Bub1 is required for maintaining cancer stem cells in breast cancer cell lines

Breast cancer is a leading cause of death among women worldwide due to therapeutic resistance and cancer recurrence. Cancer stem cells are believed to be responsible for resistance and recurrence. Many efforts to overcome resistance and recurrence by regulating cancer stem cells are ongoing. Bub1 (Budding uninhibited by benzimidazoles 1) is a mitotic checkpoint serine/threonine kinase that plays an important role in chromosome segregation. Bub1 expression is correlated with a poor clinical prognosis in patients with breast cancer. We identified that depleting Bub1 using shRNAs reduces cancer stem cell potential of the MDA-MB-231 breast cancer cell line, resulting in inhibited formation of xenografts in immunocompromised mice. These results suggest that Bub1 may be associated with cancer stem cell potential and could be a target for developing anti-breast cancer stem cell therapies.


Results
Depleting Bub1 reduces xenograft forming ability of MDA-MB-231 cells in immunocompromised mice. We used shRNAs targeting Bub1 (shBub1) to investigate the roles of Bub1 in breast cancer development and therapy. After transfection of MDA-MB-231 cells with control shRNA (shLuc), shBub1-#1 or shBub1-#2, stable clones were obtained by puromycin selection. Bub1 depletion in the stable clones was verified by western blot analysis and the reduced expression of Bub1 was maintained during this study (Fig. 1a). Bub1 depletion did not induce significant enhancement or reduction in cell proliferation (Fig. 1b). However, Bub1-depleted MDA-MB-231 cells did not generate xenografts in immunocompromised mice (Fig. 1c,d). Control cells (shLuc) formed xenografts efficiently (8/10 head) but Bub1-depleted MDA-MB-231 cells did not generate xenografts. These results suggest that depleting Bub1 reduces xenograft forming ability of MDA-MB-231 cells in immunocompromised mice.

Depleting Bub1 reduces the CSC potential of MDA-MB-231 and MCF7 cells. A xenograft assay
is widely used in immunocompromised mice to evaluate CSC potential. Thus, we investigated whether Bub1-depleted cells had reduced CSC potential. A 3D Matrigel sphere-forming assay was used to evaluate CSC potential. Bub1-depleted cells generated smaller and fewer spheres in 3D Matrigel culture than those in control cells (Fig. 2a). CD24 and CD44 surface staining was used to estimate the CSC population of breast cancer cells. FACS analysis showed that the CD24 low CD44 high population, which is believed to contain breast CSC subsets, was reduced in Bub1-depleted cells compared to that in control  (Fig. 2b). Enhanced invasion ability is a CSC characteristic. Depleting Bub1 consistently reduced invasion by MDA-MB-231 cells in the Matrigel invasion assay (Fig. 2c,d). Reduced CSC population by Bub1 depletion was further confirmed in MCF7 cells. Bub1 depletion in MCF7 cells did not result in significant reduction in cell proliferation (Fig. 3a,b). Bub1-depleted MCF7 cells were analyzed by FACS analysis using anti-CD24 and CD44 antibodies. Although MCF7 cells have much less CSC population than MDA-MB-231 cells, Bub1 depletion reduced the CD24 low CD44 high population (Fig. 3c). Taken together with the xenograft assay results, these results suggest that depleting Bub1 reduced the CSC potential of breast cancer cell lines.
Reduced CSC potential correlates with alterations in the RHAMM-GSK3β pathway. We further investigated how depleting Bub1 resulted in reduced CSC potential. RHAMM plays diverse roles on the cell surface and in the nucleus 26,44,45 . RHAMM influences maintenance of CSCs by regulating GSK3β activity and also is involved in mitotic spindle integrity 45,46 . Since Bub1 is a mitotic checkpoint protein involved in regulating the kinetochore-microtubule interaction during mitosis, we investigated whether Bub1 depletion modulate the RHAMM-GSK3β pathway. RHAMM expression was reduced and inhibitory phosphorylation of GSK3β was increased in Bub1-depleted MDA-MB-231 cells compared to those in control cells, indicating that depleting Bub1 may affect the RHAMM-GSK3β signaling pathway (Fig. 4a). These results were further confirmed in Bub1-depleted MCF7 cells (Fig. 3d). In the next step, we used RHAMM shRNAs and a GSK3β inhibitor to investigate the relationship between RHAMM and GSK3β . Depleting RHAMM with shRNAs enhanced GSK3β inhibitory phosphorylation but the GSK3β inhibitor did not modulate RHAMM expression (Fig. 4b,c). These findings suggest that depleting Bub1 decreased RHAMM expression, which, in turn, enhanced inhibitory phosphorylation of GSK3β .
Reduced CSC potential by depleting Bub1 results in enhanced sensitivity to irradiation. CSCs are a main anti-cancer therapy resistance mechanism. Thus, we investigated whether reduced CSC potential of Bub1-depleted MDA-MB-231 or MCF7 cells results in sensitization to anti-cancer therapies. Control and Bub1-depleted MDA-MB-231 or MCF7 cells were compared for their sensitivity to irradiation. Clonogenic assays showed that sensitivity to irradiation was enhanced in Bub1-depleted cells (a) Approximately 5 × 10 3 cells were cultured using a serum-free MammoCult Human Medium kit containing 2.5% Matrigel, and images were obtained 10-14 days later. (b) FACS analysis was performed to evaluate the CD24 low CD44 high CSC population. % of CD24 low CD44 high population is shown. (c,d) The Matrigel invasion assay was performed, the cells were stained with crystal violet, and images were taken for counting. The number of cells invading the Matrigel was normalized by setting the number of control cells invading the Matrigel to 100%. Data were shown as mean ± Standard Deviation (SD). Statistical significance compared to the control is shown. compared to that in control cells (Fig. 5a,c). Enhanced cell death in Bub1-depleted cells by irradiation was further confirmed by enhanced PARP and caspase-3 cleavage (Fig. 5b,d).

Discussion
CSCs, like stem cells, are maintained through a balance between symmetric and asymmetric division during mitosis. Deregulation of mitotic progression may influence the balance between symmetric and asymmetric division, resulting in changes in the CSC population. This finding suggests that mitotic kinases or mitotic checkpoint proteins may be good targets for developing anti-CSC therapies. AurA, Plk1, and BubR1 are consistently reported to be good targets for developing anti-CSC therapies [29][30][31][32][33][34] . As a mitotic checkpoint, Bub1 plays important roles in regulating mitosis by interacting with BubR1 and kinetochores. Our results support the possibility that Bub1 can be a target for developing anti-CSC therapies at least in breast cancers, where Bub1 expression correlates with poor clinical prognosis [38][39][40]42 .  Although several mitotic kinases and mitotic checkpoint proteins were reported to be involved in maintaining stem cells or cancer stem cells, specific mechanisms remains poorly understood. Ding et al. showed that brain tumor-initiating cells (BTIC) have kinetochore-microtubule (KT-MT) attachment defects and BubR1, specifically GLE2p-binding sequence (GLEBS) domain activity, is required to overcome the defects 34 . In mammary stem/progenitor cell fate determination, it was reported that AurA modulates the balance between the luminal and basal cell lineages by regulating the orientation of the mitotic spindle 47 . These results suggest that it may be important to regulate mitotic microtubules in maintaining stem cell population either in normal organs and cancers.
During mitosis, Bub1 plays several roles, such as recruiting the mitotic checkpoint proteins to the kinetochore, mounting the mitotic checkpoint response and chromosome congression. Bub1 has several protein-protein interaction domains, such as Tetratricopeptide repeat (TPR), GLEBS and a conserved region (CDI) domains involved in recruiting the mitotic checkpoint proteins to the kinetochore and the spindle checkpoint function, and Ser/Thr kinase domain involved in chromosome congression 48 . We need to figure out which domain or activity is involved in modulating CSC population. As described above, GLEBS domain of BubR1 is required to maintain brain tumor-initiating cells (BTIC) 34 . Since Bub1 also has GLEBS domain similar to that of BubR1, it may be interesting to investigate whether GLEBS domain of Bub1 is required to maintain breast cancer stem cells.
RHAMM functions at the cell surface and in the nucleus. Reduced expression of RHAMM is associated with decreased CSC self-renewal and embryonic stem cell pluripotency 46,49 . However, it is unclear whether cell surface or nuclear RHAMM is required to maintain self-renewal of stem cells or CSC. Nuclear RHAMM regulates stability of the mitotic spindle, which is involved in chromosome segregation through interactions with kinetochores. As Bub1 is located at the kinetochores during mitosis, depleting Bub1 may influence RHAMM expression by affecting the mitotic spindles and destabilizing RHAMM. Further investigation may be needed to determine how depleting Bub1 affects RHAMM expression and whether cell surface or nuclear RHAMM is important for CSC self-renewal.
As the CSC breast cancer populations are an obstacle to therapeutics, molecules required for CSC maintenance may be promising targets. Our results and previous reports support the possibility that regulation of kinetochore-mitotic microtubule (KT-MT) attachment may be involved in maintaining stem cell or CSC population and can be a target for developing anti-breast CSC therapies. 24-well plates. The cell culture inserts were rehydrated with serum-free DMEM for 1 hr. Approximately 2.5 × 10 4 cells were suspended in serum-free DMEM and seeded into the upper chambers of the cell culture inserts. DMEM with 10% fetal bovine serum was added to the lower chambers. The cell culture inserts containing cells were incubated at 37°C for 24 hr, and the inserts were removed. The cells that had passed through the membranes were fixed in 100% methanol and stained with 0.1% crystal violet for 10 min. The upper surfaces of the inserts were wiped with a swab, and the membranes were isolated from the inserts to prepare slides. Invading cells were observed and photographed using an Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan) and the Image-Pro Plus program (MediaCybernetics, Bethesda, MD, USA).

Statistics.
Results were expressed as mean ± Standard Deviation (SD). Student's t-test was used to compare values of test and control samples.