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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.


Phosphorylation of BMK1 enhances the resistance to CIBM.(a,b) A375 and SK-MEL-28 cells were transfected with a constitutively active mutant of MEK5 (HA-MEK5D) and empty vector followed by selection with puromycin. The lysates of stable vector (control) and MEK5D cells were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (c,e) A375-Ctrl (control) and A375-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. Briefly, six replicates of A375-Ctrl (control) and A375-MEK5D cells were treated with Vemurafenib or Trametinib for three days at the concentration as noted. Then MTT assays were used to build growth inhibition curves. Unless otherwise stated, three-day MTT assays were used to build growth inhibition curves in this study. (d,f) SK-MEL-28-Ctrl (control) and SK-MEL-28-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. (g) A375-Ctrl and A375-MEK5D cell combined GI50 of Vemurafenib and Trametinib as noted. The combined GI50 was assessed as described in Fig. 2d. (h) SK-MEL-28-Ctrl and SK-MEL-28-MEK5D cell combined GI50 of Vemurafenib and Trametinib.
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f4: Phosphorylation of BMK1 enhances the resistance to CIBM.(a,b) A375 and SK-MEL-28 cells were transfected with a constitutively active mutant of MEK5 (HA-MEK5D) and empty vector followed by selection with puromycin. The lysates of stable vector (control) and MEK5D cells were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (c,e) A375-Ctrl (control) and A375-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. Briefly, six replicates of A375-Ctrl (control) and A375-MEK5D cells were treated with Vemurafenib or Trametinib for three days at the concentration as noted. Then MTT assays were used to build growth inhibition curves. Unless otherwise stated, three-day MTT assays were used to build growth inhibition curves in this study. (d,f) SK-MEL-28-Ctrl (control) and SK-MEL-28-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. (g) A375-Ctrl and A375-MEK5D cell combined GI50 of Vemurafenib and Trametinib as noted. The combined GI50 was assessed as described in Fig. 2d. (h) SK-MEL-28-Ctrl and SK-MEL-28-MEK5D cell combined GI50 of Vemurafenib and Trametinib.

Mentions: To investigate the role of phospho-BMK1 in the resistance to CIBM, a constitutively active mutant of MEK5 (MEK5D) was used to phosphorylate and activate BMK1. A375 and SK-MEL-28 cells were transfected with the active mutant (HA-MEK5D) and empty vector followed by selection with puromycin. As showed in Fig. 4a,b, stable expression of MEK5D promoted the phosphorylation of BMK1 in A375 and SK-MEL-28 cells. Then the resultant stable control (empty vector) and MEK5D cells were treated with increasing concentrations of Vemurafenib or Trametinib as noted in Fig. 4c–f. After three days, the number of survival cells was assessed by MTT. Resultant data indicated that MEK5D-phosphorylated BMK1 promoted the resistance to Vemurafenib and Trametinib (Fig. 4c–f). Furthermore, the combined GI50 of Vemurafenib and Trametinib was assessed as described above to evaluate the role of BMK1. It was found that phosphorylation of BMK1 increased the combined GI50 of CIBM in A375 and SK-MEL-28 cells (Fig. 4g,h).


Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma
Phosphorylation of BMK1 enhances the resistance to CIBM.(a,b) A375 and SK-MEL-28 cells were transfected with a constitutively active mutant of MEK5 (HA-MEK5D) and empty vector followed by selection with puromycin. The lysates of stable vector (control) and MEK5D cells were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (c,e) A375-Ctrl (control) and A375-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. Briefly, six replicates of A375-Ctrl (control) and A375-MEK5D cells were treated with Vemurafenib or Trametinib for three days at the concentration as noted. Then MTT assays were used to build growth inhibition curves. Unless otherwise stated, three-day MTT assays were used to build growth inhibition curves in this study. (d,f) SK-MEL-28-Ctrl (control) and SK-MEL-28-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. (g) A375-Ctrl and A375-MEK5D cell combined GI50 of Vemurafenib and Trametinib as noted. The combined GI50 was assessed as described in Fig. 2d. (h) SK-MEL-28-Ctrl and SK-MEL-28-MEK5D cell combined GI50 of Vemurafenib and Trametinib.
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f4: Phosphorylation of BMK1 enhances the resistance to CIBM.(a,b) A375 and SK-MEL-28 cells were transfected with a constitutively active mutant of MEK5 (HA-MEK5D) and empty vector followed by selection with puromycin. The lysates of stable vector (control) and MEK5D cells were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (c,e) A375-Ctrl (control) and A375-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. Briefly, six replicates of A375-Ctrl (control) and A375-MEK5D cells were treated with Vemurafenib or Trametinib for three days at the concentration as noted. Then MTT assays were used to build growth inhibition curves. Unless otherwise stated, three-day MTT assays were used to build growth inhibition curves in this study. (d,f) SK-MEL-28-Ctrl (control) and SK-MEL-28-MEK5D cell growth inhibition curves of Vemurafenib or Trametinib as noted. (g) A375-Ctrl and A375-MEK5D cell combined GI50 of Vemurafenib and Trametinib as noted. The combined GI50 was assessed as described in Fig. 2d. (h) SK-MEL-28-Ctrl and SK-MEL-28-MEK5D cell combined GI50 of Vemurafenib and Trametinib.
Mentions: To investigate the role of phospho-BMK1 in the resistance to CIBM, a constitutively active mutant of MEK5 (MEK5D) was used to phosphorylate and activate BMK1. A375 and SK-MEL-28 cells were transfected with the active mutant (HA-MEK5D) and empty vector followed by selection with puromycin. As showed in Fig. 4a,b, stable expression of MEK5D promoted the phosphorylation of BMK1 in A375 and SK-MEL-28 cells. Then the resultant stable control (empty vector) and MEK5D cells were treated with increasing concentrations of Vemurafenib or Trametinib as noted in Fig. 4c–f. After three days, the number of survival cells was assessed by MTT. Resultant data indicated that MEK5D-phosphorylated BMK1 promoted the resistance to Vemurafenib and Trametinib (Fig. 4c–f). Furthermore, the combined GI50 of Vemurafenib and Trametinib was assessed as described above to evaluate the role of BMK1. It was found that phosphorylation of BMK1 increased the combined GI50 of CIBM in A375 and SK-MEL-28 cells (Fig. 4g,h).

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.