B-RafV600E Signaling Deregulates the Mitotic Spindle Checkpoint Through Stabilizing Mps1 Levels in Melanoma Cells

Introduction
Raf kinases, including A-Raf, B-Raf, and C-Raf, stimulate the MAP kinase signaling cascade, which involves MEK and extracellular signal-regulated kinase (ERK). While this cascade is well established in promoting cell cycle entry and progression through the G1/S transition, it also regulates later stages of the cell cycle, including the G2/M transition, mitotic spindle formation, and maintenance of the spindle assembly checkpoint arrest. Previous studies have demonstrated that the B-Raf isoform mediates mitotic activation of the MAP kinase pathway in Xenopus egg extracts. Further studies in human cells revealed that depletion of B-Raf impairs spindle formation, chromosome congression, and spindle checkpoint activation, indicating that the B-Raf/MEK/ERK cascade plays critical roles during mitosis.

Activating point mutations in the B-RAF gene are common in various human cancers, most notably in 60–70% of cutaneous melanomas, 35–60% of papillary thyroid carcinomas, and 5–20% of colorectal carcinomas. Approximately 90% of B-Raf mutations result from a single-base substitution producing a V600E mutation, leading to increased basal kinase activity of B-Raf and hyperactivation of ERK. This mutation can transform fibroblasts and melanocytes and its inhibition reduces melanoma cell proliferation and induces apoptosis, underscoring the oncogenic role of B-Raf in melanoma and other cancers.

The spindle assembly checkpoint ensures proper chromosome segregation by delaying anaphase onset until all chromosomes are correctly attached to the spindle. This involves the localization of checkpoint proteins such as MAD1, MAD2, Bub1, BubR1, Bub3, and Mps1 at the kinetochores, forming complexes that inhibit the anaphase-promoting complex (APC) until conditions are favorable for progression. Previous evidence has indicated that Mps1 is a target of ERK in activated spindle checkpoint conditions. This study investigates whether oncogenic B-RafV600E associates with and stabilizes Mps1, affecting spindle checkpoint function and mitotic progression in melanoma cells.

Results
Ectopic B-RafV600E Expression Induces Mps1 Levels in Melanoma Cell Lines
We investigated whether constitutive activation of ERK by B-RafV600E affects Mps1 expression in melanoma cells. Introduction of B-RafV600E into B-Raf wild-type melanoma cells led to a threefold increase in Mps1 and MAD1 protein levels, while Bub1 levels remained unchanged. Further analysis showed that the increase in Mps1 was specifically due to B-RafV600E expression rather than B-Raf overexpression alone. Cell lines with endogenous B-RafV600E exhibited higher Mps1 levels, indicating that the oncogenic mutant deregulates Mps1 expression.

To determine if this regulation occurs at the transcriptional level, real-time PCR was performed, showing that Mps1 mRNA levels remained unchanged despite B-RafV600E expression, suggesting post-transcriptional regulation of Mps1 by B-RafV600E.

Analysis of Mps1 and B-Raf Interactions in Human Melanoma Cells
Co-immunoprecipitation assays demonstrated that B-Raf, including the B-RafV600E mutant, associates with Mps1 in melanoma cells, with their co-localization observed at kinetochores during prometaphase and metaphase. This suggests that B-Raf and Mps1 interact in vivo during early mitosis.

B-RafV600E Signaling Through ERK Is Critical for Mps1 Expression
Reduction of B-Raf levels using siRNAs in melanoma cells led to a significant decrease in Mps1 protein levels. Inhibition of ERK using the MEK inhibitor U0126 also reduced Mps1 levels, indicating that B-RafV600E signaling upregulates Mps1 through ERK-dependent pathways.

B-RafV600E Expression Upregulates Mps1 Activity
We assessed Mps1 kinase activity and found that cells expressing B-RafV600E showed a significant increase in Mps1-associated kinase activity compared to controls, suggesting that elevated Mps1 levels result in increased functional activity.

B-RafV600E Signaling Mediates Phosphorylation of Mps1 Through ERK
Western blot analysis indicated that B-RafV600E expression causes a mobility shift in Mps1 due to hyperphosphorylation, which was reversed by phosphatase treatment and ERK inhibition, confirming that B-RafV600E induces Mps1 phosphorylation via ERK.

B-RafV600E Signaling Stabilizes hMps1 Protein
Cycloheximide chase experiments revealed that B-RafV600E expression extends the half-life of Mps1 from approximately 1.5 hours to around 12 hours, indicating that B-RafV600E signaling stabilizes Mps1 protein. Immunofluorescence analysis further showed that Mps1 is present in interphase cells in B-RafV600E mutant lines, while it is restricted to mitotic cells in B-Raf wild-type cells.

Oncogenic B-RafV600E Signaling Promotes an M-Phase Cell Cycle Delay
Flow cytometry analysis of melanoma cells expressing B-RafV600E showed an increase in the G2/M population and a reduction in G1 and S phases, indicating a mitotic delay. Immunostaining for phospho-H3 confirmed accumulation of cells in mitosis, particularly in prometaphase and metaphase, with a reduction in anaphase/telophase progression.

Depletion of Mps1 in these cells suppressed the B-RafV600E-induced mitotic delay, demonstrating that Mps1 mediates this effect.

Oncogenic B-RafV600E Signaling Prolongs Spindle Checkpoint Activation
B-RafV600E expression increased levels of spindle checkpoint proteins Mps1, MAD1, and MAD2 while maintaining Bub1 and CENP-E levels. Immunostaining revealed increased localization of MAD1 and MAD2 at kinetochores, indicating prolonged spindle checkpoint activation dependent on Mps1, as depletion of Mps1 abolished this localization.

Discussion
This study reveals that B-RafV600E signaling deregulates the mitotic spindle checkpoint by stabilizing and activating Mps1, leading to sustained checkpoint activation and mitotic delay in melanoma cells. The association and co-localization of B-Raf with Mps1 at kinetochores, along with ERK-mediated phosphorylation and stabilization of Mps1, contribute to these effects.

These findings suggest that deregulation of the spindle checkpoint by B-RafV600E may contribute to chromosomal instability in melanoma. Further studies are needed to elucidate the mechanisms by which B-RafV600E stabilizes Mps1 and to explore its potential role in promoting chromosomal segregation errors and aneuploidy in cancer.

Materials and Methods
B-RafV600E and B-RafWT plasmids were used for transfection and retroviral infection of melanoma cell lines, including SK-MEL5, SK-MEL28, A375, WM35, and Sbcl2. siRNA-mediated knockdown was employed to reduce B-Raf and Mps1 expression. MEK inhibitor U0126 was used to inhibit ERK activity.

Protein lysates were prepared for immunoblotting, and immunoprecipitation assays were conducted to analyze protein interactions. Kinase activity assays were performed to assess Mps1 functional activity, and immunofluorescence staining was used to determine protein localization during the cell cycle.Flow cytometry was utilized to analyze BAY 1217389 cell cycle distribution in melanoma cells under various treatment conditions.