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cMET Activation and EGFR-Directed Therapy Resistance in Triple-Negative Breast Cancer.

Sohn J, Liu S, Parinyanitikul N, Lee J, Hortobagyi GN, Mills GB, Ueno NT, Gonzalez-Angulo AM - J Cancer (2014)

Bottom Line: However, anti-EGFR therapies have not been effective in these patients.In western blotting, combination therapy resulted in abrogation of pAKT and pMAPK while monotherapy did not.Targeting both EGFR and MET receptors may provide an effective therapeutic strategy in TNBC.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA ; 2. Division of Medical Oncology, Department of Internal Medicine, Breast Cancer Clinic, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Background: EGFR expression and pathway activation are common in triple-negative breast cancer (TNBC). However, anti-EGFR therapies have not been effective in these patients. We aimed to study the efficacy of targeting MET in overcoming resistance to EGFR therapy in TNBC cell lines.

Methods: TNBC lines (MDA-MB-468, HCC-1395, and MDA-MB-231), and a hormone receptor-positive breast cancer line (T47D) were stimulated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF). Lines were then treated with different concentrations of EGFR inhibitors (gefitinib or cetuximab), with or without a MET tyrosine kinase inhibitor (EMD 1214063). Proliferation was measured by MTS assay, in soft agar and with a matrigel assay. Synergy was measured with Calcusyn. Protein expression and signaling were examined with immunoblotting.

Results: There was activation of ligand-receptor-downstream signaling pathways in MDA-MB-468 and HCC-1395 upon stimulation with EGF and HGF. In these cell lines, we observed synergism when combining EGFR and MET inhibitors. These results were observed across assays. In western blotting, combination therapy resulted in abrogation of pAKT and pMAPK while monotherapy did not.

Conclusion: Our data demonstrate that dual EGFR/MET inhibition is synergistic in TNBC. Targeting both EGFR and MET receptors may provide an effective therapeutic strategy in TNBC.

No MeSH data available.


Related in: MedlinePlus

Effects of combination of EGFR inhibitors with cMET inhibitor EMD 1214063 on soft agar assay. Cells were suspended in complete RPMI medium containing 0.3% soft agar and seeded in triplicate on 35-mm dishes precoated with 0.6% agar in complete growth medium mixed with corresponding drug and incubated at 37°C, 5% CO2. After 10 days, colonies were photographed and counted in 10 randomly chosen fields and expressed as means of triplicates. For staining of the colonies, 0.005% crystal violet in 20% methanol solution were added to the dishes for 30 minutes and washed until the background becomes clear. Drug concentrations were as follows; Gefitinib 1uM, EMD 121463 5uM, and Cetuximab 200 ug/ml, in MDA-MB-468; Gefitinib 5uM, EMD 121463 5uM, and Cetuximab 200 ug/ml in MDA-MB-231. Colonies were not formed in HCC 1395 and there were no difference between single agent and combination in T47D (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001 vs EMD 121463; **, P<0.05 vs EMD 121463; ***, P<0.001 vs gefitinib; #, P>0.05 vs cetuximab).
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Figure 2: Effects of combination of EGFR inhibitors with cMET inhibitor EMD 1214063 on soft agar assay. Cells were suspended in complete RPMI medium containing 0.3% soft agar and seeded in triplicate on 35-mm dishes precoated with 0.6% agar in complete growth medium mixed with corresponding drug and incubated at 37°C, 5% CO2. After 10 days, colonies were photographed and counted in 10 randomly chosen fields and expressed as means of triplicates. For staining of the colonies, 0.005% crystal violet in 20% methanol solution were added to the dishes for 30 minutes and washed until the background becomes clear. Drug concentrations were as follows; Gefitinib 1uM, EMD 121463 5uM, and Cetuximab 200 ug/ml, in MDA-MB-468; Gefitinib 5uM, EMD 121463 5uM, and Cetuximab 200 ug/ml in MDA-MB-231. Colonies were not formed in HCC 1395 and there were no difference between single agent and combination in T47D (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001 vs EMD 121463; **, P<0.05 vs EMD 121463; ***, P<0.001 vs gefitinib; #, P>0.05 vs cetuximab).

Mentions: In order to evaluate effects of drug combinations on anchorage independent growth, cells were seeded in soft agar and EGFR inhibitors (gefitinib or cetuximab), EMD 121463, or combinations were added. After 10-14 days, the colony size and number were significantly reduced in the MDA-MB-468 cell line when gefitinib or cetuximab was combined with EMD 121463 compared to single agents. In MDA-MB-231 cell line, there was a significant reduction of colony number when EMD 121463 was combined with gefitinib but not with cetuximab compared to single agents (Fig. 2).


cMET Activation and EGFR-Directed Therapy Resistance in Triple-Negative Breast Cancer.

Sohn J, Liu S, Parinyanitikul N, Lee J, Hortobagyi GN, Mills GB, Ueno NT, Gonzalez-Angulo AM - J Cancer (2014)

Effects of combination of EGFR inhibitors with cMET inhibitor EMD 1214063 on soft agar assay. Cells were suspended in complete RPMI medium containing 0.3% soft agar and seeded in triplicate on 35-mm dishes precoated with 0.6% agar in complete growth medium mixed with corresponding drug and incubated at 37°C, 5% CO2. After 10 days, colonies were photographed and counted in 10 randomly chosen fields and expressed as means of triplicates. For staining of the colonies, 0.005% crystal violet in 20% methanol solution were added to the dishes for 30 minutes and washed until the background becomes clear. Drug concentrations were as follows; Gefitinib 1uM, EMD 121463 5uM, and Cetuximab 200 ug/ml, in MDA-MB-468; Gefitinib 5uM, EMD 121463 5uM, and Cetuximab 200 ug/ml in MDA-MB-231. Colonies were not formed in HCC 1395 and there were no difference between single agent and combination in T47D (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001 vs EMD 121463; **, P<0.05 vs EMD 121463; ***, P<0.001 vs gefitinib; #, P>0.05 vs cetuximab).
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Figure 2: Effects of combination of EGFR inhibitors with cMET inhibitor EMD 1214063 on soft agar assay. Cells were suspended in complete RPMI medium containing 0.3% soft agar and seeded in triplicate on 35-mm dishes precoated with 0.6% agar in complete growth medium mixed with corresponding drug and incubated at 37°C, 5% CO2. After 10 days, colonies were photographed and counted in 10 randomly chosen fields and expressed as means of triplicates. For staining of the colonies, 0.005% crystal violet in 20% methanol solution were added to the dishes for 30 minutes and washed until the background becomes clear. Drug concentrations were as follows; Gefitinib 1uM, EMD 121463 5uM, and Cetuximab 200 ug/ml, in MDA-MB-468; Gefitinib 5uM, EMD 121463 5uM, and Cetuximab 200 ug/ml in MDA-MB-231. Colonies were not formed in HCC 1395 and there were no difference between single agent and combination in T47D (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001 vs EMD 121463; **, P<0.05 vs EMD 121463; ***, P<0.001 vs gefitinib; #, P>0.05 vs cetuximab).
Mentions: In order to evaluate effects of drug combinations on anchorage independent growth, cells were seeded in soft agar and EGFR inhibitors (gefitinib or cetuximab), EMD 121463, or combinations were added. After 10-14 days, the colony size and number were significantly reduced in the MDA-MB-468 cell line when gefitinib or cetuximab was combined with EMD 121463 compared to single agents. In MDA-MB-231 cell line, there was a significant reduction of colony number when EMD 121463 was combined with gefitinib but not with cetuximab compared to single agents (Fig. 2).

Bottom Line: However, anti-EGFR therapies have not been effective in these patients.In western blotting, combination therapy resulted in abrogation of pAKT and pMAPK while monotherapy did not.Targeting both EGFR and MET receptors may provide an effective therapeutic strategy in TNBC.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA ; 2. Division of Medical Oncology, Department of Internal Medicine, Breast Cancer Clinic, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Background: EGFR expression and pathway activation are common in triple-negative breast cancer (TNBC). However, anti-EGFR therapies have not been effective in these patients. We aimed to study the efficacy of targeting MET in overcoming resistance to EGFR therapy in TNBC cell lines.

Methods: TNBC lines (MDA-MB-468, HCC-1395, and MDA-MB-231), and a hormone receptor-positive breast cancer line (T47D) were stimulated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF). Lines were then treated with different concentrations of EGFR inhibitors (gefitinib or cetuximab), with or without a MET tyrosine kinase inhibitor (EMD 1214063). Proliferation was measured by MTS assay, in soft agar and with a matrigel assay. Synergy was measured with Calcusyn. Protein expression and signaling were examined with immunoblotting.

Results: There was activation of ligand-receptor-downstream signaling pathways in MDA-MB-468 and HCC-1395 upon stimulation with EGF and HGF. In these cell lines, we observed synergism when combining EGFR and MET inhibitors. These results were observed across assays. In western blotting, combination therapy resulted in abrogation of pAKT and pMAPK while monotherapy did not.

Conclusion: Our data demonstrate that dual EGFR/MET inhibition is synergistic in TNBC. Targeting both EGFR and MET receptors may provide an effective therapeutic strategy in TNBC.

No MeSH data available.


Related in: MedlinePlus