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Inhibition of ERK1/2 down-regulates the Hippo/YAP signaling pathway in human NSCLC cells.

You B, Yang YL, Xu Z, Dai Y, Liu S, Mao JH, Tetsu O, Li H, Jablons DM, You L - Oncotarget (2015)

Bottom Line: Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5.Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately.Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.

ABSTRACT
Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3'UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

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Analysis of Hippo/YAP pathway activity after MEK1/2 inhibition in NSCLC cells(A) Western blotting analysis of YAP expression in A549 cells treated with MEK1/2 inhibitor Trametinib or ERK inhibitor FR180204. (B) The mRNA level of YAP in A549 cells treated with MEK1/2 inhibitor Trametinib was measured using RT-PCR. (C) A dose-dependent decrease in GTIIC reporter activity of the Hippo pathway was analyzed in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA and Scheffe multiple comparisons). (D) Decreased mRNA levels of BIRC5 and Gli2, the downstream genes of the Hippo pathway, in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA, Scheffe multiple comparisons).
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Figure 7: Analysis of Hippo/YAP pathway activity after MEK1/2 inhibition in NSCLC cells(A) Western blotting analysis of YAP expression in A549 cells treated with MEK1/2 inhibitor Trametinib or ERK inhibitor FR180204. (B) The mRNA level of YAP in A549 cells treated with MEK1/2 inhibitor Trametinib was measured using RT-PCR. (C) A dose-dependent decrease in GTIIC reporter activity of the Hippo pathway was analyzed in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA and Scheffe multiple comparisons). (D) Decreased mRNA levels of BIRC5 and Gli2, the downstream genes of the Hippo pathway, in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA, Scheffe multiple comparisons).

Mentions: To investigate whether inhibiting MEK, an upstream regulator of ERK signaling, affects Hippo pathway activity, we analyzed YAP expression in NSCLC A549 cells treated with the MEK1/2 inhibitor Trametinib (N-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6, 8-dimethyl-2, 4, 7-trioxo-3, 4, 6, 7-tetrahydropyrido [4,3-d] pyrimidin-1(2H)-yl) phenyl) acetamide, GSK1120212 [29] or the ERK inhibitor FR180204. We found that YAP protein level decreased after treatment with either inhibitor, in contrast with what occurred in cells treated with DMSO (Figure 7A). The mRNA level of YAP in A549 cells after Trametinib treatment was analyzed using RT-PCR, and there was no significant difference in YAP mRNA level in the cells with Trametinib or DMSO treatment (Figure 7B, Suppl. Table S4). Together, these results suggest that MEK inhibition decreased YAP protein level, but has only a minimal effect on YAP transcription in the cells. Furthermore, we examined the reporter activity and downstream gene expression of the Hippo pathway in NSCLC cells after MEK inhibition. GTIIC reporter activity of the Hippo pathway was decreased in A549 and H2170 cells after treatment with the MEK inhibitor Trametinib, compared to that of their respective control cells (Figure 7C, P < 0.05). Similarly, the mRNA levels of Hippo downstream genes, Gli2 and BIRC5, were consistently down-regulated by MEK inhibition in A549 cells (Figure 7D, Suppl. Table S5, P < 0.05). The effect of MEK inhibition was similar to that of ERK inhibition on Hippo/YAP pathway activity. Our results suggest that MEK inhibition mediated YAP down-regulation at the protein level and thereby suppresses the reporter activity and downstream gene expression of the Hippo pathway in NSCLC cell lines.


Inhibition of ERK1/2 down-regulates the Hippo/YAP signaling pathway in human NSCLC cells.

You B, Yang YL, Xu Z, Dai Y, Liu S, Mao JH, Tetsu O, Li H, Jablons DM, You L - Oncotarget (2015)

Analysis of Hippo/YAP pathway activity after MEK1/2 inhibition in NSCLC cells(A) Western blotting analysis of YAP expression in A549 cells treated with MEK1/2 inhibitor Trametinib or ERK inhibitor FR180204. (B) The mRNA level of YAP in A549 cells treated with MEK1/2 inhibitor Trametinib was measured using RT-PCR. (C) A dose-dependent decrease in GTIIC reporter activity of the Hippo pathway was analyzed in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA and Scheffe multiple comparisons). (D) Decreased mRNA levels of BIRC5 and Gli2, the downstream genes of the Hippo pathway, in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA, Scheffe multiple comparisons).
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Figure 7: Analysis of Hippo/YAP pathway activity after MEK1/2 inhibition in NSCLC cells(A) Western blotting analysis of YAP expression in A549 cells treated with MEK1/2 inhibitor Trametinib or ERK inhibitor FR180204. (B) The mRNA level of YAP in A549 cells treated with MEK1/2 inhibitor Trametinib was measured using RT-PCR. (C) A dose-dependent decrease in GTIIC reporter activity of the Hippo pathway was analyzed in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA and Scheffe multiple comparisons). (D) Decreased mRNA levels of BIRC5 and Gli2, the downstream genes of the Hippo pathway, in A549 and H2170 cells treated with MEK1/2 inhibitor Trametinib (*P < 0.05, One-way ANOVA, Scheffe multiple comparisons).
Mentions: To investigate whether inhibiting MEK, an upstream regulator of ERK signaling, affects Hippo pathway activity, we analyzed YAP expression in NSCLC A549 cells treated with the MEK1/2 inhibitor Trametinib (N-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6, 8-dimethyl-2, 4, 7-trioxo-3, 4, 6, 7-tetrahydropyrido [4,3-d] pyrimidin-1(2H)-yl) phenyl) acetamide, GSK1120212 [29] or the ERK inhibitor FR180204. We found that YAP protein level decreased after treatment with either inhibitor, in contrast with what occurred in cells treated with DMSO (Figure 7A). The mRNA level of YAP in A549 cells after Trametinib treatment was analyzed using RT-PCR, and there was no significant difference in YAP mRNA level in the cells with Trametinib or DMSO treatment (Figure 7B, Suppl. Table S4). Together, these results suggest that MEK inhibition decreased YAP protein level, but has only a minimal effect on YAP transcription in the cells. Furthermore, we examined the reporter activity and downstream gene expression of the Hippo pathway in NSCLC cells after MEK inhibition. GTIIC reporter activity of the Hippo pathway was decreased in A549 and H2170 cells after treatment with the MEK inhibitor Trametinib, compared to that of their respective control cells (Figure 7C, P < 0.05). Similarly, the mRNA levels of Hippo downstream genes, Gli2 and BIRC5, were consistently down-regulated by MEK inhibition in A549 cells (Figure 7D, Suppl. Table S5, P < 0.05). The effect of MEK inhibition was similar to that of ERK inhibition on Hippo/YAP pathway activity. Our results suggest that MEK inhibition mediated YAP down-regulation at the protein level and thereby suppresses the reporter activity and downstream gene expression of the Hippo pathway in NSCLC cell lines.

Bottom Line: Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5.Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately.Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.

ABSTRACT
Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3'UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

Show MeSH
Related in: MedlinePlus