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Abstract

Tripartite motif-containing protein 24 (TRIM24) has currently emerged as a crucial cancer-related gene present in a wide range of human cancer types. However, the involvement of TRIM24 in acute myeloid leukemia (AML) has not been well investigated. The present study aims to investigate the significance, cellular function, and potential regulatory mechanism of TRIM24 in AML. We found that TRIM24 expression was significantly upregulated in AML compared with normal tissues. AML patients with low expression of TRIM24 had higher survival rates than those expressing TRIM24 at higher levels. High expression of TRIM24 was also detected in AML cells and its knockdown markedly restricted proliferation and promoted apoptosis in AML cells. Further investigation revealed that TRIM24 contributed to the regulation of Wnt/β-catenin signaling, which was associated with modulating the phosphorylation status of glycogen synthase kinase-3β (GSK-3β). Inactivation of GSK-3β partially reversed the TRIM24 knockdown-mediated antitumor effects observed in AML cells. Furthermore, knockdown of TRIM24 retarded the growth of AML-derived xenograft tumors in nude mice in vivo. Overall, these findings demonstrate that knockdown of TRIM24 impedes the AML tumor growth through the modulation of Wnt/GSK-3β/β-catenin signaling. These findings highlight the potential TRIM24 as an attractive anticancer target to treat AML.

Keywords

Acute myeloid leukemia β-catenin TRIM24 Wnt

Introduction

Acute myeloid leukemia (AML) is a major subtype of leukemia that occurs in adults with high incidence and mortality rates.¹ AML is heterogeneous disease characterized by the uncontrolled proliferation and inhibited apoptosis of myeloid progenitor cells within bone marrow (BM), which leads to hematopoietic dysfunction.² Currently, there is a shortage of effective anticancer drugs for the treatment of AML and the survival rate and complete remission rate of AML patients remain unsatisfactory.^3,4 Although our understanding of AML biology is currently improving, we are still far from a complete understanding of the detailed mechanism that underlies the pathology of AML. Therefore, investigation of the molecular mechanisms underlying AML progression is important and may help to identify novel targets that can be used for the development of effective anticancer therapy for the treatment of AML.

Tripartite motif-containing protein 24 (TRIM24), also named transcriptional intermediary factor 1α, is a multifunctional multidomain protein that plays a key role in various biological processes.⁵ TRIM24 possesses a nuclear receptor-interacting LXXLL motif, through which TRIM24 interacts with nuclear receptors, acting as a co-regulator of transcription.^6,7 TRIM24 is a RING-type E3 ubiquitin ligase domain-containing protein that can polyubiquitinate p53 and promote its degradation.⁸ Interestingly, studies have suggested that TRIM24 is a crucial cancer-related protein that is dysregulated in a wide range of human cancer types.⁵ High levels of expression of TRIM24 have frequently been detected in tumor tissues and have been associated with clinical stage, lymphatic metastasis, and low survival rate.^9,10 Elevated levels of TRIM24 expression promote the tumor growth and metastasis as well as chemoresistance by affecting various signaling pathways.^11
–13 The oncogenic function of TRIM24 has been demonstrated in a variety of other cancer types,^14

–17 making it as an attractive target for further study.

Wnt/β-catenin signaling is a highly conserved signaling pathway that plays a potent oncogenic role in various cancers.¹⁸ The activation of Wnt/β-catenin signaling is controlled by a signaling cascade. In general, Wnt ligands engage with co-receptors on the cell membrane in order to promote the phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the destruction complex, leading to dephosphorylation, stability, and accumulation of β-catenin. β-catenin molecules then translocate into the nucleus, where they bind T-cell factor (TCF)/lymphoid enhancer factor (LEF) co-activators to initiate the expression of target genes.¹⁸ Indeed, increased β-catenin expression is frequently observed in primary AML tissues and correlates with poor survival rates in AML patients.^19,20 Therefore, Wnt/β-catenin signaling has emerged as a potential target for the development of anti-AML treatment.^21,22

To date, the role of TRIM24 in AML remains largely unknown. The present study has been designed to explore the expression patterns and biological function of the molecule, as well as to elucidate the potential regulatory mechanism of TRIM24 in AML. Our results have demonstrated that TRIM24 expression is significantly upregulated in AML and high levels of TRIM24 expression predict shorter survival rates in AML patients. TRIM24 knockdown markedly restricted proliferation and promoted apoptosis in AML cells. Further investigation has revealed that TRIM24 contributes to the regulation of Wnt/β-catenin signaling associated with modulating the phosphorylation of GSK-3β (pGSK-3β). Further, inactivation of GSK-3β partially reversed the TRIM24 knockdown-mediated antitumor effects observed in AML cells. In addition, knockdown of TRIM24 retarded the growth of AML-derived xenograft tumors in nude mice in vivo. Overall, these findings demonstrate that downregulation of TRIM24 impedes AML tumor growth by modulating Wnt/GSK-3β/β-catenin signaling, suggesting that TRIM24 may be a potential prognostic biomarker of and therapeutic target for AML.

Materials and methods

Tissue collection and cell culture

BM tissues (n = 4) were collected from patients had been newly diagnosed with AML in The First Affiliated Hospital of Xi’an Medical University. BM tissues (n = 4) isolated from healthy donors were used as normal controls. Written informed consents were acquired from all the participants of the study with respect to tissue donation used for research purposes. This study was reviewed and approved by the Institutional Research Ethics Committee of The First Affiliated Hospital of Xi’an Medical University and experiments were conducted in accordance with the regulations and guidelines of the Declaration of Helsinki. Primary CD34⁺ blasts were selected from BM tissues using a magnetic-activated cell sorting method using a CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocols. Three human AML cell lines (AML-193, Kasumi-1, and HL-60) were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and were cultured following the recommended methods of the manufacturer. Briefly, AML-193, Kasumi-1, and HL-60 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were grown under 5% CO₂ in a humidified incubator at 37°C.

Lentivirus production and infection

Recombinant lentivirus carrying TRIM24 shRNA (LV-TRIM24 shRNA) and TRIM24 open reading frame sequences (LV-TRIM24) were purchased from GenePharma (Shanghai, China). Recombinant lentivirus were transfected into AML cells at a multiplicity of infection of 100 by using polybrene (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The infected cells were incubated for 72 h before being subjected to subsequent experiments.

RNA extraction and real-time quantitative polymerase chain reaction analysis

Tissues or cells were homogenized in TRIzol Reagent (Invitrogen; Thermo Fisher Scientific) and total RNA was extracted as per the manufacturer’s instructions. Experimental procedures were conducted in accordance with the guidelines of minimum information for publication of quantitative real-time PCR experiments (MIQE). The value of A260/A280 of RNA was 1.9. Complementary DNA (cDNA) was generated using SuperScript II Reverse Transcriptase (Invitrogen) and was subjected to polymerase chain reaction (PCR) amplification using Power SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The cDNA was amplified with appropriate primers using the standard thermal cycle program, which included the following steps: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The expression of target genes was normalized to the internal control gene, GAPDH, using the 2^−ΔΔCt method.

Protein isolation and Western blot analysis

Tissues or cells were homogenized in protein extraction lysis buffer containing a protease inhibitor cocktail (Beyotime, Shanghai, China). After centrifugation, the supernatant was collected, and protein concentrations were determined. Total proteins were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis before transferring to a polyvinylidene fluoride membrane (Millipore, Bedford, Massachusetts, USA). After being immersed in blocking buffer for 1 h at room temperature, the membrane was probed overnight at 4°C with primary antibodies for TRIM24 (Abcam, Cambridge, Massachusetts, USA), phospho-GSK-3β (Ser9) (Cell Signaling Technology, Danvers, Massachusetts, USA), active β-catenin (Cell Signaling Technology), and GAPDH (Abcam). Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; Abcam) and proteins were visualized using ECL Chemiluminescent Western Blotting Reagent (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania, USA). Band intensity was quantified by using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, Maryland, USA).

Cell counting kit-8 assay

Cell counting kit-8 (CCK-8) assay was carried out using an Enhanced CCK-8 Assay Kit (Beyotime) following the recommended protocols of the manufacturer. In brief, AML cells were counted and seeded using 5000 cells per well into 96-well tissue culture plates. After indicated treatments, cells were treated with 10 µl CCK-8 reagent per well and were continuedly cultured for 2 h at 37°C. For the measurement of cell proliferation, the optical density value of each well was assessed using an ELISA reader (Molecular Devices, Sunnyvale, California, USA). The wavelength measured was 450 nm.

Colony formation assay

AML cells infected with LV-TRIM24 shRNA or LV-TRIM24 for 72 h were resuspended, counted, and seeded using 1000 cells per well into 6-well tissue culture plates. The medium used was refreshed every 2 days and cells were allowed to grow for 2–3 weeks to form colonies. Colonies were fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet for visualization. The stained colonies were photographed and counted using an inverted microscope.

Flow cytometry analysis

The apoptosis of AML cells was assessed by flow cytometry analysis using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Beyotime). AML cells were collected, washed, and re-suspended in binding buffer. Thereafter, cells were incubated with Annexin V-FITC and PI under dark conditions for 15 min at room temperature. For measuring apoptotic cells, cell samples were analyzed using a FACSCalibur Flow Cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). Cells stained by Annexin V-FITC⁺/PI⁻ and Annexin V-FITC⁺/PI⁺ were considered as apoptotic cells.

Luciferase reporter assay

For evaluation of Wnt/β-catenin signaling, AML cells were co-transfected with a TCF reporter vector and pRL-TK Ranilla luciferase control reporter and incubated for 72 h. A Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA) was utilized to detect luciferase activities of cell lysates.

Animal experiments

BALB/c nude mice were provided by the Medical Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, China) and were raised under pathogen-free environment. The animal experiments were approved by the Institutional Research Ethics Committee of The First Affiliated Hospital of Xi’an Medical University. Mice were divided into two groups: LV-scrambled shRNA group (n = 3) and LV-TRIM24 shRNA group (n = 3). For the xenograft tumor assay, LV-scrambled shRNA- or LV-TRIM24 shRNA-transfected AML cells (approximately 1 × 10⁷) were subcutaneously injected into the right flank of nude mice. Tumors were measured by using a slide caliper every 6 days and tumor volume was calculated according to the following formula: width² × length/2.

Statistical analysis

Quantitative data obtained from experiments were calculated and expressed as mean ± standard deviation. The differences between groups were determined by Student’s t-test or one-way analysis of variance followed by the Bonferroni’s post hoc test. Data were analyzed using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, California, USA). Differences were supposed to be statistically significant when p < 0.05.

Results

High levels of expression of TRIM24 were detected in AML and were associated with poor survival rates

To determine the potential relevance of TRIM24 in AML, we analyzed TRIM24 expression in patients with AML. Using the Oncomine database, an online database consisting of open-access microarray data. We found that TRIM24 expression was significantly elevated in AML tissues compared with normal tissues (Figure 1(a)). Using PrognoScan database analysis, we found that high levels of expression of TRIM24 were able to significantly predict lower overall survival rates for AML patients (Figure 1(b)). To confirm that TRIM24 was highly expressed in AML, we examined the mRNA and protein expression of TRIM24 in BM tissues from AML patients. The results demonstrated that both the levels of mRNA and protein expression of TRIM24 were significantly upregulated in BM tissues of AML patients compared with those of healthy controls (Figure 1(c) to (e)). Overall, these findings suggest that TRIM24 is elevated in patients with AML and can be used as a potential prognostic biomarker.

Figure 1.

TRIM24 is highly expressed in AML and is associated with poor survival. (a) Expression of TRIM24 in normal BM tissues (n = 6) and AML BM tissues (n = 22) was analyzed using an Oncomine database. Fold change of 2.047 was statistically significant, p = 9.43E⁻⁷. (b) A Kaplan–Meier curve was used to compare a population of AML patients highly expressing TRIM24 (n = 19) with a population lowly expressing TRIM24 (n = 15) was obtained from PrognoScan database (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html). p = 0.008144. (c) and (d) Protein expression of TRIM24 in BM tissues from AML patients (n = 4) and normal controls (n = 4) examined using Western blot. (e) Relative mRNA expression of TRIM24 in BM tissues from AML patients (n = 4) versus normal controls (n = 4), which was determined by RT-qPCR. ***p < 0.001 versus normal controls. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; BM: bone marrow; RT-qPCR: real-time quantitative polymerase chain reaction.

Knockdown of TRIM24 shows antitumor effect in AML cells

To investigate the biological function of TRIM24 in AML cells, we detected the regulatory effect of TRIM24 on cell growth using AML cell lines. The expression of TRIM24 was significantly increased in primary blasts isolated from BM tissues of AML patients as well as in multiple AML cell lines compared with normal blasts (Figure 2(a) and (b)). Next, we performed loss-of-function of TRIM24 experiments in order to elucidate the effect of TRIM24 on the growth of AML-193 and Kasumi-1 cells. Transfection of LV-TRIM24 shRNA significantly depleted the expression of TRIM24, as confirmed by Western blot analysis (Figure 2(c)). A CCK-8 cell proliferation assay showed that knockdown of TRIM24 significantly repressed the proliferation of both AML-193 and Kasumi-1 cells (Figure 2(d)). Furthermore, the inhibitory effect of TRIM24 knockdown on cell proliferation was validated using a colony formation assay. AML cells lowly expressing TRIM24 formed fewer colonies than those that expressed TRIM24 more highly (Figure 2(e)). In addition, knockdown of TRIM24 promoted apoptosis in both AML-193 and Kasumi-1 cells (Figure 2(f)). Overall, these results indicate that knockdown of TRIM24 impedes the growth of AML cells.

Figure 2.

Knockdown of TRIM24 produces antitumor effects in AML cells. The (a) mRNA and (b) protein expression of TRIM24 in AML primary blasts and AML cell lines (AML-193, Kasumi-1, and HL-60) were determined by RT-qPCR and Western blot, respectively. n = 3, ***p < 0.001 versus normal control cells. AML-193 and Kasumi-1 cells were infected with LV-TRIM24 shRNA or LV-scrambled shRNA for 72 h, and (c) protein expression of TRIM24 was examined by Western blot. (d) The effect of TRIM24 knockdown on AML cell proliferation was evaluated using the CCK-8 assay. (e) The effect of TRIM24 knockdown on the colony-forming ability of AML cells was assessed using a colony formation assay. (f) The effect of TRIM24 knockdown on apoptosis in AML cells was measured using an Annexin V-FITC/PI assay with flow cytometry analysis. n = 3, **p < 0.01 versus LV-scrambled shRNA. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; RT-qPCR: real-time quantitative polymerase chain reaction; CCK-8: cell counting kit-8.

Ectopic expression of TRIM24 promotes the growth of AML cells

To confirm whether TRIM24 expression produces oncogenic effects in AML, we determined the effect of ectopic TRIM24 expression on AML cell growth. Upregulation of TRIM24 in LV-TRIM24-infected AML cells was confirmed by Western blot (Figure 3(a)). As expected, TRIM24 overexpression significantly promoted proliferation and colony formation in AML cells (Figure 3(b) and (c)). Moreover, TRIM24 overexpression decreased apoptosis in AML cells (Figure 3(d)). These results confirm that TRIM24 exhibits oncogenic functions in AML cells.

Figure 3.

TRIM24 overexpression produces oncogenic effects in AML cells. AML-193 and Kasumi-1 cells were infected with either LV-control or LV-TRIM24 for 72 h, and (a) protein expression of TRIM24 was determined using Western blot. (b) Cell proliferation was assessed using a CCK-8 cell proliferation assay. (c) The colony-forming ability of AML cells was evaluated using a colony formation assay. (d) Cell apoptosis was measured using an Annexin V-FITC/PI apoptosis assay. n = 3, *p < 0.05 and **p < 0.01 versus LV-control. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; CCK-8: cell counting kit-8.

TRIM24 regulates Wnt/β-catenin signaling in AML cells

To uncover the molecular mechanism of TRIM24 in regulating the growth of AML cells, we investigated the regulatory effect of TRIM24 on Wnt/β-catenin signaling. We found that knockdown of TRIM24 decreased pGSK-3β and downregulated the expression of active β-catenin (Figure 4(a) and (b)). Moreover, TRIM24 knockdown suppressed the transcriptional activity of TCF/LEF (Figure 4(c)). In contrast, TRIM24 overexpression had an opposite effect, in which TRIM24 overexpression promoted the activation of Wnt/β-catenin signaling in AML cells (Figure 4(d) to (f)). Overall, these data indicate that TRIM24 contributes to the regulation of Wnt/β-catenin signaling in AML cells.

Figure 4.

TRIM24 modulates the activation of Wnt/β-catenin signaling in AML cells. (a) and (b) The effect of TRIM24 knockdown on protein expression levels of pGSK-3β and active β-catenin was measured by Western blot. (c) The effect of TRIM24 knockdown on TCF/LEF transcriptional activity was assessed by luciferase reporter assay. (d) and (e) The effect of TRIM24 overexpression on protein levels of pGSK-3β and active β-catenin was measured by Western blot. (f) The effect of TRIM24 overexpression on TCF/LEF transcriptional activity was monitored by luciferase reporter assay. n = 3, **p < 0.01 versus LV-control. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; pGSK-3β: phosphorylation of glycogen synthase kinase-3β; TCF/LEF: T-cell factor/lymphoid enhancer factor.

Inactivation of GSK-3β abrogates TRIM24 knockdown-mediated antitumor effect

To confirm whether TRIM24 regulates Wnt/β-catenin signaling through modulation of GSK-3β activity, we next investigated the effect of GSK-3β inactivation on TRIM24 knockdown-mediated antitumor effects. These experiments revealed that treatment with the GSK-3β inhibitor, TWS119, significantly abolished the inhibitory effect of TRIM24 knockdown on the activation of Wnt/β-catenin signaling (Figure 5(a)). Moreover, TRIM24 knockdown-mediated antitumor effects were partially reversed by GSK-3β inactivation (Figure 5(b) to (d)). Overall, these results suggest that TRIM24 knockdown exhibits antitumor effect through the modulation of GSK-3β activity.

Figure 5.

TRIM24 regulates Wnt/β-catenin signaling through the modulation of GSK-3β activity. AML-193 and Kasumi-1 cells were infected with LV-TRIM24 shRNA and incubated for 72 h in the presence of 5 μM of TWS119. (a) Activation of Wnt/β-catenin signaling was monitored by measuring TCF/LEF transcriptional activity. (b) Cell proliferation was detected using a CCK-8 cell proliferation assay. (c) Colony-forming ability of AML cells was assessed using colony formation assay. (d) Cell apoptosis was analyzed by Annexin V-FITC/PI apoptosis assay. n = 3, *p < 0.05. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; GSK-3β: glycogen synthase kinase-3β; TCF/LEF: T-cell factor/lymphoid enhancer factor; CCK-8: cell counting kit-8.

TRIM24 knockdown retards the in vivo growth of AML-derived xenograft tumors

To evaluate whether TRIM24 knockdown exhibits antitumor effect in vivo, we performed a xenograft tumor formation assay in nude mice. We found that Kasumi-1 cells infected with LV-TRIM24 shRNA that were subcutaneously inoculated into nude mice formed smaller tumors compared with infected cells containing LV-scrambled shRNA (Figure 6(a)). Moreover, the expression of pGSK-3β and active β-catenin proteins were significantly decreased in tumors formed by AML cells transfected with LV-TRIM24 shRNA (Figure 6(b) and (c)). Overall, these results suggest that TRIM24 knockdown exhibits antitumor effects in vivo through downregulation of pGSK-3β and active β-catenin protein expression.

Figure 6.

TRIM24 knockdown retards AML tumor growth in vivo. (a) Growth curves of tumors generated by Kasumi-1 cells transfected with either LV-scrambled shRNA (n = 3) or LV-TRIM24 shRNA (n = 3) in nude mice in vivo. (b) and (c) Western blot analysis of pGSK-3β and active β-catenin protein expression in tumor tissues. n = 3, **p < 0.01 versus LV-scrambled shRNA. (d) Schematic diagram depicting the role of the TRIM24-mediated GSK-3β/β-catenin/TCF/LEF axis in AML progression. TRIM24: tripartite motif-containing protein 24; AML: acute myeloid leukemia; pGSK-3β: phosphorylation of glycogen synthase kinase-3β; TCF/LEF: T-cell factor/lymphoid enhancer factor.

Discussion

In this study, we have provided compelling evidence suggesting that TRIM24 acts as an oncoprotein in AML. Our results have demonstrated that elevated TRIM24 expression is found in AML tissues and cell lines. Notably, high levels of expression of TRIM24 predicted significantly lower survival rates in AML patients, indicating that TRIM24 has the potential to be used as a prognostic biomarker. Functional assays revealed that upregulation of TRIM24 promoted the tumor growth in AML cells, while knockdown of TRIM24 exhibited antitumor effects, suggesting that TRIM24 acts as an oncoprotein in AML and is an attractive anticancer target. Moreover, we elucidated that TRIM24 contributes to regulation of AML tumor growth that is associated with activation of Wnt/β-catenin signaling via inactivation of GSK-3β, highlighting the potential relevance of the TRIM24/GSK-3β/Wnt/β-catenin signaling axis in the progression of AML.

TRIM24 has been implicated in multiple pathological processes, especially in cancer.⁵ Previous reports have shown that TRIM24 is capable of promoting the degradation of the tumor suppressor, p53, and thereby acts as a critical oncoprotein.⁸ TRIM24 can activate estrogen-dependent genes that facilitate tumor development in breast cancer.²³ Aberrantly high expression of TRIM24 is associated with poor prognosis and reduced levels of survival in breast cancer patients.^23,24 TRIM24 acts as an oncogenic transcriptional activator in prostate cancer, in which the protein augments the activation of the androgen receptor to promote cell proliferation and tumor progression.^25
–27 Notably, the oncogenic function of TRIM24 has been shown in a wide range of cancer types, including glioma, squamous cell carcinoma, cervical cancer, and gastric cancer.^{10,12,14,28
–30} In accordance with these findings, our study reported an oncogenic function of TRIM24 in AML. We found that high levels of expression of TRIM24 could be detected in AML tissues and patients with higher expression of TRIM24 had a reduced overall survival rate, indicating that TRIM24 is a prognostic indicator in AML. On the other hand, our results demonstrated that knockdown of TRIM24 effectively reduced proliferation and enhanced apoptosis in AML cells. Therefore, our findings, together with previous studies, support the notion that TRIM24 may be an attractive target for the development of anticancer therapy.

Conversely, a tumor-suppressive function of TRIM24 has been reported in some other cancer types. Knockout of TRIM24 in mice leads to spontaneous liver tumor formation.^31
–33 The overexpression of TRIM24 represses the proliferation and colony formation of hepatocellular carcinoma cells.³¹ Further, TRIM24 has been shown to suppress the progression of hepatocellular carcinoma by inhibiting the activation of the retinoic acid receptor.^34,35 These findings suggest that TRIM24 has a liver-specific tumor-suppressive function. To date, the tumor-suppressive function of TRIM24 has only reported in hepatocellular carcinoma. Strikingly, there are also studies demonstrating an oncogenic function of TRIM24 in hepatocellular carcinoma. Elevated expression of TRIM24 has been detected in tumor tissues of hepatocellular carcinoma, which were correlated with advanced tumor progression, metastasis, and poor prognosis.³⁶ Moreover, TRIM24 has been reported to promote tumor progression of hepatocellular carcinoma via enhancing AMPK signaling.¹⁷ Therefore, the precise role of TRIM24 in hepatocellular carcinoma requires further study. The opposite function of TRIM24 indicates that TRIM24 may regulate tumorigenesis in a context- or tissue-dependent manner.

The relevance of TRIM24 in leukemias has been reported in several studies. High levels of expression of TRIM24 has been detected in acute lymphoblastic leukemia and chronic myeloid leukemia.^37,38 Inhibition of TRIM24 via the promotion of its degradation produces a potent anti-proliferative response in AML cells.³⁹ However, the detailed mechanistic relevance of TRIM24 remains undetermined. Herein, we have demonstrated that TRIM24 is elevated in AML and knockdown of TRIM24 decreased proliferation and increased apoptosis in AML cells. Moreover, knockdown of TRIM24 inhibited tumor growth in vivo. Our study suggests that TRIM24 plays a key role in AML progression and confirms that the protein an attractive anticancer target for the treatment of AML.

Aberrant activation of Wnt/β-catenin signaling is closely related to AML progression.^19,20 Interestingly, our results revealed that TRIM24 overexpression increased the pGSK-3β and upregulated β-catenin expression, leading to the activation of TCF/LEF-mediated transcription. Indeed, the regulation of Wnt/β-catenin signaling by TRIM24 has been reported in previous studies.^12,40 Our results confirm that TRIM24 is a positive regulator of Wnt/β-catenin signaling in AML. High levels of expression of TRIM24 contribute to AML progression, which is associated with enhanced activation of Wnt/β-catenin signaling. These findings provide new insights into understanding the molecular pathogenesis of AML.

In conclusion, we have demonstrated an oncogenic role of TRIM24 in AML and have shown that knockdown of TRIM24 blocks the tumor growth in AML both in vitro and in vivo. Our study reveals an oncogenic effect for TRIM24 in AML progression, which is associated with the upregulation of Wnt/β-catenin signaling via modulation of GSK-3β phosphorylation. This highlights a key role for the TRIM24/GSK-3β/Wnt/β-catenin signaling axis in AML. Our findings suggest that TRIM24 may serve as potentially promising a prognostic biomarker and an anticancer target for the treatment of AML.

Footnotes

Authors’ note

CL and HX contributed equally to this work and shared the first authorship.

Author contributions

CL and HX designed the work, carried out the experiment operation, and drafted the article; YS interpreted the data; and JM designed the work and revised the article. All authors have approved the final version of this article for publication.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Foundation Project of Shanxi Provincial Department of Education [Grant No. 12JK0760].

ORCID iDs

H Xin

J Mu

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Knockdown of TRIM24 suppresses growth and induces apoptosis in acute myeloid leukemia through downregulation of Wnt/GSK-3β/β-catenin signaling

Abstract

Keywords

Introduction

Materials and methods

Tissue collection and cell culture

Lentivirus production and infection

RNA extraction and real-time quantitative polymerase chain reaction analysis

Protein isolation and Western blot analysis

Cell counting kit-8 assay

Colony formation assay

Flow cytometry analysis

Luciferase reporter assay

Animal experiments

Statistical analysis

Results

High levels of expression of TRIM24 were detected in AML and were associated with poor survival rates

Knockdown of TRIM24 shows antitumor effect in AML cells

Ectopic expression of TRIM24 promotes the growth of AML cells

TRIM24 regulates Wnt/β-catenin signaling in AML cells

Inactivation of GSK-3β abrogates TRIM24 knockdown-mediated antitumor effect

TRIM24 knockdown retards the in vivo growth of AML-derived xenograft tumors

Discussion

Footnotes

Authors’ note

Author contributions

Declaration of conflicting interests

Funding

ORCID iDs

References