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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 in MTS assay. Cells were seeded in 96-well microplates in medium supplemented with 5% FBS and penicillin/streptavidin. The optimal cell number for each cell line was determined to ensure that each was in growth phase at the end of the assay (~70% confluency). Cells were allowed to attach for 24 hours. The media was changed to low FBS (2%) and drugs with different combinations were added (cetuximab 200ug/mL, gefitinib 0.25-8 umol/L and EMD 121463 2-10 umol/L). In terms of determination of drug concentration, twofold serial dilution was conducted for gefitinib and dose was increased by 2 umol/L in EMD 121463 with the upper limit of 10 umol/L because of poor solubility. Cells were incubated at 37°C for 72 hours. Growth inhibition was determined with Cell Titer Blue (Promega, Madison, WI) at a 72-hour time point according to the manufacturer's instructions. Quantification of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 530/604. Drug concentrations were as follows; Gefitinib 1uM and EMD 121463 5uM in MDA-MB-468; Gefitinib 5uM and EMD 121463 5uM in MDA-MB-231. There was no statistical difference in T47D and HCC1395 (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001).
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Figure 1: Effects of combination of EGFR inhibitors with cMET inhibitor, EMD 1214063 in MTS assay. Cells were seeded in 96-well microplates in medium supplemented with 5% FBS and penicillin/streptavidin. The optimal cell number for each cell line was determined to ensure that each was in growth phase at the end of the assay (~70% confluency). Cells were allowed to attach for 24 hours. The media was changed to low FBS (2%) and drugs with different combinations were added (cetuximab 200ug/mL, gefitinib 0.25-8 umol/L and EMD 121463 2-10 umol/L). In terms of determination of drug concentration, twofold serial dilution was conducted for gefitinib and dose was increased by 2 umol/L in EMD 121463 with the upper limit of 10 umol/L because of poor solubility. Cells were incubated at 37°C for 72 hours. Growth inhibition was determined with Cell Titer Blue (Promega, Madison, WI) at a 72-hour time point according to the manufacturer's instructions. Quantification of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 530/604. Drug concentrations were as follows; Gefitinib 1uM and EMD 121463 5uM in MDA-MB-468; Gefitinib 5uM and EMD 121463 5uM in MDA-MB-231. There was no statistical difference in T47D and HCC1395 (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001).

Mentions: To determine the sensitivity of TNBC cell lines and T47D to EGFR inhibition, MET inhibition, and combined therapy inhibition, breast cancer cell lines were treated with gefitinib (0.25-8 umol/L), EMD 121463 (2-10 umol/L), or both agents given at the same concentration in combination for 72 hours. GI 50 of these cell lines were 1-5 uM to gefitinib and 4uM- ≥10uM to EMD 121463. These cell lines were essentially resistant to both gefitinib and EMD 121463 as single agents. The inhibitory effect of combined treatment with gefitinib and EMD 121463 was significantly enhanced compared with single agent therapy in MDA-MB-468 cells (P=0.002) but not in the other cell lines (MDA-MB-231, HCC1395, and T47D) (Fig.1). However, when data were analyzed in Calcusyn software, synergism was also documented at different concentration combinations in the other cell lines (Table 1). These experiments indicate a dose dependent synergistic interaction between gefitinib and EMD 121463 in suppressing growth of triple-negative breast cancer cells.


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 in MTS assay. Cells were seeded in 96-well microplates in medium supplemented with 5% FBS and penicillin/streptavidin. The optimal cell number for each cell line was determined to ensure that each was in growth phase at the end of the assay (~70% confluency). Cells were allowed to attach for 24 hours. The media was changed to low FBS (2%) and drugs with different combinations were added (cetuximab 200ug/mL, gefitinib 0.25-8 umol/L and EMD 121463 2-10 umol/L). In terms of determination of drug concentration, twofold serial dilution was conducted for gefitinib and dose was increased by 2 umol/L in EMD 121463 with the upper limit of 10 umol/L because of poor solubility. Cells were incubated at 37°C for 72 hours. Growth inhibition was determined with Cell Titer Blue (Promega, Madison, WI) at a 72-hour time point according to the manufacturer's instructions. Quantification of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 530/604. Drug concentrations were as follows; Gefitinib 1uM and EMD 121463 5uM in MDA-MB-468; Gefitinib 5uM and EMD 121463 5uM in MDA-MB-231. There was no statistical difference in T47D and HCC1395 (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001).
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Figure 1: Effects of combination of EGFR inhibitors with cMET inhibitor, EMD 1214063 in MTS assay. Cells were seeded in 96-well microplates in medium supplemented with 5% FBS and penicillin/streptavidin. The optimal cell number for each cell line was determined to ensure that each was in growth phase at the end of the assay (~70% confluency). Cells were allowed to attach for 24 hours. The media was changed to low FBS (2%) and drugs with different combinations were added (cetuximab 200ug/mL, gefitinib 0.25-8 umol/L and EMD 121463 2-10 umol/L). In terms of determination of drug concentration, twofold serial dilution was conducted for gefitinib and dose was increased by 2 umol/L in EMD 121463 with the upper limit of 10 umol/L because of poor solubility. Cells were incubated at 37°C for 72 hours. Growth inhibition was determined with Cell Titer Blue (Promega, Madison, WI) at a 72-hour time point according to the manufacturer's instructions. Quantification of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 530/604. Drug concentrations were as follows; Gefitinib 1uM and EMD 121463 5uM in MDA-MB-468; Gefitinib 5uM and EMD 121463 5uM in MDA-MB-231. There was no statistical difference in T47D and HCC1395 (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001).
Mentions: To determine the sensitivity of TNBC cell lines and T47D to EGFR inhibition, MET inhibition, and combined therapy inhibition, breast cancer cell lines were treated with gefitinib (0.25-8 umol/L), EMD 121463 (2-10 umol/L), or both agents given at the same concentration in combination for 72 hours. GI 50 of these cell lines were 1-5 uM to gefitinib and 4uM- ≥10uM to EMD 121463. These cell lines were essentially resistant to both gefitinib and EMD 121463 as single agents. The inhibitory effect of combined treatment with gefitinib and EMD 121463 was significantly enhanced compared with single agent therapy in MDA-MB-468 cells (P=0.002) but not in the other cell lines (MDA-MB-231, HCC1395, and T47D) (Fig.1). However, when data were analyzed in Calcusyn software, synergism was also documented at different concentration combinations in the other cell lines (Table 1). These experiments indicate a dose dependent synergistic interaction between gefitinib and EMD 121463 in suppressing growth of triple-negative breast cancer cells.

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