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Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines.

Gusenbauer S, Zanucco E, Knyazev P, Ullrich A - Mol. Cancer (2015)

Bottom Line: Met activation correlates with poor patient outcome.Amphiregulin is transcriptionally upregulated and is released into the supernatant.We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany. gusenbau@biochem.mpg.de.

ABSTRACT

Background: Squamous cell carcinoma (SCC) is the most common type of tongue and larynx cancer and a common type of lung cancer. In this study, we attempted to specifically evaluate the signaling pathway underlying HGF/Met induced EGFR ligand release in SSCs. The Met proto-oncogene encodes for a tyrosine kinase receptor which is often hyperactivated in human cancers. Met activation correlates with poor patient outcome. Several studies revealed a role of Met in receptor-crosstalk inducing either activation of other receptors, or inducing their resistance to targeted cancer treatments. In an epithelial tumor cell line screen we recently showed that the Met ligand HGF blocks the EGFR tyrosine kinase and at the same time activates transcriptional upregulation and accumulation in the supernatant of the EGFR ligand amphiregulin (Oncogene 32:3846-56, 2013). In the present work we describe the pathway responsible for the amphiregulin induction.

Findings: Amphiregulin is transcriptionally upregulated and is released into the supernatant. We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF. A siRNA knockdown of Erk2 completely abolishes amphiregulin release in squamous cell carcinomas.

Conclusions: These results identify Erk2 as the key downstream signal transducer between Met activation and EGFR ligand upregulation in squamous cell carcinoma cell lines derived from tongue, larynx and lung.

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Related in: MedlinePlus

The MAPK pathway regulates amphiregulin induction and amphiregulin release upon HGF stimulation depends on amphiregulin protein synthesis. (A) Quantification of amphiregulin protein release in different SCC cell lines treated with HGF for 24 h. Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (B) Quantification of amphiregulin mRNA induction. SCC9 cells were treated with 100 ng/ml HGF for the indicated time points. Data represent the increase of amphiregulin normalized to HPRT1 cDNA. Values are shown as mean ± SD (n = 2). (C) Quantification of amphiregulin protein release. Values are shown as mean ± SD (n = 2). (D) Quantification of amphiregulin protein release. SCC9 cells were treated with 100 ng/ml HGF and with the translation inhibitors cycloheximide (=CHX; 1 μg/ml) and geneticin (=G418; 1 mg/ml) for 24 h. Ligand release into the supernatant was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (E) Quantification of amphiregulin mRNA induction. SCC9 cells were pretreated with 5 μM of the MEK inhibitor UO126 and 50 nM of the PI3K inhibitor wortmannin (=W) for 15 min before 2 h HGF treatment. (F) Quantification of amphiregulin protein release. SCC9 cells were pretreated for 15 min with UO126 and wortmannin before 24 h HGF treatment. Error bars indicate SEM of three independent experiments. Asterisks indicate statistically significant repression or induction (p < 0.05, paired t-test).
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Fig1: The MAPK pathway regulates amphiregulin induction and amphiregulin release upon HGF stimulation depends on amphiregulin protein synthesis. (A) Quantification of amphiregulin protein release in different SCC cell lines treated with HGF for 24 h. Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (B) Quantification of amphiregulin mRNA induction. SCC9 cells were treated with 100 ng/ml HGF for the indicated time points. Data represent the increase of amphiregulin normalized to HPRT1 cDNA. Values are shown as mean ± SD (n = 2). (C) Quantification of amphiregulin protein release. Values are shown as mean ± SD (n = 2). (D) Quantification of amphiregulin protein release. SCC9 cells were treated with 100 ng/ml HGF and with the translation inhibitors cycloheximide (=CHX; 1 μg/ml) and geneticin (=G418; 1 mg/ml) for 24 h. Ligand release into the supernatant was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (E) Quantification of amphiregulin mRNA induction. SCC9 cells were pretreated with 5 μM of the MEK inhibitor UO126 and 50 nM of the PI3K inhibitor wortmannin (=W) for 15 min before 2 h HGF treatment. (F) Quantification of amphiregulin protein release. SCC9 cells were pretreated for 15 min with UO126 and wortmannin before 24 h HGF treatment. Error bars indicate SEM of three independent experiments. Asterisks indicate statistically significant repression or induction (p < 0.05, paired t-test).

Mentions: Here, we attempted to specifically investigate the signaling pathway underlying HGF/Met induced EGFR ligand release in SCCs derived from different tissues. Amphiregulin protein release upon HGF stimulation could be observed in SCCs of the tongue, lung and larynx (Figure 1A). In order to investigate which signal transducer downstream of Met activation mediates the upregulation of amphiregulin, we used, due to the high amphiregulin production, SCC9 cells as a preliminary model system. The amphiregulin transcript induction peaked within the first two hours after HGF stimulation (Figure 1B). Amphiregulin protein accumulation started after 4–8 hours and peaked after 24 hours (Figure 1C). To test whether the amphiregulin release depends on new protein synthesis or on shedding of existing pro-forms, the effect of the translation inhibitors cycloheximide (=CHX) and geneticin (=G418) was investigated. Both inhibitors abrogated amphiregulin release into the supernatant, suggesting that amphiregulin release fully depends on new protein synthesis (Figure 1D). Furthermore, SCC9 cells were incubated with inhibitors for MEK and for PI3 kinase, prior to HGF stimulation. mRNA levels of amphiregulin were measured after 2 hours and protein levels were measured after 24 hours of stimulation. The inhibitor specificity and efficacy was analyzed 5 minutes after HGF stimulation and is shown in Additional file 1: Figure S1. Notably, full inhibition of amphiregulin mRNA (Figure 1E) and protein (Figure 1F) induction was achieved with the MEK inhibitor UO126, while only a minor effect was observed with the PI3K inhibitor at the protein level (Figure 1F). These experiments prove the regulation on transcript level and reveal a MAPK-pathway-dependent amphiregulin production.Figure 1


Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines.

Gusenbauer S, Zanucco E, Knyazev P, Ullrich A - Mol. Cancer (2015)

The MAPK pathway regulates amphiregulin induction and amphiregulin release upon HGF stimulation depends on amphiregulin protein synthesis. (A) Quantification of amphiregulin protein release in different SCC cell lines treated with HGF for 24 h. Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (B) Quantification of amphiregulin mRNA induction. SCC9 cells were treated with 100 ng/ml HGF for the indicated time points. Data represent the increase of amphiregulin normalized to HPRT1 cDNA. Values are shown as mean ± SD (n = 2). (C) Quantification of amphiregulin protein release. Values are shown as mean ± SD (n = 2). (D) Quantification of amphiregulin protein release. SCC9 cells were treated with 100 ng/ml HGF and with the translation inhibitors cycloheximide (=CHX; 1 μg/ml) and geneticin (=G418; 1 mg/ml) for 24 h. Ligand release into the supernatant was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (E) Quantification of amphiregulin mRNA induction. SCC9 cells were pretreated with 5 μM of the MEK inhibitor UO126 and 50 nM of the PI3K inhibitor wortmannin (=W) for 15 min before 2 h HGF treatment. (F) Quantification of amphiregulin protein release. SCC9 cells were pretreated for 15 min with UO126 and wortmannin before 24 h HGF treatment. Error bars indicate SEM of three independent experiments. Asterisks indicate statistically significant repression or induction (p < 0.05, paired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: The MAPK pathway regulates amphiregulin induction and amphiregulin release upon HGF stimulation depends on amphiregulin protein synthesis. (A) Quantification of amphiregulin protein release in different SCC cell lines treated with HGF for 24 h. Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (B) Quantification of amphiregulin mRNA induction. SCC9 cells were treated with 100 ng/ml HGF for the indicated time points. Data represent the increase of amphiregulin normalized to HPRT1 cDNA. Values are shown as mean ± SD (n = 2). (C) Quantification of amphiregulin protein release. Values are shown as mean ± SD (n = 2). (D) Quantification of amphiregulin protein release. SCC9 cells were treated with 100 ng/ml HGF and with the translation inhibitors cycloheximide (=CHX; 1 μg/ml) and geneticin (=G418; 1 mg/ml) for 24 h. Ligand release into the supernatant was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. (E) Quantification of amphiregulin mRNA induction. SCC9 cells were pretreated with 5 μM of the MEK inhibitor UO126 and 50 nM of the PI3K inhibitor wortmannin (=W) for 15 min before 2 h HGF treatment. (F) Quantification of amphiregulin protein release. SCC9 cells were pretreated for 15 min with UO126 and wortmannin before 24 h HGF treatment. Error bars indicate SEM of three independent experiments. Asterisks indicate statistically significant repression or induction (p < 0.05, paired t-test).
Mentions: Here, we attempted to specifically investigate the signaling pathway underlying HGF/Met induced EGFR ligand release in SCCs derived from different tissues. Amphiregulin protein release upon HGF stimulation could be observed in SCCs of the tongue, lung and larynx (Figure 1A). In order to investigate which signal transducer downstream of Met activation mediates the upregulation of amphiregulin, we used, due to the high amphiregulin production, SCC9 cells as a preliminary model system. The amphiregulin transcript induction peaked within the first two hours after HGF stimulation (Figure 1B). Amphiregulin protein accumulation started after 4–8 hours and peaked after 24 hours (Figure 1C). To test whether the amphiregulin release depends on new protein synthesis or on shedding of existing pro-forms, the effect of the translation inhibitors cycloheximide (=CHX) and geneticin (=G418) was investigated. Both inhibitors abrogated amphiregulin release into the supernatant, suggesting that amphiregulin release fully depends on new protein synthesis (Figure 1D). Furthermore, SCC9 cells were incubated with inhibitors for MEK and for PI3 kinase, prior to HGF stimulation. mRNA levels of amphiregulin were measured after 2 hours and protein levels were measured after 24 hours of stimulation. The inhibitor specificity and efficacy was analyzed 5 minutes after HGF stimulation and is shown in Additional file 1: Figure S1. Notably, full inhibition of amphiregulin mRNA (Figure 1E) and protein (Figure 1F) induction was achieved with the MEK inhibitor UO126, while only a minor effect was observed with the PI3K inhibitor at the protein level (Figure 1F). These experiments prove the regulation on transcript level and reveal a MAPK-pathway-dependent amphiregulin production.Figure 1

Bottom Line: Met activation correlates with poor patient outcome.Amphiregulin is transcriptionally upregulated and is released into the supernatant.We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany. gusenbau@biochem.mpg.de.

ABSTRACT

Background: Squamous cell carcinoma (SCC) is the most common type of tongue and larynx cancer and a common type of lung cancer. In this study, we attempted to specifically evaluate the signaling pathway underlying HGF/Met induced EGFR ligand release in SSCs. The Met proto-oncogene encodes for a tyrosine kinase receptor which is often hyperactivated in human cancers. Met activation correlates with poor patient outcome. Several studies revealed a role of Met in receptor-crosstalk inducing either activation of other receptors, or inducing their resistance to targeted cancer treatments. In an epithelial tumor cell line screen we recently showed that the Met ligand HGF blocks the EGFR tyrosine kinase and at the same time activates transcriptional upregulation and accumulation in the supernatant of the EGFR ligand amphiregulin (Oncogene 32:3846-56, 2013). In the present work we describe the pathway responsible for the amphiregulin induction.

Findings: Amphiregulin is transcriptionally upregulated and is released into the supernatant. We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF. A siRNA knockdown of Erk2 completely abolishes amphiregulin release in squamous cell carcinomas.

Conclusions: These results identify Erk2 as the key downstream signal transducer between Met activation and EGFR ligand upregulation in squamous cell carcinoma cell lines derived from tongue, larynx and lung.

Show MeSH
Related in: MedlinePlus