Par3 expression positively correlates with the paclitaxel efficacy in breast cancer patients.
Breast cancer specimens from 45 patients who had undergone curative resection and paclitaxel-based chemotherapy with matched adjacent normal breast tissues were collected to investigate the association between Par3 expression pattern and chemotherapeutic response of paclitaxel in breast cancer. Clinical characteristics of patients were listed in Supplementary Table 1. The typical immunostaining images were showed in Fig. 1A (lower panel). Chemotherapy-sensitive with clinical benefit (complete response [CR]/partial response [PR]/stable disease [SD] ≥ 6) months was observed in 29 patients, while chemotherapy-resistant with no clinical benefit (SD༜ 6 months/progressive disease [PD]) was achieved in 13 patients (Fig. 1A and 1E). According to the median value of immunohistochemical staining scores of Par3, the patients were classified into Par3 low expression group and high expression group. The chi-square test was employed and the results indicated that low Par3 expression positively correlated with liver metastasis phenotypes (P = 0.047, Supplementary Table 1). High level of Par3 staining in tumor tissues was observed in 18 (18/29, 57.1%) patients reaching clinical benefit (CBR), but in 3 (3/13, 23.1 %) patients in non-CBR group (Fig. 1E). The Par3 expression in tumor tissues positively and significantly correlated with the clinical response to chemotherapy (P = 0.048) (Fig. 1A and 1E). Par3 expression in tumor tissues of CBR groups was significantly higher than that in non-CBR groups (Fig. 1B). Moreover, low Par3 expression was significantly associated with shorter PFS (P = 0.014, Fig. 1C), compared with that observed in high expression group. The Par3 expression in the breast cancer was also negatively associated with OS, although without reaching statistical significance (P = 0.48, Fig. 1D).
Par3 decreases the viability and promotes the apoptosis of breast cancer cells in response to paclitaxel.
To study the role of Par3 in breast cancer cell viability, the endogenous Par3 expression was determined in eight breast cancer cell lines (Fig. S1A). For future experiments we employed SK-BR-3 and T-47D cell lines which representing human epidermal growth factor receptor 2 (HER2) + and luminal (hormone receptor [HR] + HER2-) breast cancer subtypes respectively, and their moderate expression made them viable to up- and down-regulate the expression of Par3 on one same cell line. We then over-expressed or knocked down Par3 in breast cancer cells SK-BR-3 and T-47D (Fig. S1B), and incubated the cells in medium with different concentration of paclitaxel. Cell viability was measured against a control group after 48 h and the results indicated that Par3 over-expression decreased cell viability whereas Par3 knockdown increased cell viability (Fig. 2A). The half-maximal inhibitory concentrations (IC50) of paclitaxel on control and Par3 manipulated cells were then calculated and compared. The IC50 values on Par3 over-expressed T-47D and SK-BR-3 cells were 3.5 nM (95%CI 1.1–14.9) and 185 nM (95%CI 76–390), respectively, which were dramatically decreased in comparison with the control groups (13.3 nM [95%CI 3.1–49.3] on T-47D and 460 nM [95%CI 154 − 127] on SK-BR-3) (Fig. 2A). Consistently, Par3 knockdown increased IC50 of paclitaxel (56.9 nM [95%CI 17.8-194.3] on T-47D and 1400 nM [95%CI 427–4934] on SK-BR-3), approximately 2 or 6 folds higher than those of control cells (9.9 nM [95%CI 3.5–36.7] on T-47D and 795nM [95%CI 275–2452] on SK-BR-3) (Fig. 2A). These data propose that Par3 sensitizes breast cancer cells to paclitaxel.
The flow cytometry assays were then performed to measure the cell apoptosis in breast cancer cells with different Par3 expression. The results showed that paclitaxel induced apoptosis was significantly aggravated by Par3 over-expression but attenuated by Par3 knockdown in both SK-BR-3 and T-47D breast cancer cells (Fig. 2B and 2C). Similarly, the Hoechst staining detected 80% apoptotic cells in Par3 over-expressed SK-BR-3 cells, while Par3 knockdown significantly decreased the number of apoptotic cells (30%) (Fig. 2D and 2E). Furthermore, the regulators of apoptosis were determined by western blot and the results showed that Par3 over-expression strengthened paclitaxel induced alteration of apoptotic regulators, that is, the decrease of anti-apoptotic Bcl-2 protein, the increase of pro-apoptotic Bax protein, and the cleavage of PARP, caspase-9 and − 3, were enhanced by Par3 over-expression (Fig. 2F and S2). In contrast, paclitaxel induced changes of apoptotic regulators were rescued by Par3 down-regulation (Fig. 2F and S2). Above results indicate that Par3 promotes cell apoptosis, thus increases the sensitivity of breast cancer cells to paclitaxel.
Par3 aggravates paclitaxel induced mitotic arrest at metaphase of breast cancer cells
To explore whether Par3 expression affect the proliferation of breast cancer cells, we first employed BrdU incorporation assay and found that Par3 over-expression decreased cell proliferation while Par3 knockdown promoted cell proliferation in SK-BR-3 cells subjected to paclitaxel (Fig. 3A). Next, flow cytometry results indicated that paclitaxel induced mitotic arrest in SK-BR-3 cells, which could be aggravated upon Par3 over-expression or reversed upon Par3 knockdown (Fig. 3B). We then determined the regulators of the cell cycle, cyclin B1 and A2. Cyclin B1 is the major mitotic cyclin partner. We detected an aggressive increase of cyclin B1 in paclitaxel treated group and Par3 up-regulation has a promoting role in cyclin B1 level (Fig. 3C). The increased levels of cyclin A2 in mammalian cells have been reported to delay metaphase and anaphase onset.[18, 19] Paclitaxel significantly decreased cyclin A2 level and Par3 exacerbated this effect (Fig. 3C and S3). Par3 knockdown showed a resistant role in the effect of paclitaxel (Fig. 3C and S3). These results indicate that upon paclitaxel, Par3 over-expression blocks more cells in M phase and then increases the sensitivity of tumor cells to paclitaxel (Fig. 3D).
To detail the promoting role of Par3 in paclitaxel induced mitotic arrest, we further stained α-tubulin of SK-BR-3 cells and observed microtubule structures (shown in red) undergoing marked morphological changes to mediate specific functions throughout the cell cycle (Fig. 3E and 3G). Over-expression of Par3 significantly increased metaphase cells, while Par3 knockdown decreased the cell number at metaphase (Fig. 3E and 3F). In paclitaxel treatment group, 60.20% cells at metaphase and a plus of Par3 over-expression elevated the cell number at metaphase to 70.89% (Fig. 3E and 3F). Consistently, Par3 knockdown lessened the cells at metaphase, which partially reversed the effect of paclitaxel (Fig. 3E and 3F). Moreover, a decrease of aneuploid cells was observed in Par3 over-expressing group while Par3 knockdown raised the numbers of aneuploid cells upon paclitaxel condition (Fig. 3G and 3H). Above data illuminate the promoting role of Par3 in paclitaxel induced mitotic arrest of breast cancer cells at metaphase.
Par3 stabilizes microtubules of breast cancer cells in response to paclitaxel
Paclitaxel is globally a microtubule-stabilizing drug which can interact with the microtubule system and function as antimitotic agents. Par3 enhanced mitotic arrest might have an important role in paclitaxel sensitivity of breast cancer cells. To further investigate the mechanism that Par3 sensitized breast cancer cells to paclitaxel, we examined the functional link between Par3 expression and the stability of microtubules. When we treated breast cells SK-BR-3 with paclitaxel, β-tubulin immunostaining indicated the appearance of microtubule bundles (Fig. 4A and 4C). Remarkably, more, thick, rounded tubular structures were formed and higher fluorescence density were detected in Par3 over-expressed cells (Fig. 4A and 4C), indicating excessive microtubule assembly and stabilization. Consistently, microtubule bundles and β-tubulin staining density showed a significant decrease in Par3 knockdown cells (Fig. 4A and 4C).
We then employed nocodazole, a microtubule-depolymerizing agent, to confirm the role of Par3 in the regulation of microtubule stability. Upon nocodazole treatment, a substantial fraction of cells over-expressing Par3 possessed more stabilized microtubules (Fig. 4B and 4D). In contrast, Par3 knockdown attenuated stabilized microtubules in nocodazole treated cells (Fig. 4B and 4D). Moreover, polymeric and soluble forms of α-tubulin were determined with western bolt and the results indicated that Par3 over-expression significantly reduced the soluble α-tubulin, while Par3 knockdown raised the level of soluble α-tubulin (Fig. 4E and 4F). These results indicate that Par3 increases polymeric forms of α-tubulin and stabilizes microtubule structure, which might be involved in the chemotherapeutic response of paclitaxel in breast cancer.