Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells

Abstract

Background: All-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance.

Results: We performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes.

Conclusion: Our results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.

Figure 1

ATRA activates the Akt pathway through non-genomic mechanisms in A549 cells. (A) Left, A549 cells were serum-starved for 18 h, treated or non-treated (NT) with 5 μM of ATRA, for the times indicated. Right, A549 cells were preincubated for 1 h with 3 μM of BMS493 before ATRA treatment and total extracts were prepared. The phosphorylated form of Akt and total proteins were detected by western blot using specific antibodies. The bottom graph represents the densitometric values of Akt phosphorylation of three independent experiments (means ± SEM, *P < 0.05; **P < 0.001 compared with non-treated cells (NT) (analysis of variance and Newman-Keuls test). (B) A549 cells were serum-starved and treated with ATRA with or without BMS493 for 48 h. Total proteins were detected by western blot.

Figure 2

ATRA promotes Akt activation mediated by RARα-Akt interaction. (A) RARα was immunoprecipitated from A549 cells treated or non-treated with 5 μM of ATRA for 15 min. Immunoprecipitated RARα and associated protein were detected by western blot. Control refers to immunoprecipitation performed with an Erk1 antibody. The bottom graph shows the results of densitometric analyses of Akt bound to RARα obtained from three independent experiments (means ± SEM, *P < 0.05 compared with non-treated cells (NT) assessed by t test analysis). (B) RARα was immunoprecipitated from A549 cells transfected with EGFP-APPL1 or empty vector and treated with 5 μM of ATRA for 15 min. Association of RARα with Akt was detected by western blot using specific antibodies. Image shows one representative experiment of three independent.

Figure 3

ATRA promotes recruitment of RARα to the plasma membrane. A549 cells were serum-starved and treated with 5 μM of ATRA for the times indicated. Then cells were fixed and incubated with anti-RARα and anti-Akt followed by incubation with anti-mouse Alexa Fluor 532 and Alexa Fluor 647, respectively, as described in Materials and Methods. Finally, the cells were analyzed by confocal microscopy.

Figure 4

ATRA stimulates Rac activation and promotes invasion. (A) Left, A549 cells were serum-starved for 18 h and treated with 5 μM of ATRA for the times indicated. Other cells were preincubated for 1 h with 5 μM of 15e. Activated Rac was detected with the Rac1 Activation assay kit according to the manufacturer’s instructions. Right, the graph shows the results of densitometric analysis of relative increase of Rac activation obtained in three independent experiments. (B) Cell invasion was analyzed by QCM™ 24–well Invasion Assay Kit. A549 cells were transfected with Myr-Akt, Akt-K179M or empty vector and seeded at 2.5 × 10⁵ cells/well into the upper chamber. DMEM/F12 was added to the lower chamber with or without 5 μM ATRA for 48 h. The invasive cells were detected according to the manufacturer’s instructions. The graphs shows the results of three independent experiments (means ± SEM, *P < 0.05 compared with non-treated cells (NT) (analysis of variance and Newman-Keuls test).

Figure 5

Inhibition of the PI3k/Akt pathway promotes apoptosis by activation of caspase-3. (A) Left, A549 cells were serum-starved and treated or non-treated (control) with ATRA for 48 h, during the first 12 h after treatment with ATRA, the cells were irradiated with 150 J/m² of UV-C light for 30 min. Subsequently, DNA fragmentation was detected by TUNEL according to the manufacturer’s instructions. The apoptotic cells are stained brown. Bar, 20 μm. Right, percentages of TUNEL-positive cells were quantified by counting 200 cells from four random microscopic fields (means ± SEM, *P < 0.05 compared with non-treated cells (control) assessed by t test analysis). (B) A549 cells were treated for 48 h with 5 μM of ATRA alone or combined with 5 μM of 15e. Subsequently, DNA fragmentation was detected by TUNEL. Control cells were non-treated. Percentages of TUNEL-positive cells were quantified by counting 200 cells from four random microscopic fields. Means ± SEM, *P < 0.05; **P < 0.001 compared with non-treated cells (control) (analysis of variance and Newman-Keuls test). (C) A549 cells were serum-starved and treated or non-treated (control) with 5 μM of ATRA alone or combined with 5 μM of 15e for 48 h. The cells were fixed, stained with anti-cleaved caspase-3 followed by donkey anti-goat FITC as described in Materials and Methods and analyzed by fluorescence microscopy. Bar, 20 μm.

Figure 6

Inactive form of Akt (K179M) blocks the ATRA-dependent survival effect. (A) A549 cells were transfected with Myr-Akt, Akt-K179M or empty vector and subsequently treated or non-treated with 5 μM of ATRA for 48 h. Subsequently, DNA fragmentation was detected by TUNEL according to the manufacturer’s instructions. Control cells were non-treated. The apoptotic cells are stained brown. (B) Percentages of TUNEL-positive cells were quantified by counting 100 cells from three random microscopic fields. Means ± SEM, *P < 0.05; **P < 0.001 compared with non-treated cells (NT) (analysis of variance and Newman-Keuls test). Bar, 20 μm.

Figure 7

Akt activation promotes the down-regulation of RARβ2 and p53. (A) Left, A549 cells were transfected with Myr-Akt, Akt-K179M or empty vector and subsequently treated or non-treated with 5 μM of ATRA for 48 h. Total extracts were prepared and levels of protein were detected by western blot. Right, the graph shows the results of densitometric analysis of relative RARβ2 protein expression levels, obtained in three independent experiments (means ± SEM, *P < 0.05 compared with non-treated cells (NT) transfected with empty vector (analysis of variance and Newman-Keuls test). (B) A549 cells were transfected with Myr-Akt and subsequently treated or non-treated with 5 μM of ATRA for 48 h. For the last 24 h of the 48 h treatment period, the cells were incubated with 20 μM of MG132. Total extracts were prepared and levels of protein were detected by western blot using specific antibodies. The image shows one representative experiment of three independent. (C) A549 cells were serum-starved and treated or non-treated (control) with 5 μM of ATRA alone or in combination with 5 μM of 15e for 24 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 < 0.001 compared with non-treated cells (NT) (analysis of variance and Newman-Keuls test).

Figure 8

Model depicting the molecular mechanism of ATRA resistance in lung cancer. ATRA promotes RARα recruitment to the membrane, where it activates the PI3k-Akt pathway (1–2). Akt activation promotes cellular survival and cellular invasion (3). Akt represses RARβ2 and p53 expression (4). PI3k-Akt inhibition restores sensitivity to ATRA treatment and blocks survival and invasion (5).