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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

LKB1 inactivation does not increase the sensitivity to mTOR inhibitors. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L rapamycin for 2 h. Cell extracts were subjected to immunoblotting analysis for p-4EBP1, 4EBP1, and GAPDH. Statistical significance was evaluated by comparison with the control group using Student's t-test. bP<0.05. (B) H1299 and H460 were seeded on 96-well plates and exposed to rapamycin at different concentrations. The cells were subjected to MTS assays after 72 h. (C) A549 cells were exposed to RAD001 (0, 1, 10, 100, and 1000 nmol/L) for 2 h. p-4EBP1, 4EBP1, and GAPDH were tested by immunoblotting analysis. Statistical significance was evaluated using one-way analysis of variance. bP<0.05. (D) H1299, H1792, Calu-1, H460, and A549 cells were treated with RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) and were subjected to an MTS assay after 72 h. (E) RAD001 was treated in the same cell line with concentrations of 0, 0.1, 1, 10, 100, and 1000 nmol/L and were then subjected to SRB assays after 72 h. (F) Anti-LKB1 antibody was used to detect LKB1 using GAPDH as the loading control. (G) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of rapamycin (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation. (H) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation.
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fig2: LKB1 inactivation does not increase the sensitivity to mTOR inhibitors. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L rapamycin for 2 h. Cell extracts were subjected to immunoblotting analysis for p-4EBP1, 4EBP1, and GAPDH. Statistical significance was evaluated by comparison with the control group using Student's t-test. bP<0.05. (B) H1299 and H460 were seeded on 96-well plates and exposed to rapamycin at different concentrations. The cells were subjected to MTS assays after 72 h. (C) A549 cells were exposed to RAD001 (0, 1, 10, 100, and 1000 nmol/L) for 2 h. p-4EBP1, 4EBP1, and GAPDH were tested by immunoblotting analysis. Statistical significance was evaluated using one-way analysis of variance. bP<0.05. (D) H1299, H1792, Calu-1, H460, and A549 cells were treated with RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) and were subjected to an MTS assay after 72 h. (E) RAD001 was treated in the same cell line with concentrations of 0, 0.1, 1, 10, 100, and 1000 nmol/L and were then subjected to SRB assays after 72 h. (F) Anti-LKB1 antibody was used to detect LKB1 using GAPDH as the loading control. (G) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of rapamycin (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation. (H) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation.

Mentions: mTOR is a key regulator of cell growth; thus, we hypothesized that LKB1 mutant NSCLC cells with higher mTOR activity would be more sensitive to mTOR inhibitors than were LKB1 wild-type cells. To test this possibility, we evaluated the effect of mTOR inhibitors on the proliferation of these NSCLC cells. It was observed that treatment with 10 nmol/L rapamycin significantly decreased 4EBP1 phosphorylation in both LKB1 wild-type (H1792 and Calu-1) and LKB1 mutant cells (H460 and A549) (Figure 2A), confirming the inhibition of mTOR activity by rapamycin in NSCLC cells. We then treated these cells with a gradient of concentrations of rapamycin and measured cell proliferation by MTS assay. Unexpectedly, rapamycin had little effect on the growth of either H1299 (wild-type LKB1) or H460 (mutant LKB1) cells (Figure 2B). To validate our observation, we used the mTOR specific inhibitor RAD001. As shown in Figure 2C, treatment of A549 cells with a gradient of concentrations of RAD001 determined that RAD001 decreased 4EBP1 phosphorylation in a dose-dependent manner, indicating that it effectively inhibited mTOR activity. Next, we examined its effect on cell proliferation with MTS assay. Similarly to rapamycin, RAD001 did not obviously affect the proliferation of the various NSCLC cell lines (Figure 2D). Growth inhibition by RAD001, even at concentrations up to 1000 nmol/L, was less than 50%. Moreover, some LKB1 wild-type cells, such as Calu-1, were even more responsive to RAD001 than the LKB1 mutant cells, including H460. To confirm our results, we additionally measured cell proliferation by SRB assays, and similar results were achieved (Figure 2E). Thus, no association was found between sensitivity to mTOR inhibitors and LKB1 gene status.


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)

LKB1 inactivation does not increase the sensitivity to mTOR inhibitors. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L rapamycin for 2 h. Cell extracts were subjected to immunoblotting analysis for p-4EBP1, 4EBP1, and GAPDH. Statistical significance was evaluated by comparison with the control group using Student's t-test. bP<0.05. (B) H1299 and H460 were seeded on 96-well plates and exposed to rapamycin at different concentrations. The cells were subjected to MTS assays after 72 h. (C) A549 cells were exposed to RAD001 (0, 1, 10, 100, and 1000 nmol/L) for 2 h. p-4EBP1, 4EBP1, and GAPDH were tested by immunoblotting analysis. Statistical significance was evaluated using one-way analysis of variance. bP<0.05. (D) H1299, H1792, Calu-1, H460, and A549 cells were treated with RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) and were subjected to an MTS assay after 72 h. (E) RAD001 was treated in the same cell line with concentrations of 0, 0.1, 1, 10, 100, and 1000 nmol/L and were then subjected to SRB assays after 72 h. (F) Anti-LKB1 antibody was used to detect LKB1 using GAPDH as the loading control. (G) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of rapamycin (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation. (H) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation.
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fig2: LKB1 inactivation does not increase the sensitivity to mTOR inhibitors. (A) H1792, Calu-1, A549, and H460 cells were treated with 10 nmol/L rapamycin for 2 h. Cell extracts were subjected to immunoblotting analysis for p-4EBP1, 4EBP1, and GAPDH. Statistical significance was evaluated by comparison with the control group using Student's t-test. bP<0.05. (B) H1299 and H460 were seeded on 96-well plates and exposed to rapamycin at different concentrations. The cells were subjected to MTS assays after 72 h. (C) A549 cells were exposed to RAD001 (0, 1, 10, 100, and 1000 nmol/L) for 2 h. p-4EBP1, 4EBP1, and GAPDH were tested by immunoblotting analysis. Statistical significance was evaluated using one-way analysis of variance. bP<0.05. (D) H1299, H1792, Calu-1, H460, and A549 cells were treated with RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) and were subjected to an MTS assay after 72 h. (E) RAD001 was treated in the same cell line with concentrations of 0, 0.1, 1, 10, 100, and 1000 nmol/L and were then subjected to SRB assays after 72 h. (F) Anti-LKB1 antibody was used to detect LKB1 using GAPDH as the loading control. (G) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of rapamycin (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation. (H) H1299-LKB1shRNA and H1299-PLKO.1 were treated with a gradient of concentrations of RAD001 (0, 0.1, 1, 10, 100, and 1000 nmol/L) for 72 h, and MTS assays were performed to measure cell proliferation.
Mentions: mTOR is a key regulator of cell growth; thus, we hypothesized that LKB1 mutant NSCLC cells with higher mTOR activity would be more sensitive to mTOR inhibitors than were LKB1 wild-type cells. To test this possibility, we evaluated the effect of mTOR inhibitors on the proliferation of these NSCLC cells. It was observed that treatment with 10 nmol/L rapamycin significantly decreased 4EBP1 phosphorylation in both LKB1 wild-type (H1792 and Calu-1) and LKB1 mutant cells (H460 and A549) (Figure 2A), confirming the inhibition of mTOR activity by rapamycin in NSCLC cells. We then treated these cells with a gradient of concentrations of rapamycin and measured cell proliferation by MTS assay. Unexpectedly, rapamycin had little effect on the growth of either H1299 (wild-type LKB1) or H460 (mutant LKB1) cells (Figure 2B). To validate our observation, we used the mTOR specific inhibitor RAD001. As shown in Figure 2C, treatment of A549 cells with a gradient of concentrations of RAD001 determined that RAD001 decreased 4EBP1 phosphorylation in a dose-dependent manner, indicating that it effectively inhibited mTOR activity. Next, we examined its effect on cell proliferation with MTS assay. Similarly to rapamycin, RAD001 did not obviously affect the proliferation of the various NSCLC cell lines (Figure 2D). Growth inhibition by RAD001, even at concentrations up to 1000 nmol/L, was less than 50%. Moreover, some LKB1 wild-type cells, such as Calu-1, were even more responsive to RAD001 than the LKB1 mutant cells, including H460. To confirm our results, we additionally measured cell proliferation by SRB assays, and similar results were achieved (Figure 2E). Thus, no association was found between sensitivity to mTOR inhibitors and LKB1 gene status.

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