High PSMD8 expression was correlated with poor survival in BC patients
We analyzed the expression of PSMD8 in nontumor breast tissue and tumor tissue from GEPIA and UALCAN. The level of PSMD8 expression was significantly upregulated in tumor tissue compared with normal tissue (Fig. 1A-B). Similarly, IHC staining demonstrated the same result (Fig. 1D). Moreover, the PSMD8 transcripts were positively correlated with tumor stage and lymphatic metastasis stage (Fig. 1C, E-F). Kaplan–Meier (K-M) analysis of recurrence-free survival (RFS), and overall survival (OS) showed that high expression of PSMD8 was associated with poor survival (Fig. 1G-H). In particular, a high level of PSMD8 implied worse RFS in lymphatic metastatic BC patients (Fig. 1I). Taken together, the bioinformatics analysis and IHC staining reminded us that PSMD8 was upregulated in tumor tissue and was associated with poor prognosis in BC.
PSMD8 promoted cell growth and invasion in vitro
To explore the effect of PSMD8 on the biological behavior of MDA-MB-231 and MCF7 breast cancer cell lines, we used PSMD8 siRNA and plasmids to downregulate and upregulate the expression level of PSMD8 in cells, respectively. We first displayed the efficiency of PSMD8 knockdown (KD) and overexpression (OE) (Fig. 2A and D). A CCK-8 assay was performed to test tumor proliferation after the corresponding treatments. PSMD8 knockdown attenuated tumor growth, while PSMD8 overexpression enhanced tumor growth (Fig. 2B and E). In addition, cell invasion was also decreased as PSMD8 was knocked down and increased as PSMD8 was overexpressed (Fig. 2C and F). In brief, PSMD8 promoted proliferation and invasion in BC cells.
PSMD8 triggered EMT program in BC cells
Given the correlation between EMT and tumor development, we further explored the role of PSMD8 in EMT progression. We knocked down the expression of PSMD8 and detected the protein levels of the EMT-related proteins E-cadherin (E-cad) and Snail. The WB results showed that the level of E-cad was upregulated, and snail was downregulated in PSMD8KD cells. The opposite results were observed in PSMD8OE cells (Fig. 3A-B). In parallel, the picture of IF confirmed this phenomenon as well. The level of E-cad was strengthened, and the level of snail was weakened in PSMD8KD cells (Fig. 3C). In addition, IHC was performed to detect the levels of PSMD8 and E-cad in BC tissues. The tissue with a high expression level of PSMD8 showed a low expression level of E-cad, and the tissue with low expression of PSMD8 displayed a high expression level of E-cad (Fig. 3D). Furthermore, we overexpressed PSMD8 in PSMD8KD cells and detected the protein levels of E-cad and snail. The WB results illustrated that PSMD8 overexpression reversed the effect of PSMD8 knockdown (Fig. 3E). In summary, PSMD8 promoted EMT progression in vitro.
Inhibition of PSMD8 suppressed tumor growth and lung metastasis in vivo
We established xenograft models to verify the effect of PSMD8 in vivo. First, LV-shPSMD8 was applied to MDA-MB-231 cells to establish stably silenced cells (Fig. 4A-B). A photograph of xenografts is displayed (Fig. 4C), and their weights were compared. The mean weight of the shPSMD8 group was significantly less than that of its counterpart (Fig. 4D).
Additionally, we infected mice with the treated cells in situ and detected lung metastasis. The separated lung tissue showed that the number of lung metastatic nodules was lower in the shPSMD8 group (Fig. 4E). HE staining demonstrated the same trend (Fig. 4F). In addition, we detected the levels of Ki67 and E-cad in tumors removed from xenograft models. IHC staining showed that E-cad expression increased, while the expression of Ki67 decreased after PSMD8 was silenced (Fig. 4G). In summary, this phenomenon suggested that silencing PSMD8 impaired the growth of breast tumors and decreased lung metastasis.
PSMD8 mediated the expression of KIF10
According to the TCGA and GSE1456 databases, the expression of PSMD8 was positively correlated with the expression of KIF10 (Fig. 5A-B). Furthermore, the WB results showed that the protein level of KIF10 was decreased after PSMD8 knockdown and increased after PSMD8 overexpression (Fig. 5C). Parallel effects were observed in BC patient tissues by IHC staining (Fig. 5D). Additionally, the protein levels of PSMD8 and KIF10 in xenografts were detected by IHC, which illustrated that the level of KIF10 declined in the shPSMD8 group compared with the shCtrl group (Fig. 5E). More important, K-M analysis showed that the prognosis of BC patients with high levels of PSMD8 and KIF10 was worse than the prognosis of BC patients with low levels of both PSMD8 and KIF10 in GES1456 database (Fig. 5F). Overall, the expression of KIF10 was regulated by PSMD8 and was positively correlated.
High KIF10 expression was correlated with poor survival in BC patients
Analysis from GEPIA, UALCAN and IHC staining demonstrated that the expression of KIF10 was higher in tumor tissue than in normal tissue (Fig. 6A-B and D), regardless of the subtype of BC (Fig. 6C). Moreover, the levels of KIF10 transcripts were positively associated with the BC stage and lymphatic metastasis stage (Fig. 6E-F). The K-M analysis of OS and RFS in all patients showed that the higher the expression of KIF10, the worse the prognosis was, particularly the RFS in lymphatic metastatic patients (Fig. 6G-I). In short, tumor tissue had higher KIF10 expression than normal tissue and was correlated with a poor prognosis.
KIF10 knockdown decreased cell growth and invasion in vitro
We will explore the exact effect of KIF10 on the biological functions of BC cells. The knockdown efficiency of KIF10 in BC cells is shown in Fig. 7A. CCK-8 and Transwell assays were performed to detect the proliferation and invasion abilities of BC cell lines. The results demonstrated that cell proliferation ability and the ratio of cell invasion were decreased after KIF10 knockdown (Fig. 7B-C). In addition, similar to PSMD8, the WB results showed that the expression level of E-cad was increased and the level of Snail was decreased in KIF10-knockdown cells (Fig. 7D). Furthermore, the expression of PSMD8 was positively correlated with the expression of KIF10 (Fig. 7E). These results suggested that KIF10 was capable of impairing tumor progression.
PSMD8/KIF10 axis promoted tumor development in vitro
Given that both PSMD8 and KIF10 promoted tumor development and that these two had a positive correlation, we further verified their link and effect. FCM were performed to detect apoptosis in PSMD8KD cells and KIF10KD cells under paclitaxel (PTX) conditions. Knockdown of either PSMD8 or KIF10 significantly enhanced PTX-induced the rate of cell apoptosis (Fig 8A-B), while the two proteins overexpression significantly antagonized PTX-induced apoptosis in BC cells (Fig 8C-D). Furthermore, knocking down the expression of KIF10 in PSMD8OE cells partially reversed the protective effect of PSMD8 against PTX (Fig 8C-D). Similarly, cell proliferation and invasion also decreased (Fig 8E-F). EMT-related markers were detected by the WB. Rescue experiments illustrated that the expression of E-cad increased and snail decreased (Fig 8G). In summary, the PSMD8-KIF10 axis could decrease chemosensitivity to PTX and enhance EMT progression.
Extracellular signal-regulated kinase 1/2 (ERK) signaling is essential for EMT and chemosensitivity mediated by the PSMD8/KIF10 axis in BC
To further explore the involved signaling pathways, the protein levels of p-ERK and ERK were detected. The ratio of p-ERK/ERK significantly decreased in both PSMD8KD and KIF10KD cells (Fig. 9A), while PSMD8 overexpression significantly enhanced the activation of ERK pathway (Fig. 9B). In addition, the ratio of p-ERK/ERK returned after the knockdown of KIF10 in PSMD8OE cells (Fig. 9B). Furthermore, the inhibitor of ERK signaling, U0126, was used to treat PSMD8OE cells. FMC demonstrated that overexpression of PSMD8 significantly protected tumor cells from PTX, but U0126 partially blocked this effect (Fig. 9C-D). Meanwhile, it also attenuated the cell proliferation and invasion effects of PSMD8 (Fig. 9E-F). Furthermore, the protein level of E-cad was increased, and the level of snail was decreased after U0126 treatment in PSMD8OE cells (Fig. 9G). In short, the ERK signaling pathway was involved in tumor proliferation and invasion regulated by the PSMD8/KIF10 axis. Inhibition of the ERK pathway could retard PSMD8/KIF10-induced EMT and enhance sensitivity to PTX.