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  • br Previous studies demonstrated that

    2020-07-31


    Previous studies demonstrated that aurora-A induces cell migration by upregulating the expression of the phosphatase Slingshot-1 (SSH1), which dephosphorylates cofilin and thus facilitates KX2-391 reorganization and polymerization [25,26]. Furthermore, the chaperone protein Hsp90 forms a complex with aurora-A to protect the latter from proteasomal degradation [26]. Here, we investigated whether MPT0G211 inhibited breast cancer cell migration by modulating aurora-A. As shown in Fig. 3A, MPT0G211 treatment significantly increased Hsp90 acetyla-tion, leading to aurora-A/Hsp90 complex dissociation (Fig. 3B) and decreased aurora-A levels (Fig. 3C, D). The finding that the proteosome inhibitor MG132 (Fig. 3E) or HDAC6 overexpression (Fig. 3F) could significantly prevent the MPT0G211-mediated aurora-A down-regulation suggested that MPT0G211 promotes the proteosomal de-gradation of aurora-A by inhibiting HDAC6 activity and promotes Hsp90 acetylation to induce aurora-A/Hsp90 dissociation. We further proved that MPT0G211 treatment in MDA-MB-231 TNBC cells in-creased aurora-A ubiquitination, indicating that MPT0G211 indeed promotes the proteasome degradation of aurora-A (Supplemental Fig. 3). The corresponding effect of tubastatin A was relatively less potent (Fig. 3A, B, C).
    We next identified whether MPT0G211 could influence SSH1 ex-pression and cofilin phosphorylation. Compared with ER-positive MCF-7 breast cancer cells, MDA-MB-231 cells exhibited higher levels of HDAC6, aurora-A, and SSH1 and a lower level of KX2-391 phosphorylated cofilin (Fig. 4A). Following MPT0G211 treatment, MDA-MB-231 cells ex-hibited a significant downregulation of SSH1 expression (Fig. 4B), which led to a marked increase in the phosphorylation of cofilin (non-active form; Fig. 4C). In addition, MPT0G211 treatment significantly inhibited actin polymerization (F-actin) and increased monomer actin levels (G-actin; Fig. 5A). Furthermore, the actin distribution from the leading edge of the cell (Fig. 5B, control group), a characteristic of mesenchymal cell migration, shifted to the cytoplasm in response to MPT0G211 treatment.
    4.3. MPT0G211 treatment inhibited the binding of cortactin with F-actin
    The HDAC6-mediated deacetylation of cortactin contributes to its ability to bind and stabilize F-actin, and also stimulates actin poly-merization and cell motility. By contrast, acetylated cortactin cannot bind to F-actin, leading to decreased cell motility [27]. As shown in Fig. 5C, MPT0G211 treatment significantly reduced cortactin/F-actin binding in a manner correlated with the increasing acetylation of cor-tactin (Fig. 5D). Collectively, these results indicate that MPT0G211 significantly disrupts F-actin polymerization by decreasing the expres-sion of SSH1 and phosphorylation of cofilin, thus inhibiting actin/F-actin binding.
    4.4. MPT0G211 treatment significantly attenuated breast cancer metastasis in vivo
    We next evaluated the anti-metastatic effects of MPT0G211 in vivo in a mouse model. As shown in Fig. 6A, tumor nodules were detectable
    in control animals by 73 days post-MDA-MB-231 implantation. By contrast, significantly reduced numbers of nodules (Fig. 6A) and lung weights (Fig. 6B) were observed in the MPT0G211 group. Interestingly, the combination of MPT0G211 with paclitaxel caused more significant reductions in the number of tumor nodules (Fig. 6A) and lung weight (Fig. 6B), but did not affect body weight (Fig. 6C) when compared with each treatment alone.
    An immunohistochemical analysis of the lungs revealed that serious pulmonary parenchyma that caused by cancer cells in the control group (Fig. 6D). By contrast, MPT0G211 significantly alleviated this alveolar damage and led to marked increases in α-tubulin acetylation and cofilin phosphorylation. In the combination group, the integrity of the alveolar tissue was retained, and cancer cell invasion was almost undetectable. These results demonstrate that MPT0G211 treatment significantly in-hibits TNBC tumor metastasis in vivo. Taken together, our results sug-gest that the potent HDAC6 inhibitor, MPT0G211, significantly inhibits the metastatic characteristics of MDA-MB-231 TNBC cells both in vitro and in vivo.
    5. Discussion
    Tumor metastasis requires cell migration. HDAC6 possesses two catalytically active domains and deacetylates highly abundant sub-strates, such as Hsp90 and cortactin, to mediate diverse and complex pathways related to both cytoskeleton remodeling and cell migration. These mediatory targets include a reduction in the ability of breast cancer metastasis suppressor 1 (BRMS1) to inhibit metastasis via Hsp90 deacetylation and increased BRMS1 degradation [28], and the regula-tion of pseudopodial (invadopodial) invasive activity and extracellular matrix (ECM) decomposition to promote the cancer cell invasion [15]. A previous study of the less aggressive cell line MCF-7 found that HDAC6 overexpression could increase the cell migration ability by four-fold [29]. Furthermore, Park and colleagues demonstrated higher levels of HDAC6 expression in MDA-MB-231 cells, compared with MCF-7 cells [30]. These data are consistent with the results of the present study (Fig. 4A).