Integrative radiogenomic profiling of squamous cell lung cancer

Abstract

Radiotherapy is one of the mainstays of anticancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is still not well defined. Here, we report the development of a high-throughput platform for measuring radiation survival in vitro and its validation in comparison with conventional clonogenic radiation survival analysis. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiotherapy, to identify parameters that predict radiation sensitivity. We showed that activation of NFE2L2, a frequent event in lung squamous cancers, confers radiation resistance. An expression-based, in silico screen nominated inhibitors of phosphoinositide 3-kinase (PI3K) as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1-mutant cells to radiation. We then combined results from this high-throughput assay with single-sample gene set enrichment analysis of gene expression data. The resulting analysis identified pathways implicated in cell survival, genotoxic stress, detoxification, and innate and adaptive immunity as key correlates of radiation sensitivity. The integrative and high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solid-tumor-derived cell lines should facilitate tumor radiogenomics and the discovery of genotype-selective radiation sensitizers and protective agents.

Fig. 1

The high-throughput platform accurately profiles lung squamous cancer cell lines. (a) (Top) R² values were calculated comparing the proliferating fraction PFx from high-throughput profiling and SFx from clonogenic assay. R² values depicted in red if P < 0.05. (Bottom) Scatter plots and linear regression for PF4 with SF2, SF5, or SF8. (b) Integral survival was calculated for each cell line, n ≥ 2). Error bars represent SEM. (c) Integral survival was calculated for proliferation assays for each cell line at days 4, 6, 8, and 9. Separately, integral survival was calculated for clonogenic survival assay. Data represents the average integral survival value for each cell line, n ≥ 2.

Fig. 2

NFE2L2 activation regulates radiation resistance and is a target for radiotherapeutic sensitization. (a) NRF2 scores from lung SqCC cell lines and integral survival were plotted. (b) Average integral survival values calculated from high-throughput assays were plotted as function of high NRF2 score defined as greater than the median (0.10) and/or the presence of alteration in the coding region of NFE2L2 or KEAP1. (c) Schematic depiction of the functional domains of NRF2. The Neh2 domain contains the two KEAP1 association motifs, DLG and ETGE. (d) NRF2 protein level in cell lines: RERF-LC-AI (−/+ tBHQ), LC-1/SQSF, and HCC15; Actin was used as a loading control. Immunblot analysis of NRF2 protein in LC-1/SQ-SF (e) and RERFLC-AI (h) cells infected with control shRNA (shNTC) and shNRF2-1 and shNRF2-2 after induction with Doxycycline for 24 hours; Actin was used as a loading control. LC-1/SQSF (f) and RERF-LC-AI (i) clones infected with shNTC, shNRF2-1, or shNRF2-2 were measured for clonogenic survival after induction with Doxycycline. LC-1/SQSF clones infected with shNTC or shNRF2-1 (g) and RERF-LC-AI clones infected with shNTC, shNRF2-1, or shNRF2-2 (j) were treated as control (0 Gy) or with radiation (2, 4, 6 Gy) after induction with Doxycycline for 24 hours. Data points represent mean values of duplicates (clonogenic survival) or six replicates (clonogenic survival after radiation) and are representative of three independent experiments. Error bars represent SEM. Cropped blots were imported directly into Adobe Illustrator CS6; no adjustments of brightness, contrast, or color balance were applied.

Fig. 3

Inhibition of PI3K antagonizes NRF2. (a) The rank, cmap name, connectivity score for each of the selected chemicals is shown. (b) The ‘‘barview’’ is constructed from horizontal lines, each representing an individual treatment instance, ordered by their corresponding connectivity scores with the NFE2L2 signature (+1, top; −1, bottom). All instances in the data set are colored in black. Colors applied to the remaining instances reflect the sign of their scores (green, positive; gray, null; red, negative). (c) LC-1/SQSF clones containing the ARE luciferase reporter were treated with LY 294002, NVP-BKM 120, and TGX-221 for 24 hours. Cellular viability was measured at 48 hours. Data points represent mean values of triplicates and error bars represent SD. The experiment was performed three times with comparable results. (d) The pan-PI3K inhibitor NVP-BKM 120 decreases NRF2 protein level. LC-1/SQSF cells were treated with control (DMSO), LY 294002, NVP-BKM 120, or TGX-221 and HCC15 cells were treated with DMSO and NVP-BKM120 for 24 hours and lysates were subjected to immunoblot analysis for the NRF2 protein. RERF-LC-AI cells were treated with DMSO and NVP-BKM120 for 24 hours followed by induction with 10 µM tBHQ for 24 hours before lysates were subjected to immunoblot analysis for the NRF2 protein. Actin was used as a loading control. Cropped blots were imported directly into Adobe Illustrator CS6; no adjustments of brightness, contrast, or color balance were applied.

Fig. 4

Inhibition of PI3K effects radiosensitization in cell lines with a NFE2L2 pathway alteration. (a) RERF-LC-AI, SQ-1, LC-1/SQSF, HCC15, or A549 cells were incubated with NVP-BKM120 for 24 hours and treated as control (0 Gy) or with radiation. Survival is measured by clonogenic assay. Data points represent mean values of duplicates and error bars represent SD. The experiment was performed three times with comparable results. In the experiment shown in panel (a), surviving fraction after exposure to 2 and 4 Gy (RERF-LC-AI, LC-1/SQSF, and HCC-15), 4 and 6 Gy (A549), and 1 and 2 Gy (SQ-1) radiation for cells incubated with DMSO alone are as follows: LC-1/SQSF, 0.71 ± 0.11 and 0.37 ± 0.08; HCC15, 0.75 ± 0.9 and 0.42 ± 0.12; RERF-LC-AI, 0.52 ± 0.06 and 0.33 ± 0.01; A549, 0.46 ± 0.09 and 0.24 ± 0.06; SQ-1, 0.65 ± 0.16 and 0.48 ± 0.06. (b) NFE2L2/KEAP1 genotype of cell lines tested for radiosensitization.