Glucocorticoid receptor status is a principal determinant of variability in the sensitivity of non-small-cell lung cancer cells to pemetrexed

Abstract

Introduction: Pemetrexed is an S-phase targeted drug in front-line or maintenance therapy of advanced nonsquamous non-small-cell lung cancer (NSCLC) but methods are needed for predicting the drug response. Dexamethasone is typically administered the day before, the day of, and the day after pemetrexed. As dexamethasone strongly regulates many genes including p53 through the glucocorticoid receptor (GR), we hypothesized that dexamethasone influences tumor response to pemetrexed.

Methods: Eight nonsquamous NSCLC cell line models with varied p53 and GRα/GRβ status were used for gene expression and cell-cycle analyses and for loss- or gain-of-function experiments.

Results: In three cell lines dexamethasone profoundly, but reversibly, suppressed the fraction of S-phase cells. Dexamethasone also reversibly repressed expression of thymidylate synthase and dihydrofolate reductase, which are primary targets of pemetrexed but are also quintessential S-phase enzymes as well as the S-phase-dependent expression of thymidine kinase 1. Dexamethasone also decreased expression of the major pemetrexed transporters, the reduced folate carrier and the proton coupled folate transporter. Only cells expressing relatively high GRα showed these dexamethasone effects, regardless of p53 status. In cells expressing low GRα, the dexamethasone response was rescued by ectopic GRα. Further, depletion of p53 did not attenuate the dexamethasone effects. The presence of dexamethasone during pemetrexed treatment protected against pemetrexed cytotoxicity in only the dexamethasone responsive cells.

Conclusions: The results predict that in nonsquamous NSCLC tumors, reversible S-phase suppression by dexamethasone, possibly combined with a reduction in the drug transporters, attenuates responsiveness to pemetrexed and that GR status is a principal determinant of tumor variability of this response.

Figure 1. Differential effects of Dex on the expression of genes involved in pemetrexed action in A549 vs. H1299 cells

A549 cells (Panels A, C) and H1299 cells (Panels B, D) were plated at 20 percent confluence in media containing charcoal-stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100 nM) for 48 h. Cells were harvested and total RNA extracted to measure mRNA levels by real time RT-PCR (Panels A and B). Whole cell lysates were also extracted from the cells to measure the protein levels of TS and DHFR by western blot; GAPDH was used as the loading control (Panels C and D). * P < 0.01. All of the mRNA measurements were carried out using biological triplicate samples.

Figure 2. Effect of Dex on cell cycle phase distribution in A549 and H1299 cells

A549 cells (Panel A) and H1299 cells (Panel B) were plated at 20 percent confluence in media containing charcoal-stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100 nM) for 48 h and harvested for flow cytometry analysis. The experiments were repeated at least three times. Within each experiment, the percent error for replicate samples was < 10 percent. The P values for Dex induced changes noted in the text were < 0.0001.

Figure 3. Reversibility of Dex effects in A549 cells

A549 cells were plated at 20 percent confluence in media containing charcoal-stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100 nM). After 48 h, Dex was removed by washing the cells twice and replacing with fresh media. Cells were cultured for additional periods of 24 h and 48 h. Cells were harvested for flow cytometry analysis (Panel A). The experiments were repeated at least three times. Within each experiment, the percent error for replicate samples was < 10 percent. The P values for Dex induced changes noted in the text were < 0.0001. In parallel, cells were harvested for mRNA measurement by real time RT-PCR (Panel B). * P < 0.01. All of the mRNA measurements were carried out using biological triplicate samples.

Figure 4. Effect of restoring GRα on responsiveness to Dex in H1299 cells

H1299-GRα cells were generated as described under Materials and Methods. RNA extracted from A549 cells, H1299 cells and H1299-GRα cells was used to measure the relative mRNA levels for GRα (Panel A). H1299-GRα cells were plated at 20 percent confluence in media containing charcoal-stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100 nM) for 48 h. Cells were harvested for mRNA measurement by real time RT-PCR (Panel B). * P < 0.01. In parallel, cells were harvested for cell cycle analysis (Panel C). The experiments were repeated at least three times. Within each experiment, the percent error for replicate samples was < 10 percent. The P values for Dex induced changes noted in the text were < 0.0001. All of the mRNA measurements were carried out using biological triplicate samples.

Figure 5. Effect of knocking down p53 on responsiveness to Dex in H226 cells

The relative p53 expression levels in the parental H226 cells vs. H226-p53KD cells were determined by real time RT-PCR (Panel A). H226-p53KD cells (Panel B) or the parental H226 cells (Panel C) were plated at 20 percent confluence in media containing charcoal-stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100 nM) for 48 h and then harvested for mRNA measurement by real time RT-PCR. * P < 0.01. All of the mRNA measurements were carried out using biological triplicate samples.

Figure 6. Differential effects of Dex on growth inhibition by pemetrexed in A549 vs. H1299 cells

A549 cells (Panel A) and H1299 cells (Panel B) were plated in six replicate wells in 96 well plates in media containing charcoal-stripped serum as described under Materials and Methods for MTT assays. 24 h after plating, the cells were treated with vehicle (ethanol) or Dex (100 nM) for 72 h – 96 h. The cells were exposed to pemetrexed (5 μM) or vehicle (water) for a 24 hour window in the midst of the Dex (or vehicle) treatment. MTT assays were performed on the indicated days, beginning with the day of pemetrexed treatment (Day 0). At each time point, the ratio of the absorbance for pemetrexed treatment to its vehicle control was used to determine the percentage of viable cells.

Figure 7. Influence of Dex on inhibition of colony formation by pemetrexed in relation to GRα status

A549 cells (Panels A, D), H1299 cells (Panels B, E) and H1299-GRα cells (Panels C, F) were first plated at 20 percent confluence in 10 cm dishes in media containing charcoal-stripped serum for hormone depletion. 24 h later cells were treated with vehicle (ethanol) or Dex (100 nM) for 72 h – 96 h. The cells were exposed to pemetrexed (5 μM) or vehicle (water) for a 24 hour window in the midst of the Dex (or vehicle) treatment. The cells were then plated for the colony formation assay as described under Materials and Methods. After the colonies were formed, they were stained with crystal violet. The colony counts from replicate wells are represented in form of histograms (Panels D, E and F). *,** P < 0.0001.