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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

Erk2 is required for HGF-induced amphiregulin production. (A) Erk1 and Erk2 siRNA knockdown in SCC9 cells. MAD-NT CM was used as HGF source. For the Erk2 knockdown two siRNAs (Erk2#1 and Erk2#2) were used. Knockdown was verified with Erk1/2 immunoblot. (B) Erk2 siRNA knockdown in different SCC cell lines. Non-targeting siRNA was used as negative control. Amphiregulin release was measured with sandwich ELISA. Error bars indicate SEM of three independent experiments. Asterisks indicate a statistically significant decrease (p < 0.05, paired t-test). Knockdown was verified with Erk1/2 immunoblot. Tubulin served as loading control.
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Fig3: Erk2 is required for HGF-induced amphiregulin production. (A) Erk1 and Erk2 siRNA knockdown in SCC9 cells. MAD-NT CM was used as HGF source. For the Erk2 knockdown two siRNAs (Erk2#1 and Erk2#2) were used. Knockdown was verified with Erk1/2 immunoblot. (B) Erk2 siRNA knockdown in different SCC cell lines. Non-targeting siRNA was used as negative control. Amphiregulin release was measured with sandwich ELISA. Error bars indicate SEM of three independent experiments. Asterisks indicate a statistically significant decrease (p < 0.05, paired t-test). Knockdown was verified with Erk1/2 immunoblot. Tubulin served as loading control.

Mentions: To evaluate which MAPK is responsible for the EGFR ligand induction, siRNA knockdown experiments of Erk1 and Erk2 were performed. The CM of MAD-NT cells was used as HGF source. A Knockdown of Erk2 dramatically reduced the release of amphiregulin, whereas a knockdown of Erk1 showed no effect. To exclude the possibility of an off-target effect, two different siRNAs for Erk2 were used. Similar results were obtained with both Erk2 siRNAs. Interestingly a double knockdown of Erk1 and Erk2 further reduced the production of amphiregulin, indicating that Erk1 could partly compensate for the loss of Erk2 (Figure 3A). Moreover Erk2 depletion blocks HGF induced amphiregulin release in tongue-, lung- and larynx-derived SCC cell lines. Notably, although in NCI-H2882 cells an Erk2 knockdown dramatically reduced basal amphiregulin production, HGF stimulation was still able to augment amphiregulin release similar to baseline levels (Figure 3B). Altogether these data suggest that in different human SCCs the upregulation and release of amphiregulin upon HGF stimulation is mediated via Erk2.Figure 3


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)

Erk2 is required for HGF-induced amphiregulin production. (A) Erk1 and Erk2 siRNA knockdown in SCC9 cells. MAD-NT CM was used as HGF source. For the Erk2 knockdown two siRNAs (Erk2#1 and Erk2#2) were used. Knockdown was verified with Erk1/2 immunoblot. (B) Erk2 siRNA knockdown in different SCC cell lines. Non-targeting siRNA was used as negative control. Amphiregulin release was measured with sandwich ELISA. Error bars indicate SEM of three independent experiments. Asterisks indicate a statistically significant decrease (p < 0.05, paired t-test). Knockdown was verified with Erk1/2 immunoblot. Tubulin served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4359546&req=5

Fig3: Erk2 is required for HGF-induced amphiregulin production. (A) Erk1 and Erk2 siRNA knockdown in SCC9 cells. MAD-NT CM was used as HGF source. For the Erk2 knockdown two siRNAs (Erk2#1 and Erk2#2) were used. Knockdown was verified with Erk1/2 immunoblot. (B) Erk2 siRNA knockdown in different SCC cell lines. Non-targeting siRNA was used as negative control. Amphiregulin release was measured with sandwich ELISA. Error bars indicate SEM of three independent experiments. Asterisks indicate a statistically significant decrease (p < 0.05, paired t-test). Knockdown was verified with Erk1/2 immunoblot. Tubulin served as loading control.
Mentions: To evaluate which MAPK is responsible for the EGFR ligand induction, siRNA knockdown experiments of Erk1 and Erk2 were performed. The CM of MAD-NT cells was used as HGF source. A Knockdown of Erk2 dramatically reduced the release of amphiregulin, whereas a knockdown of Erk1 showed no effect. To exclude the possibility of an off-target effect, two different siRNAs for Erk2 were used. Similar results were obtained with both Erk2 siRNAs. Interestingly a double knockdown of Erk1 and Erk2 further reduced the production of amphiregulin, indicating that Erk1 could partly compensate for the loss of Erk2 (Figure 3A). Moreover Erk2 depletion blocks HGF induced amphiregulin release in tongue-, lung- and larynx-derived SCC cell lines. Notably, although in NCI-H2882 cells an Erk2 knockdown dramatically reduced basal amphiregulin production, HGF stimulation was still able to augment amphiregulin release similar to baseline levels (Figure 3B). Altogether these data suggest that in different human SCCs the upregulation and release of amphiregulin upon HGF stimulation is mediated via Erk2.Figure 3

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