Limits...
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

Phosphorylation of BMK1 is associated with the resistance to CIBM.(a) The Thr-Glu-Tyr (TEY) activation motif of BMK1 shows significant similarity with ERK1/2. (b) Phylogentic tree analysis indicates that BMK1 is the most closely related to ERK1/2. (c) Phospho-BMK1 was enhanced by the combined inhibition of BRAF and MEK1/2. Phospho-BMK1 was evaluated in A375-P resistant cells, which were built as described above. (d) Several BRAF V699E mutant melanoma cell lines were used to evaluate the phosphorylation of BMK1 in drug resistant cells, which were built as described above. (e) The phosphorylation of BMK1 is increased in A375-P xenograft model, compared with A375 xenograft model. Briefly, A375 cells were suspended in DMEM and injected subcutaneously into the flank of Nod/Scid mice. After 2 weeks, the mice were grouped and treated with/without Vemurafenib and Trametinib. The phospho-BMK1 in resulting individual A375 (without Vemurafenib and Trametinib treatment) and A375-P (with Vemurafenib and Trametinib treatment) tumors was evaluated by western blot as noted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384194&req=5

f3: Phosphorylation of BMK1 is associated with the resistance to CIBM.(a) The Thr-Glu-Tyr (TEY) activation motif of BMK1 shows significant similarity with ERK1/2. (b) Phylogentic tree analysis indicates that BMK1 is the most closely related to ERK1/2. (c) Phospho-BMK1 was enhanced by the combined inhibition of BRAF and MEK1/2. Phospho-BMK1 was evaluated in A375-P resistant cells, which were built as described above. (d) Several BRAF V699E mutant melanoma cell lines were used to evaluate the phosphorylation of BMK1 in drug resistant cells, which were built as described above. (e) The phosphorylation of BMK1 is increased in A375-P xenograft model, compared with A375 xenograft model. Briefly, A375 cells were suspended in DMEM and injected subcutaneously into the flank of Nod/Scid mice. After 2 weeks, the mice were grouped and treated with/without Vemurafenib and Trametinib. The phospho-BMK1 in resulting individual A375 (without Vemurafenib and Trametinib treatment) and A375-P (with Vemurafenib and Trametinib treatment) tumors was evaluated by western blot as noted.

Mentions: As mentioned above, BMK1 shows the most similar to ERK1/2 (Fig. 3a,b). Meanwhile, BMK1 is well known to promote cell survival and share a bunch of substrates with ERK1/21112. In addition, among the detected upregulated phospho-proteins in this study, BMK1 showed the most significant ability to promote the proliferation of A375-P cells (Fig. 2f). Hence, our study was focused on BMK1.


Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma
Phosphorylation of BMK1 is associated with the resistance to CIBM.(a) The Thr-Glu-Tyr (TEY) activation motif of BMK1 shows significant similarity with ERK1/2. (b) Phylogentic tree analysis indicates that BMK1 is the most closely related to ERK1/2. (c) Phospho-BMK1 was enhanced by the combined inhibition of BRAF and MEK1/2. Phospho-BMK1 was evaluated in A375-P resistant cells, which were built as described above. (d) Several BRAF V699E mutant melanoma cell lines were used to evaluate the phosphorylation of BMK1 in drug resistant cells, which were built as described above. (e) The phosphorylation of BMK1 is increased in A375-P xenograft model, compared with A375 xenograft model. Briefly, A375 cells were suspended in DMEM and injected subcutaneously into the flank of Nod/Scid mice. After 2 weeks, the mice were grouped and treated with/without Vemurafenib and Trametinib. The phospho-BMK1 in resulting individual A375 (without Vemurafenib and Trametinib treatment) and A375-P (with Vemurafenib and Trametinib treatment) tumors was evaluated by western blot as noted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384194&req=5

f3: Phosphorylation of BMK1 is associated with the resistance to CIBM.(a) The Thr-Glu-Tyr (TEY) activation motif of BMK1 shows significant similarity with ERK1/2. (b) Phylogentic tree analysis indicates that BMK1 is the most closely related to ERK1/2. (c) Phospho-BMK1 was enhanced by the combined inhibition of BRAF and MEK1/2. Phospho-BMK1 was evaluated in A375-P resistant cells, which were built as described above. (d) Several BRAF V699E mutant melanoma cell lines were used to evaluate the phosphorylation of BMK1 in drug resistant cells, which were built as described above. (e) The phosphorylation of BMK1 is increased in A375-P xenograft model, compared with A375 xenograft model. Briefly, A375 cells were suspended in DMEM and injected subcutaneously into the flank of Nod/Scid mice. After 2 weeks, the mice were grouped and treated with/without Vemurafenib and Trametinib. The phospho-BMK1 in resulting individual A375 (without Vemurafenib and Trametinib treatment) and A375-P (with Vemurafenib and Trametinib treatment) tumors was evaluated by western blot as noted.
Mentions: As mentioned above, BMK1 shows the most similar to ERK1/2 (Fig. 3a,b). Meanwhile, BMK1 is well known to promote cell survival and share a bunch of substrates with ERK1/21112. In addition, among the detected upregulated phospho-proteins in this study, BMK1 showed the most significant ability to promote the proliferation of A375-P cells (Fig. 2f). Hence, our study was focused on BMK1.

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