Syntelin inhibits triple-negative breast cancer cell proliferation and metastasis

Dear Editor, The hallmarks of cancer comprise several distinct biological characteristics acquired during the multistep development of human tumors with the unique feature of genomic instability (Shen, 2011). These cancer characteristics include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (Chen et al., 2011; Song et al., 2018). Triple-negative breast cancer (TNBC), an aggressive disease with increased risks for visceral metastases, has a poor prognosis due to unavailable and viable therapeutic targets (Bianchini et al., 2016). A TNBC diagnosis indicates that cancer cells test negative for three key receptors: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Bianchini et al., 2016). The absence of these three receptors renders existing hormone and targeted therapies ineffective. Kinesin proteins serve as potential targets for cancer therapy due to their aberrant overexpression, association with malignancy, and drug resistance in solid tumors (Rath and Kozielski, 2012). Centromere-associated protein E (CENP-E), a mitotic kinesin, is an essential protein that regulates metaphase chromosome alignment and central spindle assembly (Huang et al., 2019; Liu et al., 2020). Recent genomic analyses suggest the function of CENP-E in TNBC progression (Kung et al., 2014), which prompted us to evaluate the efficacy of the CENP-E chemical inhibitor syntelin (Figure 1A) in TNBC chemotherapy. Syntelin is a first-in-class chemical inhibitor of CENP-E that blocks the release of ADP and locks CENP-E–microtubule interaction (Ding et al., 2010), which results in syntelic attachment of sister kinetochores steadily linked to microtubules and subsequent mitotic arrest (Liu et al., 2020). Here, we report the chemotherapeutic efficacy of syntelin in TNBC. Our preliminary screen of CENP-E in various breast cancer cell lines indicated its high protein levels in TNBC cell lines, such as MDA-MB231 cells (Supplementary Figure S1A), consistent with the aforementioned genetic analyses and TCGA database (Kung et al., 2014). To evaluate whether syntelin inhibits MDA-MB-231 cell proliferation, we carried out time-course analyses using an MTT assay that involves the conversion of the water-soluble yellow dye MTT to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is then solubilized and the concentration is determined by optical density at 570 nm. As shown in Figure 1B, syntelin and taxol exhibited little effect on MDA-MB-231 cell growth at an initial interval of 0.5 h. However, a long-term treatment of 12–15 h resulted in a significant inhibition of cell proliferation compared to the control group (P < 0.001; Figure 1B and Table 1) without altering CENP-E protein level (Supplementary Figure S1B). Previous studies have established the dynamic distribution pattern of CENP-E in HeLa cells (Yao et al., 1997). We next sought to test the subcellular distribution of CENP-E in MDA-MB-231 cells. As shown in Supplementary Figure S2A, CENP-E is associated with kinetochore in control metaphase cells (top panel) and relocated to the central spindle and midbody (third panel), similar to what was observed in other cell types. However, the majority of syntelin-treated cells were arrested in the prometaphase with misaligned chromosomes, consistent with the role of CENP-E seen in HeLa cells. As a control, taxol stabilized the microtubule polymers and blocked cell division (Supplementary Figure S2A). Our statistical analyses show that syntelin treatment resulted in a significant increase in cells bearing misaligned chromosomes (33% 6 4.2%; P < 0.01; Supplementary Figure S2B). To evaluate the precise impact of syntelin treatment on MDA-MB-231 cell division, real-time imaging analyses were carried out. As shown in Figure 1C, control cells progress through mitotic anaphase within 35 min, while syntelin-treated cells remain in metaphase after 70 min, indicating that syntelin blocks metaphase– anaphase transition. Given the observed inhibition of MDAMB-231 cell proliferation and division by syntelin, we sought to understand whether inhibition of CENP-E alters cell fate. Specifically, we probe whether syntelin alters necrosis vs. apoptosis using apopxin and 7-AAD as the reporter of apoptosis and necrosis, respectively. As shown in Figure 1D, syntelin-treated cells exhibited positive staining of apopxin without labeling of 7-AAD, while taxol treatment resulted in both increased apoptosis and necrosis judged by the intensities of apopxin and 7-AAD labeling, respectively. Our statistical analyses, from three independent experiments, show that taxol induced necrosis >5-fold