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. 2012 Nov 20;109(47):19408-13.
doi: 10.1073/pnas.1216363109. Epub 2012 Nov 5.

Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins

William W Lockwood  1 , Kreshnik Zejnullahu, James E Bradner, Harold Varmus
Affiliations

Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins

William W Lockwood et al. Proc Natl Acad Sci U S A. .

Abstract

Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones and facilitate transcription of target genes. Inhibitors targeting the activity of BET proteins have shown potent antiproliferative effects in hematological cancers through the suppression of c-MYC and downstream target genes. However, as the epigenetic landscape of a cell varies drastically depending on lineage, transcriptional coactivators such as BETs would be expected to have different targets in cancers derived from different cells of origin, and this may influence the activity and mechanism of action of BET inhibitors. To test this hypothesis, we treated a panel of lung adenocarcinoma (LAC) cell lines with the BET inhibitor JQ1 and found that a subset is acutely susceptible to BET inhibition. In contrast to blood tumors, we show that LAC cells are inhibited by JQ1 through a mechanism independent of c-MYC down-regulation. Through gene expression profiling, we discovered that the oncogenic transcription factor FOSL1 and its targets are suppressed by JQ1 in a dose-dependant manner. Knockdown of BRD4 also decreased FOSL1 levels, and inhibition of FOSL1 phenocopied the effects of JQ1 treatment, suggesting that loss of this transcription factor may be partly responsible for the cytotoxic effects of BET inhibition in LAC cells, although ectopic expression of FOSL1 alone did not rescue the phenotype. Together, these findings suggest that BET inhibitors may be useful in solid tumors and that cell-lineage-specific differences in transcriptional targets of BETs may influence the activity of inhibitors of these proteins in different cancer types.

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Conflict of interest statement

Conflict of interest statement: Dana Farber Cancer Institute has licensed drug-like derivatives of JQ1 from the J.E.B. laboratory to Tensha Therapeutics for clinical development as anticancer agents.

Figures

Fig. 1.
Fig. 1.
JQ1 treatment inhibits the growth of a subset of lung cancer cell lines. (A) Average JQ1 IC50 values for the indicated 19 lung cancer cell lines. Cells were treated with increasing doses of JQ1 for 72 h and the number of viable cells was determined using Alamar Blue. Red bars signify sensitive cell lines (IC50 values <5 μM); blue bars mark the insensitive cell lines (IC50 values >10 μM). Error bars denote the SDs of independent experiments. (B) Putative driver mutations and JQ1 IC50 values for the 19 cell lines tested. Values are presented as averages ± SD of two to four experiments. (C) Long-term colony formation assay of cell lines deemed sensitive (H23 and A549) and insensitive (H1650) in the short-term viability assays. Cells (2,000–8,000 per six-well plate) were grown in the absence or presence of 500 nM of JQ1 for 10 d, stained with crystal violet, and photographed. (D) Cell cycle analysis of H23 (sensitive) and H460 (insensitive) cells treated with 1 μM JQ1 for 24 h. BrdU incorporation (represented by the proportion of cells stained with an anti-BrdU antibody labeled with APC fluorescent dye) indicates cells in S phase. Numbers in red represent the average percentage of cells in S phase ± SD (n = 2). (E) Induction of apoptosis by JQ1 in H1975 cells. Cells were treated with 1 μM JQ1 for 48 h and flow cytometry was performed using 7-aminoactinomycin D (7-AAD) and annexin V staining. Values represent the average percentage of annexin V+ cells (early (Q4) + late (Q3) apoptotic) ± SD (n = 2). (F) Induction of cleaved PARP1 in JQ1 sensitive (H1975) but not insensitive (H460) cell lines. Cells were treated with the indicated concentrations of JQ1 for 24 and 48 h and assessed for full-length and cleaved PARP1 protein levels by Western blot using an anti-PARP antibody. GAPDH serves as a loading control.
Fig. 2.
Fig. 2.
Growth inhibition by JQ1 in lung adenocarcinoma cells is not dependent on c-MYC down-regulation. (A) Quantitative RT-PCR for c-MYC RNA levels in JQ1-treated cell lines. The MM control cell line RPMI-8226 (blue) and sensitive lung cancer cell lines (red) were treated with 1 μM JQ1 for 6 h before RNA extraction and analysis. Data are presented as the average ratio of MYC expression for each cell line relative to its corresponding DMSO-treated control (mean ± SEM). Asterisks denote the level of statistical significance (*P < 0.05, **P < 0.01, ***P < 0.005; two-tailed t test). (B) c-MYC protein levels in JQ1-treated sensitive lung cancer cell lines. Cells were treated with DMSO (−) or 5 μM JQ1 (+) for 6 h before lysis and Western blot analysis with an anti–c-MYC antibody. GAPDH serves as a loading control. (C) c-MYC protein levels are stable over time in response to JQ1 treatment. H1975 cells were treated with the indicated doses of JQ1, then c-MYC and PARP levels were evaluated at 24 and 48 h. Cleavage of PARP occurs in the absence of c-MYC down-regulation. (D) Dose-dependent effects of JQ1 treatment on c-MYC protein levels in sensitive lung cancer cell lines (red bars) and the control MM cell line (blue bar). Cells were treated with the indicated doses of JQ1 for 24 h before analysis. Again, cleavage of PARP occurs without c-MYC down-regulation.
Fig. 3.
Fig. 3.
JQ1 represses expression of FOSL1 and produces a downstream gene expression signature in drug-sensitive lung cancer cell lines. (A) Venn diagram depicting the overlap of significantly differentially expressed genes (Benjamini–Hochberg corrected P ≤ 0.01) after exposure to 1 μM JQ1 for 6 h in two sensitive (H23 and H1975) and one insensitive (H460) lung cancer cell lines. The red font highlights the number of genes differentially expressed in both sensitive cell lines but not the insensitive cell line. (B) Heatmap representation of the top-20 down-regulated (blue) and top-20 up-regulated (red) genes following JQ1 treatment in sensitive and insensitive lung cancer cell lines. Genes are ranked by the differential expression score, which is shown in the Left column (details in SI Materials and Methods). Data presented are mean normalized by row for each cell line. FOSL1 (arrow) is down-regulated by JQ1 treatment. (C) Ingenuity Pathway Analysis of transcription factor programs significantly deregulated (z score ± 2.0, P < 0.05) by JQ1 treatment of drug-sensitive lung cancer cell lines. The 298 genes highlighted in A, along with their differential expression scores, revealed that a gene expression program consistent with inhibition of FOS genes is induced by JQ1 treatment. The number of genes predicted to be regulated by each transcription factor program that are also deregulated by JQ1 treatment are presented along with the corresponding gene symbols. (D) Gene set enrichment analysis plot displaying the down-regulation of genes with AP-1 DNA binding motifs after JQ1 treatment in drug-sensitive cell lines. The 298 genes from A are ranked according to their differential expression score from highest to lowest along the x axis. The overrepresentation of genes with AP-1 sites (represented by the black lines) at the bottom of the ranked gene list suggests that there is a correlation between genes with this binding motif and JQ1 down-regulated genes. The green line represents the running enrichment score. Additional details are provided in Fig. S4 and SI Materials and Methods. (E) Quantitative RT-PCR for FOSL1 (red) and c-MYC (blue) RNA levels in JQ1-treated cell lines. Data are presented as the average ratio of each gene’s expression for each cell line, relative to corresponding DMSO-treated controls (mean ± SEM). All adenocarcinoma cell lines displayed are sensitive to JQ1 except H460. The MM cell line RPMI-8226 is also depicted. Asterisks denote the level of statistical significance (*P < 0.05, **P < 0.01, ***P < 0.005; two-tailed t test). (F) Analysis of FOSL1 and c-MYC protein levels in JQ1-treated sensitive (red) and insensitive (blue) lung cancer cell lines. Cells were treated with DMSO (−) or 5 μM JQ1 (+) for 6 h before assay with anti-FOSL1 and anti–c-MYC antibodies as in Fig. 2. (G) FOSL1 protein levels diminish with the duration of JQ1 treatment. H23 cells were treated with the indicated doses of JQ1, and FOSL1 proteins were assessed at 24 and 48 h. c-MYC proteins are shown for comparison. (H) Dose-dependent effects of JQ1 treatment on FOSL1 protein levels in a drug-sensitive and a drug-resistant lung cancer cell line. c-MYC protein levels are shown for comparison. Cells were treated with the indicated doses for 6 h before analysis. GAPDH serves as a loading control for all Western blots.
Fig. 4.
Fig. 4.
FOSL1 knockdown phenocopies the effects of JQ1 treatment and BRD4 knockdown in lung cancer cell lines. (A) Knockdown of BRD4 decreases the expression of FOSL1 in JQ1-sensitive cell lines. FOSL1 (blue) and BRD4 (red) mRNA levels were assessed by quantitative RT-PCR 72 h after transfection of the indicated siRNAs. Data presented are the average ratio of each gene’s expression relative to the levels observed in cells transfected with nontargeting (NonT) siRNA (mean ± SEM, n = 3). (B) Knockdown of BRD4 decreases FOSL1 protein levels. H1975 and A549 were transfected with the indicated siRNAs and FOSL1 and c-MYC levels were assessed by Western blot after 72 h as described in Fig. 3. GAPDH serves as a loading control. BRD4 knockdown decreases FOSL1 but not c-MYC levels, mimicking JQ1 treatment. (C) Knockdown of FOSL1 and BRD4 decreases viability of JQ1-sensitive lung cancer cell lines. Cell lines were transfected with the indicated siRNAs and viability was assessed by Alamar Blue after 72 h. Data are presented as the average viability of cells treated with each siRNA or JQ1, relative to cells transfected with the nontargeting (NonT) siRNA or DMSO (mean ± SEM, n = 3). KIF11 and KRAS siRNAs serve as positive controls. Asterisks denote the level of statistical significance (*P < 0.05, **P < 0.01, ***P < 0.005; one-tailed, one-sample t test).
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