Background
Colorectal cancer (CRC) is the third most common cancer in men and the second in women throughout the world. More than 700,000 patients died from CRC annually, thus making CRC the fourth most common cause of cancer-related death [
1]. CRC will account for more than 1.1 million deaths and 2.2 million new diagnosed cases per year worldwide by 2030 [
2]. It is highly desirable to identify precise biomarkers, which will help the diagnosis and treatment of CRC, and further facilitate the prediction or monitoring of cancer recurrence.
Tripartite motif-containing proteins (TRIM), containing more than 70 members, play critical roles in immune responses, carcinogenesis and chemoresistance [
3‐
5]. Tripartite motif-containing protein 6 (TRIM6) is a member of TRIM family proteins.
TRIM6 gene locates to chromosome 11p15, where it resides within a TRIM gene cluster that includes TRIM5, TRIM21, TRIM22, TRIM34, and a TRIM pseudogene [
6]. Like other TRIM family proteins, TRIM6 has a tripartite motif and possesses E3-ubiquitin ligase activity [
7]. Previous studies have revealed the roles of TRIM6 in viral infection and inflamation responses. Rajsbaum et al. reported that TRIM6 can active IκB kinase-ε (IKKε) and promote the induction of downstream type I interferon (IFN-I) stimulated genes (ISGs), thus facilitating viral control [
8]. The pathogenic Nipah virus (family
Paramyxoviridae) can inhibit IKKε signaling via targeting the degradation of TRIM6 [
9], which further demonstrated the antiviral responses of TRIM6. On the contrary, another study reported that TRIM6 can enhance Ebola virus replication by promoting the ubiquitination of an important viral protein VP35 [
7]. However, little attention has been focused on the possible functions of TRIM6 on carcinogenesis.
In the present study, we reported that TRIM6 expression was significantly elevated in CRC samples and explored the correlation between TRIM6 expression and clinical parameters of CRC patients. Knocking down TRIM6 expression suppressed CRC cell proliferation and induced cell cycle arrested at G2/M phase. Mechanistically, we used immunoprecipitation followed by proteomics analysis to explore potential interaction proteins affecting TRIM6 functions. TIS21, an antiproliferative protein involved in the regulation of G2/M arrest [
10], was identified as an interaction partner of TRIM6. Our study has revealed the clinical significance of TRIM6 in the progression of CRC and may provide a novel therapy target for CRC patients.
Materials and methods
CRC tissue samples
This study was approved by the Institutional Review Board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Two cohorts of patients treated at Shanghai Jiao Tong University Affiliated Sixth People’s Hospital were enrolled in this study after written consent informed was collected. Cohort 1 included 35 CRC patients treated between 2016 and 2017, and 35 pairs of fresh CRC specimens and their adjacent mucosa tissues were obtained from these patients, and stored at − 80 °C until analysis. Cohort 2 contained 90 CRC patients treated between 2010 and 2012 with clinical information and prognosis information (Table
1), and paraffin-embedded CRC specimens were available for immunohistochemical (IHC) staining.
Table 1
Clinicopathological characteristics and TRIM6 expression (n = 90)
Gender |
Male | 48 | 53.3 |
Female | 42 | 46.7 |
Age (years) |
≥ 65 | 29 | 32.2 |
< 65 | 61 | 67.8 |
Tumor size (cm) |
≥ 4.0 | 60 | 66.7 |
< 4.0 | 30 | 33.3 |
Pathologic differentiation |
Well/Moderate | 75 | 83.3 |
Poor | 15 | 16.7 |
Clinical stage |
I/II | 43 | 47.8 |
III | 47 | 52.2 |
Metastasis |
yes | 17 | 18.9 |
no | 63 | 81.1 |
Vital status (at followed-up) |
Alive | 42 | 46.7 |
Dead | 48 | 53.3 |
CEA levels (ng/mL) |
≥ 3.5 | 58 | 64.4 |
< 3.5 | 32 | 35.6 |
TRIM24 expression |
Low | 34 | 37.8 |
High | 56 | 62.2 |
Cell lines
Human normal colorectal mucosa cell line, FHC, and CRC cell lines, LOVO, Sw620, HCT-8 and HCT116, were purchased from Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and grown in a 37 °C incubator at 5% CO2 using DMEM medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Life Technology, Grand Island, NY, USA).
Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from tissues with Trizol reagent (Life Technology). One μg RNA was processed to cDNA synthesis with the reverse transcription kit (Fermentas, Hanover, MD, USA). Real-time PCR reactions were performed in triplicates with SYBR Green mix (Thermo Fisher Scientific (Rockford, IL, USA) on ABI-7300 system (Applied Biosystem, Foster City, CA) according to the manufacturer’s instructions. The primer sequences were listed in Additional file
1: Table S1. The relative mRNA levels were normalized to that of GAPDH.
Immunohistochemical staining
Paraffin-embedded CRC specimens were cut at 4 μm, and deparaffinizaion and rehydration was performed with xylene, a solution of xylene and ethanol and a series ethanol solutions. Antigen retrieval was carried out by boiling slides in 0.01 M sodium citrate buffer (pH 6) at 100 °C for 15–20 min. Subsequently, the slides were block with 3% hydrogen peroxide for 30 min and then with 5% bovine serum albumin (BSA) for 2 h at room temperature (RT). After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.
Western blotting
Cells or tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Beyotime Biotech., Shanghai, China) on ice for 30 min, and the lysates were collected by centrifugation. Equal amounts of protein were mixed with lamini loading buffer, boiled for 5 min, separated by 10% or 15% sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The membrane was blocked in 5% non-fat milk for 1 h at room temperature, and then incubated with the primary antibody, anti-TRIM6 (1:1000, Proteintech, Chicago, IL, USA), anti-TIS21 (1:200, Abcam), anti-cleaved-caspase3 (1:1000, Abcam), anti-Cyclin B1 (1:500, Cell Signaling Technology, Danvers, MA, USA), anti-c-Myc (1:1000, Abcam), anti-forkhead box M1 (FoxM1) (1:1000, Cell Signaling Technology), anti-p-FoxM1(Thr600) (1:1000, Cell Signaling Technology), anti-p-FoxM1(Ser35) (1:1000, Cell Signaling Technology), and anti-GAPDH (1:2000, Cell Signaling Technology) overnight at 4 °C. After incubation with HRP-conjugated secondary antibodies (Beyotime Biotech.), the signal was detected using an ECL kit (Pierce, Rockford, IL, USA).
Construction of plasmids
TRIM6, TRIM6 with substitute mutation of C15A and FoxM1 cDNA was cloned into the pcDNA3.1-myc vector (Life Technology) to express Myc-tagged TRIM6, TRIM6 E3 catalytic mutant (C15A) and FoxM1, respectively. The coding sequence for TRIM6, wild-type TIS21 (WT) or TIS21 with substitute mutations of K5R, K51R or K150R was cloned into the pCMV-Tag2 plasmid (Stratagene, La Jolla, CA, USA) to expressed FLAG-tagged TRIM6, WT and mutant TIS21. TRIM6 and FoxM1 cDNA was cloned into pGEX-2 T vector to produce GST fusion protein. The plasmids were verified by double enzyme digestion and DNA sequencing.
Construction of shRNAs targeted TRIM6
shRNAs specially targeting TRIM6 (shTRIM6–1, shTRIM6–2 and shTRIM6–3) were designed and synthesized as listed in Additional file
1: Table S2. Scrambled shRNA sequence was also generated as negative control (shNC). After annealing, the double-stranded DNA was ligated into AgeI/EcoRI digested pLKO.1 plasmid (Addgene, Cambridge, MA, USA) and verified by DNA sequencing. Lentivirus was produced by transfection of plasmids into 293 T cells with Lipofectamine 2000 (Life Technology) following the manufacture’s instruction as previously described [
11].
Cell counting kit-8 (CCK-8) and bromodeoxyuridine (BrdU) incorporation assays
CCK-8 and BrdU incorporation assays were performed to analyze cell proliferation. Cells were plated into 96-well plate at a density of 3000 per well and cultured at 37 °C overnight. The cells were infected with TRIM6 shRNA/shNC, or transfected with pcDNA3.1-myc-TIRM6/ pCMV-Tag2-TIS21 as indicated in the figure legends, and cultured for 0 h, 24 h, 48 h and 72 h. For CCK-8 assay, the medium was replaced by DMEM medium containing 10% CCK-8 solution (SAB biotech. College Park, MD, USA and cultured at 37 °C for 1 h. Absorbance at 450 nm was determined using a microplate reader.
BrdU Cell Proliferation ELISA Kit (Abcam) was used for BrdU incorporation assay. In brief, cultures were labeled with BrdU for 2 h, incubated with fixing solution and then with mouse anti-BrdU for 1 h. After incubation with HRP-conjugated goat anti-mouse antibody, the cells were stained with the peroxidase substrate. Quantification of BrdU-labeled cells was measured using a microplate reader.
Cell cycle analysis
Cells were plated in 6-well plates and treated as indicated in the figure legends. After 48 h, the cells were collected, fixed with ice-cold ethanol at 4 °C overnight, and labelled with propidium iodine (PI, Sigma-Aldrich). Cell cycle was analyzed with flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.
Chemosensitivity assay
To test whether TRIM6 influences the IC50 (drug concentration producing 50% growth inhibition) of 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), HCT-8 and HCT116 were infected with TRIM6 shRNA/shNC, and treated with 300, 400, 500 or 600 μM of 5-FU (Xdhelp, Shanghai, China) or 40, 60, 80 or 100 μM of L-OHP (Sanofi Aventis, Shanghai, China). After 24 h of culture, CCK-8 assay was performed to calculate the IC50.
To test the effect of TRIM6 on L-OHP, 5-FU-induced apoptosis, HCT-8 and HCT116 were infected with TRIM6 shRNA/shNC, and treated with 400 μM 5-FU (Xdhelp, Shanghai, China), 64 μM L-OHP (Sanofi Aventis, Shanghai, China) or vehicle (DMSO) for 24 h. The cells were collected and stained with Annxin V/PI (KeyGEN Biotech, Nanjing, China). Cell apoptosis was assessed by flow cytometry.
Primary CRC cell isolation and treatment
Primary CRC cells were isolated from 12 patients who were admitted to Shanghai Jiao Tong University Affiliated Sixth People’s Hospital as previously described [
12] after written informed consent was obtained. TRIM6 expression was determined by qRT-PCR. The cells seeded on 96-well plates were exposed to 400 μM 5-FU, 64 μML-OHP, 2 μM Thiostrepton (TST, Sigma-Aldrich, St. Louis, MO, USA) or vehicle (DMSO) for 48 h. CCK-8 assay was performed as mentioned above and the percentage of proliferation inhibition was calculated with the following formula: Inhibition rate (%) = (OD
vehicle– OD
treatment)/OD
vehicle.
Immunoprecipitation (IP) and liquid chromatography/mass spectrometry (LC/MS) analysis
pCMV-Tag2-TIRM6 or pCMV-Tag2 vector was transfected into 293 T cells, and 48 h later, 293 T cells were harvested and extracted in RIPA buffer. The overexpression of FLAG-TRIM6 was confirmed by western blotting. Following pre-cleared with IgG and protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 2 h, extracts were incubated with anti-FLAG beads (Sigma-Aldrich) overnight at 4 °C. The immunoprecipitated protein complexes were eluted with FLAG peptide (Sigma-Aldrich), resolved on SDS-PAGE, and stained with Coomassie Brilliant Blue. Several differential bands were excise, digested with trypsin, and analyzed by LC/MS.
Co-IP experiments
Cell lysates prepared from HCT-8 and HCT116 cells with RIPA buffer were incubated with anti-TRIM6 (Bioss Inc.), anti-TIS21 (Santa Cruz Biotechnology) or control IgG (Santa Cruz Biotechnology) for 2 h at 4 °C and then with protein A/G Plus agarose (Santa Cruz Biotech.) for 2 h at 4 °C. The immunoprecipitated proteins were analyzed by western blotting analysis.
GST-pull down assay
GST fusion proteins of TRIM6 and TIS21, and GST protein were produced in Escherichia coli and conjugated to glutathione 4B beads (GE Healthcare, Pittsburgh, PA, USA). HCT-8 cell lysate was incubated with GST fusion proteins or GST protein for 2 h at 4 °C. The beads were washed three times with RIPA buffer, boiled with SDS sample buffer, and analyzed by western blotting.
Half-life of TIS21
HCT-8 cells were transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc (Vector) for 24 h, and exposed to 20 mM cycloheximide (CHX, Sigma-Aldrich). Cell lysate was prepared at 0, 3 and 6 h after exposure and subjected to western blotting analysis.
Ubiquitination analysis
Cell lysates prepared from HCT-8 cells transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc-TRIM6 (C15A) were reacted with anti-TIS21 or control IgG. The immunoprecipitated complexes were subjected to western blotting analysis using anti-ubiquitin (Abcam).
The 293 T cells were transfected with plasmids expressing myc-TRIM6, His-ubiquitin and FLAG-TIS21 (WT, K5R, K51R or K150R). Two days later, cells were harvested and sonicated in buffer A (20 mM imidazole, 5 M guanidine-HCl, 100 mM Na2HPO4/NaH2PO4, pH 8.0). Cell lysates were incubated with nickelnitrilotriacetic acid beads (Qiagen) at room temperature for 1 h. The beads were washed three times with buffer A, twice with buffer B (20 mM imidazole, 1 M guanidine-HCl, 100 mM Na2HPO4/NaH2PO4, pH 8.0), and then twice with buffer C (20 mM imidazole, 25 mM Tris, pH 6.5). The immunoprecipitated proteins were analyzed by western blotting analysis with anti-FLAG (Abcam).
Immunofluorescence
HCT-8 or HCT116 cells cultured on the coverslips were washed twice in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 30 min, and then blocked with 5% BSA at RT for 1 h. The cells were incubated with rabbit anti-TRIM6 (Bioss Inc.) and mouse anti-TIS21 (Novus Biologicals, Inc.; Littleton, CO, USA) overnight at 4 °C. Cells were washed three times with PBS, and then incubated with the Alexa Fluor 555-labeled goat anti-rabbit IgG(H+L) (Beyotime Biotech.) and Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) (Beyotime Biotech.) at room temperature for 1 h. After washing thrice with PBS, 4′-6-diamidino-2-phenylindole (DAPI, Beyotime Biotech.) was used to stain nuclei.
In vivo tumorigenicity assay
All procedures were approved by Animal Care and Use Committee, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Male nude mice (4–6 weeks old) were housed under specific pathogen-free conditions. Cell suspensions of HCT-8 expressing shNC or shTRIM6 cells (5 × 106) were injected subcutaneously into the nude mice (6 mice for each group, randomly assigned). On the 33th day after inoculation, the tumors were resected, photographed and weighed.
A xenograft model was established to evaluate the outcome of TST treatment. Nude mice (34 mice for each cell line, randomly assigned) were subcutaneously injected with HCT116 or SW620 cells (5 × 106 cells per mouse). On the 12th day after inoculation, the mice were randomly divided into two groups (n = 17 per group), and administrated with TST (500 mg/ kg /day) or vehicle by intraperitoneal injection every three days. On the 33th day after transplantation, 5 mice of each group were sacrificed and xenografts were weighed. Overall survival analysis was performed on the remaining mice (n = 12 per group).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 software (San Diego, CA, USA). Statistically significant differences were determined by Student’s t test (two groups), and one-way ANOVA test (more than two groups). P < 0.05 was regarded as statistically significant.
Discussion
TRIM proteins are well known to involve in immune responses and carcinogenesis [
23]. The current study demonstrated the upregulated expression of TRIM6 in CRC tissues and suggested that TRIM6 expression may be an independent prognostic marker for CRC (Fig.
1). In vitro (Fig.
2 and Fig.
6) and in vivo functional experiments (Fig.
7) demonstrated that TRIM6 promoted cell cycle progression and proliferation of CRC cells. Taken together, the current study implies that TRIM6 plays oncogenic role in CRC development.
Next, we tried to explore the mechanisms by which TRIM6 influences CRC progression. We identified TIS21 as a candidate associated protein of TRIM6. Our data suggest that TRIM6 overexpression decreased TIS21 stability and increased TIS21 ubiquitination, which ascribed to the E3-ubiquitin ligase activity of TRIM6 (Fig.
5). TIS21 functions as an anti-proliferative transcriptional cofactor in fibroblast cells (NIH3T3), mouse embryo fibroblasts, breast cancer cells, prostate cancer cells and hepatocellular carcinoma (HCC) cells [
17,
24‐
26]. In the current study, TIS21 overexpression reversed the proliferative effects of TRIM6 overexpression in CRC cells, indicating that TIS21 was a downstream regulator of TRIM6 (Fig.
6). Western blotting analysis and Pearson correlation analysis revealed a negative correlation between the protein expression of TRIM6 and TIS21 protein in clinical samples, which further validated the in vitro findings (Fig.
8).
Aberrant cell cycle activity is a main characteristic of cancer cells [
27]. TRIM6 knockdown in CRC cells caused cell arrested at G2/M phase. In HCC cell line, forced expression of TIS21 significantly induced the G2/M arrest by inhibiting the activity of FoxM1 [
17]. FoxM1, a forkhead box family transcription factor, involves in the regulation of DNA replication, mitosis and cell proliferation by binding to promoters of target genes [
17]. In the current study, we demonstrated that TRIM6 overexpression elevated the expression and phosphorylation of FoxM1, which was blocked by TIS21 overexpression. FoxM1 overexpression reversed the effects of TRIM6 knockdown on CRC cell proliferation and cell cycle progression (Fig.
6). Cyclin B1 and c-Myc, which are required for mitotic initiation, are known as target genes of FoxM1 [
28,
29]. Here, TRIM6 knockdown decreased the expression of both proteins, which was partial reversed by FoxM1 overexpression (Fig.
6j). On the contrary, TRIM6 overexpression displayed the reverse effect and such effect was blocked by TIS21 overexpression (Fig.
6f). FoxM1 expression is found to be upregulated in a number of human cancers, including CRC [
30]. In general, FoxM1 overexpression is closely related to high proliferation rate and late tumor stage, and may serve as a prognostic marker for numerous human cancers [
30‐
34]. Targeting FoxM1 may be a promising strategy for cancer [
35]. Consistent with the previous report [
30], we found that FoxM1 protein expression was increased in CRC tissues. More importantly, TRIM6 protein expression was positively correlated with the expression and phosphorylation of FoxM1. Taken together, TRIM6 may exert oncogenic role by regulating TIS21/FoxM1 in CRC cells. Park TJ et al. reported that TIS21 had little effect on FoxM1 expression but suppressed FoxM1 activation by binding to the CDK1/Cyclin B1 complex in HCC cells [
17]. Overexpression of FoxM1 in HCC cells led to a decreased protein level of TIS21 by promoting Skp2 (S phase kinase associated protein 2)-mediated TIS21 ubiquitination [
36]. Their studies revealed a regulatory loop between TIS21 and FoxM1 in HCC cells. Our current study also observed the decreased protein expression of TIS21 following the overexpression of FoxM1 in CRC cells (Fig.
6j). However, our data showed that TRM6/TIS21 affected both expression and activity of FoxM1. These different results may due to the different types of cell lines. Further experiments are required to investigate the detailed mechanisms how TIS21 downregulates FoxM1 protein expression in CRC cell lines.
Chemotherapy is one of the standard treatment options for cancer patients, but chemoresistance limits its effectiveness. The mechanism of chemoresistance is far from fully understood. Other members of TRIM family proteins, such as TRIM14, TRIM24 and TRIM98 have been reported to enhance chemoresistance in certain human cancers [
3‐
5]. Here, we found that knockdown of TRIM6 significantly enhanced the anti-proliferative effect of 5-FU and L-OHP (Fig.
3), and primary CRC cells with higher level of TRIM6 was more resistant to 5-FU and L-OHP (Fig.
9c). Targeting FoxM1 may have promising therapeutic benefits for cancer treatment [
35]. Thiostrepton (TST), a specific inhibitor for FoxM1, shows anti-cancer activity in many human cancers [
19‐
22]. In the current study, primary CRC cells with higher level of TRIM6 was more sensitive to TST (Fig.
9c). TST had better efficacy in treating xenografts from HCT116 cells, which displayed higher level of TRIM6, than those from SW620 cells, which showed lower level of TRIM6 (Fig.
9d-g). Collectively, these data indicate that TRIM6 expression levels influence the anti-cancer efficacy of different drugs, which may be taken into account before these drugs were applied to CRC patients.
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