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Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression

View Article: PubMed Central - PubMed

ABSTRACT

Background: Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated.

Methods: To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines.

Results: The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines.

Conclusions: Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment.

Trial registration: Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck.

No MeSH data available.


EGF and HBEGF induced AREG expression and actuated MAPK and PI3K/Akt pathways in OSCC cell lines. a Both EGF and HBEGF stimulated AREG mRNA expression after two h, peaked at four h and disappeared after 24 h (left panel, Student’s t-test, ***p < 0.001). This was accompanied by increased AREG protein concentration in the supernatant during the following 48 h (right panel; untreated cells were used as control group; Student’s t-test, ***p < 0.001). A representative experiments in the D2 cell line is shown. b EGF induced phosphorylation of the EGF receptor, ERK, p38, JNK, Akt and ErbB2 could be completely inhibited by specific kinase inhibitors. Western blotting from a representative experiment using the D2 cell line
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Fig4: EGF and HBEGF induced AREG expression and actuated MAPK and PI3K/Akt pathways in OSCC cell lines. a Both EGF and HBEGF stimulated AREG mRNA expression after two h, peaked at four h and disappeared after 24 h (left panel, Student’s t-test, ***p < 0.001). This was accompanied by increased AREG protein concentration in the supernatant during the following 48 h (right panel; untreated cells were used as control group; Student’s t-test, ***p < 0.001). A representative experiments in the D2 cell line is shown. b EGF induced phosphorylation of the EGF receptor, ERK, p38, JNK, Akt and ErbB2 could be completely inhibited by specific kinase inhibitors. Western blotting from a representative experiment using the D2 cell line

Mentions: Since cancer extracellular matrix releases EGF and HBEGF [25], and AREG correlated to HBEGF expression, we hypothesized that extracellular EGF and/or HBEGF induced AREG overexpression in OSCC. We therefore treated three OSCC cell lines, C12, D2 and E10, with EGF and HBEGF and observed that both factors induced AREG mRNA and protein increase in the same manner (Fig. 4a). Thus, we used EGF stimulation to examine how AREG overexpression is controlled in OSCC. As shown in Fig. 4b, EGF-stimulation induced Erk1/2, p38, JNK, Akt, EGFR and ErbB2 phosphorylation. The activation of each of these pathways could be blocked by specific kinase inhibitors.Fig. 4


Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression
EGF and HBEGF induced AREG expression and actuated MAPK and PI3K/Akt pathways in OSCC cell lines. a Both EGF and HBEGF stimulated AREG mRNA expression after two h, peaked at four h and disappeared after 24 h (left panel, Student’s t-test, ***p < 0.001). This was accompanied by increased AREG protein concentration in the supernatant during the following 48 h (right panel; untreated cells were used as control group; Student’s t-test, ***p < 0.001). A representative experiments in the D2 cell line is shown. b EGF induced phosphorylation of the EGF receptor, ERK, p38, JNK, Akt and ErbB2 could be completely inhibited by specific kinase inhibitors. Western blotting from a representative experiment using the D2 cell line
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5037594&req=5

Fig4: EGF and HBEGF induced AREG expression and actuated MAPK and PI3K/Akt pathways in OSCC cell lines. a Both EGF and HBEGF stimulated AREG mRNA expression after two h, peaked at four h and disappeared after 24 h (left panel, Student’s t-test, ***p < 0.001). This was accompanied by increased AREG protein concentration in the supernatant during the following 48 h (right panel; untreated cells were used as control group; Student’s t-test, ***p < 0.001). A representative experiments in the D2 cell line is shown. b EGF induced phosphorylation of the EGF receptor, ERK, p38, JNK, Akt and ErbB2 could be completely inhibited by specific kinase inhibitors. Western blotting from a representative experiment using the D2 cell line
Mentions: Since cancer extracellular matrix releases EGF and HBEGF [25], and AREG correlated to HBEGF expression, we hypothesized that extracellular EGF and/or HBEGF induced AREG overexpression in OSCC. We therefore treated three OSCC cell lines, C12, D2 and E10, with EGF and HBEGF and observed that both factors induced AREG mRNA and protein increase in the same manner (Fig. 4a). Thus, we used EGF stimulation to examine how AREG overexpression is controlled in OSCC. As shown in Fig. 4b, EGF-stimulation induced Erk1/2, p38, JNK, Akt, EGFR and ErbB2 phosphorylation. The activation of each of these pathways could be blocked by specific kinase inhibitors.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis&nbsp;and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated.

Methods: To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines.

Results: The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26&nbsp;% if tumor expressed none to one EGFR-ligand, to 45&nbsp;% in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines.

Conclusions: Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment.

Trial registration: Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck.

No MeSH data available.