Differential response to ablative ionizing radiation in genetically distinct non-small cell lung cancer cells

Abstract

Stereotactic ablative radiotherapy (SABR) has emerged as a highly promising treatment for medically inoperable early-stage non-small cell lung cancer patients. Treatment outcomes after SABR have been excellent compared to conventional fractionated radiotherapy (CFRT). However, the biological determinants of the response to ablative doses of radiation remain poorly characterized. Furthermore, there's little data on the cellular and molecular response of genetically distinct NSCLC subtypes to radiation. We assessed the response of 3 genetically distinct lung adenocarcinoma cell lines to ablative and fractionated ionizing radiation (AIR and FIR). We studied clonogenic survival, cell proliferation, migration, invasion, apoptosis and senescence. We also investigated the effect of AIR and FIR on the expression of pro-invasive proteins, epithelial-to-mesenchymal transition (EMT), extracellular signal-regulated kinases (ERK1/2) and the transmembrane receptor cMET. Our findings reveal that AIR significantly reduced cell proliferation and clonogenic survival compared to FIR in A549 cells only. This differential response was not observed in HCC827 or H1975 cells. AIR significantly enhanced the invasiveness of A549 cells, but not HCC827 or H1975 cells compared to FIR. Molecular analysis of pathways involved in cell proliferation and invasion revealed that AIR significantly reduced phosphorylation of ERK1/2 and upregulated cMET expression in A549 cells. Our results show a differential proliferative and invasive response to AIR that is dependent on genetic subtype and independent of intrinsic radioresistance. Further examination of these findings in a larger panel of NSCLC cell lines and in pre-clinical models is warranted for identification of biomarkers of tumor response to AIR.

Keywords: Ablative radiotherapy; adenocarcinoma; cMET; invasion; lung cancer; proliferation.

Figure 1.

Proliferation and clonogenic surival of NSCLC cells treated with AIR or FIR. (A-C) MTT proliferation assay 5 d after exposure of cells to AIR and FIR of 8Gy and 12Gy. Data is normalized to respective untreated controls. Error bars represent SEM of 3 independent experiments. (D) Clonogenic cell survival of NSCLC cells exposed to doses of 2Gy-10Gy. Data was fitted using the linear-quadratic model (α/β = 10).

Figure 2.

Analysis of modes of cell death after AIR. (A) Time course analysis of Trypan blue in NSCLC cell lines after exposure to 12Gy. (B) Quantitative analysis of senescence-associated β-galactosidase activity 5 d after exposure to AIR. (C) Analysis of polyploidy after exposure to AIR. Bars represent SEM of 3 independent experiments. Captions show representative images from AIR-treated cells (magnification x100).

Figure 3.

Cell cycle analysis of NSCLC cells 24 hours after exposure to AIR as assessed using flow cytometry. Right panels show Western blot expression of proteins involved in cell cycle (p53 and p21) and apoptosis (PARP). Actin was used as loading control.

Figure 4.

Migration and invasion of NSCLC cells in modified Boyden chambers 5 d after exposure to AIR and FIR. (A-C) Migration of NSCLC cells through uncoated Boyden chambers. (D-F) Invasion of NSCLC cells through matrigel-coated Boyden chambers. Data is normalized to respective controls. Bars represent SEM from 3 independent experiments. Representative images of invaded cells are shown at the bottom (magnification x200).

Figure 5.

Wound healing assay of A549 cells 3 d after exposure to AIR and FIR. (A) Representative wound-healing images taken at 0h, 24h and 48h with respective wound-closure ratios. Magnification x100. (B) Wound-closure analysis after AIR and FIR normalized to 0% at 0h. Experiments were repeated 3 times. Bars represent standard deviation from one representative experiment.

Figure 6.

Western blot analysis of pro-invasive proteins in A549 cells treated with AIR or FIR. (A) Representative blots of membranes probed with antibodies directed against c-MET, ERK1/2, phosphor ERK1/2, vimentin, E-cadherin, FAK, phospho FAK, AKT, phospho AKT, mTOR and phospho mTOR. Actin was used as loading control. (B) Quantification of protein expression relative to baseline expression from untreated A549 cells. Bars represent SEM from 3 independent experiments.