黑色素瘤中癌基因B-Raf^(V600E)对B-Raf/ERK/Mps1负反馈抵抗作用的机制

Mechanism of Oncogenic B-Raf^(V600E) Abrogates B-Raf/ERK/Mps1 Signaling Pathway in Melanoma Cells

目的明确癌基因B-RafV600E在Mps1和B-RafWT/MEK/ERK通路之间自动调节的负反馈回路中抵抗作用的具体机制。方法 (1)Sbcl2转染B-RafWT和Mps1-KD,Western blot方法检测p-ERK水平;(2)向B-Raf野生型SK-MEL31、Sbcl2、WM35细胞及V600E突变型SK-MEL28、A375细胞中过表达Mps1,Western blot方法检测p-ERK水平;(3)在SK-MEL31、Sbcl2、WM35细胞中敲低AKT,转染Mps1,Western blot方法检测p-ERK水平;(4)在SK-MEL31、Sbcl2、WM35细胞中敲低内源性B-Raf,过表达外源性RafV600E,Western blot方法检测p-AKT水平;敲低SK-MEL-28、A375细胞中RafV600E,Western blot方法检测p-A K T水平。结果 (1)M p s 1激酶和B-RafWT/MEK/ERK通路之间的自动负反馈通路不依赖Mps1激酶的活性;(2)在野生型SK-MEL31、Sbcl2、WM35细胞中外源性Mps1的表达可诱导AKT磷酸化,抑制ERK活性;V600E突变型SK-MEL28、A375细胞中外源性Mps1的表达不能诱导AKT磷酸化,亦不影响ERK活性。(3)敲低野生型黑色素瘤细胞中的AKT后,Mps1和B-RafWT/MEK/ERK之间的负反馈作用消失。(4)癌基因B-RafV600E通过抑制AKT的磷酸化,进而抵抗Mps1激酶与B-Raf/MEK/ERK通路之间的负反馈调节作用。结论 Mps1和B-RafWT/MEK/ERK通路之间的自动负反馈通路不依赖Mps1激酶的活性,且癌基因B-RafV600E对B-Raf/MEK/ERK/Mps1负反馈通路的抵抗作用是通过抑制AKT的磷酸化实现的。

Objective To clarify the specific mechanism of oncogenic B-RafV600 E presenting resistance to the auto-regulatory negative feedback loop of Mps1 and B-RafWT/MEK/ERK signaling pathway. Methods(1) Sbcl2 cells were transfected with B-RafWT or Mps1-KD and the activity of p-ERK was analyzed by Western blot.(2)The p-ERK levels were detected by Western blot in SK-MEL31, Sbcl2 and WM35 cells which harbor B-RafWT, meanwhile the expression of p-ERK was also detected in SK-MEL28 and A375 cells which harbor B-RafV600 E.(3) AKT was knocked down in SK-MEL31, Sbcl2 and WM35 cells with B-Raf wild-type genetic background, then transfected with Mps1, and the activity of p-ERKwas analyzed by Western blot.(4) The endogenous wild-type B-Raf in Sbcl2, WM35 and SK-MEL31 cells were knocked down, then exogenous B-RafV600 E was transfected into the cells. The expression of p-AKT was detected by Western blot. Meanwhile we knocked down B-RafV600 E in A375 and SK-MEL-28 melanoma cells for assessing the expression of p-AKT. Results(1) The auto-regulatory negative feedback loop of Mps1 on the B-RafWT/MEK/ERK signaling pathway was independent of kinase activity of Mps1.(2) The expression of exogenous Mps1 could enhance AKT phosphorylation and suppress ERK activity in melanoma cells harboring B-RafWT but not B-RafV600 E.(3) Knockdown of AKT in different melanoma cell lines with B-Raf wild-type genetic background leaded to Mps1 losing the ability of regulating the feedback loop.(4) AKT phosphorylation was suppressed by B-RafV600 E, which contributed to the abrogation of the regulatory negative feedback loop of Mps1 on the MAPK signaling pathway. Conclusion The auto-regulatory negative feedback loop of Mps1 on the B-RafWT/ERK signaling pathway is independent of kinase activity of Mps1. Oncogenic B-RafV600 E could surppress AKT phosphorylation to abrogate the regulatory negative feedback loop of B-Raf/MEK/ERK/Mps1.