Background
Tribbles homolog 2, a member of the tribbles family (TRIB1, TRIB2, TRIB3), is first identified in
Drosophila as mitosis blocker that regulates embryo and germ cell development [
1]. It comprises an N-terminal domain, a C-terminal domain, and a central pseudokinase domain that contains a Ser/Thr protein kinase-like domain but lacks ATP affinity and catalytic activity [
2]. In the absence of kinase activity, TRIB2 functions as a scaffold protein to regulate different signaling pathway in fundamental biological processes as well as in pathological conditions, including cancer [
3]. TRIB2 plays a crucial role in regulating various cellular processes in cancer, such as proliferation, apoptosis and drug resistance [
4‐
6]. Currently, the role of TRIB2 in cancer remains controversial. TRIB2 is overexpressed in human acute myeloid leukemia (AML) and accelerates AML progression via the inactivity of C/EBPα [
7]. In liver cancer, TRIB2 functions as an adaptor protein and promotes YAP protein stabilization through the E3 ubiquitin ligase βTrCP, contributing to cancer cell proliferation and transformation [
8]. In contrast, Mara et al. reported that TRIB2 might counteract the chemotherapy resistance and propagation in myeloid leukemia via activation of p38; in liver cancer, TRIB2 inhibits Wnt-signaling by regulating the degradation of key factors, such as βTrCP, COP1 and Smurf1 [
6,
9]. Interestingly, recent literature has reported that high-TRIB2 expression correlated with a worse clinical outcome of colorectal cancer (CRC) [
10]. However, the biological role of TRIB2 and its underlying mechanism in CRC are not fully understood.
Cellular senescence is a state of growth arrest and characterized as some phenotypic alterations, such as remodeled chromatin, reprogrammed metabolism, morphology changes and up-regulated senescence-associated β-galactosidase (SA-β-gal) activity [
11,
12]. Various intrinsic and extrinsic insults could trigger cellular senescence, including oxidative stress, mitochondrial dysfunction, DNA damage and therapeutic drugs or radiation [
13]. Substantial evidence has shown that disruption of senescence accelerates and induction of senescence inhibits cancer development [
14]. Therefore, senescence might be a promising target for tumor therapy.
The cyclin-dependent kinase inhibitor p21 (CDKN1A or p21
WAF1/Cip1), a member of the Cip/Kip family, is a critical regulator of cell cycle exit and cellular senescence through blocking the activities of cyclin-dependent kinases (CDK), including CDK1 and CDK2 [
15‐
17]. Microarray-based studies indicate that p21 is positively correlated with genes involved in cellular senescence [
18]. Currently, induction of p21 expression by a variety of stimuli is thought to be the driver of senescence initiation [
19]. The tumor suppressor protein p53 is the major transcription regulator for p21 and multiple proteins involved in regulating cellular senescence work through p53/p21 pathway. Besides, many other transcription factors like Smad3, BRCA1, CHK2 and transcription factor activating enhancer-binding protein 4 (AP4), have been reported to control p21 expression [
20,
21]. As a member of the basic helix-loop-helix transcription factors superfamily, AP4 activates or represses a series of genes by recognizing and binding to the E-box sequence CAGCTG in the promoter [
22]. It has been reported that AP4 occupies the four CAGCTG motifs in the promoter of p21 and subsequently repressing its transcription activity to contribute to cancer cell proliferation and cell cycle arrest [
21,
23].
In the present study, we found that TRIB2 was overexpressed in colorectal cancer and inversely correlated with survival rate of CRC patients. Down-regulation of TRIB2 inhibited cancer cells proliferation, induced cell cycle arrest and promoted senescence in CRC cells. Moreover, TRIB2 physically interacted with AP4 and the TRIB2-AP4 interaction enhanced AP4-mediated transcriptional activity. Using rescue experiments, we demonstrated TRIB2 negatively regulated cellular senescence through cooperating with AP4 to repress p21 expression. Thus, our study identifies a novel mechanism mediated by TRIB2/AP4/P21 axis in regulating cellular senescence, and suggests that TRIB2 might be a new target in clinical practice for CRC treatment.
Materials and methods
Colorectal cancer samples
Primary tumor samples and the corresponding adjacent normal tissues were obtained from CRC patients who received surgical resection at Tongji Hospital (Wuhan, China), between January 2017 and January 2018, after their written informed consent. None of the patients received chemotherapy or radiotherapy before surgery. This study was approved by the Huazhong University of Science and Technology Ethics Committee.
Cell lines, antibodies and reagents
The cell lines HEK 293 T, SW48 and LoVo were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS) at 5% CO2 and 37 °C. The antibodies against p53 (sc-47698) and p21 (sc-397) were purchased from Santa Cruz Company (Santa Cruz, CA, USA), the antibody against GAPDH (A00227) were purchased from Boster Company (Boster, Wuhan, China), the antibody to TRIB2 (A11661) were purchased from Abclonal Company (Abclonal, Wuhan, China) and the antibody to AP4 (ab28512) were purchased from Abcam Company (Abcam, MA, USA). Doxorubicin (Dox), used to establish the cellular senescent model, was obtained from Calbiochem (La Jolla, CA, USA).
Cell viability assay
Cell viability was determined by CCK8 assays. Briefly, colon cancer cells were seeded in 96-well plates (5 × 103 cells/well) and treated with corresponding processes. CCK8 was added into the wells for 3 h at indicated times. The absorbance in each well at wavelength of 450 nm (A450) was measured with a Thermomax microplate reader.
Cell cycle analysis
Cells were trypsinized, washed with cold phosphate-buffered saline (PBS) and fixed in 80% ethanol overnight at − 20 °C. Cells were then washed twice with PBS, and stained with PI at room temperature for 1 h. Cell cycle distribution was measured by the Becton-Dickinson FACScan System (Franklin Lakes, NJ, USA).
Apoptosis assay
Cells were trypsinized, washed with cold phosphate-buffered saline (PBS), stained with Annexin V-FITC and propidiumiodide (PI) using an Annexin V-FITC/PI-staining kit (BD Pharmingen, San Diego, CA, USA) and placed at room temperature for 30 min. The apoptosis of cells was measured by flow cytometry.
Senescence-associated β-galactosidase staining
Tumor cells were transfected with or without siRNA or plasmid, treated with doxorubicin and cultured for 48 h. Cells were washed with PBS for 3 times and stained with freshly prepared SA-β-Gal staining solution following the protocol provided by the manufacturer (Beyotime Biotechnology Ltd., Shanghai, China). The stained cells were detected with a microscope and the percentage of senescence cells was quantified by calculating the percentage of SA-β-Gal-positive cells in randomly selected fields (n = 3).
Western blot analysis and Immunoprecipitation (IP)
Cancer cells were collected, washed twice with cold PBS and lysed in NP-40 lysis buffer for 30 min at 4 °C. Protein concentration was measured using bicinchoninic acid assay kit (Thermo). Protein extracts were separated by electrophoresis in an 8~12% premade sodium dodecyl sulfate-polyacrylamide minigel (Tris-HCL SDS-PAGE) and transferred to a PVDF membrane. The membrane was incubated with indicated antibodies and detected by using a chemiluminescence method. For immunoprecipitation, total cell lysates were incubated with appropriate antibodies overnight and subsequently rotated with protein A/G beads for 2~4 h at 4 °C. Beads were washed three times using NP-40 lysis buffer, mixed with 2 × SDS sample buffer and boiled for 5~10 min. The co-precipitates were analyzed by western blot analysis.
Immunohistochemistry (IHC)
The procedures followed standard manufacturer’s protocols as described previously. Two pathologists reviewed and scored IHC staining for each sample independently. IRS system was used to quantify IHC staining. The percentage of positively stained tumor cells was scored as follows: 1 (< 10%), 2 (10–50%), 3 (50–75%) and 4 (> 75%). Staining intensity was scored 0–3: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow brown); 3, strong staining (brown). The staining index ranged from 0 to 12, which was calculated by multiplying the score of the percentage of positive tumor cells and the staining intensity.
GST pull-down assay
GST-TRIB2 fusion proteins and His-AP4 proteins were expressed in BL21 and purified using Glutathione Sepharose 4B beads (Amersham Pharmacia, Piscataway, NJ, USA) or Ni beads (GE Healthcare, CA, USA), respectively. Purified His-AP4 protein was incubated with GST or GST-TRIB2 fusion proteins bound to Glutathione Sepharose beads at 4 °C overnight. Beads-associated proteins were detected by western blot. Expression of GST fusion proteins was confirmed by Coomassie Blue staining.
Luciferase activity assay
Cancer cells were seeded in 12-well plates (2 × 105 cells/well) and co-transfected with luciferase reporter constructs contained p21 promoter (p21-Luc), TK-Renilla expression plasmids, along with indicated expression plasmids or siRNA by lipofectamine 2000 reagent. After 48 h of transfection, luciferase activity assay was performed using the dual luciferase assay kit according to the manufacturer’s instruction (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. All the experiments were carried out three times.
ChIP assay
ChIP assay was carried out using the ChIP assay kit according to the protocol. Briefly, 1 × 107 cancer cells were harvested and treated with 1% formaldehyde for 10 min at 37 °C to cross-link. To stop the reaction, glycine was added to the cell suspension at a final concentration of 0.125 M. Chromatin was sheared on ice by sonication to generate DNA fragments with a bulk size of 200~1000 bp. After centrifugation, the cell lysates were incubated with indicated antibody overnight and subsequently with protein G-agarose beads for 2~4 h at 4 °C with agitation. Beads were washed and eluted, and the cross links were reversed by incubation at 65 °C for 4 h. Purified DNA was used to analyze the binding of Flag-AP4 or His-TRIB2 to p21 promoter locus by PCR reactions. The sequences of oligonucleotides used as qChIP primers: p21 promoter forward 5’-TGTGTCCTCCTGGAGAGTGC-3′ and p21 promoter reverse 5’-CAGTCCCTCGCCTGCGTTG-3′.
RNA interference
Short interfering RNA (siRNA) oligonucleotide duplexes targeting TRIB2, AP4 and p53 used in this study were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China). The sequences of TRIB2 (#1 and #2), AP4 and p53 are as follows.
siTRIB2 #1: 5′- CGTGGACTCTAGTATGTAAAT -3′,
siTRIB2 #2: 5′- GCGTTTCTTGTATCGGGAAAT -3’.
siAP4#1: 5′- GTGATAGGAGGGCTCTGTAG -3’.
siAP4#2: 5’-GCAGAGCATCAACGCGGGATT -3′.
sip53: 5′- GACTCCAGTGGTAATCTAC -3′.
A nonsense siRNA with no homology to the known genes in human cells was used as negative control. siRNA transfections of cancer cells were performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the knockdown efficiency was verified 48 h after transfection. All the siRNAs were used at a final concentration of 100 nM.
Quantitative real-time PCR
Total RNA was extracted using the TRIzol (Invitrogen, Carlsbad, CA, USA) method in accordance with the manufacture’s protocol. Reverse transcription of total RNA was performed to generate cDNA. Real-time PCR was carried out using the Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green Real-time PCR Master Mix (TOYOBO, Shanghai, China). The primer sequences for real-time PCR analysis are listed as follows.
TRIB2 | ATGAACATACACAGGTCTACCCC | GGGCTGAAACTCTGGCTGG |
p21 | CGATGGAACTTCGACTTTGTCA | GCACAAGGGTACAAGACAGTG |
GAPDH | GGAGCGAGATCCCTCCAAAAT | GGCTGTTGTCATACTTCTCATGG |
p53 | CAGCACATGACGGAGGTTGT | TCATCCAAATACTCCACACGC |
AP4 | GAGGGCTCTGTAGCCTTGC | GAATCCCGCGTTGATGCTCT |
Animal study
Cancer cells were collected, washed with PBS, resuspended in culture medium and mixed with Matrigel (BD Biosciences) at the ratio of 1:1. The nude mice were randomly divided into two groups and subcutaneously injected with the prepared cells above (1 × 105 cells/mouse). After a week of injection, tumor size was measured with calipers every 4 days. At the end of the experiment, all mice were sacrificed and the tumors were isolated and weighed.
Statistical analysis
All statistical analyses were performed using SPSS 24.0 (SPSS Inc.). All data were quantified as mean ± SD. Two-tailed Student’s t-test was used to evaluate the differences between two groups. Survival curves were plotted using Kaplan-Meier methodology with log-rank test on univariate analysis. On multivariate analysis, Cox proportional hazards model was used for analyzing prognostic factors. The cut-offs for the expression levels were identified by reference to the Human Protein Atlas (
www.proteinatlas.org). Values of
p < 0.05 were considered as statistically significant in all cases.
Discussion
Tribbles, members of the pseudokinase family, were demonstrated to regulate many cellular functions, including glucose and lipid metabolism, apoptosis, adipocyte differentiation and inflammation. Despite possessing no kinase activity, tribbles act as molecule adaptors to regulated protein-protein interaction [
2]. Recently, growing evidence suggests that tribbles act as pro-oncogenes and play critical roles in some kinds of malignant tumors [
7,
30,
31]. For instance, elevated TRIB1 expression is able to induce AML via activation of ERK and degradation of C/EBPα; TRIB3 regulates MAPK- and TGF-β-mediated Notch activation to promote cancer progression in breast cancer cells and promotes APL progression via interacting with and inhibiting degradation of oncoprotein PML-RARα [
32,
33]; TRIB2 is also reported to contribute to acute myeloid leukemia (AML) and hematopoietic development [
34]. In the present study, we demonstrated that TRIB2 expression in human CRC tissues was much higher than that in adjacent tissues and negatively correlated with prognosis of CRC patients, which is consistent with the results reported by Hill, R. et al. [
10]. Moreover, we identified TRIB2 as an independent prognostic factor affecting the survival of CRC patients through multivariate analysis, further illustrating its important clinical role in CRC patients. These results indicated that TRIB2 might be an available prognostic marker for CRC patients.
Studies on the mechanism of regulating the expression of TRIB2 have been widely conducted, and transcriptional regulation is likely to be pivotal in TRIB2 expression. The aberrant expression of some oncogenes has been reported to account for elevated TRIB2. Wang et al. showed that the TRIB2 is a direct target of Wnt/TCF pathway in liver cancer [
8]. In AML cells, dysregulated C/EBPalpha and E2F1 contributes to up-regulation of TRIB2 [
35]. In human T cell acute lymphoblastic leukemia (T-ALL), NOTCH, PITX and TAL1 are also found to up-regulate TRIB2 [
36]. However, it is unclear why TRIB2 is overexpressed in CRC patients. It would be interesting to further investigate the mechanism of TRIB2 up-regulation in CRC.
To further explore the oncogenic role of TRIB2 in tumorigenesis, we performed a series of experiments in CRC cell lines and found that ectopic expression of TRIB2 dramatically blocked cellular senescence. Cellular senescence is identified as a state of irreversible cell-cycle arrest induced by a series of insults, and induction of senescence is considered as a potent strategy for suppressing cancer progression with minor side effects [
37]. As the critical marker for cellular senescence, p21 has been proved to drive senescence initiation. Various stimuli including ROS, mitochondrial dysfunction, irradiation and chemotherapeutic drugs induce cellular senescence through DNA damage response by activating ATM and eventually activating p53/p21 pathway [
38]. Although p21 is mainly regulated by p53 at the transcription level, multiple transcription factors (BRCA1, Smad3, AP4 and c-myc) have also been reported to control the transcriptional activation or repression of p21 [
39‐
41].In our study, TRIB2 could negatively regulate p21 promoter activities and expression, and inhibit cellular senescence in CRC cells. Earlier reports of Richard et al. have showed that in melanoma cells TRIB2 inhibits p53 and p21 expression in an AKT-dependent manner [
4]. However, in the current study we did not detect significant changes of p53 expression in SW48 cells when TRIB2 was silenced. Experiments in CRC cells, which were transfected with p53- and or not TRIB2-specific siRNA, suggested that TRIB2 knockdown could still transcriptionally increase p21 expression even in the absence of p53. These results revealed that TRIB2 regulated p21 expression in a p53-independent manner, which is opposite with previous study. We speculated that TRIB2 may have distinct functions depending on etiologic background of different cancer types.
To extensively study the mechanisms responsible for TRIB2-mediated regulation of cellular senescence, we analyzed the BioGRID database and found TRIB2 directly interacted with transcription factor AP4. AP4, belonging to the basic helix-loop-helix transcription factors (bHLH-LZ) superfamily, is identified as an oncogene and accelerates development and progression in a variety of human cancers [
23,
42‐
45]. Moreover, AP4 is frequently up-regulated in these malignancies and positively correlates with poor prognosis. The functions of AP4 in modulating tumorigenic properties have been extensively studied. It plays a critical role in regulating stem-like phenotypes and epithelial-mesenchymal transition (EMT) through directly targeting genes such as CD44, LGR5, VIM and E-cadherin [
45]. Mechanically, AP4 recognizes and binds to consensus E-box motif CAGCTG in promoters of downstream target genes [
22]. There is evidence that AP4 directly binds to p21 promoter and transcriptionally represses p21 expression [
21]. Our results suggest that TRIB2 regulates p21 expression by enhancing the functions of AP4, through protein-protein interaction. Similar to our results, Nobumichi et al. showed that TRIB3, another member of the tribbles family, interacts with transcription factor ATF4 and then regulates transcriptional activity of ATF4 [
29]. In addition, TRIB3 alters PPARγ transcriptional activity through directly binding to PPARγ during adipocyte differentiation [
28]. The above evidence indicates that it is a common phenomenon for the tribble family members to influence the activity of transcription factors through protein-protein interaction. However, the exact mechanism of how TRIB2 regulate the transcription activities of AP4 remains to be clarified. It is likely that TRIB2 acts as scaffold to recruit some corepressor (s) or trigger histone modification. Future studies are needed to determine the specific mechanism.