All-Trans Retinoic Acid Induces Proliferation, Survival, and Migration in A549 Lung Cancer Cells by Activating the ERK Signaling Pathway through a Transcription-Independent Mechanism

Abstract

All-trans retinoic acid (ATRA) has been used as an antineoplastic because of its ability to promote proliferation, inhibition, and differentiation, primarily in leukemia; however, in other types of cancer, such as lung cancer, treatment with ATRA is restricted because not all the patients experience the same results. The ERK signaling pathway is dysregulated in cancer cells, including lung cancer, and this dysregulation promotes proliferation and cell invasion. In this study, we demonstrate that treatment with ATRA can activate the ERK signaling pathway by a transcription-independent mechanism through a signaling cascade that involves RARα and PI3K, promoting growth, survival, and migration in lung cancer cells. Until now, this mechanism was unknown in lung cancer cells. The inhibition of the ERK signaling pathway restores the beneficial effects of ATRA, reduces proliferation, increases apoptosis, and blocks the cell migration process in lung cancer cells. In conclusion, our results suggest that the combination of ATRA with ERK inhibitor in clinical trials for lung cancer is warranted.

Figure 1

ATRA activates the ERK pathway through nongenomic mechanisms in A549 cells. A549 cells were serum-starved for 18 h, treated or nontreated (NT) with 5 μM of ATRA for the times indicated. The phosphorylated form of ERK and total proteins were detected by western blot using specific antibodies.

Figure 2

Effect of AGN193109 and Ro 41-5253 inhibitors on ATRA-induced ERK activation. A549 cells were serum-starved for 18 h, treated or nontreated (NT) with 5 μM of ATRA for 15 minutes. Cells were preincubated for 1 h with 10 μM of AGN193109 or 20 μM of Ro 41-5253 alone or in combination with ATRA. The phosphorylated form of ERK was detected by western blot using specific antibodies. β-Actin was used as the loading control. The graph represents the densitometric values of ERK phosphorylation in three independent experiments (means ± SEM, ^*** P < 0.0001; ^**** P < 0.00001 compared with NT cells, analysis of variance and Newman-Keuls test).

Figure 3

Effect of PI3K inhibitor wortmannin on ATRA-induced ERK phosphorylation. A549 cells were serum-starved for 18 h and treated or nontreated (NT) with 5 μM of ATRA at different times. Cells were preincubated for 1 h with 10 μM of wortmannin (Wm) alone or in combination with ATRA. The phosphorylated form of ERK and Akt and total proteins were detected by western blot using specific antibodies. The graph represents the densitometric analysis of ERK and Akt phosphorylation in three independent experiments (means ± SEM, ^* P < 0.05; ^** P < 0.001; analysis of variance and Newman-Keuls test).

Figure 4

ERK activation is associated with proliferation on ATRA-resistant lung cancer cells. A549 cells were serum-starved and treated or nontreated (NT) with 5 μM of ATRA alone or in combination with 25 μM of PD98059 for 48 h. The proliferative effect was assessed by BrdU labeling according to the manufacturer's instructions. The graph shows the results of three independent experiments (means ± SEM, ^* P < 0.05; ^*** P < 0.0001 compared with NT cells, analysis of variance, and Newman-Keuls test).

Figure 5

Pharmacologic inhibition of MEK-ERK in combination with ATRA promotes apoptosis. A549 cells were serum-starved and treated or nontreated (NT) with 5 μM of ATRA alone or in combination with 25 μM of PD98059 for 48 h. Cells irradiated with 20 J/m² of UV light for 2 min were used as a positive control for apoptosis (+). DNA fragmentation was detected by TUNEL. The apoptotic cells are stained brown. Percentages of TUNEL-positive nuclei were quantified by counting 50 cells from five random microscopic fields (means ± SEM, ^** P < 0.001; ^*** P < 0.0001 compared with NT cells, analysis of variance, and Newman-Keuls test).

Figure 6

The inhibitor PD98059 with ATRA blocks the process of cell migration mediated by ATRA. A549 cells were serum-starved for 24 h and treated with 12 μM mitomycin C for 2 h. After starvation, cells were scratch-wounded and treated or nontreated (NT) with 5 μM of ATRA alone or in combination with 25 μM of PD98059 for 48 h. Pictures were taken at 48 h after wounding and quantification of wound area was documented from five random microscopic fields (means ± SEM, ^** P < 0.001; ^*** P < 0.0001 compared with NT cells, analysis of variance, and Newman-Keuls test).