Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells

Abstract

Purpose: The epidermal growth factor receptor (EGFR) is recognized as a key mediator of proliferation and progression in many human tumors. A series of EGFR-specific inhibitors have recently gained Food and Drug Administration approval in oncology. These strategies of EGFR inhibition have shown major tumor regressions in approximately 10% to 20% of advanced cancer patients. Many tumors, however, eventually manifest resistance to treatment. Efforts to better understand the underlying mechanisms of acquired resistance to EGFR inhibitors, and potential strategies to overcome resistance, are greatly needed.

Experimental design: To develop cell lines with acquired resistance to EGFR inhibitors we utilized the human head and neck squamous cell carcinoma tumor cell line SCC-1. Cells were treated with increasing concentrations of cetuximab, gefitinib, or erlotinib, and characterized for the molecular changes in the EGFR inhibitor-resistant lines relative to the EGFR inhibitor-sensitive lines.

Results: EGFR inhibitor-resistant lines were able to maintain their resistant phenotype in both drug-free medium and in athymic nude mouse xenografts. In addition, EGFR inhibitor-resistant lines showed a markedly increased proliferation rate. EGFR inhibitor-resistant lines had elevated levels of phosphorylated EGFR, mitogen-activated protein kinase, AKT, and signal transducer and activator of transcription 3, which were associated with reduced apoptotic capacity. Subsequent in vivo experiments indicated enhanced angiogenic potential in EGFR inhibitor-resistant lines. Finally, EGFR inhibitor-resistant lines showed cross-resistance to ionizing radiation.

Conclusions: We have developed EGFR inhibitor-resistant human head and neck squamous cell carcinoma cell lines. This model provides a valuable preclinical tool to investigate molecular mechanisms of acquired resistance to EGFR blockade.

Fig. 1. Growth profile of EGFR inhibitor-resistant cells

Cetuximab-resistant (Cet-R), gefitinib-resistant (Gef-R), erlotinib-resistant (Erl-R) cells and their corresponding parental SCC-1 controls were treated with increasing amounts of EGFR inhibitors. Following 72 hours incubation, the numbers of viable cells in each well were determined by a proliferation assay as described in “Materials and Methods”. Results were expressed as the percentage of cell growth relative to controls. Each point represents mean ± SD of three determinations.

Fig. 2. EGFR inhibitor-resistant cells maintain resistance as tumor xenografts

Parental or EGFR inhibitor-resistant cells (Cet-R, Gef-R or Erl-R) were injected subcutaneously into the dorsal flank of athymic mice. Following the establishment of tumor, mice were treated with cetuximab, gefitinib or erlotinib as described in “Materials and Methods”. Tumor volume was monitored and the growth of tumor was expressed as the fold increase of tumor volume following initiation of drug treatment (n=8).

Fig. 3. EGFR signaling profile of EGFR inhibitor-resistant cells

Parental and EGFR inhibitor-resistant cells (Cet-R, Gef-R or Erl-R) were exposed to different concentration of corresponding inhibitors for 2 hrs followed by 45 min of EGF (10 μg/ml) stimulation. Following harvesting, cells were lysed and processed for immunoblotting using antibodies directed against p-EGFR, p-AKT, p-MAPL and p-STAT3 as described in “Materials and Methods”. The α-Tubulin (α-Tu) serves as a loading control.

Fig. 4. Apoptosis response of EGFR inhibitor-resistant cells following EGFR inhibitors treatment

Apoptosis was examined by flow cytometry using Annexin and PI staining as described in “Materials and Methods”. Parental and EGFR inhibitor-resistant cells (Cet-R, Gef-R or Erl-R) were exposed to different concentrations of cetuximab, gefitinib or erlotinib for 72 hrs. The percentage of cells in early apoptotic population was analyzed by Cell Quest software (Becton Dickinson, San Diego, CA). *, p<0.05. Similar results were obtained in replicate experiments

Fig. 5. Angiogenesis potential of EGFR inhibitor-resistant cells

Parental or EGFR inhibitor-resistant (Cet-R, Gef-R or Erl-R) cells were implanted into dorsal Matrigel plugs (upper panel) prepared in athymic mice as described in “Materials and Methods”. Following 10 days after implantation the matrigel plugs were removed and examined by fluorescence microscope. Pictures in the middle panel demonstrate green fluorescent blood vessels in Matrigel plug. The intensity of fluorescence was further quantified and shown in the bottom panel. Results were obtained from four mice in two independent experiments. Bars, SD.

Fig. 6. Radiation response of EGFR inhibitor-resistant cells

Radiosensitivity of EGFR inhibitor-resistant (Cet-R, Gef-R or Erl-R) and parental tumor cells were examined by clonogenic survival analysis following 3, 6 or 9 Gy of radiation as described in “Materials and Methods”. Results were expressed as the percentage of colony formation relative to controls. Data points, means of duplicates with five cultures per replicate per point; bars, SD.

Fig. 7. Radiation-induced apoptosis profile of EGFR inhibitor-resistant cells

Parental and EGFR inhibitor-resistant cells (Cet-R, Gef-R or Erl-R) were exposed to 0, 6 or 20 Gy of radiation. Twenty-four hours later, cells were harvested and processed for Annexin V/PI staining followed by flow cytometry analysis as described in “Materials and Methods”. The percentage of cells in early apoptotic population was analyzed by Cell Quest software (Becton Dickinson, San Diego, CA). Similar results were obtained in replicate experiments