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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

Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease (p < 0.05, paired t-test). (D) Western blot analysis of SCC9 cells stimulated for 10 min with HGF, MAD-NT CM, MAD-NT CM plus a HGF-blocking antibody and of SCC9 cells pretreated with the Met inhibitor PHA-665752. Immunoblots for pSAPK/JNK, pAkt, pp38 MAPK and pErk1/2 are shown. Tubulin served as loading control.
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Fig2: Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease (p < 0.05, paired t-test). (D) Western blot analysis of SCC9 cells stimulated for 10 min with HGF, MAD-NT CM, MAD-NT CM plus a HGF-blocking antibody and of SCC9 cells pretreated with the Met inhibitor PHA-665752. Immunoblots for pSAPK/JNK, pAkt, pp38 MAPK and pErk1/2 are shown. Tubulin served as loading control.

Mentions: Consistent with our previous findings, although the EGFR ligand amphiregulin is present in the cell culture medium, the EGF receptor does not become phosphorylated on tyrosine residues after HGF treatment [32] (Figure 2A). Therefore, in order to test if the released amphiregulin is capable of activating EGFR, the following two-step experiment was performed: first, SCC9 cells were stimulated with HGF. After 24 hours the conditioned medium (=CM) was collected. Second, fresh, untreated SCC9 cells were stimulated for three minutes with this CM, lysed and immunobloted for phospho-tyrosine and EGFR. If active EGFR ligands were shed, an activation of the EGFR should occur now. Indeed, strong EGFR and HER2 phosphorylation was observed and a blocking antibody experiment (verification of antibody specificity in Additional file 2: Figure S2) revealed amphiregulin to be the major contributor of activation (Figure 2B).Figure 2


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)

Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease (p < 0.05, paired t-test). (D) Western blot analysis of SCC9 cells stimulated for 10 min with HGF, MAD-NT CM, MAD-NT CM plus a HGF-blocking antibody and of SCC9 cells pretreated with the Met inhibitor PHA-665752. Immunoblots for pSAPK/JNK, pAkt, pp38 MAPK and pErk1/2 are shown. Tubulin served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359546&req=5

Fig2: Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease (p < 0.05, paired t-test). (D) Western blot analysis of SCC9 cells stimulated for 10 min with HGF, MAD-NT CM, MAD-NT CM plus a HGF-blocking antibody and of SCC9 cells pretreated with the Met inhibitor PHA-665752. Immunoblots for pSAPK/JNK, pAkt, pp38 MAPK and pErk1/2 are shown. Tubulin served as loading control.
Mentions: Consistent with our previous findings, although the EGFR ligand amphiregulin is present in the cell culture medium, the EGF receptor does not become phosphorylated on tyrosine residues after HGF treatment [32] (Figure 2A). Therefore, in order to test if the released amphiregulin is capable of activating EGFR, the following two-step experiment was performed: first, SCC9 cells were stimulated with HGF. After 24 hours the conditioned medium (=CM) was collected. Second, fresh, untreated SCC9 cells were stimulated for three minutes with this CM, lysed and immunobloted for phospho-tyrosine and EGFR. If active EGFR ligands were shed, an activation of the EGFR should occur now. Indeed, strong EGFR and HER2 phosphorylation was observed and a blocking antibody experiment (verification of antibody specificity in Additional file 2: Figure S2) revealed amphiregulin to be the major contributor of activation (Figure 2B).Figure 2

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