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A positive feedback loop between HER2 and ADAM12 in human head and neck cancer cells increases migration and invasion.

Rao VH, Kandel A, Lynch D, Pena Z, Marwaha N, Deng C, Watson P, Hansen LA - Oncogene (2011)

Bottom Line: HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers.Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression.Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA.

ABSTRACT
Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.

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HER2 and EGFR up-regulated ADAM12 in HNSCC cellsA–B) The low ADAM12 expressing cell line UM-SCC 74A was transfected with HER2 or with the empty vector pcDNA. Real-time RT-PCR was performed using primers for ADAM12L, ADAM12S, or HER2 (A). HER2 protein was assessed using immunoblotting (B). C–D) The high ADAM12 expressing cell line UM-SCC 74B and the low ADAM12 expressing cell line UM-SCC 74A (as a negative control) were treated with the vehicle DMSO or with the HER2 inhibitor AG825 at the indicated concentrations for 24 h. Phosphorylation of HER2 (P-HER2) on immunoblot (C) and ADAM12L and ADAM12S transcripts following real-time RT-PCR (D) are shown. E–F) UM-SCC 74A and UM-SCC 74B cells were treated with the EGFR inhibitor AG1478 or with the vehicle DMSO alone. Immunoblotting for phospho-EGFR (P-EGFR)(E) and real-time RT-PCR for ADAM12L and ADAM12S (F) are shown. *Indicates a significant difference compared to the empty vector (A) or vehicle-treated UM-SCC 74B (D,F) controls using a Student's t-test, where p ≤ 0.05.
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Figure 3: HER2 and EGFR up-regulated ADAM12 in HNSCC cellsA–B) The low ADAM12 expressing cell line UM-SCC 74A was transfected with HER2 or with the empty vector pcDNA. Real-time RT-PCR was performed using primers for ADAM12L, ADAM12S, or HER2 (A). HER2 protein was assessed using immunoblotting (B). C–D) The high ADAM12 expressing cell line UM-SCC 74B and the low ADAM12 expressing cell line UM-SCC 74A (as a negative control) were treated with the vehicle DMSO or with the HER2 inhibitor AG825 at the indicated concentrations for 24 h. Phosphorylation of HER2 (P-HER2) on immunoblot (C) and ADAM12L and ADAM12S transcripts following real-time RT-PCR (D) are shown. E–F) UM-SCC 74A and UM-SCC 74B cells were treated with the EGFR inhibitor AG1478 or with the vehicle DMSO alone. Immunoblotting for phospho-EGFR (P-EGFR)(E) and real-time RT-PCR for ADAM12L and ADAM12S (F) are shown. *Indicates a significant difference compared to the empty vector (A) or vehicle-treated UM-SCC 74B (D,F) controls using a Student's t-test, where p ≤ 0.05.

Mentions: To test the hypothesis that HER2 signaling upregulates ADAM12, the low HER2-expressing cell line UM-SCC 74A was transfected with a HER2 mammalian expression construct. Immunoblotting and real-time RT-PCR analyses confirmed increased HER2 following transfection of UM-SCC 74A (Fig. 3A,B). HER2 transfection significantly increased transcripts for both ADAM12L and ADAM12S in the UM-SCC 74A cells (Fig. 3A). AG825, a potent and relatively specific inhibitor of HER2 (Madson et al. 2006), reduced HER2 activity in UM-SCC 74B cells, as detected by HER2 phosphorylation on immunoblot (Fig. 3C). Inhibition of HER2 also reduced ADAM12L and ADAM12S transcripts in a dose-dependent manner, such that ADAM12 transcripts in the high-expressing UM-SCC 74B cell line after treatment with the highest dose were similar to the levels in untreated low-expressing cells (Fig. 3D). Collectively, these experiments demonstrated that HER2 induces both ADAM12L and ADAM12S expression in oral SCC cells. Because EGFR and HER2 can heterodimerize to transduce signals, we also investigated whether EGFR influences ADAM12. EGFR activity was inhibited in UM-SCC 74B cells using AG1478, as shown in Fig. 3E, lanes 5–8. Inhibition of EGFR also reduced ADAM12 transcripts in UM-SCC 74B cells (Fig. 3F), similarly to inhibition of HER2.


A positive feedback loop between HER2 and ADAM12 in human head and neck cancer cells increases migration and invasion.

Rao VH, Kandel A, Lynch D, Pena Z, Marwaha N, Deng C, Watson P, Hansen LA - Oncogene (2011)

HER2 and EGFR up-regulated ADAM12 in HNSCC cellsA–B) The low ADAM12 expressing cell line UM-SCC 74A was transfected with HER2 or with the empty vector pcDNA. Real-time RT-PCR was performed using primers for ADAM12L, ADAM12S, or HER2 (A). HER2 protein was assessed using immunoblotting (B). C–D) The high ADAM12 expressing cell line UM-SCC 74B and the low ADAM12 expressing cell line UM-SCC 74A (as a negative control) were treated with the vehicle DMSO or with the HER2 inhibitor AG825 at the indicated concentrations for 24 h. Phosphorylation of HER2 (P-HER2) on immunoblot (C) and ADAM12L and ADAM12S transcripts following real-time RT-PCR (D) are shown. E–F) UM-SCC 74A and UM-SCC 74B cells were treated with the EGFR inhibitor AG1478 or with the vehicle DMSO alone. Immunoblotting for phospho-EGFR (P-EGFR)(E) and real-time RT-PCR for ADAM12L and ADAM12S (F) are shown. *Indicates a significant difference compared to the empty vector (A) or vehicle-treated UM-SCC 74B (D,F) controls using a Student's t-test, where p ≤ 0.05.
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Figure 3: HER2 and EGFR up-regulated ADAM12 in HNSCC cellsA–B) The low ADAM12 expressing cell line UM-SCC 74A was transfected with HER2 or with the empty vector pcDNA. Real-time RT-PCR was performed using primers for ADAM12L, ADAM12S, or HER2 (A). HER2 protein was assessed using immunoblotting (B). C–D) The high ADAM12 expressing cell line UM-SCC 74B and the low ADAM12 expressing cell line UM-SCC 74A (as a negative control) were treated with the vehicle DMSO or with the HER2 inhibitor AG825 at the indicated concentrations for 24 h. Phosphorylation of HER2 (P-HER2) on immunoblot (C) and ADAM12L and ADAM12S transcripts following real-time RT-PCR (D) are shown. E–F) UM-SCC 74A and UM-SCC 74B cells were treated with the EGFR inhibitor AG1478 or with the vehicle DMSO alone. Immunoblotting for phospho-EGFR (P-EGFR)(E) and real-time RT-PCR for ADAM12L and ADAM12S (F) are shown. *Indicates a significant difference compared to the empty vector (A) or vehicle-treated UM-SCC 74B (D,F) controls using a Student's t-test, where p ≤ 0.05.
Mentions: To test the hypothesis that HER2 signaling upregulates ADAM12, the low HER2-expressing cell line UM-SCC 74A was transfected with a HER2 mammalian expression construct. Immunoblotting and real-time RT-PCR analyses confirmed increased HER2 following transfection of UM-SCC 74A (Fig. 3A,B). HER2 transfection significantly increased transcripts for both ADAM12L and ADAM12S in the UM-SCC 74A cells (Fig. 3A). AG825, a potent and relatively specific inhibitor of HER2 (Madson et al. 2006), reduced HER2 activity in UM-SCC 74B cells, as detected by HER2 phosphorylation on immunoblot (Fig. 3C). Inhibition of HER2 also reduced ADAM12L and ADAM12S transcripts in a dose-dependent manner, such that ADAM12 transcripts in the high-expressing UM-SCC 74B cell line after treatment with the highest dose were similar to the levels in untreated low-expressing cells (Fig. 3D). Collectively, these experiments demonstrated that HER2 induces both ADAM12L and ADAM12S expression in oral SCC cells. Because EGFR and HER2 can heterodimerize to transduce signals, we also investigated whether EGFR influences ADAM12. EGFR activity was inhibited in UM-SCC 74B cells using AG1478, as shown in Fig. 3E, lanes 5–8. Inhibition of EGFR also reduced ADAM12 transcripts in UM-SCC 74B cells (Fig. 3F), similarly to inhibition of HER2.

Bottom Line: HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers.Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression.Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA.

ABSTRACT
Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.

Show MeSH
Related in: MedlinePlus