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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

Rescue experiment evaluating AKT and MEK transfection after therapy inhibition. AKT and MEK inserted in pCMV5-expression vector with a selection marker of ampicillin (100ug/ml) was gently diluted (0.01ug/ul) with serum-free media. Diluted DNA (100ul) was mixed with X-tremeGENE DNA transfection reagent (3ul) and incubated for 15 minutes at room temperature. These mixtures were added into the 60mm dish culturing MDA-MB-468 on day 1 and incubated overnight with drugs including gefitinib (1uM) and EMD 121463 (5uM) alone or in combination; they were treated with serum-free media on day 2. Cells were harvested for western blotting after treatment of EGF 20ng/mL and/or recombinant human HGF 75ng/mL for 10 minutes before cell lysis.
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Figure 5: Rescue experiment evaluating AKT and MEK transfection after therapy inhibition. AKT and MEK inserted in pCMV5-expression vector with a selection marker of ampicillin (100ug/ml) was gently diluted (0.01ug/ul) with serum-free media. Diluted DNA (100ul) was mixed with X-tremeGENE DNA transfection reagent (3ul) and incubated for 15 minutes at room temperature. These mixtures were added into the 60mm dish culturing MDA-MB-468 on day 1 and incubated overnight with drugs including gefitinib (1uM) and EMD 121463 (5uM) alone or in combination; they were treated with serum-free media on day 2. Cells were harvested for western blotting after treatment of EGF 20ng/mL and/or recombinant human HGF 75ng/mL for 10 minutes before cell lysis.

Mentions: Then, we investigated the changes in downstream signaling for each cell line before and after treatment of drug alone and in combination. In western blotting, both gefitinib and cetuximab reduced pEGFR and EMD1214063 abrogated pMET, respectively in MDA- MB-468 as well as in HCC-1395. Either drug alone was insufficient to alter pAKT, pMAPK and pS6 levels. Strikingly, however the combination of these two drugs significantly reduced or abrogated the expression of downstream signaling molecules. These results suggest that each inhibitor has effects on their respective direct targets but that these effects are not translated into blocking of survival and proliferative pathways. However, the combination of EGFR and MET inhibition could abrogate the activation of essential molecules required for cell growth control. Interestingly, in MDA-MB-231 and T47D, there were no effects on PI3K or MAPK signaling not only after the single agent therapy but also even after combination treatment (Fig. 4). Next, we checked if we could reverse the inhibition of these drugs on downstream molecules when we transfect either AKT or MEK into the cells. As shown in Figure 5, transfection of wild type AKT and/or wild type MEK into MDA-MB-468 cells increased pAKT and pMEK respectively (Fig 5). Surprisingly, in transfected cells, the combination of EGFR or MET inhibition failed to decrease pAKT in AKT transfected cells and pMEK in MEK transfected cells. This effect was also seen when AKT and MEK were cotransfected (Fig. 5).


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)

Rescue experiment evaluating AKT and MEK transfection after therapy inhibition. AKT and MEK inserted in pCMV5-expression vector with a selection marker of ampicillin (100ug/ml) was gently diluted (0.01ug/ul) with serum-free media. Diluted DNA (100ul) was mixed with X-tremeGENE DNA transfection reagent (3ul) and incubated for 15 minutes at room temperature. These mixtures were added into the 60mm dish culturing MDA-MB-468 on day 1 and incubated overnight with drugs including gefitinib (1uM) and EMD 121463 (5uM) alone or in combination; they were treated with serum-free media on day 2. Cells were harvested for western blotting after treatment of EGF 20ng/mL and/or recombinant human HGF 75ng/mL for 10 minutes before cell lysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4216798&req=5

Figure 5: Rescue experiment evaluating AKT and MEK transfection after therapy inhibition. AKT and MEK inserted in pCMV5-expression vector with a selection marker of ampicillin (100ug/ml) was gently diluted (0.01ug/ul) with serum-free media. Diluted DNA (100ul) was mixed with X-tremeGENE DNA transfection reagent (3ul) and incubated for 15 minutes at room temperature. These mixtures were added into the 60mm dish culturing MDA-MB-468 on day 1 and incubated overnight with drugs including gefitinib (1uM) and EMD 121463 (5uM) alone or in combination; they were treated with serum-free media on day 2. Cells were harvested for western blotting after treatment of EGF 20ng/mL and/or recombinant human HGF 75ng/mL for 10 minutes before cell lysis.
Mentions: Then, we investigated the changes in downstream signaling for each cell line before and after treatment of drug alone and in combination. In western blotting, both gefitinib and cetuximab reduced pEGFR and EMD1214063 abrogated pMET, respectively in MDA- MB-468 as well as in HCC-1395. Either drug alone was insufficient to alter pAKT, pMAPK and pS6 levels. Strikingly, however the combination of these two drugs significantly reduced or abrogated the expression of downstream signaling molecules. These results suggest that each inhibitor has effects on their respective direct targets but that these effects are not translated into blocking of survival and proliferative pathways. However, the combination of EGFR and MET inhibition could abrogate the activation of essential molecules required for cell growth control. Interestingly, in MDA-MB-231 and T47D, there were no effects on PI3K or MAPK signaling not only after the single agent therapy but also even after combination treatment (Fig. 4). Next, we checked if we could reverse the inhibition of these drugs on downstream molecules when we transfect either AKT or MEK into the cells. As shown in Figure 5, transfection of wild type AKT and/or wild type MEK into MDA-MB-468 cells increased pAKT and pMEK respectively (Fig 5). Surprisingly, in transfected cells, the combination of EGFR or MET inhibition failed to decrease pAKT in AKT transfected cells and pMEK in MEK transfected cells. This effect was also seen when AKT and MEK were cotransfected (Fig. 5).

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