Breaking Bad: Uncoupling of Modularity in Centriole Biogenesis and the Generation of Excess Centrioles in Cancer.

Centrosomes are tiny yet complex cytoplasmic structures that perform a variety of roles related to their ability to act as microtubule-organizing centers. Like the genome, centrosomes are single copy structures that undergo a precise semi-conservative replication once each cell cycle. Precise replication of the centrosome is essential for genome integrity, because the duplicated centrosomes will serve as the poles of a bipolar mitotic spindle, and any number of centrosomes other than two will lead to an aberrant spindle that mis-segregates chromosomes. Indeed, excess centrosomes are observed in a variety of human tumors where they generate abnormal spindles in situ that are thought to participate in tumorigenesis by driving genomic instability. At the heart of the centrosome is a pair of centrioles, and at the heart of centrosome duplication is the replication of this centriole pair. Centriole replication proceeds through a complex macromolecular assembly process. However, while centrosomes may contain as many as 500 proteins, only a handful of proteins have been shown to be essential for centriole replication. Our observations suggest that centriole replication is a modular, bottom-up process that we envision akin to building a house; the proper site of assembly is identified, a foundation is assembled at that site, and subsequent modules are added on top of the foundation. Here, we discuss the data underlying our view of modularity in the centriole assembly process, and suggest that non-essential centriole assembly factors take on greater importance in cancer cells due to their function in coordination between centriole modules, using the Monopolar spindles 1 protein kinase and its substrate Centrin 2 to illustrate our model.

Novel pyrrolopyrimidines as Mps1/TTK kinase inhibitors for breast cancer.

New targeted therapy approaches for certain subtypes of breast cancer, such as triple-negative breast cancers and other aggressive phenotypes, are desired. High levels of the mitotic checkpoint kinase Mps1/TTK have correlated with high histologic grade in breast cancer, suggesting a potential new therapeutic target for aggressive breast cancers (BC). Novel small molecules targeting Mps1 were designed by computer assisted docking analyses, and several candidate compounds were synthesized. These compounds were evaluated in anti-proliferative assays of a panel of 15 breast cancer cell lines and further examined for their ability to inhibit a variety of Mps1-dependent biological functions. The results indicate that the lead compounds have strong anti-proliferative potential through Mps1/TTK inhibition in both basal and luminal BC cell lines, exhibiting IC50 values ranging from 0.05 to 1.0µM. In addition, the lead compounds 1 and 13 inhibit Mps1 kinase enzymatic activity with IC50 values from 0.356µM to 0.809µM, and inhibited Mps1-associated cellular functions such as centrosome duplication and the spindle checkpoint in triple negative breast cancer cells. The most promising analog, compound 13, significantly decreased tumor growth in nude mice containing Cal-51 triple negative breast cancer cell xenografts. Using drug discovery technologies, computational modeling, medicinal chemistry, cell culture and in vivo assays, novel small molecule Mps1/TTK inhibitors have been identified as potential targeted therapies for breast cancers.

Antizyme restrains centrosome amplification by regulating the accumulation of Mps1 at centrosomes.

Extra centrosomes are found in many tumors, and their appearance is an early event that can generate aberrant mitotic spindles and aneuploidy. Because the failure to appropriately degrade the Mps1 protein kinase correlates with centrosome overproduction in tumor-derived cells, defects in the factors that promote Mps1 degradation may contribute to extra centrosomes in tumors. However, while we have recently characterized an Mps1 degradation signal, the factors that regulate Mps1 centrosomal Mps1 are unknown. Antizyme (OAZ), a mediator of ubiquitin-independent degradation and a suspected tumor suppressor, was recently shown to localize to centrosomes and modulate centrosome overproduction, but the known OAZ substrates were not responsible for its effect on centrosomes. We have found that OAZ exerts its effect on centrosomes via Mps1. OAZ promotes the removal of Mps1 from centrosomes, and centrosome overproduction caused by reducing OAZ activity requires Mps1. OAZ binds to Mps1 via the Mps1 degradation signal and modulates the function of Mps1 in centrosome overproduction. Moreover, OAZ regulates the canonical centrosome duplication cycle, and reveals a function for Mps1 in procentriole assembly. Together, our data suggest that OAZ restrains the assembly of centrioles by controlling the levels of centrosomal Mps1 through the Cdk2-regulated Mps1 degradation signal.

Mps1 as a link between centrosomes and genomic instability.

Centrosomes are microtubule-organizing centers that must be precisely duplicated before mitosis. Centrosomes regulate mitotic spindle assembly, and the presence of excess centrosomes leads to the production of aberrant mitotic spindles which generate chromosome segregation errors. Many human tumors possess excess centrosomes that lead to the production of abnormal spindles in situ. In some tumors, these extra centrosomes appear before aneuploidy, suggesting that defects in centrosome duplication might promote genomic instability and tumorigenesis. The Mps1 protein kinase is required for centrosome duplication, and preventing the proteasome-dependent degradation of Mps1 at centrosomes increases its local concentration and causes the production of excess centrosomes during a prolonged S-phase. Here, we show that Mps1 degradation is misregulated in two tumor-derived cell lines, and that the failure to appropriately degrade Mps1 correlates with the ability of these cells to produce extra centrosomes during a prolonged S-phase. In the 21NT breast-tumor derived cell line, a mutant Mps1 protein that is normally constitutively degraded can accumulate at centrosomes and perturb centrosome duplication, suggesting that these cells have a defect in the mechanisms that target Mps1 to the proteasome. In contrast, the U2OS osteosarcoma cell line expresses a nondegradable form of Mps1, which we show causes the dose-dependent over duplication of centrioles even at very low levels of expression. Our data demonstrate that defects in Mps1 degradation can occur through multiple mechanisms, and suggest that Mps1 may provide a link between the control of centrosome duplication and genomic instability.

Preventing the degradation of mps1 at centrosomes is sufficient to cause centrosome reduplication in human cells.

Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells.

Human Mps1 protein kinase is required for centrosome duplication and normal mitotic progression.

The mitotic spindle is essential for the maintenance of genetic stability, and in budding yeast its assembly and function depend on the Mps1 protein kinase. Mps1p is required for centrosome duplication and the spindle checkpoint. Several recent reports demonstrate that vertebrate Mps1 proteins regulate the spindle checkpoint, but reports conflict regarding their role in centrosome duplication. Here we provide multiple lines of evidence that the human Mps1 protein (hMps1) is required for centrosome duplication. A recently described rabbit polyclonal antibody against hMps1 specifically recognizes centrosomes in a variety of human cell types. Overexpression of a dominant-negative version of hMps1 (hMps1KD) can prevent centrosome duplication in a variety of cell types, and active hMps1 accelerates centrosome reduplication in U2OS cells. Finally, we demonstrate that disruption of hMps1 function with pools of hMps1-specific small interfering RNAs causes a pleiotropic phenotype resulting from the combination of severe mitotic abnormalities and failures in centrosome duplication. This approach demonstrates that hMps1 is required for centrosome duplication and for the normal progression of mitosis, and suggests that the threshold level of hMps1 function required for centrosome duplication is lower than that required for hMps1 mitotic functions.

Centrosomes and tumour suppressors.

Centrosomes are microtubule organising centres that act as spindle poles during mitosis. Recent work implicates centrosomes in many other processes, and shows that centrosome defects can cause genetic instability. Many regulators of mammalian centrosome function were predicted from studies of model systems. Surprisingly, some well-known tumour suppressors have recently been found at centrosomes, where they influence centrosome duplication and function, suggesting that control of centrosome function is central to genetic stability.