TRIM25 promotes cell proliferation and cell cycle progression of BC cells
The dysregulation of BRD7 expression in breast cancer is an important mechanism leading to tumor occurrence, development, and PTX resistance. Our previous study showed that BRD7 was an unstable protein in breast cancer, and that the ubiquitin-proteasome pathway was involved in the regulation of BRD7 protein instability [12]. To further reveal the mechanism of BRD7's involvement in development and PTX chemotherapy sensitization in breast cancer, we screened and identified a potential E3 ubiquitin ligase, TRIM25, interacting with BRD7 by IP-MS in our previous study [12]. In order to determine the biological functions of TRIM25, we first constructed TRIM25-overexpressing and TRIM25 knockdown cells using MDA-MB-231 and MCF7 cell lines, the efficiency of which was confirmed by western blot assay (Fig. 1A, B). The Cell Counting Kit-8 (CCK-8) assay was performed to explore the effect of TRIM25 on the proliferation of breast cancer cells. The results showed that the overexpression of TRIM25 significantly promoted the proliferation of BC cells, while the knockdown of TRIM25 reduced the proliferation ability of BC cells (Fig. 1C, D). Furthermore, the colony formation assay showed that knockdown of TRIM25 decreased colony formation capacity, while the overexpression of TRIM25 led to the opposite result (Fig. 1E, F). Next, we analyzed the effect of TRIM25 on the cell cycle. As shown in Fig. 1G, H, the overexpression of TRIM25 resulted in an increased percentage of BC cells in the G2/M phase compared with the control, while the inhibition of TRIM25 decreased the percentage of cells in the G2/M phase in breast cancer cells. These results indicate that TRIM25 facilitates the cell cycle progression and proliferation of BC cells, suggesting a tumor-promoting role of TRIM25 in breast cancer.
We then investigated the role of TRIM25 in the process of paclitaxel resistance in BC cells. The TRIM25-knockdown or TRIM25-overexpressing BC cells were treated with different concentrations of PTX for 48 h, or treated with the same concentration of PTX for different times. The CCK-8 assay was used to evaluate cell viability. The downregulation of TRIM25 increased the sensitivity of BC cells to PTX (Fig. 2A, B), while the upregulation of TRIM25 decreased the sensitivity of cells to PTX (Fig. S1A, B). Subsequent colony formation assays also confirmed that the downregulation of TRIM25 enhanced the inhibitory effect of PTX on the colony-formation ability of BC cells (Fig. 2C), while overexpression of TRIM25 had the opposite result (Fig. S1C). We also performed cell apoptosis analysis, which showed that TRIM25 knockdown significantly increased PTX-induced apoptosis of BC cells (Fig. 2D), whereas overexpression of TRIM25 reduced PTX-induced apoptosis (Fig. S1D). Western blot analysis showed that TRIM25 knockdown promoted the expression of cleaved-PARP, while the expression of cleaved-PARP was decreased in the TRIM25 overexpression group. Meanwhile, PTX treatment enhanced the protein expression of cleaved-PARP compared was enhanced compared to that in untreated groups (Figs. 2E, S1E).
In order to further substantiate that TRIM25 was indeed related to paclitaxel resistance in breast cancer cells, a paclitaxel-resistant MDA-MB-231 cell line (MDA-MB-231-PR) was used for the following study. According to the result shown in Fig. S2A, compared with the parental MDA-MB-231 cells, PTX-resistant cells had a significantly higher IC50 value after a 48-h exposure to paclitaxel. The IC50 values of paclitaxel in the parental cell lines and resistant cell lines were 52.84 nM and 582.2 nM, respectively. We subsequently established TRIM25 overexpressed and knockdown paclitaxel-resistant MDA-MB-231-PR cell lines (Fig. S2B). Cell viability assays were performed on these MDA-MB-231-PR cells following treatment with varying concentrations and different durations of paclitaxel. The results demonstrated that TRIM25 overexpression significantly reduced the sensitivity of MDA-MB-231-PR cells to PTX. Conversely, TRIM25 knockdown produced the opposite effect (Fig. S2C, D). Furthermore, under PTX treatment, TRIM25 overexpression markedly enhanced the colony formation capacity of MDA-MB-231-PR cells and inhibited apoptosis. In contrast, TRIM25 knockdown suppressed colony formation and promoted apoptosis in these cells (Fig. S2E, F). Further analysis revealed that in MDA-MB-231-PR cells, TRIM25 overexpression downregulated the expression level of cleaved-PARP protein upon paclitaxel treatment, while TRIM25 knockdown resulted in a significant upregulation of cleaved-PARP expression in MDA-MB-231-PR cells (Fig. S2G). Collectively, these results demonstrate that TRIM25 plays a critical role in conferring resistance to paclitaxel in breast cancer cells.
Since our previous study demonstrated that TRIM25, a latent E3 ubiquitin ligase, is a potential interacting protein of BRD7 [12], we further verified the protein interaction between TRIM25 and BRD7. First, the co-immunoprecipitation (co-IP) assay confirmed that endogenous TRIM25 and BRD7 proteins could be co-immunoprecipitated with each other (Fig. 3A). Next, we determined the colocalization of TRIM25 and BRD7 in BC cells. Immunofluorescence assay in MDA-MB-231 and MCF7 cells showed that TRIM25 was mainly co-located with BRD7 in the nucleus (Fig. 3B). To determine the domain of TRIM25 that interacts with BRD7, we constructed a series of HA-tagged BRD7 deletion mutants and Flag-tagged TRIM25 deletion mutants (Fig. 3C, D). The results of co-IP confirmed that the TRIM25-PRYSPAY domain interacts with the N-terminal region of BRD7 in MCF7 cells (Fig. 3E, F). Taken together, these results indicate that TRIM25 directly interacts with BRD7 in breast cancer cells.
The interaction between the E3 ubiquitin ligase TRIM25 and BRD7 leads us to presume that TRIM25 decreases BRD7 protein level by ubiquitin-proteasome degradation. Here, we found that the overexpression and knockdown of TRIM25 did not influence BRD7 mRNA levels in either MDA-MB-231 or MCF7 cells (Fig. S3A, B), but TRIM25 negatively regulated the protein expression of BRD7 (Figs. 4A and S3C), indicating that TRIM25 might regulate the level of BRD7 protein by affecting its stability. Furthermore, we utilized cycloheximide (CHX), a protein synthesis inhibitor, to evaluate the influence of TRIM25 on BRD7 protein stability. We found that TRIM25 knockdown significantly increased the half-life of BRD7 (Fig. 4B). Additionally, treatment of BC cells with the proteasome inhibitor MG132 eliminated the effects of TRIM25 overexpression on BRD7 protein, which indicated that TRIM25 downregulation of BRD7 protein in BC cells was mediated by the proteasome pathway (Fig. 4C).
Next, we examined the influence of TRIM25 on BRD7 ubiquitination. Our data showed that the downregulation of TRIM25 markedly reduced the ubiquitination of endogenous BRD7 in BC cells (Fig. 4D), whereas overexpression of TRIM25 enhanced the BRD7 ubiquitination level (Fig. S3D). Next, we investigated the specific subtypes of ubiquitin chains involved in BRD7 ubiquitination mediated by TRIM25. K48-linked and K63-linked ubiquitination stand out as the most prevalent degradation-related mechanisms among ubiquitin-mediated processes. Our result demonstrated that TRIM25 specifically promoted K48-linked ubiquitination of BRD7 (Fig. 4E). Considering that TRIM25 belongs to the TRIM family, most of whose members are RING-type E3 ligases, we sought to determine whether TRIM25 negatively regulated BRD7 protein levels via its E3 ligase activity. We co-transfected MCF7 cells with Flag-TRIM25 plasmid or Flag-TRIM25ΔRING mutant plasmid, along with HA-Ub plasmid. The results showed that the expression of Flag-TIM25, but not that of Flag-TRIM25ΔRING, increased the ubiquitination of BRD7 in MCF7 cells (Fig. 4F). Consistent with this observation, the protein stability of BRD7 was significantly reduced in cells overexpressing TRIM25, but not in those transfected with Flag-TRIM25ΔRING plasmid (Fig. S4A). Thus, these results indicated that TRIM25 promotes ubiquitination of BRD7 in a manner dependent on its RING domain. To identify potential ubiquitination sites on BRD7, we used the UBPRED website to predict six possible ubiquitination sites within the N-terminal domain of BRD7 (K21, K28, K52, K103, K119, and K127) (Fig. S4B). We then mutated lysine (K) residues to arginine (R) residues and evaluated the effects of these mutations on TRIM25-mediated ubiquitination of BRD7. Ubiquitination analysis demonstrated that transient transfection with wild-type HA-BRD7 or K21R, K28R, K52R, K103R, and K127R mutants, but not the K119R mutant, increased TRIM25-mediated BRD7 ubiquitination (Fig. S4C). Collectively, our experiments revealed that TRIM25 facilitated the degradation of BRD7 by inducing K48-linked ubiquitination at the K119 site.
We demonstrated that TRIM25 directly bound to BRD7 and mediated K48-linked ubiquitination and degradation of the BRD7 protein, so we were interested in defining the downstream molecular pathway of the TRIM25/BRD7 axis. In our previous study, Y-box binding protein-1 (YB1), a transcriptional activator, was identified as a novel interacting protein of BRD7 in breast cancer, and BRD7 suppressed cell proliferation and EMT-mediated invasion and metastasis in breast cancer by negatively regulating YB1 [8]. Therefore, we speculated that TRIM25 participated in the malignant progression of breast cancer by regulating the BRD7/YB1 axis. To validate this hypothesis, we detected the protein expression levels of BRD7 and YB1 in TRIM25-overexpressing and TRIM25-knockdown BC cells. Our results showed that overexpression of TRIM25 efficiently reduced the protein levels of BRD7 but promoted the protein levels of YB1. In contrast, TRIM25 knockdown promoted the protein levels of BRD7 but reduced the protein levels of YB1 (Fig. 5A, B), supporting the finding that TRIM25 modulates the BRD7/YB1 axis to promote BC progression.
Bcl-2 has been reported to play an important role in paclitaxel-mediated apoptosis and anti-tumor pathway [25, 26], therefore, it is an important effector protein in paclitaxel-mediated tumor chemotherapy. We used the JASPAR database to predict that there was direct binding of the YB1 protein to the promoter region of Bcl-2, which carries 3 putative YB1-binding sites along the Bcl-2 promoter region, with a relative score higher than 0.85 (Fig. S5A). Since YB1 is a transcription factor, we further explored the effect of YB1 on the protein and mRNA expression of Bcl-2 using qPCR and western blot in MDA-MB-231and MCF7 cells. The results showed that the overexpression of YB1 increased the mRNA and protein levels of Bcl-2, whereas the knockdown of YB1 reduced the protein and mRNA levels of Bcl-2 (Fig. 5C-E). To investigate whether YB1 directly targets the promoter of Bcl-2 in BC cells, the CHIP-qPCR assay was performed. Specifically, to detect whether YB1 could directly bind to the promoter sequence of Bcl-2, we designed three pairs of primers according to the relative score of the potential binding sequence of YB1 and Bcl-2 promoters according to JASPAR database (P1, P2 and P4), meanwhile we designed two pairs of primers as negative control (P3 and P5) according to the sequence of Bcl-2 promoter (Fig. S5A, B). ChIP-qPCR results showed significant enrichment of YB1 at the Bcl-2 promoter regions -782 to -790 and -927 to -919 in MCF7 cells (Fig. 5F). These results proved that YB1 acts as a transcription factor to promote the expression of Bcl-2. In addition, we further tested the effect of BRD7 expression on YB1/Bcl-2 signaling axis by western blot and found that restoring YB1 reversed BRD7-mediated inhibition of Bcl-2 protein (Fig. 5G). Meanwhile, restoring BRD7 expression partially rescued the inhibitory effect of TRIM25 knockdown on YB1 and Bcl-2 (Fig. 5H). Altogether, these data demonstrated that TRIM25, functioning as an E3 ubiquitin ligase, decreased the stability of BRD7 protein through the ubiquitin-proteasome pathway, and inhibited the negative regulation of BRD7 on YB1/Bcl-2 transcriptional axis activity, thus promoting malignant progression and paclitaxel resistance of breast cancer.
To inquire whether the effect of TRIM25 on BC progression and PTX resistance was mediated by BRD7, we carried out several rescue experiments. Firstly, we used siBRD7 to restore the expression of BRD7 in TRIM25-knockdown BC cells, as shown in Fig. 6A, the protein level of BRD7 was successfully restored after transfection with siBRD7 in MDA-MB-231 and MCF7 cells. CCK-8 and colony formation assays showed that TRIM25 downregulation reduced the ability of BC cells to proliferate, while restoration of BRD7 recovered this ability (Fig. 6B, C). Additionally, cell cycle analysis demonstrated that BRD7 restoration reversed the cell cycle arrest in MDA-MB-231 and MCF7 caused by TRIM25 knockdown (Fig. S6A). Our results showed that TRIM25 knockdown decreased the expression of CDK4 and CDK1, and increased the protein levels of P21 as well as cleaved-PARP in both MDA-MB-231 and MCF7 cell lines, whereas the expression levels of these cell cycle and apoptosis-related markers were significantly reversed after BRD7 restoration (Fig. 6D).
In order to further confirm the important role of the TRIM25/BRD7 axis in paclitaxel resistance of breast cancer, we detected the differential expression of TRIM25 and BRD7 between PTX-resistant BC cells and their parent cells. As shown in Fig. S7A, TRIM25 was upregulated and BRD7 was downregulated in MDA-MB-231-PR cells as compared with the parent MDA-MB-231 cells, while TRIM25 knockdown in MDA-MB-231-PR reversed these effects. Cell cycle analysis showed that the ratio of cells in G2/M phase in MDA-MB-231-PR cells was significantly increased compared to that in their parent cells, while TRIM25 knockdown significantly reduced the G2/M phase ratio in MDA-MB-231-PR cells (Fig. S7B). These results suggested that TRIM25 knockdown could reverse the cell cycle progression in PTX-resistant breast cancer cells. In a word, the TRIM25/BRD7 signaling axis is indeed related to the paclitaxel resistance in breast cancer.
Based on these findings, we conducted several rescue experiments to further investigate whether TRIM25 mediates PTX resistance via BRD7. The CCK-8, colony formation, and apoptosis assays were conducted to validate the effects of BRD7 restoration in the TRIM25 knockdown-mediated PTX chemosensitivity in MDA-MB-231 and MCF7 cells. We found that TRIM25 knockdown could increase the PTX chemosensitivity compared with that in the control group, and restoration of BRD7 reversed the PTX chemosensitivity induced by TRIM25 silent expression (Figs. 6E-G, S6B). Similarly, in MDA-MB-231-PR cells, the results of CCK-8 assays, colony formation assays, and apoptosis experiments all demonstrated that restoring the expression of BRD7 reversed the TRIM25 silencing-mediated enhancement of PTX chemosensitization (Fig. S8A-D). Collectively, our data revealed that BRD7 is essential for TRIM25-induced malignant progression and PTX chemoresistance of breast cancer cells.
We further investigated the effect of the TRIM25/BRD7 axis on tumor growth and PTX resistance in vivo. A nude mouse tumor model was established by using the MCF7 breast cancer cell line, and the mice were divided into six groups, including the siNC, siTRIM25, siTRIM25 plus siBRD7, siNC plus PTX, siTRIM25 plus PTX, and siTRIM25 plus siBRD7 plus PTX groups. For the PTX treatment groups, ten days after subcutaneous implantation, the mice were intraperitoneally injected with PTX (15 mg/kg), and then treated once every two days for a total of four times. The results showed that compared with the siNC group, TRIM25 knockdown slowed tumor growth and reduced tumor weight, whereas the recovery expression of BRD7 reversed the inhibitory effect of TRIM25 knockdown (Fig. 7A-C, S9A). Additionally, in the PTX treatment groups, compared with the untreated groups, the tumor was significantly smaller, indicating the anti-tumor effect of PTX on breast cancer. Meanwhile, compared with the siNC plus PTX group, TRIM25 knockdown significantly increased the anti-tumor effect of PTX, and restoring BRD7 expression could significantly reverse the effect of TRIM25 knockdown on paclitaxel chemotherapy (Fig. 7A-C, S9B). Furthermore, immunohistochemical staining (IHC) indicated that in both the PTX untreated and treated groups, TRIM25 knockdown downregulated the expression of YB1, Ki67, and Bcl-2 but upregulated the expression of BRD7 and cleaved-PARP. Meanwhile, in the PTX untreated groups, TRIM25 knockdown downregulated the expression of CDK4 and CDK1 but upregulated the expression of p21. However, after the expression of BRD7 resumed, the expression of BRD7, YB1, Ki67, CDK4, CDK1, Bcl-2, p21, and cleaved-PARP were partially reversed, which was consistent with the change trend observed in vitro (Fig. 7D, S9C). Taken together, these data validate that TRIM25 promotes breast cancer tumorigenesis and PTX chemotherapy resistance by negatively regulating the stability of BRD7 protein in vivo.
To better understand the potential clinical correlation between TRIM25 and BRD7, we detected the expression of TRIM25 and BRD7 in 34 normal breast tissues and 219 breast cancer biopsies. The results of IHC showed that the expression level of TRIM25 in breast cancer tissues was significantly higher than that in normal breast tissues (Fig. 8A, B). By analyzing the expression of TRIM25 across different clinical stages, we found that its expression in stage III and IV breast cancer tissues was higher than in stage I and II tissues, indicating a positive correlation with disease stage (Fig. 8B). We further explored the expression correlation between BRD7 and TRIM25. As shown in Fig. 8C, the expression level of TRIM25 was negatively correlated with that of BRD7 in BC samples (Pearson correlation coefficient r = -0.1697, P = 0.0131). Additionally, we evaluated the prognostic value of TRIM25 and BRD7 expression via survival analysis and found that a higher TRIM25 expression was correlated with shorter overall survival (Fig. 8D). Moreover, BC patients with low expression of TRIM25 combined with high expression of BRD7 showed significantly prolonged overall survival (Fig. 8E). Next, we analyzed the correlation between TRIM25 expression and prognosis of breast cancer patients treated with PTX, and found that the breast cancer patients treated with PTX with lower expression of TRIM25 had a longer overall survival (Fig. 8F). In addition, we analyzed the correlation between TRIM25 and BRD7 expression and clinicopathological features in BC patients. The results showed that the expression of TRIM25 was correlated with the clinical stage, tumor size, and distant metastasis of breast cancer patients, but not with the age and lymph node metastasis, while the combined expression of TRIM25 and BRD7 correlated with the clinical stage, tumor size, lymph node metastasis, and distant metastasis of breast cancer patients (Table 3). Overall, these data identified that the TRIM25/BRD7 axis could guide the early diagnosis and treatment in breast cancer patients and provide a potential therapeutic target for breast cancer therapy.