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

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.

Multifaceted Roles of TRIM Proteins in Colorectal Carcinoma

“…Knockdown of TRIM59 for example can inhibit malignant processes including evasion of apoptosis in p53 wild-type cells (HCT116) as well as in the p53 mutant CRC cell line SW480 [ 78 ]. These findings fit to our previous studies which demonstrated that knockdown of TRIM25 via upregulation of the pro-apoptotic caspase-2 similarly sensitizes wild-type p53 (RKO) and p53 mutated (DLD-1) CRC cells to DNA-damage-induced apoptosis [ 64 ] thus inferring that some TRIMs exert anti-apoptotic programs which do not rely on the inhibition of p53. Although not particularly highlighted in CRC, many of the TRIMs previously described as modulators of other main oncogenic signaling pathways—including NF-κB, STAT3, Wnt/β-catenin, TGFβ, and PI3 kinase—may indirectly affect the outcome of apoptosis and consequently, the responses of colon carcinoma cells toward chemotherapeutic drugs ( Figure 1 ).…”

“…Therefore, the TRIM25 -mediated inhibition of these functions may be highly relevant for the malignant phenotype and increased therapy resistance of CRC cells. It is worth noting that, in a clear contrast to previous studies by Zhang et al [ 62 ] who demonstrated that TRIM25 dampened the p53-mediated DNA damage-induced apoptosis in the CRC cell line HCT116, the translation inhibitory effects by TRIM25 were independent of the p53 status of the investigated CRC cell lines [ 64 ]. This implies that TRIM25 may exert a broad anti-apoptotic program through diverse mechanisms.…”

“…Previous studies furthermore demonstrated that several TRIM proteins, including TRIM25 represent important hubs connecting the ubiquitin protein modification pathway directly with different RNA functions, especially mRNA stability and mRNA translation. Interestingly, several of these TRIMs contain typical zinc finger DNA-binding domain analogues to TRIM25 implicating that these TRIMs, in addition to posttranslational protein modifications, can directly modulate gene expression at the transcriptional and/or post-transcriptional level [ 29 , 64 , 90 ]. Given the pleiotropic roles exhibited by TRIMs in the control of oncogenic signaling pathways and their frequent deregulation in human cancers, TRIMs represent attractive drug targets for novel anticancer therapies.…”

“…Using RNA affinity chromatography, we previously identified TRIM25 as a novel caspase-2 mRNA-binding protein in human CRC cell lines, which reduces protein expression of caspase-2 mainly through interfering with caspase-2 translation [ 64 ]. Functionally, silencing of TRIM25 caused a significant elevation in caspase-2 protein expression concomitant with an increased sensitivity towards intrinsic apoptosis of CRC cells induced by the topoisomerase inhibitors doxorubicin and etoposide [ 64 ]. Our data highlight the assigned role of caspase-2 to act as a damage sensor, driving cells into apoptosis at the last resort to genotoxic insults [ 143 , 144 ].…”

Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

“…By unveiling this TRIM25 unexpected function, a novel mechanism of drug resistance in human colorectal carcinoma cells associated with the activity of TRIM25 as an inhibitor of chemotherapeutic drug-induced apoptosis was described. 60 Analogously to DNA, RNA molecules, interact with proteins to perform a variety of functions in living cells. Pisa et al employed AP-MS to isolate novel cap-binding factors that might be involved in translational control of specific mRNAs within the growing Drosophila oocyte, by using m7GTPderivatized Sepharose beads as bait.…”