B. METHODS
4. Effect of EGF on colony dispersion
photography at 6 hours, 12 hours, 18 hours, and 24 hours.
5. Wound healing assay
FaDu cells were plated in culture plates at a density of approximately 1ⅹ105/well in the absence of serum. Confluent cell monolayers were then deprived of growth factors for 48 hours and a cell-free area was introduced by scraping the monolayer with a sterile pipette tip followed by extensive washing to remove cellular debris. In vitro reepithelialization was monitored by the repopulation of the cleared area with cells over time. To confirm the effect
of EGF, cells were treated with EGF(0, 10ng/㎖, 30ng/㎖). Wound healing was documented by photography at 12, 24, 36 and 48 hours.
For objective measurements, distance between both side was measured by computer calculator at five points(per one field) in 48 hours. The average distance at seven fields was compared each other( control vs EGF treatment group and EGF 10ng/㎖ vs EGF 30ng/㎖).
6. Effect of EGF on Invasion
Transwell chambers (Costar) were used to verify the degree of invasiveness depending on the administration of EGF. First, type I collagen (6 ㎍/filter) melted in EMEM 100 ㎕ was poured into the upper part of a polyethylene filter (8 ㎛ pore-sized); coating was carried out in a laminar flow hood for one night. 500 ㎕ of 0.5% FBS medium was put into the lower part of each well, and the wells were adjusted to EGF densities of 0, 10, and 30 ng/㎕.
After preprocessing with mitomycin C (8 ㎍/㎖) for 30 minutes, 105 cells (in 100 ㎕ of growth medium) were attached to the top of the filter of the upper well. After this chamber was cultivated in 5% CO2 at 37℃ for 48 hours, the filter of the upper well was removed and the cells passed through the pore. The attached cells on the lower part were dyed with hematoxylin and counted using a light microscope.
7. RT-PCR of MMP-2 and MMP-9
Using 50-100 ㎎ of frozen FaDu cells, RNA was prepared as described above. The primer pairs used for MMP-2 and MMP-9 were as follows;
MMP-2-F, 5'-ACCTGGATGCCGTCGTGGAC-3'; and MMP-2-R,
5'-TGTGGCAGCACCAGGGCAGC-3'; and MMP-9-F, 5'-GGGGAAGATGCTGCTGTTCA-3' and MMP-9-R, 5'-GGTCCCAGTGGGGATTTACA-5'-GGGGAAGATGCTGCTGTTCA-3'. After denaturation for 3 minutes at 96℃, the samples were amplified by PCR for 30 cycles of 30 seconds at 96℃, 30 seconds at 55℃, and 30 seconds in 72℃, with extension for 5 minutes at 72℃ .
8. Zymography of MMP-2 and MMP-9
Cultures were deprived of growth factors and serum for 48 hours prior to treatment with EGF (0, 10 ng/㎖, 30 ng/㎖) for the indicated periods of time. After quantification of the
protein in the supernatant, 30 ㎍ of protein from each sample was mixed with 15 ㎕ APMA and the samples were activated for 1 hour at 37℃. Cytoplasm samples were obtained after 24 hours using the MightySlim™ SX 250 (Hoefer, San Francisco, CA, USA) process. Each 10 ㎕ sample was placed in sample buffer for 10 minutes, and then electrophoresed in a polyacrylamide gel at 125 V for 120 minutes at 4℃ using the Novex XCell II. The gel was incubated in renaturation buffer for 60 minutes at room temperature, and then incubated in 100 ㎖ of developing buffer at 37℃ for 18 hours with light shaking.
The gel was stained with Coomassie blue for 3 hours and washed with water. After decolorization in 400 ㎖ methanol, 100 ㎖ acetic acid, and 500 ㎖ distilled water, cell images were taken every 10 minutes using an image analyzer.
9. Statistical Methods
Student’s t test and one-way ANOVA test (for the invasion assay) were used for statistical analyses of the data. Patient survival rates were calculated using the Kaplan-Meier method, and statistically significant differences in survival were identified using the log-rank test.
All statistical analyses were conducted using SPSS 10.0 statistical software (SPSS, Chicago, IL). A p value of less than 0.05 was considered statistically significant.
III. Results
A. Expression of EGFR in Human Hypopharyngeal Cancer Tissue
EGFR overexpression was observed in 63.2%(36/59) of hypopharyngeal cancer tissues. In most of the specimens, EGFR expression was primarily seen in the cell membrane and the cytoplasm of cancer cells (Fig. 1A). The rate of EGFR expression was 52.9% in patients with tumors less than 4 cm (stage T1 or T2) and 67.5% in tumors more than 4 cm (stage T3 and T4). Thus, it could be observed that the expression of EGFR increased according to tumor size. However, there was no statistical significance (Table 1). In cases of lymph node metastasis, EGFR expression was observed in 36 out of 57 (63.1%). But, a statistically nonsignificant increase in EGFR expression was seen in 27 out of 41 cases (65.9%) where there was lymph node metastasis. EGFR expression seemed to be increased in cases of distant metastasis (90%), however there was no significant difference compared to those without distant metastasis (57.4 %). EGFR expression was positive in 16.7% (1/6) and 68.6% (35/51) in early and advanced stage carcinoma, respectively, demonstrating that there was a significant increase in EGFR expression according to tumor stage (p<0.05).
Pathologic cell differentiation did not seem to affect EGFR expression. There was no statistically significant difference between groups with and without recurrence (60%, 50%).
Of the 57 patients, 20 (35.1%) died during the follow up period. The cause of death was local recurrence in 11 cases, distant metastasis in 9 cases, and both in no case. Survival analysis was performed using the Kaplan-Meier method and the statistical significance was evaluated by the log-rank test(Fig. 1B). There showed a difference of survival rate between the groups with negative and positive EGFR expression, but the p-value was 0.053, resulting in no statistical significance.
Fig. 1. A: Immunohistochemistry: Paraffin sections of hypopharyngeal carcinomas immunostained for EGFR : EGFR staining was primarily observed in the membrane of the cancer cells, but was seen occasionally seen in the both membrane and cytoplasm of cancer cells(200x). A: no staining, B: definite but weak staining (±), C: moderate staining (+), D:
strong staining (++) B: Survival rate: Five-year survival of 59 patients with hypopharyngeal carcinoma according to EGFR expression. Survival was significantly poorer in patients with positive EGFR expression compared with the negative EGFR expression (p = 0.053).
Table 1. Correlation between the Expression Pattern of EGFR and Clinicopathologic stage carcinoma, respectively, demonstrating that there was a significant increase in EGFR expression according to tumor stage (p<0.05). It was seemed that other variables(T, N, M, grade, recurrence) were correlated with expression of EGFR, but there was not significant.
# calculated by Pearson Chi-Square's test (p<0.05)
* calculated by Fisher's exact test (p<0.05)
B. Western Blot in Hypopharyngeal Cancer Tissue and RT-PCR and Western Blot in human Hypopharyngeal Cancer Cell Line
Western blotting was performed on the 3 patients, and EGFR was strongly expressed in the carcinoma cells and not in the normal tissues (Fig. 2).
Fig. 2. Expression of EGFR in human pharyngeal cancer tissues. Western blotting demonstrated that EGFR was strong expressed in carcinomas in comparison with normal tissues.
The expression of EGFR mRNA by FaDu cells was detected by RT-PCR, and the EGFR protein content was detected by Western blotting. However, EGF was not detected by either RT-PCR or Western blotting (Fig. 3).
Fig. 3. Analysis of expression of EGF and EGFR in the FaDu cell line. A: RT-PCR:
The expression of EGF and EGFR mRNA was measured by RT-PCR in the hypopharyngeal cancer line FaDu. B: Western blotting: Detection of the EGFR protein in FaDu cells by Western blotting. EGF was not detected by either RT-PCR or Western blot analysis.
C. Effect of EGF on proliferation of FaDu cells
EGF stimulated statistically significant increase in proliferation of FaDu cells on 3rd and 5th days respectively (p<0.05). It showed the result that the case of EGF 30ng/ ㎖ considerably increased the proliferation than the case of control and 10ng/㎖ on 3rd days (Fig. 4).
Fig. 4. Proliferative acitivity of EGF. Proliferative assay of FaDu cells after treatment with EGF for 5 days. Exogenous EGF significantly enhanced the growth of FaDu in a dose-dependent manner(* : p<0.05. calculated by one-way ANOVA).
D. Effect of EGFR on colony dispersion
In the control, there was no significant colony dispersion of the FaDu cells. In EGF treated groups, the FaDu cells dissociated 24 hours after treatment (Fig. 5). After 6 hours, the formation of actin microspikes (filopodia) and membrane ruffling (lamellipodia) were observed, and cell shape changed to the spindle-like features (Fig. 4). This result suggests that EGF may be associated with scattering. Although we did not measure the objective scattering effect according to the concentration of EGF, 30 ng/㎖ seemed to produce a more potent scattering effect than 10 ng/㎖ 24 hours after EGF stimulation.
Fig. 5. Scatter activity of EGF. FaDu cells were cultured (A) without treatment (control), (B) with EGF 10 ng/㎖, or (C) EGF 30 ng/㎖ for 24 hours (Original magnification, 150x).
In control group, significant colony dispersion of FaDu cells didn’t happen. In EGF treated groups, EGF significantly increased dispersion of FaDu cells. EGF 30 ng/㎖ seemed to be more potent scattering effect than EGF 10 ng/㎖ at 24 hours after EGF stimulation.
E. Effect of EGF on Wound Healing assay
In order to assess the contributions of EGF to both migratory and proliferative activities, we performed the in vitro wound healing assay of FaDu cells with EGF(0, 10ng/㎖, 30ng/㎖).
Exogenous EGF significantly enhanced the migration and proliferation of FaDu in dose dependant manner (Fig. 6). EGF 10 ng/㎖ led to a mean 1.65±0.93-fold increase in cell migration (p < 0.05 versus control), and EGF 30 ng/㎖ led to a mean 4.42±2.59-fold increase in cell migration (p < 0.05 versus control) . EGF 30 ng/㎖ led to a mean 2.67±1.22-fold increase in cell migration (p < 0.05 versus EGF 10 ng/㎖).
Fig. 6. Wound healing assay of EGF. Wound healing assay of FaDu cells after treatment of EGF. Exogenous EGF enhanced the migration and proliferation of FaDu in dose-dependant manner (p<0.05. calculated by one-way ANOVA). A: microscopic findings, B:
graph
F. Effect of EGFR on Cell Invasion
The effect of EGF on FaDu cell invasion was evaluated using a Type 1 collagen-coated Transwell invasion assay. EGF at 10 ng/㎖ led to a mean 4±1.5-fold increase in cell invasion (p < 0.05 versus control), and EGF at 30 ng/㎖ led to a mean 8.5±2-fold increase in cell invasion (p < 0.05 versus control) (Fig. 7). EGF 30 ng/㎖ led to a mean 2.5±0.5-fold increase in cell invasion (p < 0.05 versus EGF 10 ng/㎖).
Fig. 7. Invasion assay of EGF. A,B: Invasion assay of FaDu cells using a Transwell chamber after treatment with EGF. FaDu cells seeded on the upper membrane in the presence (10, 30 ng/㎖) or absence of recombinant EGF in the lower compartment. After a 48-hour incubation, plugged cells in the 8-µm pore or cells attached to the undersurface or the membrane were counted. The bars show the SD of triplicate samples. Data are representative of 3 separate experiments with similar results. EGF significantly promoted the invasion ability of FaDu cells in a dose-dependent manner. *p < 0.05 versus untreated cells. ** p < 0.05 versus cells treated with 10. ng/㎖
G. RT-PCR of MMP-2 and MMP-9
RT-PCR of FaDu cells after EGF treatment for 24 hours showed a marked increase in MMP-2. There was no significant difference between EGF at 10 ng/㎖ and EGF at 30 ng/
㎖. In the case of MMP-9 in FaDu cells after EGF treatment, RT-PCR showed a slight increase in the level of MMP-9 for the EGF treatment group as compared to the control group (Fig. 8A).
Fig. 8. Induction of MMP-2 and MMP-9 activities by EGF. A: RT-PCR of MMP-2 and MMP-9 in FaDu cells. Detection of expression of MMP-2 and MMP-9 in FaDu cells treated for 24 hours with 0, 10, and 30 ng/㎖ EGF. Exogenous EGF slightly enhanced MMP-2 and MMP-9 expression. B: Zymography. FaDu cells were serum-deprived for 48 hours, and then incubated with fresh medium that contained EGF (10 or 30 ng/㎖). The conditioned media were collected after 24 and 48 hours. The samples were fractionated on a polyacrylamide gel that contained 0.1% gelatin, and a zymogram was developed as described in the Materials and Methods section. The 92- and 72-kDa gelatinase activity bands, as determined relative to the molecular weight standards, are indicated by the solid arrows. The level of MMP-2 and MMP-9 activity was increased after EGF treatment for 24 hours as compared to the control group. However, there was no significant difference between the 10 and 30 ng/㎖ EGF treatment groups.
H. Zymography of MMP-2 and MMP-9
MMP-2 activity was slightly increased after 24 hours in the EGF treatment group as compared to the control group. There was no significant difference between EGF at 10 ng/
㎖ and EGF at 30 ng/㎖. MMP-9 activity after EGF treatment was increased at 24 hours, although there was no significant difference between EGF at 10 ng/㎖ and EGF at 30 ng/㎖.
(Fig. 8B).
IV. Discussion
EGFR is a glycoprotein of 170 kDa, encoded by a gene located on chromosome 7p12 (Davies, et al,1980). The EGFR is a member of the erbB family of receptor tyrosine kinase proteins, which also includes HER2/neu (erbB2), HER3 (erbB3), and HER4 (erbB4). These receptors are composed of an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain and, with the exception of HER2, all bind to receptor-specific ligands. Phosphorylation of the tyrosine kinase domain followed by homodimerization or heterodimerization between different receptors of the same family leads to protein activation(Lemmon and Schlessinger,1994). Receptor dimerization is promoted by ligand binding, high receptor density from over-expression, and mutations in the kinase domain. Protein activation on the cell surface of cancer cells is believed to promote signaling cascades, cell growth, differentiation, cell survival (apoptosis), drug and radiation sensitivity, cell cycle progression, and angiogenesis(Ono and Kuwano,2006).
In resting, nontransformed cells, EGFR signaling is tightly controlled. However, oncogenic activation of this pathway occurs as a result of EGFR mutation, overexpression, structural rearrangements, and/or relief of its normal autoinhibitory and regulatory constraints(Arteaga,2001). The evidence to support a role for the EGFR proto-oncogene in transformation was provided by the demonstration that the EGFR is the cellular homolog (proto-oncogene) of the avian erythroblastosis virus v-erbB oncogene (AEV). AEV encodes a C-terminus truncated form of erbB1 (v-erbB) that lacks the EC domain and exhibits several intracellular mutations, resulting in ligand-independent dimerization and phosphorylation(Downward, et al,1984). Binding of EGF-superfamily growth factors to EGF-R activates four major pathways, that is, the phosphatidylinositol-3 kinase (PI-3 kinase), signal transducer and activator of transcription (STAT), phospholipase C-protein kinase C (PLC-PKC), and Ras-mitogen-activated protein kinase (Ras-MAPK) pathways(Prenzel, et al,2001;Yarden and Sliwkowski,2001). All these pathways have been implicated in growth control and survival. In addition, EGF-R-mediated activation of PLC and MAPKs have been linked to migration and invasion (Chen, et al,1994;Cheresh, et al,1999;Glading, et al,2000;Xie, et al,1998).
Several other human cancers, including cancers of colon, pancreas, breast, ovary, bladder, kidney, and gliomas, display EGFR RNA and/or protein overexpression. This occurs with or without EGFR gene amplification and often is associated with increased expression of TGF or amphiregulin(Ekstrand, et al,1991;Hirai, et al,1998;Klijn, et al,1992;Rubin Grandis, et al,1998;Rusch, et al,1993;Salomon, et al,1995;Tateishi, et al,1990;Yamanaka, et al,1993;Yonemura, et al,1992). In some of these studies, EGFR-positive tumors that coexpressed receptor ligands exhibited higher proliferation and tumor grade and a worse survival than EGFR-expressing tumors without coexpression of receptor ligands(Hirai, et al,1998;Tateishi, et al,1990;Yonemura, et al,1992).
Growth and differentiation of HNSCC are regulated by several growth factors and their surface receptors. EGFR is up-regulated in several carcinomas. It is a transmembrane glycoprotein encoded by c-erb-B2 proto-oncogene. This protein is expressed at a low level in many normal human tissues, but activation of the c-erb-B2 oncogene results in its over-expression, seen in many human cancers. The density of EGFR varies from none on lymphoid cells to a high of 250 000/cell on keratinocytes(Cowley, et al,1984;Cowley, et al,1986;Gusterson, et al,1985). In HNSCC, EGFR is not only an independent prognostic factor of outcome in multivariate analysis, but also a first choice therapeutic target. The recent demonstration of a significant survival benefit when combining cetuximab with external radiation therapy is a major breakthrough in the management of HNSCC, establishing a new treatment option for locally advanced HNSCC. This trial provided also an important proof of principle that targeting a pertinent signaling pathway can enhance the radiation response of tumors. However, the improvement in the loco-regional control rate has been modest (within the range achieved with concurrent radiotherapy and chemotherapy) and more than half of patients receiving radiotherapy plus cetuximab still experienced local-regional relapse (Bonner, et al,2006). Therefore, there is a need to further improve outcome. Ongoing clinical efforts are devoted to address whether the addition of cetuximab to concurrent chemoradiation can yield a better outcome (i.e., RTOG study 0522). At this point, it has to be reminded that cancer cells rely on several, sometimes, redundant activation pathways; EGFR is only one of them. The risk of treatment failure is real, if only one receptor is targeted, hence the interest in combining broader range tyrosine
kinase inhibitors such as CI-1033, which targets all four members of the Erb family (pan ErbB)(Zimmermann, et al,2006). In head and neck cancer patients receiving 5-fluorouracil it was known that EGFR remains the tumor parameter with the highest prognostic impact(Etienne, et al,1999). The relationship between EGFR over-expression, increased tumour size and/or local extent of primary tumors (T classification) has been described in previous studies(Putti, et al,2002). And in other study multivalate analysis showed that N status was the most powerful clinical prognostic factor and the EGFR level of expression appears to be a major prognostic factor in the population of patients with an resectable larynx and hypopharynx cancer treated by induction chemotherapy and radiotherapy. In several studies it was suggested that EGFR expression correlates with prognosis in advanced laryngeal carcinoma (stage III and IV)(Smith, et al,2001). On the other hand, several other studies also showed no significant correlation between EGFR expression and tumour prognosis in HNSCC(Kusukawa, et al,1996). Logically, it is justified to target the cellular factor linked with the unfavorable tumor outcome at a selected group of patients.
Currently, two options are under clinical development using specific inhibitors of the EGFR tyrosine kinase enzyme and monoclonal antibodies directed at the external domain of the EGFR. The inhibitors of the EGFR tyrosine kinase stop the autophosphorylation of the EGFR, and block the mitotic signal driven by EGFR activation. Among them, ZD1839 (Iressa™) and OSI774 (tarceva™) have demonstrated activity alone and in combination with cytotoxic drugs in a variety of cell lines and xenograft tumors and promising results have been obtained in early clinical studies(Ciardiello, et al,2000;LoRusso, et al,2003;Magne, et al,2003;Magne, et al,2002;Magne, et al,2002;Magne, et al,2003;Soulieres, et al,2004). Monoclonal antibodies targeting EGFR are available (C225, Cetuximab™) and have demonstrated their effectiveness against a human tumor xenograft in animal models. Early clinical trials have shown significant clinical activity in combination with cisplatin or with radiotherapy in head and neck cancer patients(Baselga, et al,2000;Herbst and Hong,2002;Robert, et al,2001;Shin, et al,2001).
In this study, immunohistochemical staining was performed on 57 cases of hypopharynx cancer to analyze the expression of EGFR. Overexpression of EGFR was noted in 63.2% of the cases. EGFR expression was significantly increased in the cases of advanced stage
tumors. Furthermore, in cases of overexpression of EGFR, rate of distant metastasis seemed to increase, and we believe that a statistically significant correlation may exist with a larger study population. From the fact that EGFR was more strongly expressed in the cancer cells, it is thought that EGFR activation is associated with the progression of cancer. In this study, even in the cases where EGFR expression was not observed, it is surmised that though EGFR were present, but could not be observed because it was too small of an amount in the paraffin embedded tissue to be immunohistochemically stained. Therefore, instead of using only immunohistochemical staining, further studies measuring the concentration of EGFR in the tissue or using Western blotting to determine the relationship between the concentration of EGF and EGFR, and their expression are warranted. Although there was no statistical significance between the expression of EGFR and the survival rate in this study, as the p-value was 0.053, it is believed that correlation may be confirmed by using a larger study population.
Degradation of ECM, which is a necessary step in tissue remodeling processes, such as wound healing and embryonal development has been attributed to proteolytic activity of MMPs. The role of EGF-R-mediated signals in regulating matrix degradation has not been
Degradation of ECM, which is a necessary step in tissue remodeling processes, such as wound healing and embryonal development has been attributed to proteolytic activity of MMPs. The role of EGF-R-mediated signals in regulating matrix degradation has not been