Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells

Abstract

To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a >1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium.

Fig. 1

a, b Histological analysis of NHTBE and H292 cells grown in ALI culture. a NHTBE cells were grown under ALI conditions in the presence of retinoic acid (5×10⁻⁸ M) for 28 days, then fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. For the detection of mucous goblet cells, the section was also stained with Alcian Blue-Periodic Acid Schiff s (arrows). b Histological section of H292 cells grown in RPMI1640 supplemented with 10% fetal bovine serum for 10 days in ALI culture were prepared and stained with hematoxylin and eosin as described above for NHTBE cells

Fig. 2

a–f Scatter plots of microarray signal intensity data. a Replicates of NHTBE plotted against each other. b Replicates of H292. c Replicate 1 of NHTBE versus replicate 1 of H292. d Replicate 1 of NHTBE versus replicate 2 of H292. e Replicate 2 of NHTBE versus replicate 1 of H292. f Replicate 2 of NHTBE versus replicate 2 of H292. r is the correlation coefficient

Fig. 3

Western analysis of selected gene products in various cell lines to validate that the differential expression of genes detected in H292 cells is reflected at the translation level. All of the tested proteins were evaluated in H292 cells and in NHTBE cells (β-actin was used as an internal control)

Fig. 4

a–d FACS analysis of the cell cycle, apoptosis, and proliferation in NHTBE and H292 cells. Panels a and b show the results of repeated experiments, which indicate that there were significant differences between the two cell lines. a NHTBE cells. A significantly higher percentage of the NHTBE cells is in the M4 (apoptosis or necrosis) or G2 phase. b H292 cells. A significantly higher percentage of H292 cells is in S phase. c Summary of the statistical analysis. d Comparisons of rates of apoptosis and cell proliferation between the NHTBE and H292 cells indicate that the H292 cells are more active in proliferation and less active in apoptosis. Thus, about 19.75% of the H292 cells progressed from the G1 checkpoint to S phase, but only about 5.16% of the NHTBE cells were in S phase. Moreover, only 2.05% of the H292 cells, compared with 27.04% of the NHTBE cells, were found to be apoptotic

Fig. 5

a, b Measurement of apoptosis based on the loss of mitochondrial membrane potential. Cells were treated with MTGreen (which stains all mitochondria) and CMXRos (which enters mitochondria that show a loss of membrane potential). Panels a and b show combination dot plots for total NHTBE (a) and H292 (b) cells stained with both agents. Areas marked R2 encompass the apoptotic cells, which show a loss of mitochondrial membrane potential. The Table below the graphs summarizes the data obtained from the membrane potential analysis. The values in the MTGreen and CMXRos columns represent the total events (numbers of cells) detected in the MTGreen and CMXRos channels. The MTGreen value is a measure of the number of mitochondria per cell, the CMXRos value is a measure of the induction of apoptosis, as indicated by a drop in mitochondrial membrane potential. The values in the LMP column indicate the percentages of apoptotic cells (those located in the R2 region) in the two dot plots. NS and S represent nonsignificant and significant differences between the NHTBE and H292 cells

Fig. 6

Schematic representation of the genetic networks that are altered in H292 cells. The network consists of the pathways mediating WNT responses, cell cycle regulation, apoptosis (which includes mitochondrially and TNF-regulated pathways), and cell proliferation. The genes in green showed no change, the genes in red were upregulated, and the genes in blue were downregulated in H292 cells. The numbers in black indicate the LBFC (see Materials and methods), while the numbers marked with asterisks were obtained from qRT-PCR results. DVL, human Disheveled protein; CK1 casein kinase 1; CK2, casein kinase 2; GSK, glycogen synthase kinase 3 beta; PP2A, protein phosphatase 2A; CBP, CREB-binding protein; TCF, T-cell factor; TGF-B1, transforming growth factor-beta 1; RAS, Ras; GADD, growth arrest and DNA-damage-inducible gene; E2F2, E2F transcription factor 2; MCM, minichromosome maintenance deficient; TNFR, TNF receptor; NF-κB, nuclear factor-κB; HSP70, heat shock protein 70 kD; HSP90, heat shock protein 90 kD; APAF1, apoptotic protease activating factor 1; BAD, Bcl-XL/Bcl-2-associated death gene; BAG1. BCL2-associated athanogene 1