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

www.nature.com/scientificreports OPEN Bub1 is required for maintaining cancer stem cells in breast cancer cell lines received: 28 May 2015 accepted: 06 October 2015 Published: 02 November 2015 Jeong Yoon Han1,*, Yu Kyeong Han1,*, Ga-Young Park1,2, Sung Dae Kim1 & Chang Geun Lee1 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. Breast cancer is one of the most common cancers and is a leading cause of death among women worldwide due to therapeutic resistance and cancer recurrence1–3. Several possible mechanisms have been proposed to explain resistance of cancers, including breast cancer to anti-cancer therapies and cancer recurrence. One is the cancer stem cell (CSC) theory in which small cancer cell subsets, which are capable of self-renewal, become more tumorigenic and resistant to anti-cancer therapeutics than others4–6. Thus, many efforts have been devoted to develop CSC-targeted therapies7–10. Several markers and methods have been suggested to define breast CSCs, such as surface expression of CD24lowCD44high, CXCR4+, Hoechst 33342 exclusion activity (side population), and aldehyde dehydrogenase activity11–15. Among them, CD24/CD44 staining is the most widely used method to identify breast CSCs. CD44, a type I transmembrane glycoprotein, is the major cellular receptor for hyaluronan (HA). Interactions between CD44 and HA are critical for expansion, differentiation, and pluripotency of stem cells including CSCs and also for cancer progression by modulating cell adhesion, migration, and invasion16–19. CD44 cooperates with HA-mediated motility (RHAMM or CD168) to deliver signals from extracellular HA, which is overexpressed in many advanced cancers including breast cancer20–23. RHAMM is an HA cell surface receptor and is also a cytoplasmic and nuclear protein that interacts with interphase microtubules, the mitotic spindle, and centrosomes24–26. Mitotic kinases and mitotic checkpoint proteins play key roles in cell division and maintenance of chromosome stability. Thus, mis-regulation of these processes may result in tumorigenesis. Furthermore, mitosis plays important roles balancing stem cells between self-renewal and differentiation to progenitor cells by regulating symmetric and asymmetric division27,28. These proteins have been shown to play an important role maintaining the CSC population and could be a potential therapeutic target for CSC-targeted therapies. Plk1 is a key mitotic regulator that has been identified as a potential therapeutic target for eliminating breast and colon CSCs29,30. CSCs are maintained by Aurora kinase A (AurA) and inhibiting AurA has been viewed as effective approach to target the CSC population in various cancers31–33. Glioblastoma initiating cells have lethal kinetochore-microtubule attachment defects that are 1 Research Center, Dongnam Institute of Radiological & Medical Sciences, Busan 619-953, ROK. 2Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, ROK. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.G.L. (email: ) Scientific Reports | 5:15993 | DOI: 10.1038/srep15993 1 www.nature.com/scientificreports/ Figure 1. Depleting Bub1 reduces xenograft forming ability of MDA-MB-231 cells in immunocompromised mice. (a) The levels of Bub1 expression in the Bub1-depleted MDA-MB-231 stable cell lines were measured by Western blot analysis. (b) 2 ×  103 cells of each cell lines were seeded onto 6 well plates. Cell numbers were counted at the indicated timpoints. Data were shown as mean values ±   SD. (c,d) Control (shLuc) and Bub1-depleted (shBub1 #1 & #2) MDA-MB-231 cells were injected subcutaneously into the right flanks of Balb/c-nu mice and allowed to form xenografts. Tumor formation was examined 30 days later. suppressed by BubR1. Thus, BubR1 may be a therapeutic target for glioblastoma CSCs34. Besides the involvement of mitotic kinases and mitotic checkpoint proteins in the maintenance of CSCs, genomic instability and polyploidy, which are induced by defects in mitotic kinases or mitotic checkpoint proteins, contribute to generating cancer-stem-like cells35,36. Bub1 is a mitotic checkpoint protein that is overexpressed in breast cancer and other cancers, and its expression correlates with a poor clinical prognosis37–43. We identified that depleting Bub1 in the MDA-MB-231 a breast cancer cell line prevents generation of xenografts in immunocompromised mice due to reduced CSC potential in Bub1-depleted cells and resulted in radiosensitization. Reduced CSC population and radiosensitization by Bub1 depletion were also observed in the MCF7 cells. These results suggest that Bub1, like other mitotic regulators, such as AurA, BubR1, and Plk1, is associated with CSC potential and may be a target for developing of anti-CSC therapy. 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. (...truncated)