antibody to c-Met for 1 h, and goat antirabbit IgG-horseradish peroxidase conjugate (170-6515; Bio-Rad, Hercules, CA) for 1 h. Membranes were developed in chemiluminescent substrate and exposed to films. In experiments using phospho-specific antibodies, membranes were first blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 for 1 h. Diluted antibodies (1:1000) were added and incubated overnight in 4°C. After washed in Tris-buffered saline with 0.1% Tween 20, membranes were incubated in the appropriate secondary antibody-peroxidase-conjugate. Signals were developed using SuperSignal West Pico chemiluminescent substrate (catalogue no. 34080; Pierce). These membranes were stripped and hybridized with nonphospho-specific antibodies.

For immunoprecipitation experiments, 500 µg of cell extracts were precleared with 20 µl of protein A-agarose conjugate for 30 min in 4°C. Ten µl of c-Met antibody-protein A-agarose conjugate was then added to the supernatant, and the incubation was continued 4°C overnight with rotation. The next morning, the pellet was collected by centrifugation and washed four times in lysis buffer. After final washing, the pellet was resuspended in 10 µl of 2 x SDS sample buffer, and Western blot analysis was performed using phosphotyrosine-specific antibody (4G10) as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Elevated Serum Levels of HGF, IL-8, and VEGF in Patients with Head and Neck Cancer.
We detected previously (21) significantly higher levels of serum IL-8 and VEGF in patients with head and neck cancer relative to normal subjects and demonstrated expression of IL-8 and VEGF in specimens of HNSCC patients by immunohistochemical staining. When we examined whether the serum of the same group of HNSCC patients contained other factors that can potentially regulate tumor cytokine production, we also found that serum HGF was significantly increased as compared with control subjects (Table 1)Citation . An intermediate linear correlation was observed between the serum HGF level versus the serum levels of IL-8 and VEGF, suggesting HGF, IL-8, and VEGF expression in tumor patients may be related.4 In vitro, HNSCC cells produced various levels of IL-8 and VEGF in their culture supernatants (19 , 20) , but we did not detect significant expression of HGF by HNSCC cell lines by ELISA, Western blot analysis, or Northern blot analysis (data not shown). By immunostaining, we observed that in the head and neck tumor samples, tumor stroma were positive for HGF immunostaining. A detailed study will be published separately.4


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  		Table 1 Serum levels of chemokine and proangiogenic cytokinesa

 
HGF Induces IL-8 and VEGF Angiogenesis Factor Production in UMSCC Culture Supernatants.
On the basis of the above observation and our previous results showing that tumor cells are one of the major sources for IL-8 and VEGF angiogenesis factor production in head and neck tumor patients, we hypothesized that HGF may be an inducer of IL-8 and VEGF production by HNSCC. To test this hypothesis, we first examined whether exogenous HGF could induce IL-8 and VEGF production in culture supernatants. To determine whether keratinocytes or HNSCC respond over a range of HGF dosages, we first treated human keratinocytes, UMSCC-9, and UMSCC-11A cells by HGF in a dose range from 10 to 200 ng/ml, collected media, and assayed for IL-8 (Fig. 1Citation , upper panels) and VEGF by ELISA (Fig. 2Citation , upper panels). We observed that, under these experimental conditions, normal keratinocytes produced minimal levels of IL-8 and did not respond to HGF treatment up to 200 ng/ml (Fig. 1)Citation . However, HGF induced IL-8 by more than 5-fold in both UMSCC-9 (from 50 pg/ml to about 250 pg/ml) and in UMSCC-11A (from 500 pg/ml to 2800 pg/ml). HGF-induced IL-8 production reached maximum levels above 40 ng/ml. Subsequently, we tested HGF induction of IL-8 in an additional six UMSCC lines using the dosage of HGF from 1–40 ng/ml. We observed 2–5-fold induction of IL-8 production by HGF in most UMSCC lines tested (Fig. 1Citation , middle and lower panels). Similar results were obtained for VEGF production. As shown in Fig. 2Citation , normal keratinocytes produced a baseline level of VEGF of about 300 pg/ml, and HGF at concentrations up to 200 ng/ml induced no significant increase in VEGF production by normal keratinocytes. The same concentrations of HGF in UMSCC-9 and -11A cells induced a significant increase of VEGF production that reached a plateau at a concentration of HGF of 10 ng/ml (Fig. 2Citation , upper panels). Six additional UMSCC cell lines treated with HGF at concentrations of HGF between 1 ng/ml to 40 ng/ml exhibited about a 2-fold increase of VEGF production.



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  		Fig. 1. Constitutive and HGF-induced IL-8 protein production in primary keratinocytes and HNSCC cells. Primary HKCs and a panel of UMSCC cells were cultured in 24-well plates at 5 x 104 cells/well 1 day before treatment (except for UMSCC-46, which was plated at 105 cells/well because of lower adhesion and growth ability). The cells were treated with recombinant human HGF at concentrations as indicated for 24 h. The culture supernatants were collected, and IL-8 was quantified by ELISA. Error bar, SD.

 


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  		Fig. 2. Constitutive and HGF-induced VEGF protein production in primary keratinocytes and HNSCC cells. VEGF in cultured supernatants was tested by ELISA under the same experimental conditions described as Fig. 1Citation .

 
HGF Induces IL-8 and/or VEGF mRNA Expression in UMSCC Cells.
Next, we tested whether HGF induced IL-8 and VEGF mRNA expression. Expression of IL-8 and VEGF mRNA in normal HKCs and UMSCC lines after HGF treatment was analyzed by Northern blot. As shown in Fig. 3Citation , variable levels of constitutive IL-8 and VEGF mRNA were detected in HNSCC lines. Two VEGF mRNA transcripts of approximately 5.5 kb and 2 kb, respectively, were detected, consistent with results described previously (41) in carcinoma cells. After treatment for 4 h by recombinant human HGF at 40 ng/ml, which is in the range of the ED50 tested by the manufacturer (R&D Systems), three of six HNSCC lines showed increased IL-8 mRNA, whereas no induction was observed in normal HKCs. HGF induced the expression of both VEGF mRNA species in five of six HNSCC cells but not in normal HKCs. We tested three independent clones of primary HKCs for HGF induction, and no significant increase of IL-8 or VEGF mRNA was detected under the experimental conditions used (Fig. 3Citation and data not shown).



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  		Fig. 3. Constitutive and HGF-induced IL-8 and VEGF mRNA expression in primary keratinocytes and HNSCC cells. Primary HKCs and a panel of HNSCC cells were treated with 40 ng/ml of recombinant HGF for 4 h, and RNA was isolated and detected with 32P-labeled cDNA probes for IL-8, VEGF, and glyceraldehyde-3-phosphate dehydrogenase by Northern blotting.

 
Results for UMSCC-1, -9, and -11A cells obtained by both ELISA and Northern blot analyses allowed us to compare the relative induction of cytokine protein and mRNA of IL-8 and VEGF by HGF. All of the three cell lines showed induction of both cytokine mRNA and protein in response to HGF. The level of induction of both mRNA and protein observed in UMSCC-1 and -11A appeared proportionate, whereas less protein was secreted by UMSCC-9 cells. Because it has been reported previously (36) that IL-8 expression in cultured cells can be affected by cell density, we repeated the above experiment with cells near 90% confluence to examine whether the relatively low increment in HGF-induced IL-8 secretion relative to mRNA expression by UMSCC-9 was reproducible. This apparent difference in the magnitude of induction of IL-8 by HGF analyzed by Northern and ELISA in UMSCC-9 was reproducible when using a higher density of cells (data not shown), suggesting that additional factors may contribute to regulation of the low basal and HGF-inducible secretion of IL-8 by UMSCC-9. We also repeated these experiments in media without serum and obtained similar results with higher constitutive and HGF-inducible IL-8 and VEGF production (data not shown). Thus, HGF can induce IL-8 and/or VEGF expression in a wide panel of UMSCC cell lines, supporting the hypothesis that HGF can serve as a factor involved in regulation of expression of these angiogenesis factors by tumor cells.

Expression and Phosphorylation of c-Met after Stimulation of Keratinocytes and HNSCC with HGF.
To explore whether the difference in response to HGF observed between primary keratinocytes and HNSCC is because of differences in expression of c-Met as suggested in previous studies (11 , 12) , we examined c-Met expression by Western analysis in a panel of cell lines selected to represent cells that were nonresponsive, low responsive, and high responsive to HGF for IL-8 and VEGF production. We also included two cell lines, Rhek-1A and Rhek-1A ras, to represent immortalized and transformed keratinocytes. As shown in Fig. 4ACitation , similar levels of p140 c-Met protein were observed in HKCs, transformed keratinocytes, and HNSCC cell lines, except for UMSCC-11B, which lacked detectable expression of p140 c-Met protein entirely. Thus, differences in the level of c-Met protein expression alone did not correlate with the differences in response between keratinocytes and SCC lines to HGF under the experimental conditions used.



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  		Fig. 4. c-Met protein expression and phosphorylation in primary and transformed HKC and HNSCC cells. A, whole cell extracts were harvested from cells in log growth phase at around 90% confluent. Western analysis was performed using anti-c-Met (top) and anti-Erk antibodies (bottom). B, cells were treated with HGF at concentrations indicated for 30 min and harvested for immunoprecipitation using c-Met antibody-protein A-agarose conjugates followed by Western blot with phosphotyrosine specific antibody (top; designated as Py-c-Met). Blots were stripped and reprobed with antibody to c-Met (bottom).

 
As a measure of c-Met activation, we compared basal and HGF-induced c-Met phosphorylation in primary keratinocytes, UMSCC-9, and UMSCC-11A by immunoprecipitation of the c-Met protein and Western blot with phosphotyrosine-specific antibody. As shown in Fig. 4BCitation , c-Met phosphorylation was detected in lysates from all of the three cell lines in response to 10 ng/ml HGF in 30 min. However, HGF at 10 and 40 ng/ml induced stronger phosphorylation of p140 c-Met in HNSCC cells as compared with primary keratinocytes. No significant differences were present either at a higher dose of HGF (160 ng/ml) or at later time points (data not shown). These results indicate that increased induction of IL-8 and VEGF production by HGF in HNSCC relative to keratinocytes may be related, only in part, to a quantitative difference in c-Met phosphorylation and activation in response to HGF.

HGF-induced Activation of the MEK and PI3K Pathways Contributes to Production of IL-8 and VEGF.
HGF-induced expression of IL-8 and VEGF could be mediated by one or more signal pathways. We recently found evidence that epidermal growth factor receptor-mediated activation of mitogen-activated protein kinase and PI3K pathways contributes to basal expression of IL-8 and VEGF in HNSCC5 . Therefore, we examined the effects of HGF on phosphorylation of mitogen-activated protein kinases Erk, JNK, p38, and PI3K substrate Akt in primary keratinocyte, UMSCC-9, and UMSCC-11A cells to determine whether HGF and c-Met modulate these pathways. Phosphorylation of Erk, JNK, p38, and Akt was examined using antibodies specific for phosphorylated kinases by Western blot analysis in whole cell extracts harvested from cells treated with 40 ng/ml HGF for 30 min. Fig. 5ACitation shows that increased phosphorylation of Erk and Akt is detected in all of the three cell lines after HGF treatment. No change in phosphorylation of JNK and p38 is observed with HGF treatment (data not shown). These results provided evidence that HGF may activate Erk and Akt, which are kinases downstream of MEK and PI3K. To test whether inhibition of MEK and PI3K can inhibit activation of Erk and Akt, we examined whether the MEK inhibitor UO126 and the PI3K inhibitor LY294002 block HGF-induced phosphorylation of Erk and Akt in the highly inducible line UMSCC-11A. Fig. 5BCitation shows that UO126 inhibited HGF-induced Erk phosphorylation with no obvious effect on Akt phosphorylation, whereas LY294002 specifically inhibited Akt phosphorylation in UMSCC-11A cells. Both inhibitors showed dose-dependent effects on their appropriate downstream targets, and no cross-talk between MEK and PI3K pathways was detected under the experimental conditions used. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and trypan blue exclusion experiments in these cell lines treated with MEK and PI3K inhibitors and demonstrated that neither inhibitor had any significant effect on cell growth or viability at these concentrations during the assay (data not shown).



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  		Fig. 5. HGF-induced phosphorylation of Erk and Akt was blocked by inhibitors for MEK and PI3K. A, primary HKC, UMSCC-9, and UMSCC-11A cells at 90% confluency of log growth phase were treated with 40 ng/ml of HGF for 30 min and harvested for Western blot analysis using anti-phospho-Erk, Erk, phospho-Akt, or Akt antibodies. B, UMSCC-11A cells were pretreated with MEK inhibitor UO126 at 10 µM and 30 µM or with PI3K inhibitor LY294002 at 10 µM and 30 µM for 1 h at 37°C. After treatment with 40 ng/ml of HGF for 30 min, cells were harvested for Western analysis using anti-phospho-Erk and anti-phospho-Akt antibodies.

 
We used the same concentration of the inhibitors to examine HGF-induced IL-8 or VEGF production in UMSCC-11A cells by ELISA. As shown in Fig. 6Citation , UO126 alone significantly but incompletely inhibited both constitutive and HGF-inducible IL-8 and VEGF production. LY294002 alone is able to completely block constitutive and partially block HGF-induced IL-8 and VEGF production. The combination of the two inhibitors completely inhibited HGF-induced IL-8 and VEGF production, indicating both MEK and PI3K are activated and contribute to the expression of basal and HGF-inducible production of IL-8 and VEGF. The similar induction of Erk and Akt phosphorylation in primary and malignant keratinocytes suggests that Erk and Akt are not sufficient to account for the preferential induction of IL-8 and VEGF by HGF in UMSCC cells and that other pathways downstream of MEK and PI3K may contribute to the differential expression of these angiogenesis factors.



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  		Fig. 6. Inhibitors for MEK and PI3K blocked constitutive and HGF-induced IL-8 and VEGF production in UMSCC-11A cells. Cells were plated in 24-well plates for overnight as described in Fig. 1BCitation . After being pretreated with two concentrations of UO126 (UO) or LY294002 (LY2) alone or in combination at 37°C for 1 h, the cells were treated with () or without ({square}) 40 ng/ml of HGF for 24 h. Culture supernatants were harvested and quantified for IL-8 (A) and VEGF (B) by ELISA. Numbers after the inhibitor names denote inhibitor concentrations used in µM. No Tx, no treatment; error bar, SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the important activities of HGF in promoting tumorigenesis and metastasis is to stimulate angiogenesis by a direct effect on endothelial growth and tube formation (13 , 14) . In this study, we provided evidence for an additional novel mechanism by which HGF promotes angiogenesis, which is its ability to induce expression of proangiogenic factors by tumor cells, such as HNSCC. We showed in the current study that recombinant HGF preferentially induced IL-8 and VEGF mRNA expression and protein production in a panel of HNSCC when compared with primary keratinocytes, although similar levels of c-Met expression were detected in both HNSCC and keratinocytes (Figs. 1Citation 2Citation 3Citation 4)Citation . HGF-induced IL-8 and VEGF gene expression were correlated with HGF-induced phosphorylation of c-Met receptor. HGF induced phosphorylation of Erk and Akt kinases in UMSCC cells (Fig. 5)Citation , which are substrates of the MEK and PI3K pathways. Blocking MEK and PI3K pathways by specific inhibitors resulted in a complete inhibition of IL-8 and VEGF (Fig. 6)Citation , indicating that these pathways are important in basal and HGF-induced expression of these angiogenesis factors. Our study provides evidence that HGF may be one of the important upstream signals leading to aberrant cytokine gene expression in HNSCC cells and suggests that HGF may be a critical factor in forming a paracrine stimulatory loop between host cells and tumor cells.

HGF has been shown to directly stimulate angiogenesis both in vitro and in vivo. In vitro, HGF stimulates endothelial chemotaxis, migration, proliferation, and capillary tube formation (13) . In a xenograft model, tumors from HGF-transfected lines demonstrate increased blood vessel density (18) . A correlation between HGF immunostaining and increased neovascularization has been reported in different tumor types (42 , 43) , demonstrating that the presence of HGF in the tumor microenvironment is associated with tumor angiogenesis. Because fibroblasts and smooth muscle cells are a major source of HGF (19 , 20) and the cell population responsible for HGF production in the tumor environment in many cases is not clear, a paracrine mechanism of HGF-induced angiogenesis between fibroblasts and endothelial cells has been proposed (16) . The role of HGF in angiogenesis involving tumor and endothelial cells is less well understood.

Our data showing that HGF, IL-8, and VEGF are detected together in serum from patients (Table 1)Citation and that HGF is able to induce the production of angiogenic factors IL-8 and VEGF by HNSCC cells (Figs. 1Citation 2Citation 3)Citation provide a foundation for the hypothesis that HGF participates in paracrine mechanisms that involve fibroblast, tumor, and endothelia. In support of the evidence in Table 1Citation , our preliminary data demonstrate the colocalization of HGF/Met staining with IL-8 and VEGF in tumor cells by immunostaining and that fibroblasts isolated from human SCC produce HGF, providing a potential source of HGF within the tumor environment.4 Our clinical study also indicates that higher levels of serum HGF, IL-8, and VEGF were correlated with disease recurrence and poor prognosis.4 Because it is difficult to examine the effects of HGF on proinflammatory cytokine expression and tumor growth in patients and in xenograft models, we have undertaken studies (24) in a syngeneic murine model in which we have shown that production of the IL-8 homologue KC by tumor cells promotes growth and metastasis. Preliminary studies6 provide evidence that enforced expression of HGF by these SCC cells promotes increased expression of proinflammatory cytokine KC and increased growth in vitro and in vivo. Ongoing studies are determining the relative contributions of fibroblast HGF and tumor KC (or IL-8) and VEGF to tumor angiogenesis and growth.

We have provided evidence using a panel of eight UMSCC cell lines that HGF can induce increased IL-8 and/or VEGF production in HNSCC with a high frequency (Figs. 1Citation and 2Citation ); HGF induced IL-8 in a dose-dependent manner between 1 ng to 200 ng/ml (Fig. 1)Citation . No induction was observed in UMSCC-11B cells (Fig. 3)Citation , consistent with the loss of c-Met receptor by this cell line. The induction of IL-8 in the majority of cell lines was found to be between 2–5-fold, except in UMSCC-74A, which was about 20%. HGF-induced IL-8 production reached a plateau around a concentration of 40 ng/ml, which is the ED50 for functional assays defined by the manufacturer (R&D Systems). The panel of eight UMSCC cell lines also produced various levels of VEGF protein, from 170 pg/ml to more that 1000 pg/ml, which were further induced about 2-fold by HGF (Fig. 2)Citation . Numerous previous studies (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) have shown that elevated expression of IL-8 and VEGF in cultured tumor lines and in tumor specimens plays an important role in angiogenesis and pathogenesis of cancer. Our laboratory showed previously (21, 22, 23, 24 , 44) that modulation of IL-8 expression by HNSCC cells has an important effect on tumor growth in vitro and in vivo, and its homologue KC promotes angiogenesis, growth, and metastasis of murine SCC.

Although the effects of HGF on IL-8 and VEGF expression by HNSCC have not been reported previously, HGF-induced VEGF production has been observed previously (45 , 46) in cultured primary keratinocytes and SCC A431 cells. Wojta et al. (46) showed that primary keratinocytes produced significantly lower VEGF (300 pg/105 cells/24 h) compared with SCC A431 cells (4000 pg/105 cells/24 h). They reported a 3–4-fold induction of VEGF by HGF in A431 cells and a 2.5-fold induction of VEGF in normal keratinocytes. In our current study, the baseline level of VEGF production by primary cultured keratinocytes (300–400 pg/ml/24 h/0.5–1 x 105 cells) were similar to the level reported by Wojta et al. (46) . However, we did not observe a significant induction of VEGF in keratinocytes at the same cell density as in HNSCC cells when we cultured the cells in media with serum or in serum-free keratinocyte culture medium supplemented with EGF and pituitary extract. Using an increased cell density, we observed a slight induction of VEGF (by less than a fold) in normal keratinocytes (data not shown), suggesting VEGF induction by HGF in keratinocytes is partly related to cell density. In addition, the elevated baseline levels and the preferential induction of IL-8 and VEGF by HGF in cultured HNSCC cells were not attributable to serum factors alone. When we repeated the experiments in medium without serum, both constitutive and the inducible production of IL-8 in UMSCC-9 was the same, but the baseline IL-8 production in UMSCC-11A was decreased. In serum-free conditions, increased baseline level production of VEGF in UMSCC-9 was observed, and the baseline and induced levels of VEGF were the same in UMSCC-11A cells (data not shown).

We detected increased serum levels of HGF and VEGF by less than 2-fold when compared between HNSCC patients and normal subjects (Table 1)Citation . A more significant increase in serum IL-8 was observed that is not proportional to the serum HGF level, suggesting other factors may play a role in regulating serum IL-8 production in HNSCC patients. Given the differences in basal expression of IL-8 and VEGF and lack of requirement for HGF (or c-Met) in some cell lines such as UMSCC-11B, HGF does not appear to be the exclusive regulatory mechanism for IL-8 and VEGF production in HNSCC cells. In fact, we have explored and recently demonstrated that in UMSCC-11B cells that lack c-Met and other lines that produce higher basal levels, that IL-1R and EGFR activation is significantly augmented and contributes to the elevation in basal expression of IL-8 and VEGF observed (47) .5 Bancroft et al.5 found that basal levels of IL-8 and VEGF production, because of autocrine EGFR activation in HNSCC cells, is also mediated by activation of MEK and PI3K pathways, explaining the suppression of both basal (EGFR-mediated) and HGF-induced activation observed in UMSCC-11A (Fig. 6)Citation . Enhanced autocrine activation of IL-1R, EGFR, and possibly other events downstream of HGF could promote proangiogenic cytokine production in SCC cells and lead to host growth factor (and HGF) independent growth.

HGF binding to c-Met induces receptor phosphorylation at multiple sites within the intracellular domains of the receptor ß-subunit. Through two autophosphorylation sites mapped in the COOH-terminal region, c-Met has been shown to interact with downstream target proteins Grb2, Src, PI3K, PLCr, and SHP2 via their SH2 domain (48, 49, 50, 51, 52, 53, 54) . In addition, the Mr 110,000 adapter protein GAB1 was found to bind c-Met through a novel Met-binding domain and, upon activation, mediate interaction with GRB2 and p85 subunit of PI3K (55) . Several of these effector proteins induce phosphorylation of MEK/Erk and Akt (48 , 54 , 56) , which can lead to activation of gene expression and biological effects of HGF. Both Erk and Akt are shown to be involved in regulating IL-8 and VEGF expression by different stimuli (57, 58, 59, 60) . Our current study showed that HGF activated both Erk and Akt (Fig. 5A)Citation , which are downstream of MEK and PI3K. Inhibitors blocking MEK and PI3K also inhibited both IL-8 and VEGF induction by HGF in a dose-dependent manner (Fig. 6)Citation . Because these inhibitors are specific for MEK and PI3K, both of which have multiple downstream targets, our data suggest that: (a) activation of Erk and Akt may not be sufficient to account for preferential induction of IL-8 and VEGF by HGF in HNSCC; and (b) other factors downstream of MEK and PI3K may be activated in HNSCC cells but are limited in keratinocytes.

Several previous reports (7, 8, 9, 10, 11, 12) showed that the level of c-Met protein was elevated in tumor cells as compared with their neighboring normal counterpart. We did not detect a significant difference in the total protein levels of p140 c-Met receptor and downstream Erk and Akt kinases after HGF stimulation in normal keratinocytes and HNSCC tumor cells in culture (Figs. 4Citation and 5Citation ). However, we did observe a difference in inducibility of c-Met phosphorylation between keratinocytes and HNSCC cells, which is correlated with the preferential cytokine production in response to HGF in HNSCC lines (Figs. 1Citation and 2Citation ). We speculate that either a suppressive mechanism(s) is present in normal cells, which is lost in tumors, or preferential activation of transcription factors further downstream of MEK and PI3K may contribute to the difference. Given that Erk and Akt phosphorylate and activate multiple transcription factors in the nucleus, we favor the idea that the difference in transcription activities in tumor cells may contribute to the preferential induction of IL-8 and VEGF by HGF. In our initial effort to look for such candidates, we observed a difference between keratinocyte and HNSCC in the expression of a nuclear factor that is a known target for Erk. Studies are under way to explore its function in IL-8 and VEGF preferential expression in HNSCC cells.

On the basis of previous and current studies, we hypothesized a potential paracrine regulatory mechanism of HGF/Met interaction in tumor environment. In our model, tumor-produced inflammatory or angiogenic cytokines, such as IL-1{alpha}, platelet-derived growth factor, fibroblast growth factor, and other factors could stimulate stromal cells to produce HGF. HGF promotes angiogenesis by directly stimulating endothelial cells or indirectly by stimulating tumor cells to produce angiogenic factors IL-8 and VEGF through MEK and PI3K signal transduction pathways, as demonstrated in the present study. These angiogenesis factors are able to promote angiogenesis in vivo. We believe that the paracrine mechanism involves at least three cell types (tumor cell, stroma, endothelia) and multiple routes. Understanding such paracrine mechanisms and developing reagents for blocking each route are essential for targeting angiogenesis in anticancer therapy.


   ACKNOWLEDGMENTS
 
We thank Drs. Glenn Merlino and Donald Bottaro of NCI, Dr. James Battey of the National Institute on Deafness and Other Communication Disorders for reviewing the manuscript, and Dr. George Vande Woude of NCI for providing recombinant human HGF.


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

1 Supported by the National Institute on Deafness and Other Communication Disorders Intramural Research Project Z01-DC-0016. Back

2 To whom requests for reprints should be addressed, at NIH, Building 10, Room 5D55, MSC-1419, 10 Center Drive, Bethesda, MD 20892-1419. Phone: (301) 402-4216; E-mail: dongg{at}nidcd.nih.gov Back

3 The abbreviations used are: HGF, hepatocyte growth factor/scatter factor; SCC, squamous cell carcinoma; HNSCC, head and neck SCC; UMSCC, University of Michigan SCC; IL, interleukin; VEGF, vascular endothelial growth factor; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3'-kinase; NCI, National Cancer Institute; HKC, human adult keratinocytes; KGM, keratinocyte growth media; Erk, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; EGFR, epidermal growth factor receptor. Back

4 Z. Chen, J. S. Wolf, D. Duffey, N. T. Yeh, C. Van Waes, and G. Dong. Elevation of serum HGF and HGF/c-Met mediated paracrine interaction between tumor cells and fibroblasts in head and neck cancer patients, manuscript in preparation. Back

5 C. C. Bancroft, J. Yeh, Z. Chen, J. B. Sunwoo, G. Dong, C. Park, N. Yeh, S. Jackson, and C. Van Waes. Epidermal growth factor receptor coactivates the NF-{kappa}B and AP-1 signal pathways and expression of angiogenesis factors IL-8 and VEGF in human head and neck squamous cell carcinoma lines, submitted for publication. Back

6 Z. Chen, E. Loukinova, N. T. Yeh, C. Van Waes, and G. Dong. HGF induced aggressive malignant phenotypes through over-expression of C-Met onco-protein in highly metastatic marine squamous cell carcinoma, manuscript in preparation. Back

Received 6/ 2/00. Accepted 5/29/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Oncogene addiction: setting the stage for molecularly targeted cancer therapy
Genes & Dev., December 15, 2007; 21(24): 3214 - 3231.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
J. Friedman, L. Nottingham, P. Duggal, F. G. Pernas, B. Yan, X. P. Yang, Z. Chen, and C. Van Waes
Deficient TP53 Expression, Function, and Cisplatin Sensitivity Are Restored by Quinacrine in Head and Neck Cancer
Clin. Cancer Res., November 15, 2007; 13(22): 6568 - 6578.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
W. L. Bigbee, J. R. Grandis, and J. M. Siegfried
Multiple Cytokine and Growth Factor Serum Biomarkers Predict Therapeutic Response and Survival in Advanced-Stage Head and Neck Cancer Patients
Clin. Cancer Res., June 1, 2007; 13(11): 3107 - 3108.
[Full Text] [PDF]