Basal-Type Breast Cancer Stem Cells Over-Express Chromosomal Passenger Complex Proteins

(1) Aim: In the present paper we analyzed the transcriptome of CSCs (Cancer Stem Cells), in order to find defining molecular processes of breast cancer. (2) Methods: We performed RNA-Seq from CSCs isolated from the basal cell line MDA-MB-468. Enriched processes and networks were studied using the IPA (Ingenuity Pathway Analysis) tool. Validation was performed with qRT-PCR and the analysis of relevant genes was evaluated by overexpression, flow cytometry and in vivo zebrafish studies. Finally, the clinical relevance of these results was assessed using reported cohorts. (3) Results: We found that CSCs presented marked differences from the non-CSCs, including enrichment in transduction cascades related to stemness, cellular growth, proliferation and apoptosis. Interestingly, CSCs overexpressed a module of co-regulated Chromosomal Passenger Proteins including BIRC5 (survivin), INCENP and AURKB. Overexpression of BIRC5 increased the number of CSCs, as assessed by in vitro and in vivo zebrafish xenotransplant analyses. Analysis of previously published cohorts showed that this co-regulated module was not only overexpressed in basal breast tumors but also associated with relapse-free and overall survival in these patients. (4) Conclusions: These results underline the importance of Cancer Stem Cells in breast cancer progression and point toward the possible use of chromosomal passenger proteins as prognostic factors.

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

Bir1 Is Required for the Tension Checkpoint

The Saccharomyces cerevisiae chromosomal passenger proteins Ipl1 (Aurora B) and Sli15 (INCENP) are required for the tension checkpoint, but the role of the third passenger, Bir1, is controversial. We have isolated a temperature-sensitive mutant (bir1-107) in the essential C-terminal region of Bir1 known to be required for binding to Sli15. This allele reveals a checkpoint function for Bir1. The mutant displays a biorientation defect, a defective checkpoint response to lack of tension, and an inability to detach mutant kinetochores. Ipl1 localizes to aberrant foci when Bir1 localization is disrupted in the bir1-107 mutant. Thus, one checkpoint role of Bir1 is to properly localize Ipl1 and allow detachment of kinetochores. Quantitative analysis indicates that the chromosomal passengers colocalize with kinetochores in G1 but localize between kinetochores that are under tension. Bir1 localization to kinetochores is maintained in an mcd1-1 mutant in the absence of tension. Our results suggest that the establishment of tension removes Ipl1, Bir1, and Sli15, and their kinetochore detachment activity, from the vicinity of kinetochores and allows cells to proceed through the tension checkpoint.

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6–related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.

Shugoshin 2 Regulates Localization of the Chromosomal Passenger Proteins in Fission Yeast Mitosis

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells.

The role of centromere-binding factor 3 (CBF3) in spindle stability, cytokinesis, and kinetochore attachment

The spindle midzone is critical for spindle stability and cytokinesis. Chromosomal passenger proteins relocalize from chromosomes to the spindle midzone after anaphase onset. The recent localization of the inner-kinetochore, centromere-binding factor 3 (CBF3) complex to the spindle midzone in budding yeast has led to the discovery of novel functions for this complex in addition to its essential role at kinetochores. In G1/S cells, CBF3 components are detected along dynamic microtubules, where they can "search-and-capture" newly replicated centromeres. During anaphase, CBF3 is transported to the microtubule plus-ends of the spindle midzone. Consistent with this localization, cells containing a mutation in the CBF3 subunit Ndc10p show defects in spindle stability during anaphase. In addition, ndc10-1 cells show defects during cytokinesis, resulting in a defect in cell abscission. These results highlight the importance of midzone-targeted proteins in coordinating mitosis with cell division. Here we discuss these findings and explore the significance of CBF3 transport to microtubule plus-ends at the spindle midzone.Key words: spindle midzone, passenger protein, inner centromere protein (INCENP), microtubule plus-end.