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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.


Inhibition of BMK1 by shRNAi suppresses the resistance to CIBM.(a) Control and shRNA-mediated knockdown cells were built as described in our previous study22. Then the A375 cell lysates were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (b,c) A375-Ctrl (control) and A375-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (d) A375-Ctrl and A375-shBMK1 cell combined GI50 of Vemurafenib and Trametinib. (e) Colony formation assay of the indicated cell lines. Cells were treated with/without the combination of 2 μM Vemurafenib and 10 nM Trametinib as noted. Resultant cells were stained with crystal violet. (f,g) SK-MEL-28-Ctrl (control) and SK-MEL-28-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (h) SK-MEL-28-Ctrl and SK-MEL-28-shBMK1 cell combined GI50 of Vemurafenib and Trametinib.
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f5: Inhibition of BMK1 by shRNAi suppresses the resistance to CIBM.(a) Control and shRNA-mediated knockdown cells were built as described in our previous study22. Then the A375 cell lysates were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (b,c) A375-Ctrl (control) and A375-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (d) A375-Ctrl and A375-shBMK1 cell combined GI50 of Vemurafenib and Trametinib. (e) Colony formation assay of the indicated cell lines. Cells were treated with/without the combination of 2 μM Vemurafenib and 10 nM Trametinib as noted. Resultant cells were stained with crystal violet. (f,g) SK-MEL-28-Ctrl (control) and SK-MEL-28-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (h) SK-MEL-28-Ctrl and SK-MEL-28-shBMK1 cell combined GI50 of Vemurafenib and Trametinib.

Mentions: To further investigate the role of BMK1, BMK1 was knocked down in A375 cells using shRNA as described in previous study (Fig. 5a)22. Then resultant control and shBMK1 cells were treated with increasing concentrations of Vemurafenib or Trametinib. Compared with the control cells, shBMK1 cells were more sensitive to BRAF or MEK inhibitor (Fig. 5b,c). Then combined GI50 of CIBM was assessed as described above to evaluate the role of BMK1. As shown in Fig. 5d, MEK5D-phosphorylated BMK1 enhanced combined GI50, while shRNA-mediated knockdown of BMK1 decreased combined GI50. Moreover, colony formation assay was carried out to confirm the role of BMK1 in drug resistance. Consistent with the role of BMK1 in combined GI50, MEK5D-phosphorylated BMK1 promoted the drug resistance to CIBM, while shRNA-mediated knockdown of BMK1 decreased the drug resistance (Fig. 5e). Then we expanded these result to SK-MEL-28 cells. Consistent with the results of A375, phospho-activation of BMK1 enhanced drug resistance, while knockdown of BMK1 decreased drug resistance (Fig. 5f–h).


Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma
Inhibition of BMK1 by shRNAi suppresses the resistance to CIBM.(a) Control and shRNA-mediated knockdown cells were built as described in our previous study22. Then the A375 cell lysates were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (b,c) A375-Ctrl (control) and A375-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (d) A375-Ctrl and A375-shBMK1 cell combined GI50 of Vemurafenib and Trametinib. (e) Colony formation assay of the indicated cell lines. Cells were treated with/without the combination of 2 μM Vemurafenib and 10 nM Trametinib as noted. Resultant cells were stained with crystal violet. (f,g) SK-MEL-28-Ctrl (control) and SK-MEL-28-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (h) SK-MEL-28-Ctrl and SK-MEL-28-shBMK1 cell combined GI50 of Vemurafenib and Trametinib.
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Related In: Results  -  Collection

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f5: Inhibition of BMK1 by shRNAi suppresses the resistance to CIBM.(a) Control and shRNA-mediated knockdown cells were built as described in our previous study22. Then the A375 cell lysates were analyzed by western blot using anti-BMK1 and anti-ACTIN antibodies as noted. (b,c) A375-Ctrl (control) and A375-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (d) A375-Ctrl and A375-shBMK1 cell combined GI50 of Vemurafenib and Trametinib. (e) Colony formation assay of the indicated cell lines. Cells were treated with/without the combination of 2 μM Vemurafenib and 10 nM Trametinib as noted. Resultant cells were stained with crystal violet. (f,g) SK-MEL-28-Ctrl (control) and SK-MEL-28-shBMK1 cell growth inhibition curves of Vemurafenib or Trametinib as noted. (h) SK-MEL-28-Ctrl and SK-MEL-28-shBMK1 cell combined GI50 of Vemurafenib and Trametinib.
Mentions: To further investigate the role of BMK1, BMK1 was knocked down in A375 cells using shRNA as described in previous study (Fig. 5a)22. Then resultant control and shBMK1 cells were treated with increasing concentrations of Vemurafenib or Trametinib. Compared with the control cells, shBMK1 cells were more sensitive to BRAF or MEK inhibitor (Fig. 5b,c). Then combined GI50 of CIBM was assessed as described above to evaluate the role of BMK1. As shown in Fig. 5d, MEK5D-phosphorylated BMK1 enhanced combined GI50, while shRNA-mediated knockdown of BMK1 decreased combined GI50. Moreover, colony formation assay was carried out to confirm the role of BMK1 in drug resistance. Consistent with the role of BMK1 in combined GI50, MEK5D-phosphorylated BMK1 promoted the drug resistance to CIBM, while shRNA-mediated knockdown of BMK1 decreased the drug resistance (Fig. 5e). Then we expanded these result to SK-MEL-28 cells. Consistent with the results of A375, phospho-activation of BMK1 enhanced drug resistance, while knockdown of BMK1 decreased drug resistance (Fig. 5f–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.