High expression of TRIM25 predicts recurrence in patients with CRC
We analyzed publicly available CRC mRNA expression profiles (GSE20842) obtained from the NCBI and found that the mRNA expression of TRIM25 is elevated in CRC tissues compared with normal tissues (Fig. 1A). To confirm our in silico observations, we detected TRIM25 protein expression in CRC samples. Immunohistochemistry (IHC) analysis demonstrated that TRIM25 protein level in CRC tissues was significantly higher than that in adjacent normal tissues (Fig. 1B and C). To explore the potential role of TRIM25 in CRC therapy, we initially evaluated TRIM25 expression in 26 primary tumor tissues from patients with stage III CRC that were treated with oxaliplatin-based chemotherapy. The results showed that TRIM25 protein level was significantly higher in oxaliplatin resistant patients who developed recurrence during follow-up period (Fig. 1D). We further analyzed TRIM25 expression in 223 paraffin-embedded human CRC specimens from patients with Xelox or FOLFOX treatment after surgery (patient characteristics were summarized in Supplementary Table 1). Representative IHC staining confirmed that TRIM25 expression was markedly increased in CRC patients with tumor relapse (Fig. 1E). Patients with high TRIM25 expression demonstrated an observably higher recurrence rate than those with low TRIM25 expression (28.9% vs. 15.0%, P = 0.012, Fig. 1F and Table 1). Furthermore, compared with low TRIM25 expression, high TRIM25 expression was significantly associated with worse overall survival and disease-free survival (P = 0.006, Fig. 1G and Supplementary Figure S1). Collectively, the above observations suggest that elevated expression of TRIM25 contributes to the progression of CRC and is associated with the failure of OXA-based chemotherapy.
TRIM25 confers OXA resistance in CRC cells in vitro
We then examined whether TRIM25 was, in fact, associated with resistance to OXA-based therapy in preclinical models. Firstly, we established stable TRIM25 knockdown and overexpressing cells from the SW48 and SW480 CRC cell lines (Fig. 2A). The CCK8 assay showed that the IC50 values for OXA were decreased in TRIM25-knockdown cells and were increased in TRIM25-overexpressing cells (Fig. 2B and C). Consistently, in the presence of OXA, TRIM25 knockdown dramatically inhibited while TRIM25 overexpression enhanced the colony-formation ability of SW48 and SW480 cells (Fig. 2D). Moreover, compared with control cells, knockdown of TRIM25 resulted in significantly increased OXA-induced apoptosis of CRC cells, while overexpression of TRIM25 reduced OXA-induced apoptosis (Fig. 2E). Measurement of cleaved caspase 3 and cleaved PARP further confirmed that knockdown of TRIM25 increased OXA sensitivity and upregulation of TRIM25 conferred OXA resistance (Fig. 2F). Altogether, these results reveal that TRIM25 confers OXA resistance in CRC cells.
TRIM25 promotes stem cell properties of CRC cells
As reported previously, stemness is believed to be responsible for chemotherapy resistance, thus we hypothesized that TRIM25 is involved in regulating CRC stemness. A couple of experiments were then performed to test our hypothesis. We conducted sphere formation assay, and found a decrease in sphere numbers and sizes in TRIM25 knockdown cells compared with the corresponding control cells; while overexpression of TRIM25 enhanced the sphere formation ability of CRC cells (Fig. 3A). In addition, the expression of stem cell related molecules such as EpCAM, SOX2, CD133 and CD44 in SW48 and SW480 cells was markedly reduced after TRIM25 inhibition, while the expression increased after TRIM25 overexpression (Fig. 3B). Furthermore, limiting dilution analysis (LDA) in vivo confirmed the markedly reduced stem cell frequency in TRIM25 knockdown SW480 cells (Fig. 3C), with forming smaller and lighter tumors than the control SW480 cells (Fig. 3D and E). And no visible tumors could be formed in nude mice when 1×104 TRIM25 knockdown SW480 cells were inoculated. These findings indicate the crucial role of TRIM25 in promoting stem cell properties of CRC cells.
TRIM25 regulates EZH2 stability in CRC cells
As the catalytic subunit of PRC2, EZH2 plays an essential role in tumor progression. Previous study has revealed that targeting EZH2 inhibits CSC self-renewal and enhances the sensitivity of CRC to OXA [13-14]. In the present study, we found that knockdown of TRIM25 decreased the protein level of EZH2 (Fig. 4A). However, there was no significant effect on EZH2 mRNA levels in both SW48 and SW480 cells (Fig. 4B). These results suggested that TRIM25 may affect the stability of EZH2. To substantiate this assumption, we treated TRIM25-knockdown or control CRC cells with the protein synthesis inhibitor cycloheximide (CHX) and the proteasome inhibitor MG132. The results showed that knockdown of TRIM25 shortened the half-life of endogenous EZH2 protein in CRC cells after CHX treatment (Fig. 4C and D). Besides, the expression of EZH2 was modestly increased in TRIM25 knockdown SW48 cells treated with MG132, and the same results were obtained in SW480 cells (Fig. 4E). Finally, we detected EZH2 protein in the same cohort of CRC samples by IHC staining, and found that EZH2 high expression was significantly positively correlated with TRIM25 high expression (Fig. 4F). CRC samples with high TRIM25 expression showed a higher rate of high EZH2 expression, whereas samples with low TRIM25 expression exhibited lower rate of high EZH2 expression (63.9% vs. 45.7%, P = 0.009, Fig. 4G). CRC patients with high expression of TRIM25 as well as high expression of EZH2 had the shortest overall survival (P 0.001, Supplementary Figure S2) and disease-free survival (P 0.001, Fig. 4H) compared with patients with low TRIM25 expression or low EZH2 expression. Taken together, these observations suggest that TRIM25 regulates EZH2 expression in CRC cells through reducing degradation of EZH2 protein.
TRIM25 blocks TRAF6-mediated ubiquitination of EZH2
Since TRIM25 is an E3 ligase, we wondered if TRIM25 modulates EZH2 stability through the ubiquitin–proteasome pathway. First, we analyzed the interaction of TRIM25 with EZH2 in CRC cells. By co-immunoprecipitation and western blot, we verified the interaction between TRIM25 and EZH2 at both exogenous and endogenous protein levels (Fig. 5A and B). Then, we performed in vivo ubiquitination assays in HEK293T cells transfected with siTRIM25, Flag-EZH2, His-tagged ubiquitin wild type (WT) or mutation plasmids (K48 or K63 mutants). As shown in Fig. 5c, knockdown of TRIM25 increased the poly-ubiquitination of EZH2, indicating that EZH2 is not a substrate of TRIM25 E3 ligase. Interestingly, EZH2 polyubiquitination enhanced by knockdown of TRIM25 was mainly extended through K63-linkage instead of K48-linkage.
TNF receptor associated factor 6 (TRAF6) is a member of the TNF receptor associated factor (TRAF) protein family, and functions as an E3 ubiquitin ligase and a scaffold protein. It has been reported that TRAF6 mediated the K63-linked ubiquitination of EZH2 in prostate cancer [26]. We then speculated whether TRIM25 is involved in TRAF6-mediated ubiquitination of EZH2. We transfected siNC and siTRIM25 in SW480 cells, and the expression of TRAF6 was detected in EZH2 immunoprecipitates. We interestingly found that knockdown of TRIM25 enhanced the interaction between EZH2 and TRAF6 (Fig. 5D). To further confirm the role of TRIM25 in TRAF6-mediated EZH2 ubiquitination, we blocked TRAF6 in TRIM25 knockdown SW480 cells. Further co-immunoprecipitation and immunoblotting analysis showed that knockdown TRIM25-induced EZH2 ubiquitination and degradation could be rescued by siTRAF6 (Fig. 5E and F). Taken together, these results suggest that TRIM25 stabilizes EZH2 most probably by preventing TRAF6 bind to EZH2.
EZH2 is required for TRIM25-induced OXA-resistance in CRC both in vitro and in vivo
To further assess whether TRIM25 mediates OXA-resistance via EZH2, we blocked EZH2 in TRIM25-overexpressing cells by shRNA or EZH2 inhibitor (UNC1999). Colony formation assay, CCK8 assay and Annexin V/PI apoptosis assay showed that inhibition of EZH2 significantly rescues the effect of TRIM25 overexpression on both the growth and anti-apoptotic capacity of CRC cells treated with OXA (Fig. 6A, B and C). Besides, the sphere formation ability of SW48 and SW480 cells overexpressing TRIM25 was markedly suppressed by EZH2 inhibition (Fig. 6D).
Moreover, nude mouse xenograft model was used to evaluate the effect of EZH2 inhibition on TRIM25-induced OXA-resistance in vivo. Consistent with in vitro findings, the results showed that inhibition of EZH2 by shEZH2 or UNC1999 resulted in a significant reduction in tumor volume and weight when treated with OXA (Fig. 6E and F). And further TUNEL staining showed that inhibition of EZH2 resulted in more OXA-induced apoptosis by comparison with TRIM25-overespressing cells in vivo, as demonstrated by a higher proportion of TUNEL positively stained cells after OXA treatment (Fig. 6G and H). Overall, EZH2 is essential for TRIM25-induced OXA-resistance in CRC, and inhibition of EZH2 is expected to overcome TRIM25-induced OXA-resistance in clinical practice.