**4. Discussion**

DTX has been the first-line therapy for metastatic CRPC patients since 2004. Recent clinical studies have reported that early DTX treatment combined with androgen deprivation therapy results in improved overall survival in comparison to androgen deprivation therapy alone in patients with metastatic hormone-sensitive PCa [28,29]. This finding suggests that the beneficial effect of DTX may not be restricted to CRPC and that DTX treatment is becoming increasingly more important in PCa [30]. Although DTX treatment improves overall survival, disease relapse eventually occurs due to the development of DTX resistance [31]. Several factors have been shown to be involved in DTX resistance [30,32]. Loss of p53 leads to DTX resistance, and p53 status is an essential determinant of DTX sensitivity [33]. Recent evidence has shown that alteration of β-tubulin isotypes is correlated with DTX resistance [34]. In addition, the expression of multidrug-resistant proteins such as ABCB1 is upregulated in a DTX-resistant PCa cell line. However, these above molecules have not been utilized clinically. Therefore, there is an urgen<sup>t</sup> need to clarify the mechanisms of DTX resistance. A recent study reported that the expression of KIFC1 is upregulated in DTX-resistant breast cancer cell lines compared with that of DTX-sensitive cell lines. What is more, overexpression of KIFC1 increased the pools of free tubulin and promoted DTX resistance in breast cancer [16]. This evidence suggests that KIFC1 may antagonize the effect of DTX at least through the dissociation of tubulin from microtubules. In the present study, the expression of KIFC1 was upregulated in DTX-resistant PCa cell lines. Knockdown of KIFC1 re-sensitized the DTX-resistant cells to DTX treatment in DU145 and C4-2 cells. To date, some preclinical studies have addressed the finding that anti-apoptotic proteins regain sensitivity to DTX [30]. ABT-263, which is a Bcl-2 inhibitor, restored DTX sensitivity in DTX-resistant cells in PCa [35]. Furthermore, glucocorticoid receptor antagonism also re-sensitizes DTX resistance through a reduction of BcL-xL expression [36]. In the present study, knockdown of KIFC1 suppressed the expression of Bcl-2, cleaved PARP and cleaved caspase-3, and enhanced the expression of Bax. This result indicates a potential mechanistic explanation for the restoration of DTX sensitivity in PCa.

A recent study reported that CW069 was identified as a highly selective small-molecule KIFC1 inhibitor using a chemogenomics-based approach [26]. CW069 increases multipolar spindle formation and inhibits cell viability in cancer cells in breast cancer. However, to date, there have been few reports on CW069 [26,37]. In the present study, CW069 treatment selectively damaged parental and DTX-resistant PCa cells but had little effect on cell viability in RWPE-1 cells. The result that CW069 re-sensitized DTX-resistant cell lines to DTX treatment has potential clinical implications. In addition, the synergistic effect was found in the combination of DTX and CW069. In current cancer treatments, different types of chemotherapeutic agents are combined to improve efficacy and to minimize toxicity. Our previous study showed that the expression of KIFC1 was higher in PCa tissues than in various normal tissue samples [17]. Collectively, these results sugges<sup>t</sup> that a combination of DTX and CW069 may be a promising therapy for CRPC patients that causes fewer adverse effects.

There are some limitations in this study. First, so far, three KIFC1 inhibitors (CW069, AZ82, and SR31527) have been reported [26,38,39]. Although these three drugs have been shown to lead to multipolar mitosis and decrease cell viability in human cancer, their effects were somewhat different because each drug binds to a different allosteric site on KIFC1 [38]. Furthermore, using a unique assay, a recent study showed that these drugs might not be specific to KIFC1 [40] Therefore, further study using these three drugs in PCa will be necessary in the future to verify our current findings. Second, recent studies have shown that cross-resistance of DTX cells were resistant to both DTX and cabazitaxel [41,42]. However, in this study, we focused on the role of KIFC1 and KIFC1 inhibitor (CW069) on only DTX resistance in PCa. In the near future, we will investigate the role of KIFC1 on cross-taxan resistance in PCa.
