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LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro.

Xiao P, Sun LL, Wang J, Han RL, Ma Q, Zhong DS - Acta Pharmacol. Sin. (2015)

Bottom Line: The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells.Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors.Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Tianjin Medical University General Hospital, Tianjin 300052, China.

ABSTRACT

Aim: Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.

Methods: Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.

Results: The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor LY294002. Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

Conclusion: LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.

No MeSH data available.


Related in: MedlinePlus

Effect of RAD001 on AKT phosphorylation. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L RAD001 for 2 h. p-AKT and AKT were analyzed by Western blot using GAPDH as a loading control. Relative changes in p-AKT protein expression were tested in various groups. Statistical significance was evaluated by comparison with the control group using Student's t test. bP<0.05 vs control. (B) H1792 and A549 cells were treated with the control, RAD001 (10 nmol/L) alone, LY294002 (10 μmol/L) alone and LY294002 (10 μmol/L) in combination with RAD001 (10 nmol/L) for 2 h. p-AKT and AKT were analyzed by Western blot. GAPDH was served as the loading control. Statistical significance was evaluated using one-way analysis of variance. bP<0.05 vs RAD001 alone.
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fig3: Effect of RAD001 on AKT phosphorylation. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L RAD001 for 2 h. p-AKT and AKT were analyzed by Western blot using GAPDH as a loading control. Relative changes in p-AKT protein expression were tested in various groups. Statistical significance was evaluated by comparison with the control group using Student's t test. bP<0.05 vs control. (B) H1792 and A549 cells were treated with the control, RAD001 (10 nmol/L) alone, LY294002 (10 μmol/L) alone and LY294002 (10 μmol/L) in combination with RAD001 (10 nmol/L) for 2 h. p-AKT and AKT were analyzed by Western blot. GAPDH was served as the loading control. Statistical significance was evaluated using one-way analysis of variance. bP<0.05 vs RAD001 alone.

Mentions: We next sought to examine the molecular mechanism underlying the resistance to mTOR inhibition in NSCLC cells with or without LKB1 inactivation. Because AKT signaling is known to be a major survival pathway, we examined the potential involvement of AKT activation in RAD001 treatment. NSCLC cells (H1792, Calu-1, A549, and H460) were treated with RAD001 (10 nmol/L) for 2 h, and p-AKT was analyzed by Western blot. It was observed that RAD001 treatment led to a significant increase in p-AKT, suggesting that mTOR inhibition induced AKT activation (Figure 3A).


LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro.

Xiao P, Sun LL, Wang J, Han RL, Ma Q, Zhong DS - Acta Pharmacol. Sin. (2015)

Effect of RAD001 on AKT phosphorylation. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L RAD001 for 2 h. p-AKT and AKT were analyzed by Western blot using GAPDH as a loading control. Relative changes in p-AKT protein expression were tested in various groups. Statistical significance was evaluated by comparison with the control group using Student's t test. bP<0.05 vs control. (B) H1792 and A549 cells were treated with the control, RAD001 (10 nmol/L) alone, LY294002 (10 μmol/L) alone and LY294002 (10 μmol/L) in combination with RAD001 (10 nmol/L) for 2 h. p-AKT and AKT were analyzed by Western blot. GAPDH was served as the loading control. Statistical significance was evaluated using one-way analysis of variance. bP<0.05 vs RAD001 alone.
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Related In: Results  -  Collection

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fig3: Effect of RAD001 on AKT phosphorylation. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L RAD001 for 2 h. p-AKT and AKT were analyzed by Western blot using GAPDH as a loading control. Relative changes in p-AKT protein expression were tested in various groups. Statistical significance was evaluated by comparison with the control group using Student's t test. bP<0.05 vs control. (B) H1792 and A549 cells were treated with the control, RAD001 (10 nmol/L) alone, LY294002 (10 μmol/L) alone and LY294002 (10 μmol/L) in combination with RAD001 (10 nmol/L) for 2 h. p-AKT and AKT were analyzed by Western blot. GAPDH was served as the loading control. Statistical significance was evaluated using one-way analysis of variance. bP<0.05 vs RAD001 alone.
Mentions: We next sought to examine the molecular mechanism underlying the resistance to mTOR inhibition in NSCLC cells with or without LKB1 inactivation. Because AKT signaling is known to be a major survival pathway, we examined the potential involvement of AKT activation in RAD001 treatment. NSCLC cells (H1792, Calu-1, A549, and H460) were treated with RAD001 (10 nmol/L) for 2 h, and p-AKT was analyzed by Western blot. It was observed that RAD001 treatment led to a significant increase in p-AKT, suggesting that mTOR inhibition induced AKT activation (Figure 3A).

Bottom Line: The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells.Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors.Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Tianjin Medical University General Hospital, Tianjin 300052, China.

ABSTRACT

Aim: Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.

Methods: Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.

Results: The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor LY294002. Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

Conclusion: LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.

No MeSH data available.


Related in: MedlinePlus