Limits...
Uncoupling of the LKB1-AMPKalpha energy sensor pathway by growth factors and oncogenic BRAF.

Esteve-Puig R, Canals F, Colomé N, Merlino G, Recio JA - PLoS ONE (2009)

Bottom Line: Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation.These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells.Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.

View Article: PubMed Central - PubMed

Affiliation: Animal Models and Cancer Laboratory, Medical Oncology Research Program, Vall d'Hebron Research Institute-VHIO, Vall d'Hebron Hospital, Barcelona, Spain.

ABSTRACT

Background: Understanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1(Ser428) becomes phosphorylated in a RAS-Erk1/2-p90(RSK) pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown.

Methodology/principal findings: Using the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1(Ser428) (Ser431 in the mouse) is constitutively phosphorylated in BRAF(V600E) mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAF(V600E) mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation. Notably, the inhibition of the RAS pathway in BRAF(V600E) mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKalpha metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1.

Conclusions/significance: These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.

Show MeSH

Related in: MedlinePlus

LKB1Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAFV600E mutant melanoma cells and mouse tumor samples.(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAFV600E), SKMel28 (BRAFV600E) and UACC903 (BRAFV600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1Ser428, p-Erk1/2Thr202/Tyr204 and p-90RSK Thr359/Ser363 is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1Ser431 and p-Erk1/2Thr202/Tyr204 levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651576&req=5

pone-0004771-g002: LKB1Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAFV600E mutant melanoma cells and mouse tumor samples.(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAFV600E), SKMel28 (BRAFV600E) and UACC903 (BRAFV600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1Ser428, p-Erk1/2Thr202/Tyr204 and p-90RSK Thr359/Ser363 is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1Ser431 and p-Erk1/2Thr202/Tyr204 levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.

Mentions: Next, we investigated if this post-translational modification was broader in scope. We tested several growth factors related to cancer development in human and mouse melanoma cell lines to check whether or not these ligands were able to induce LKB1Ser428 phosphorylation. As suggested by previous investigations [18], all ligands tested, including HGF, EGF, basic fibroblast growth factor (FGF2), insulin like growth factor 1 (IGF-1), platelet derived growth factor (PDGF), tumor necrosis factor-alpha (TNF-α), herregulin, insulin and phorbol-ester tumor promoter TPA were able to induced LKB1Ser428 phosphorylation in a cell type dependent manner (Fig. 2A). In all cases, the ligands that activated Erk1/2 and p90RSK kinases led to the phosphorylation of LKB1Ser428 (Fig. 2A).


Uncoupling of the LKB1-AMPKalpha energy sensor pathway by growth factors and oncogenic BRAF.

Esteve-Puig R, Canals F, Colomé N, Merlino G, Recio JA - PLoS ONE (2009)

LKB1Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAFV600E mutant melanoma cells and mouse tumor samples.(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAFV600E), SKMel28 (BRAFV600E) and UACC903 (BRAFV600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1Ser428, p-Erk1/2Thr202/Tyr204 and p-90RSK Thr359/Ser363 is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1Ser431 and p-Erk1/2Thr202/Tyr204 levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651576&req=5

pone-0004771-g002: LKB1Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAFV600E mutant melanoma cells and mouse tumor samples.(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAFV600E), SKMel28 (BRAFV600E) and UACC903 (BRAFV600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1Ser428, p-Erk1/2Thr202/Tyr204 and p-90RSK Thr359/Ser363 is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1Ser431 and p-Erk1/2Thr202/Tyr204 levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.
Mentions: Next, we investigated if this post-translational modification was broader in scope. We tested several growth factors related to cancer development in human and mouse melanoma cell lines to check whether or not these ligands were able to induce LKB1Ser428 phosphorylation. As suggested by previous investigations [18], all ligands tested, including HGF, EGF, basic fibroblast growth factor (FGF2), insulin like growth factor 1 (IGF-1), platelet derived growth factor (PDGF), tumor necrosis factor-alpha (TNF-α), herregulin, insulin and phorbol-ester tumor promoter TPA were able to induced LKB1Ser428 phosphorylation in a cell type dependent manner (Fig. 2A). In all cases, the ligands that activated Erk1/2 and p90RSK kinases led to the phosphorylation of LKB1Ser428 (Fig. 2A).

Bottom Line: Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation.These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells.Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.

View Article: PubMed Central - PubMed

Affiliation: Animal Models and Cancer Laboratory, Medical Oncology Research Program, Vall d'Hebron Research Institute-VHIO, Vall d'Hebron Hospital, Barcelona, Spain.

ABSTRACT

Background: Understanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1(Ser428) becomes phosphorylated in a RAS-Erk1/2-p90(RSK) pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown.

Methodology/principal findings: Using the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1(Ser428) (Ser431 in the mouse) is constitutively phosphorylated in BRAF(V600E) mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAF(V600E) mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation. Notably, the inhibition of the RAS pathway in BRAF(V600E) mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKalpha metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1.

Conclusions/significance: These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.

Show MeSH
Related in: MedlinePlus