The Molecular Mechanisms Of Mps1Mutants Causing Chromosome Instability

Background Activating BRAF mutations that deregulate the MAPK pathwaycommonly occur in cancer. The B-RAF mutation have been found in many humancancers, such as70%of Melanoma,50%of thyroid carcinoma,20%of coloncarcinoma. More than90%of the oncogenic B-RAF mutations occur as V600E. Anovel role of the B-RAF gene in mitosis has been implicated in melanoma in2008,.The B-RAF mutation was sufficient to induce spindle abnormalities andchromosomal instability, resulting in euploidy in human melanocytes or inimmortalized human mammary epithelial cells. Then, Mps1was identified as a newtarget of B-RAF^V600Eby MS. B-RAF^V600Esignaling prolongs activation of the spindlecheckpoint through stabilizing Mps1levels in melanoma cells. The previous studyhas found that phosphorylation of Mps1at residue S281by B-RAF^V600Esignalingprevents the degradation of Mps1by the anaphase promoting complex/cyclosome（APC/C）, thus allowing the Mps1protein to accumulate in excess at centrosomes.However, Whether an Mps1mutant that mimics phosphorylation at S281is sufficientto trigger centrosome amplification, leading to chromosome instability in humanmelanoma cells is a question worth exploring.Objective: To detect the effects of Mps1mutant on centrosome duplication and spindle checkpoint function. And elucidate the molecular mechanism of oncogene B-RAFV600Eresults in chromosome instability in melanoma cells.Methods: In order to detect the effects of Mps1mutant on centrosome duplication andspindle checkpoint function, we applied the following approaches: （1）HU-arrest assay was used to block SK-MEL31cells at S phase and immunofluorescence was used to analyze the effect of Mps1mutant on centrosome duplication.（2）MBP was used as the substrate for measuring kinase activity to analyze the effectof Mps1mutant on Mps1kinase activity.（3）Metaphase-spreads analysis was used to analyze the effect of Mps1mutant on chromosome instability.（4）Purchasing the skin cancer tissue microarray （ME207） to analyze association between B-RAF^V600Esignaling pathway and pS281-Mps1in skin cancer tissue chip.Result （1） To determine whether the observed centrosome amplification could bedue to a defect in centrosome duplication, we transfected S-phase arrested cells withthe various Mps1constructs, and assessed whether Mps1mutants led to centrosomere-duplication. We found that centrosome number was similar to controls in wildtype-, S821E-, and AAA-expressing cells. However, excess centrosomes wereobserved in roughly55%,30%, or70%of S281E-,436E-, and EEE-expressing cells,respectively. This explicitly demonstrates that mimicking phosphorylation of Mps1atS281and/or S436E causes centrosome re-duplication, and suggests that thecentrosome amplification observed in Fig.3A-B occurs through a defect incentrosome duplication.（2） We tested whether the phosphorylation of Mps1at S281, S436, and S821affectsMps1kinase activity. For comparison, a kinase-dead （KD）, was used as a negativecontrol. As expected, Mps1-WT phosphorylated MBP efficiently whereas Mps1-KDwas inactive. Mps1S281E and S821E have kinase activity comparable to wild type.However, while the S281A and S821A cause only modest reductions in kinaseactivity, S436A kinase activity was noticeably reduced, suggesting thatphosphorylation of S436is important for the kinase activity of Mps1in vitro.Moreover, S436E and EEE mutants have higher kinase activity as compared withMps1-WT. （3） To determine whether the amplified centrosomes and the resultant spindleabnormalities induced by phospho-mimetic mutation of Mps1give rise tochromosome missegregation, we examined chromosome segregation in abnormalities.In contrast to vector control cells, expression of the Mps1-EEE mutant in either Sbcl2or SK-MEL-31cells resulted in a high frequency of chromosome segregationanomalies, including lagging chromosomes and chromosome bridges. It stands toreason that the high incidence of chromosome mis-segregation observed in theMps1-EEE mutant expressing cells would result in aneuploidy. To test for this directly,chromosome counts on metaphase spreads were done in vector control or Mps1-EEEmutant expressing Sbcl2or SK-MEL-31cells. A mode of46chromosomes wasobserved for vector control cells, indicating that most of the cells in culture arediploid. In contrast, ectopic expression of the Mps1-EEE mutant resulted in theabsence of a chromosome mode and a wider distribution of chromosome counts.Taken together, we conclude that phospho-mimetic mutation of Mps1（EEE） inducesaneuploidy in human melanoma cells.（4） We evaluated the correlation between the level of S281-phosphorylated Mps1and the activity of B-RAF^V600Esignaling by IHC using a tissue microarray of clinicalsamples （ME207）. Analysis of this tissue array revealed that the percentage ofp-MAPK positive cases （positive plus strong positive） significantly increased fromnormal skin （0%） to malignant melanomas （48%） and malignant metastases （69%; P<.001）. Likewise, p-S281Mps1immunoreactivity dramatically increased fromnormal skin （0%） to malignant melanomas （54%） and malignant metastases （91%; P<.001）. Furthermore, we found that the samples showing strong p-MAPK stainingalso showed strong p-S281Mps1staining while samples with weak p-MAPK stainingshowed weak p-S281Mps1staining, suggesting that p-S281Mps1staining positivelycorrelated with p-MAPK in human melanoma tissues （r=0.25, p=0.002, Spearmantest）.Conclusion Based on our findings, B-RAF^V600Esignaling would also phosphorylateMps1at S436, and that S436E has higher kinase activity. The failure to properly degrade Mps1leads to the accumulation of excess Mps1with high kinase activity inthe vicinity of centrosomes, resulting in centrosome amplification, multipolar spindles,and chromosome mis-segregation, which are potential mechanisms for aneuploidy andchromosome instability in cancer.