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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.


Related in: MedlinePlus

The intracellular pathways in EGF-induced AREG expression. The EGF-induced AREG mRNA (a) and protein (b) expression was reduced after EGFR-kinase inhibition in all cell lines, while it showed no reduction after ErbB2-kinase inhibition and was differently affected by the MAPK inhibitors. a Whereas MEK-inhibition reduced EGF-induced AREG mRNA expression in all the three cell lines, p38- and JNK-inhibition did not. PI3K-inhibition increased EGF-induced AREG expression in all cell lines. b EGF-induced AREG protein secretion could be totally blocked by EGFR- inhibition. Whereas MEK- and JNK-inhibition profoundly decrease EGF-induced AREG expression in all cell lines, p38-inhibition had no effect. The PI3K-inhibitor reduced EGF-induced AREG production in the conventional OSCC cell line D2 and E10, but had no effect on the basaloid OSSC cell line C12. The ErbB-2- inhibition inhibited the AREG increase in E10 cell line, only. *represents significant difference with EGF stimulation groups without inhibitors (student’s t-test)
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Fig5: The intracellular pathways in EGF-induced AREG expression. The EGF-induced AREG mRNA (a) and protein (b) expression was reduced after EGFR-kinase inhibition in all cell lines, while it showed no reduction after ErbB2-kinase inhibition and was differently affected by the MAPK inhibitors. a Whereas MEK-inhibition reduced EGF-induced AREG mRNA expression in all the three cell lines, p38- and JNK-inhibition did not. PI3K-inhibition increased EGF-induced AREG expression in all cell lines. b EGF-induced AREG protein secretion could be totally blocked by EGFR- inhibition. Whereas MEK- and JNK-inhibition profoundly decrease EGF-induced AREG expression in all cell lines, p38-inhibition had no effect. The PI3K-inhibitor reduced EGF-induced AREG production in the conventional OSCC cell line D2 and E10, but had no effect on the basaloid OSSC cell line C12. The ErbB-2- inhibition inhibited the AREG increase in E10 cell line, only. *represents significant difference with EGF stimulation groups without inhibitors (student’s t-test)

Mentions: AREG mRNA level showed a peak four h after EGF-stimulation (Fig. 4a), which was then used to examine the dynamics in EGF-induced AREG mRNA expression. Whereas the EGFR-kinase inhibitor (AG1478) attenuated EGF-induced AREG mRNA expression, the ErbB2 kinase inhibitor (AG825) did not (Fig. 5a). Whereas the MAPK/ERK kinase (MEK) inhibitor (PD98050) reduced EGF-induced AREG mRNA expression, the PI3K inhibitor (LY294002) increased it in all three cell lines. The p38 inhibitor (SB203580) revealed a differential response pattern as it increased EGF-induced AREG mRNA expression in the cell lines C12 (basaloid SCC) and E10, only. The JNK-inhibition (SP600125) had similar effect but only in the two conventional OSCC cell lines D2 and E10.Fig. 5


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
The intracellular pathways in EGF-induced AREG expression. The EGF-induced AREG mRNA (a) and protein (b) expression was reduced after EGFR-kinase inhibition in all cell lines, while it showed no reduction after ErbB2-kinase inhibition and was differently affected by the MAPK inhibitors. a Whereas MEK-inhibition reduced EGF-induced AREG mRNA expression in all the three cell lines, p38- and JNK-inhibition did not. PI3K-inhibition increased EGF-induced AREG expression in all cell lines. b EGF-induced AREG protein secretion could be totally blocked by EGFR- inhibition. Whereas MEK- and JNK-inhibition profoundly decrease EGF-induced AREG expression in all cell lines, p38-inhibition had no effect. The PI3K-inhibitor reduced EGF-induced AREG production in the conventional OSCC cell line D2 and E10, but had no effect on the basaloid OSSC cell line C12. The ErbB-2- inhibition inhibited the AREG increase in E10 cell line, only. *represents significant difference with EGF stimulation groups without inhibitors (student’s t-test)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: The intracellular pathways in EGF-induced AREG expression. The EGF-induced AREG mRNA (a) and protein (b) expression was reduced after EGFR-kinase inhibition in all cell lines, while it showed no reduction after ErbB2-kinase inhibition and was differently affected by the MAPK inhibitors. a Whereas MEK-inhibition reduced EGF-induced AREG mRNA expression in all the three cell lines, p38- and JNK-inhibition did not. PI3K-inhibition increased EGF-induced AREG expression in all cell lines. b EGF-induced AREG protein secretion could be totally blocked by EGFR- inhibition. Whereas MEK- and JNK-inhibition profoundly decrease EGF-induced AREG expression in all cell lines, p38-inhibition had no effect. The PI3K-inhibitor reduced EGF-induced AREG production in the conventional OSCC cell line D2 and E10, but had no effect on the basaloid OSSC cell line C12. The ErbB-2- inhibition inhibited the AREG increase in E10 cell line, only. *represents significant difference with EGF stimulation groups without inhibitors (student’s t-test)
Mentions: AREG mRNA level showed a peak four h after EGF-stimulation (Fig. 4a), which was then used to examine the dynamics in EGF-induced AREG mRNA expression. Whereas the EGFR-kinase inhibitor (AG1478) attenuated EGF-induced AREG mRNA expression, the ErbB2 kinase inhibitor (AG825) did not (Fig. 5a). Whereas the MAPK/ERK kinase (MEK) inhibitor (PD98050) reduced EGF-induced AREG mRNA expression, the PI3K inhibitor (LY294002) increased it in all three cell lines. The p38 inhibitor (SB203580) revealed a differential response pattern as it increased EGF-induced AREG mRNA expression in the cell lines C12 (basaloid SCC) and E10, only. The JNK-inhibition (SP600125) had similar effect but only in the two conventional OSCC cell lines D2 and E10.Fig. 5

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.


Related in: MedlinePlus