TRIM25 regulates oxaliplatin resistance in colorectal cancer by promoting EZH2 stability

Abstract

Resistance to chemotherapy remains the major cause of treatment failure in patients with colorectal cancer (CRC). Here, we identified TRIM25 as an epigenetic regulator of oxaliplatin (OXA) resistance in CRC. The level of TRIM25 in OXA-resistant patients who experienced recurrence during the follow-up period was significantly higher than in those who had no recurrence. Patients with high expression of TRIM25 had a significantly higher recurrence rate and worse disease-free survival than those with low TRIM25 expression. Downregulation of TRIM25 dramatically inhibited, while overexpression of TRIM25 increased, CRC cell survival after OXA treatment. In addition, TRIM25 promoted the stem cell properties of CRC cells both in vitro and in vivo. Importantly, we demonstrated that TRIM25 inhibited the binding of E3 ubiquitin ligase TRAF6 to EZH2, thus stabilizing and upregulating EZH2, and promoting OXA resistance. Our study contributes to a better understanding of OXA resistance and indicates that inhibitors against TRIM25 might be an excellent strategy for CRC management in clinical practice.

Fig. 1. High expression of TRIM25 is associated with recurrence in colorectal cancer.

A Analysis of TRIM25 expression in CRC and adjacent normal tissues from publicly available GEO data set (GSE20842), as assessed using a t test. B Representative IHC images of TRIM25 in colorectal tumor or normal tissues. Scale bar, 20 μm. c IHC scores of TRIM25 levels in paired normal and CRC samples are shown as a symbol-line plot. Expression data for normal and tumor tissue from a given individual are linked with dashed lines. Differences were assessed using a paired two-tailed t test. D Western blotting analysis showing the level of TRIM25 in 26 primary tumor tissues from patients with stage III CRC who were treated with OXA-based chemotherapy, including 13 samples from patients who developed recurrence and 13 samples from patients who did not develop recurrence after surgical resection. E Representative IHC images of TRIM25 in 223 CRC tissues with or without recurrence. Scale bar, 200 μm. F Relationship between TRIM25 expression and CRC recurrence, as assessed using a χ² test. G Comparison of disease-free survival between patients with high or low TRIM25 expression, as assessed using a log-rank test.

Fig. 2. TRIM25 influences the sensitivity of CRC cells to oxaliplatin.

A Western blotting analysis of TRIM25 in SW48 and SW480 cells transfected with shTRIM25 or a TRIM25-expressing plasmid. GAPDH was used as the loading control. B–C A CCK8 assay was used to measure the viability of the indicated cells treated with different concentrations of OXA for 48 h. D Colony-formation ability (left panel: representative images; right panel: quantification of colony numbers) of the indicated cells treated with OXA (2 μM). E Annexin V-FITC and PI staining showing apoptosis in the indicated CRC cells treated with OXA (30 μM) for 48 h. Left panel: representative images; right panel: Quantification of apoptotic cells. F Western blotting analysis of cleaved caspase 3 and cleaved PARP in the indicated cells treated with or without OXA (30 μM) for 48 h. Data are represented as the mean ±SD of three independent experiments. *P < 0.05, **P < 0.01, **P < 0.001.

Fig. 3. TRIM25 promotes stem cell properties of CRC cells.

A Representative images (left panel) and quantification (right panel) of the in vitro sphere formation assay of TRIM25-knockdown and TRIM25-overexpressing SW48 and SW480 cells. Scale bar, 200 μm. Data are represented as the mean ±SD of three independent experiments. B Relative mRNA expression of EpCAM, SOX2, CD133, and CD44 in TRIM25-knockdown or TRIM25-overexpressing SW48 and SW480 cells. Data are represented as the mean ±SD of three independent experiments. C Limiting dilution data showing the stem cell frequency of the indicated group of SW480 cells from a tumorigenic mouse model. D Growth curves of each group of tumors. Indicated numbers of SW480 cells were inoculated into the nude mice and tumor volumes were measured every 4 days. Data are represented as the mean ±SD. E Each group of tumors was removed and weighted. Data represent the mean ±SD. *P < 0.05, **P < 0.01, ***P < 0.001 for indicated comparison.

Fig. 4. TRIM25 regulates EZH2 stability in CRC cells.

A Western blotting analysis of EZH2 in control and TRIM25-knockdown SW48 and SW480 cells. B RT-PCR analysis of EZH2 mRNA in control and TRIM25-knockdown SW48 and SW480 cells. C EZH2 protein levels in control and TRIM25-knockdown SW48 or SW480 cells were detected at the indicated time points after CHX (20 μg/mL) treatment. D The relative level of EZH2 at each time point was normalized by the level of GAPDH. E Western blotting showing the effect of MG132 (10 μM) on EZH2 levels in control and TRIM25-knockdown SW48 or SW480 cells. F Representative IHC images of TRIM25 and EZH2 in two cases of CRC. Scale bar, 100 μm. G Relationship between TRIM25 expression and EZH2 expression in the same cohort of CRC samples, as assessed using the χ² test. H Comparison of disease-free survival between patients with different TRIM25 and EZH2 expression patterns, as assessed using the log-rank test.

Fig. 5. TRIM25 blocks TRAF6-mediated ubiquitination of EZH2.

A Endogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-TRIM25 or anti-EZH2 antibodies in SW480 cells. B Exogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-Flag or anti-HA antibodies in HEK293T cells co-transfected with Flag-EZH2 and HA-TRIM25. C Immunofluorescence images showing colocalization of endogenous EZH2 and TRIM25 in SW480 cells. D Flag-EZH2, siTRIM25, His-tagged ubiquitin wild-type (WT) or mutation plasmids (K48 or K63 mutants) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies was performed to detect the ubiquitination level of EZH2. E Immunoprecipitation assay using IgG and anti-EZH2 antibodies in SW480 cells transfected with siNC or siTRIM25, followed by western blotting analysis with the indicated antibodies. WCE: Whole-cell extracts. F Flag-EZH2, siTRIM25, siTRAF6, His-tagged ubiquitin wild-type (WT) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies and western blotting with anti-His antibodies were performed to detect the ubiquitination level of EZH2 under different conditions. G Western blotting analysis of EZH2 in SW480 cells transfected with siTRIM25 and/or siTRAF6.

Fig. 6. EZH2 is required for TRIM25-induced OXA resistance in CRC.

A Colony-formation ability of the indicated cells treated with OXA (2 μM) for 14 days. Left panel: representative images; right panel: quantification of colony number. B CCK8 assay showing the IC50 values for OXA in the indicated cells treated with different concentrations of OXA or a combination with UNC1999 (1 μM) for 48 h. C Annexin V-FITC and PI staining showing apoptosis in the indicated CRC cells treated with OXA (30 μM) or a combination with UNC1999 (5 μM) for 48 h. Left panel: representative images; right panel: Quantification of apoptotic cells. D Representative images (left panel) and quantification (right panel) of the in vitro sphere formation assay using the indicated CRC cells. Scale bar, 200 μm. E Growth curves and tumor volume of xenograft tumors derived from TRIM25-overexpressing SW480 cells treated with control (PBS), shEZH2, or UNC1999 (n = 6 mice for each group). All mice were intraperitoneally injected with OXA (5 mg/kg, twice a week) one week after CRC cells inoculation. F Tumor weight of xenografts in each group. G Apoptotic cells in paraffin-embedded tumor sections derived from each group of SW480 cells-bearing mice were visualized using TUNEL staining. H Quantification of TUNEL-positive cells in the indicated group of tumor sections (n = 6). Data are represented as the mean ±SD. *P < 0.05, **P < 0.01, ***P < 0.001 for indicated comparison.

Fig. 7. The working model depicting the role of TRIM25 in CRC cells.

TRIM25 stabilizes EZH2 by blocking TRAF6-mediated ubiquitination for EZH2 degradation, and sustains stem cell properties to promote the resistance of CRC to OXA.