Yonsei Med J.  2017 Jan;58(1):9-18. 10.3349/ymj.2017.58.1.9.

EGF Induced RET Inhibitor Resistance in CCDC6-RET Lung Cancer Cells

• Chang H
• Sung JH
• Moon SU
• Kim HS
• Kim JW
• Lee JS

• KMID: 2374183
• DOI: http://doi.org/10.3349/ymj.2017.58.1.9

Abstract

PURPOSE
Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes.
MATERIALS AND METHODS
The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined.
RESULTS
CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors.
CONCLUSION
EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Keyword

Lung cancer; RET oncogene; tyrosine kinase inhibitor; resistance; epidermal growth factor

MeSH Terms

Adenocarcinoma/drug therapy/*genetics
Cell Line, Tumor
Cetuximab/pharmacology
Drug Resistance, Neoplasm/drug effects/*genetics
Epidermal Growth Factor/metabolism/*pharmacology
*Gene Rearrangement
Hepatocyte Growth Factor/*pharmacology
Humans
Indoles/pharmacology
Lung Neoplasms/drug therapy/*genetics
MAP Kinase Signaling System
*Mutation
Niacinamide/analogs & derivatives/pharmacology
Phenylurea Compounds/pharmacology
Piperidines/pharmacology
Protein Kinase Inhibitors/therapeutic use
Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics
Pyrroles/pharmacology
Quinazolines/pharmacology
RNA, Small Interfering/pharmacology
Receptor, Epidermal Growth Factor/genetics/metabolism
Signal Transduction/drug effects
fms-Like Tyrosine Kinase 3/metabolism
Cetuximab
Epidermal Growth Factor
Hepatocyte Growth Factor
Indoles
Phenylurea Compounds
Piperidines
Protein Kinase Inhibitors
Proto-Oncogene Proteins c-ret
Pyrroles
Niacinamide
Quinazolines
RNA, Small Interfering
Receptor, Epidermal Growth Factor
fms-Like Tyrosine Kinase 3

Figure

• 

  Fig. 1 EGFR ligand reduced the sensitivity of LC-2/ad cells to RET inhibitors in vitro. CCDC6-RET lung cancer cells were sensitive to RET inhibitors sunitinib, E7080, vandetanib, and sorafenib. LC-2/ad cells were pretreated with or without EGF (100 ng/mL) or HGF (50 ng/mL) for 24 hours and incubated with several concentrations of RET inhibitors sunitinib, E7080, vandetanib, or sorafenib. Cell growth was measured after 72 hours by celltiter-glo luminescent cell viability assay kits (Promega). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. HGF, hepatocyte growth factor; EGFR, epidermal growth factor receptor; RET, rearranged during transfection.

• 

  Fig. 2 Dose-dependent effects of EGF. LC-2/ad cells were incubated with several concentrations of EGF for 24 hours. LC-2/ad cells were incubated with RET inhibitors sunitinib (0.66 µM), E7080 (0.77 µM), vandetanib (0.22 µM) or sorafenib (1.1 µM). Cell growth was determined after 72 hours by celltiter-glo luminescent cell viability assay kit (Promega). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. EGF, epidermal growth factor; RET, rearranged during transfection.

• 

  Fig. 3 LC-2/ad, CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). EGF receptor (EGFR) ligand, EGF, or activated EGFR triggered resistance to RET inhibitors by transducing bypass survival signaling through ERK and AKT. LC-2/ad cells were treated with or without gefitinib (1 µM) and/or EGF (100 ng/mL) for 2 hours with or without RET inhibitor at half-maximum inhibitory concentration (IC50) for 1 hour. Cells were lysed and indicated proteins were detected by western blots. Shown are representative of three independent experiments. EGFR, epidermal growth factor receptor; RET, rearranged during transfection.

• 

  Fig. 4 Abrogation of EGF-triggered resistance to RET inhibitors by EGFR-TKI. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). LC-2/ad cells were incubated with or without gefitinib (1 µM) for 2 hours and/or EGF (100 ng/mL) for 24 hours with or without RET inhibitors at half-maximum inhibitory concentration (IC50), with cell growth determined after 72 hours. Cell growth was measured by celltiter-glo luminescent cell viability assay kit (Promega). *p<0.05 (one-way ANOVA). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. EGFR, epidermal growth factor receptor; RET, rearranged during transfection; TKI, tyrosine kinase inhibitor.

• 

  Fig. 5 Reduction of RET-fusion lung cancer cell sensitivity to RET inhibitors by co-culture with HUVEC endothelial cells. EGFR-specific siRNAs were transfected into LC-2/ad cells. After 24 hours, cells were cultured with or without HUVEC cells. After 24 hours, cells were incubated with RET inhibitors sunitinib, E7080, vandetanib, or sorafenib) at half-maximum inhibitory concentration (IC50) for kinase inhibitors for 72 hours. Cell growth was determined by celltiter-glo luminescent cell viability assay kit (Promega). *p<0.05 (one-way ANOVA). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. EGFR, epidermal growth factor receptor; RET, rearranged during transfection; HUVEC, human umbilical vein endothelial cell; siRNA, small interfering RNA.

• 

  Fig. 6 Reduction of RET-fusion lung cancer cell sensitivity to RET inhibitors by co-culture with HUVEC endothelial cells. LC-2/ad cells were incubated with or without Cetuximab (2 µg/mL). After 24 hours, cells were cultured with or without HUVEC cells. After 24 hours, cells were incubated with RET inhibitors for 72 hours. Cell growth was determined as above. *p<0.05 (one-way ANOVA). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. HUVEC, human umbilical vein endothelial cell; RET, rearranged during transfection.

• 

  Fig. 7 Reduction of RET-fusion lung cancer cell sensitivity to RET inhibitors by co-culture with HUVEC endothelial cells. LC-2/ad cells were incubated with or without gefitinib (1 µM). After 24 hours, cells were cultured with or without HUVEC cells and incubated with RET inhibitors as above. Cell growth was determined as above. *p<0.05 (one-way ANOVA). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. HUVEC, human umbilical vein endothelial cell; RET, rearranged during transfection.

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