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Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma

View Article: PubMed Central - PubMed

ABSTRACT

Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated. Here, we show that BMK1 pathway contributes to the drug resistance to CIBM. Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM. Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated. Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells. It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance. Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM. Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells. Further kinome-scale siRNA screening demonstrated that SRC\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM. Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma.

No MeSH data available.


Related in: MedlinePlus

Phosphoproteomic profiling of CIBM.(a) Mitogen-activated protein kinase (MAPK) signalling cascades in mammalian cells. (b) Experimental scheme for phosphoproteomic profiling. Light cells were pretreated with 2 μM Vemurafenib and 10 nM Trametinib for 1 h. After treated with serum for 1.5 hrs, heavy and light cell lysates were combined in a 1:1 ratio. This was followed by mass spectrometer analysis, which resulted in about 6,400 phosphosites identified. (c) Distribution of the ratio of phosphopeptides detected in phosphoproteomic profiling. (d,e) A375 and SK-MEL-28 cells were serum starved overnight followed by treatment with/without 2 μM Vemurafenib and 10 nM Trametinib for 1 hr as noted. Then cells were stimulated with serum for 1.5 hrs and phosphorylated BMK1 was detected by mobility retardation27. ACTIN, JUN, phospho-JUN, ERK1/2 and phospho-ERK1/2 (T202/Y204) were detected by the antibody as noted. (f) Number of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified in this study. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (g) Percentage of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (h) Number of gene enriched according to pathways by DAVID Bioinformatics Resources 6.7 using default setting. (i) Network scheme of MAPK pathways shows the emergent signaling, which occurs when BRAF-MEK is inhibited.
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f1: Phosphoproteomic profiling of CIBM.(a) Mitogen-activated protein kinase (MAPK) signalling cascades in mammalian cells. (b) Experimental scheme for phosphoproteomic profiling. Light cells were pretreated with 2 μM Vemurafenib and 10 nM Trametinib for 1 h. After treated with serum for 1.5 hrs, heavy and light cell lysates were combined in a 1:1 ratio. This was followed by mass spectrometer analysis, which resulted in about 6,400 phosphosites identified. (c) Distribution of the ratio of phosphopeptides detected in phosphoproteomic profiling. (d,e) A375 and SK-MEL-28 cells were serum starved overnight followed by treatment with/without 2 μM Vemurafenib and 10 nM Trametinib for 1 hr as noted. Then cells were stimulated with serum for 1.5 hrs and phosphorylated BMK1 was detected by mobility retardation27. ACTIN, JUN, phospho-JUN, ERK1/2 and phospho-ERK1/2 (T202/Y204) were detected by the antibody as noted. (f) Number of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified in this study. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (g) Percentage of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (h) Number of gene enriched according to pathways by DAVID Bioinformatics Resources 6.7 using default setting. (i) Network scheme of MAPK pathways shows the emergent signaling, which occurs when BRAF-MEK is inhibited.

Mentions: In addition, four MAP kinase pathways have been identified in mammal: ERK1/2, BMK1 (mitogen-activated protein kinase 7 or big mitogen-activated protein kinase 1), p38 (mitogen-activated protein kinase 14) and JNK (mitogen-activated protein kinase 8) pathways (Fig. 1a)11. Activated by growth factors, ERK1/2 and BMK1 pathways promote cell proliferation and survival. While JNK and p38 pathways regulate cell death and proliferation1112. Among the MAPKs, BMK1 is the most similar to ERK1/2. Hence, it is not surprising that BMK1 share a bunch of substrates with ERK1/21112. In response to extracellular signals, BMK1 is specifically activated by MEK5 (mitogen-activated protein kinase kinase 5) and translocates to the cell nucleus and regulates gene expression by phosphorylating1213. Like ERK1/2 pathway, BMK1 pathway has been reported to play critical roles in the multi properties of human malignancies, including tumorigenesis, chemoresistance14, proliferation15 and metastasis16. In the previous study13, a small molecular inhibitor of BMK1, XMD8-92, was developed. This BMK1 inhibitor, XMD8-92, can efficiently suppress the proliferation of multi types of cancer cells1317.


Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma
Phosphoproteomic profiling of CIBM.(a) Mitogen-activated protein kinase (MAPK) signalling cascades in mammalian cells. (b) Experimental scheme for phosphoproteomic profiling. Light cells were pretreated with 2 μM Vemurafenib and 10 nM Trametinib for 1 h. After treated with serum for 1.5 hrs, heavy and light cell lysates were combined in a 1:1 ratio. This was followed by mass spectrometer analysis, which resulted in about 6,400 phosphosites identified. (c) Distribution of the ratio of phosphopeptides detected in phosphoproteomic profiling. (d,e) A375 and SK-MEL-28 cells were serum starved overnight followed by treatment with/without 2 μM Vemurafenib and 10 nM Trametinib for 1 hr as noted. Then cells were stimulated with serum for 1.5 hrs and phosphorylated BMK1 was detected by mobility retardation27. ACTIN, JUN, phospho-JUN, ERK1/2 and phospho-ERK1/2 (T202/Y204) were detected by the antibody as noted. (f) Number of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified in this study. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (g) Percentage of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (h) Number of gene enriched according to pathways by DAVID Bioinformatics Resources 6.7 using default setting. (i) Network scheme of MAPK pathways shows the emergent signaling, which occurs when BRAF-MEK is inhibited.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384194&req=5

f1: Phosphoproteomic profiling of CIBM.(a) Mitogen-activated protein kinase (MAPK) signalling cascades in mammalian cells. (b) Experimental scheme for phosphoproteomic profiling. Light cells were pretreated with 2 μM Vemurafenib and 10 nM Trametinib for 1 h. After treated with serum for 1.5 hrs, heavy and light cell lysates were combined in a 1:1 ratio. This was followed by mass spectrometer analysis, which resulted in about 6,400 phosphosites identified. (c) Distribution of the ratio of phosphopeptides detected in phosphoproteomic profiling. (d,e) A375 and SK-MEL-28 cells were serum starved overnight followed by treatment with/without 2 μM Vemurafenib and 10 nM Trametinib for 1 hr as noted. Then cells were stimulated with serum for 1.5 hrs and phosphorylated BMK1 was detected by mobility retardation27. ACTIN, JUN, phospho-JUN, ERK1/2 and phospho-ERK1/2 (T202/Y204) were detected by the antibody as noted. (f) Number of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified in this study. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (g) Percentage of phosphosites (p-S, p-T and p-Y), phosphopeptides (p-Pep), and phosphoproteins (p-Pro) identified. p-S: phosphorylated serine; p-T: phosphorylated threonine; p-Y: phosphorylated tyrosine. (h) Number of gene enriched according to pathways by DAVID Bioinformatics Resources 6.7 using default setting. (i) Network scheme of MAPK pathways shows the emergent signaling, which occurs when BRAF-MEK is inhibited.
Mentions: In addition, four MAP kinase pathways have been identified in mammal: ERK1/2, BMK1 (mitogen-activated protein kinase 7 or big mitogen-activated protein kinase 1), p38 (mitogen-activated protein kinase 14) and JNK (mitogen-activated protein kinase 8) pathways (Fig. 1a)11. Activated by growth factors, ERK1/2 and BMK1 pathways promote cell proliferation and survival. While JNK and p38 pathways regulate cell death and proliferation1112. Among the MAPKs, BMK1 is the most similar to ERK1/2. Hence, it is not surprising that BMK1 share a bunch of substrates with ERK1/21112. In response to extracellular signals, BMK1 is specifically activated by MEK5 (mitogen-activated protein kinase kinase 5) and translocates to the cell nucleus and regulates gene expression by phosphorylating1213. Like ERK1/2 pathway, BMK1 pathway has been reported to play critical roles in the multi properties of human malignancies, including tumorigenesis, chemoresistance14, proliferation15 and metastasis16. In the previous study13, a small molecular inhibitor of BMK1, XMD8-92, was developed. This BMK1 inhibitor, XMD8-92, can efficiently suppress the proliferation of multi types of cancer cells1317.

View Article: PubMed Central - PubMed

ABSTRACT

Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated. Here, we show that BMK1 pathway contributes to the drug resistance to CIBM. Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM. Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated. Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells. It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance. Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM. Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells. Further kinome-scale siRNA screening demonstrated that SRC\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM. Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma.

No MeSH data available.


Related in: MedlinePlus