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. 2019 Dec 12;8(12):1622.
doi: 10.3390/cells8121622.

Identification of TRIM25 as a Negative Regulator of Caspase-2 Expression Reveals a Novel Target for Sensitizing Colon Carcinoma Cells to Intrinsic Apoptosis

Affiliations

Identification of TRIM25 as a Negative Regulator of Caspase-2 Expression Reveals a Novel Target for Sensitizing Colon Carcinoma Cells to Intrinsic Apoptosis

Usman Nasrullah et al. Cells. .

Abstract

Colorectal cancer (CRC) is one of the most common cancers that is characterized by a high mortality due to the strong metastatic potential of the primary tumor and the high rate of therapy resistance. Hereby, evasion of apoptosis is the primary underlying cause of reduced sensitivity of tumor cells to chemo- and radiotherapy. Using RNA affinity chromatography, we identified the tripartite motif-containing protein 25 (TRIM25) as a bona fide caspase-2 mRNA-binding protein in colon carcinoma cells. Loss-of-function and gain-of-function approaches revealed that TRIM25 attenuates the protein levels of caspase-2 without significantly affecting caspase-2 mRNA levels. In addition, experiments with cycloheximide revealed that TRIM25 does not affect the protein stability of caspase-2. Furthermore, silencing of TRIM25 induced a significant redistribution of caspase-2 transcripts from RNP particles to translational active polysomes, indicating that TRIM25 negatively interferes with caspase-2 translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly increased the sensitivity of colorectal cells to drug-induced intrinsic apoptosis as implicated by increased caspase-3 cleavage and cytochrome c release. Importantly, the apoptosis-sensitizing effects by transient TRIM25 knockdown were rescued by concomitant silencing of caspase-2, demonstrating a critical role of caspase-2. Inhibition of caspase-2 by TRIM25 implies a survival mechanism that critically contributes to chemotherapeutic drug resistance in CRC.

Keywords: TRIM25; caspase-2; chemotherapy resistance; colon carcinoma cells; intrinsic apoptosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TRIM25 is a bona fide caspase-2 mRNA-binding protein in the human colon carcinoma cell line DLD-1 (A) and RKO (B). RNP-IP assays from total cell lysates of colon carcinoma cells were performed and followed by RT-PCR. TRIM25-bound mRNA was precipitated by the addition of monoclonal TRIM25-specific antibodies (anti-TRIM25) or, alternatively, the same amount of mouse IgG (IgG) as a negative control. RNA samples were analyzed by semiquantitative RT-PCR using primersets encompassing the coding region of caspase-2. The specific immunoprecipitation (IP) of TRIM25 in both cell lines was validated by Western blot analysis (W.b.) as shown on the right-hand side. Input levels of caspase-2 mRNA were monitored by RT-PCR (input). Graphs shown in the lower parts of the panels show means ± SD (n = 3) and depict the relative caspase-2 binding to TRIM25 (filled bars) in relation to IgG (open bars). *** p ≤ 0.001 vs. IgG.
Figure 2
Figure 2
TRIM25 reduces abundance of caspase-2 protein in RKO cells but not in HEK cells. Subconfluent RKO (A) or HEK293 (B) cells mock transfected (-) or transfected with the indicated amount of Flag-tagged human TRIM25 (pCMV-Flag-TRIM25) or, alternatively, with the same amount of empty pCMV-Flag vector (pCMV-Flag). Then, 48 h after transfection, the amounts of endogenous and ectopic TRIM25 and caspase-2 were monitored by Western blot analysis and β-actin was used as a loading control. Graphs at the bottom show means ± SD (n = 3) and depict the relative abundance of TRIM25 (open bars) and caspase-2 (filled bars) protein in both transfected cell populations. ** p ≤ 0.005, *** p ≤ 0.001 (TRIM25), and # p ≤ 0.05 (Casp2), respectively, vs. empty vector transfected.
Figure 3
Figure 3
Silencing of TRIM25 increases caspase-2 protein levels in colon carcinoma cells. Subconfluent DLD-1 (A) or RKO (B) cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of TRIM25 (siTRIM25). Then, 48 h after transfection, cells were harvested and total protein lysates (20 µg) were subjected to SDS-PAGE. The abundance of TRIM25 and caspase-2 was analyzed by Western blot analysis and β-actin was used as a loading control. Graphs at the bottom show means ± SD (n = 3) and depict the relative protein levels of TRIM25 (open bars) and caspase-2 (filled bars) protein. ** p ≤ 0.005, (TRIM25) and ## p ≤ 0.005 (Casp2), respectively, vs. control siRNA-transfected (siCtrl.) cells.
Figure 4
Figure 4
TRIM25 silencing-mediated increase in caspase-2 depends on de novo protein synthesis. Subconfluent DLD-1 (A) or RKO (B) cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of TRIM25 (siTRIM25) for 24 h before translation was blocked by the addition of cycloheximide (CHX, 25 µg/mL). After an additional 24 h, cells were harvested for total protein extraction. Total protein lysates (20 µg) were subjected to SDS-PAGE before the abundance of TRIM25 and caspase-2 was analyzed by Western blot analysis, with β-actin used as a loading control. Graphs at the bottom show means ± SD (n = 3) and depict the relative protein levels of TRIM25 (open bars) and caspase-2 (filled bars) protein at 24 h after CHX application. * p ≤ 0.05 (TRIM25) vs. control siRNA-transfected (siCtrl.) cells. (C). Subconfluent DLD-1 cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of TRIM25 (siTRIM25) for 48 h before fractions of translational inactive RNPs were separated from translational active polysomes by ultracentrifugation. The amount of caspase-2 mRNA was determined by qRT-PCR using primer pairs that were complementary and specific to the coding region of caspase-2. The graph shows means ± SD (n = 3) and depicts the amount of caspase-2 mRNA levels normalized to GAPDH mRNA in polysomes relative to those in RNP fractions from control siRNA- (open bars) and TRIM25 siRNA-transfected cells (filled bars). * p ≤ 0.05 (TRIM25) vs. control siRNA-transfected (siCtrl.) cells.
Figure 5
Figure 5
Knockdown of TRIM25 causes an increase in chemotherapeutic drug-induced intrinsic apoptosis in colon carcinoma cells. DLD-1 cells (A) or RKO cells (B) were either transfected with control siRNA (siCtrl.) or with siRNA duplexes against TRIM25 (siTRIM25) for 24 (A) or 48 (B) h before stimulation with doxorubicin (Doxo.) or etoposide (Etopo.) for a further 24 h. Thereafter, the cells were lysed for total protein extraction and caspase-3 cleavage and knockdown of TRIM25 were determined by Western blot, with β-actin used for equal loading. Graphs at the bottom summarize the densitometric analysis of cleaved caspase-3 (Casp3*) levels in relation to full-length caspase-3. Data show means ± SD (n = 3) * p ≤ 0.05, ** p ≤ 0.01 siTRIM25 (filled bars) vs. siCtrl. (open bars) transfected cells.
Figure 6
Figure 6
Silencing of TRIM25 via increased mitochondrial cytochrome c release leads to a sensitization of doxorubicin-induced caspase-3 cleavage. DLD-1 cells (A) or RKO cells (B) were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes targeting TRIM25 (siTRIM25) for 48 h before being stimulated with the indicated doses of doxorubicin (Doxo.). After a further 24 h, cells were harvested for cytoplasmic cell lysates. The content of cytoplasmic cytochrome c (Cyt c), mature caspase-8, and cleavage of caspase-3 as well as the knockdown efficiency of TRIM25 was subsequently determined by Western blot using β-actin as a loading control. Data are representative of two independent experiments giving similar results.
Figure 7
Figure 7
Silencing of caspase-2 prevents doxorubicin-induced apoptosis upon TRIM25 knockdown. DLD-1 (A) or RKO (B) cells were transfected with control siRNA (siCtrl.) or with siRNA duplexes either targeting caspase-2 (siCasp2) or TRIM25 (siTRIM25) or, alternatively, simultaneously transfected with TRIM25 plus caspase-2-specific siRNAs for 24 (A) or 48 (B) h before cells were exposed to doxorubicin for a further 24 h. Thereafter, cells were collected for total protein extraction and activation of caspase-3 cleavage and knockdown efficiency of TRIM25 or caspase-2 determined by Western blot using β-actin as a control for equal protein loading. Graphs summarize a densitometric analysis of cleaved caspase-3 (Casp3*) levels in relation to full-length caspase-3. Values show means ± SD (n = 3) *** p ≤ 0.005 siTRIM25 vs. siCtrl. and # p ≤ 0.05, ## p ≤ 0.01 siTRIM25/Casp2 vs. siTRIM25. (C, left panel). Subconfluent RKO cells were transfected for 48 h with 6 µg of Flag-tagged human TRIM25 (pCMV-Flag-Trim25) or, alternatively, with the same amount of empty pCMV-Flag vector (pCMV-Flag) before being treated with either the vehicle (-) or with the indicated chemotherapeutic drugs. After a further 24 h, cells were harvested for total protein extraction and the cleavage of caspase-3 as well as the overexpression of TRIM25 was determined by Western blot using β-actin as a loading control. (C, right panel). RNP-IP assays from total cell lysates of RKO cells expressing either Flag-tagged human TRIM25 (pCMV-Flag-TRIM25) or pCMV-Flag (pCMV-Flag). TRIM25-bound mRNA was isolated by the use of anti-Flag-M2 magnetic beads followed by RT-PCR. For PCR, primer pairs, complementary and specific to the coding region of caspase-2, were used and the length of specific PCR products determined by a DNA ladder. Data shown are representative of three independent experiments giving similar results.
Figure 8
Figure 8
Doxorubicin induces TRIM25 binding to the 5′UTR of caspase-2 and reduces recruitment of caspase-2 mRNA at polysomes. (A). Representative biotin pull-down assays showing doxorubicin-induced TRIM25 affinity to the 5′UTR of caspase-2. The biotinylated transcript, which encompasses 241 nucleotides of the 5′UTR of the human caspase-2 (5′UTR-Casp2), was incubated with either total (left) or cytoplasmic (right) cell lysates from vehicle-treated DLD-1 cells (−) or from cells treated with 10 µg/mL of doxorubicin (+) for 6 h. RNA binding of TRIM25 to the pull-down material was assessed by Western blot analysis and an equal input TRIM25 levels were assayed by Western blot analysis (input). Blots shown are representative of two independent experiments with similar results. (B). DLD-1 cells were stimulated for 6 h with either vehicle or with 10 µg/mL doxorubicin (Doxo.) before intracellular TRIM25 was analyzed by confocal microscopy. Cell nuclei were visualized by DAPI staining (blue panel). Bar: 20 µm. (C). DLD-1 cells were stimulated for 6 h either with vehicle (−) or doxorubicin (+) before fractions of translational inactive RNPs were separated from translational active polysomes by ultracentrifugation. The amount of caspase-2 mRNA and GAPDH mRNA was determined by semiquantitative RT-PCR using primer pairs that were complementary and specific to the coding region of both genes The graph on the right panel shows means ± SD (n = 3) and depicts the amount of caspase-2 mRNA levels normalized to GAPDH mRNA in polysomes (open bars) relative to those in RNP (grey bars) fractions. ** p ≤ 0.01, doxorubicin vs. vehicle-treated cells.

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