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β-catenin signaling pathway
Biomedicine & Pharmacotherapy 107 (2018) 1548–1555
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DIXDC1 promotes the growth of acute myeloid leukemia cells by upregulating the Wnt/β-catenin signaling pathway
T
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Hong Xina, Chengliang Lib, , Minjuan Wangc a
Department of Cardiovasology, The First Affiliated Hospital of Xi'an Medical University, No. 48 Fenghao West Road, Xi'an, 710077, China Department of Hematology, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China c Department of General Practice and Geriatrics, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute myeloid leukemia DIXDC1 Wnt/β-catenin
Accumulating evidence suggests that dysregulation of Dishevelled-Axin domain-containing 1 (DIXDC1) is involved in the progression and development of various cancers. However, little is known about the relevance of DIXDC1 in acute myeloid leukemia (AML). In this study, we aimed to investigate the expression status and potential biological function of DIXDC1 in AML. Our results showed that DIXDC1 expression was highly upregulated in AML cell lines and primary AML blasts compared with normal blasts. Knockdown of DIXDC1 by siRNA-mediated gene silencing significantly inhibited proliferation, induced cell cycle arrest, and promoted apoptosis of AML cells in vitro. By contrast, DIXDC1 overexpression promoted proliferation, accelerated cell cycle progression, and reduced apoptosis of AML cells. Moreover, we found that DIXDC1 knockdown decreased the expression of β-catenin and restricted the activation of Wnt signaling. In addition, DIXDC1 knockdown decreased the expression of Wnt/β-catenin target genes, including cyclin D1 and c-myc, while DIXDC1 overexpression had the opposite effect. Notably, β-catenin knockdown partially reversed the oncogenic effect of DIXDC1 in AML cells. Taken together, these results demonstrate that DIXDC1 promotes the growth of AML cells, possibly through upregulating the Wnt/β-catenin signaling pathway. Our study suggests that DIXDC1 may serve as a potential therapeutic target for the treatment of AML.
1. Introduction Acute myeloid leukemia (AML) is a common type of hematological malignancy, which is characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow and peripheral blood [1,2]. In China, the incidence and mortality rates of AML have increased in recent years [3]. Despite advances in cancer therapy strategies, an effective treatment for AML is still lacking [4]. Genetic alterations and environmental exposures are involved in the development and progression of AML [5,6]. However, the precise molecular mechanisms underlying AML pathogenesis remains largely unknown. Therefore, identification of novel and key molecules that drive the development and progression of AML will aid the elucidation of the molecular pathogenesis of AML and development of effective target therapies. Dishevelled-Axin domain-containing 1 (DIXDC1), the human homolog of Coiled-coil-Dishevelled-Axin1 (Ccd1), is emerging as a key
player in tumorigenesis [7,8]. DIXDC1 is identified as a scaffolding protein consisting of three protein-protein interaction domains: a DIX domain, a coiled-coil (CC) domain, and an actin-binding calponin homology domain [9,10]. An increasing number of studies have suggested that DIXDC1 is a positive regulator of Wnt/β-catenin signaling, which activates TCF-dependent transcription through interaction with Dishevelled and Axin [11,12]. DIXDC1 plays an important role in regulating neural development, neural differentiation, and nerve injury [13–17]. However, the dysregulation of DIXDC1 is associated with the development and progression of numerous cancers. A growing amount of evidence has demonstrated that DIXDC1 is highly expressed in a variety of cancer types, including colon cancer [18], gastric cancer [19], lung cancer [8], and pancreatic ductal adenocarcinoma [20]. High levels of DIXDC1 are correlated with tumor size, tumor invasion depth, and tumor node metastasis and predict poor overall survival [20,21]. Notably, DIXDC1 has been reported to promote the growth, migration, and invasion of cancer cells associated with activation of
Abbreviations: AML, acute myeloid leukemia; DIXDC1, Dishevelled-Axin domain containing 1; FBS, fetal bovine serum; CCK-8, cell counting kit-8; RT-qPCR, realtime quantitative polymerase chain reaction ⁎ Corresponding author. E-mail address: [email protected] (C. Li). https://doi.org/10.1016/j.biopha.2018.08.144 Received 19 June 2018; Received in revised form 17 August 2018; Accepted 28 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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various oncogenic signaling pathways, such as Wnt/β-catenin and PI3K/Akt [8,19]. However, whether DIXDC1 is involved in AML development and progression remains unknown. The canonical Wnt/β-catenin signaling pathway plays an important role in regulating development, morphogenesis, differentiation, and proliferation [22]. Under resting conditions, β-catenin is phosphorylated by the destruction complex APC/Axin/GSK-3β and undergoes ubiquitination and proteasome-mediated degradation [23]. When the Wnt ligand binds to the Frizzled and LRP co-receptors, Dishevelled is recruited to the plasma membrane and forms a heteromeric complex with Axin [23]. Consequently, the promotional effect of the destruction complex APC/Axin/GSK-3β on β-catenin phosphorylation is dismissed, leading to β-catenin accumulation and nuclear translocation to activate TCF-dependent transcription of Wnt target genes [23]. Wnt/β-catenin signaling is involved in the pathological processes of various diseases, including cancer [24,25]. Aberrant activation of Wnt/β-catenin signaling and its downstream effectors is frequently occurred in AML [26]. A better understanding of the regulatory mechanism of Wnt/β-catenin signaling in AML may provide novel insights into the development of effective therapies for AML. In this study, we aimed to investigate the expression, biological function, and regulatory mechanism of DIXDC1 in AML. We found that DIXDC1 expression was increased in primary AML blasts and AML cell lines. DIXDC1 Knockdown inhibited AML cell growth, while DIXDC1 overexpression promoted AML cell growth. We further demonstrated that the regulatory effect of DIXDC1 on AML cell growth was associated with its promotional effect on activation of Wnt/β-catenin signaling. Overall, these findings suggest that DIXDC1 may serve as a novel therapeutic target for AML.
concentrations were measured using a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China). Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After incubation with 5% non-fat milk in Tris-buffered Saline with Tween 20 (TBST) for 45 min at 37 °C, the membrane was incubated with primary antibodies against DIXDC1 (Abcam, Cambridge, MA, USA), β-catenin (Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-myc (Santa Cruz Biotechnology) and GAPDH (Abcam) overnight at 4 °C. Afterwards, the membrane was washed three times with TBST and incubated with HRP-conjugated secondary antibody (Abcam) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The protein band intensities were measured using Image-Pro Plus 6.0. GPADH was used as the loading control.
2. Materials and methods
Cell proliferation was measured by using a cell counting kit-8 (CCK8) assay. In brief, cells were seeded into 96-well plates at 5 × 103 cells/ well and cultured overnight. After transfection for 24, 48, and 72 h, 10 μl of CCK8 reagents (Beyotime Biotechnology) were added to each well. After incubation at 37 °C for 2 h, the absorbance at 450 nm was detected using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).
2.4. Cell transfection DIXDC1 siRNA and control siRNA were synthesized by GenePharma (Shanghai, China). β-catenin siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ORF of DIXDC1 was inserted into the pcDNA3.1 plasmid to generate a pcDNA3.1/DIXDC1 expression plasmid. Transfection of siRNA or plasmid into cells was performed using RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The transfection efficacy was determined by RT-qPCR and western blot analysis. 2.5. Cell proliferation assay
2.1. Cell culture Acute myeloid leukemia (AML) cell lines (HL-60, Kasumi-1, and AML-193) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to the manufacturer’s instructions. Briefly, HL-60, Kasumi-1, and AML-193 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. CD34+ blasts were isolated from bone marrow of AML patients and normal volunteers by mini-magnetic activated cell sorting method using CD34 MicroBead Kit (Miltenyi Biotec, Shanghai, China) according to the manufacturer’s instructions. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
2.6. Colony formation assay The 6-well plates were precoated with growth medium containing 0.6% agar. Cells (48 h post transfection) were resuspended in medium containing 0.3% agarose and seeded in 6-well plates at 1000 cells/well. After a 14-day incubation, the number of colonies was counted under an inverted microscope.
2.2. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
2.7. Cell cycle analysis
Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to cDNA by using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA, USA). RT-qPCR was carried out with Power SYBR Green PCR Master Mix (Applied Biosystems) and the following thermal cycling protocols: 95 °C, 10 min; 40 cycles of 95 °C, 15 s, and 60 °C, 1 min. The RT-qPCR primer sequences were as follows: DIXDC1 (F: 5′-TGCATGTTATGGAGACGCAG AAG-3′ and R: 5′-AGGTGCTGCTGACAGTTGGAGA-3′); Cyclin D1 (F: 5′-CGTGGCCTCTAAGATGAAGGA-3′ and R: 5′-CGGTGTAGATGCACAG CTTCTC-3′); c-myc (F: 5′-GCTGCTTAGACGCTGGATTT-3′ and R: 5′-CACCGAGTCGTAGTCGAGGT-3′); GAPDH (F: 5′-ACAGTCAGCCGCA TCTTCTT-3′ and R: 5′-GACAAGCTTCCCGTTCTCAG-3′). GAPDH was used as the internal control. Relative gene expression was calculated using the 2−ΔΔCt method.
Cells (1 × 106 cells) were harvested after 48 h of transfection and fixed with 75% cold ethanol at 4 °C for 24 h. After washing with phosphate buffer saline (PBS), 0.5 ml of PI/RNase Staining Solution (Thermo Fisher Scientific) was added to each sample and incubated for 30 min at room temperature in the dark. Afterwards, each sample was analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA), and data were analyzed by using BD CellFIT software. 2.8. Cell apoptosis assay Cell apoptosis was determined by measurement of caspase-3 activity with the Caspase 3 Activity Assay Kit (Beyotime Biotechnology). Briefly, 2 × 106 cells were collected from each sample after the indicated treatments and washed with PBS. Cells were then lysed with 100 μl of lysis buffer followed by centrifugation at 16,000 g for 15 min. The supernatants were harvested and treated with Ac-DEVD-pNA (2 mM) for 1–2 h at 37 °C. The absorbance at 405 nm was measured using an ELISA reader (Molecular Devices).
2.3. Western blot analysis Total proteins were extracted with RIPA lysis buffer, and protein 1549
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AML cells.
2.9. Luciferase reporter assay Cells were seeded into 96-well plates and cotransfected with the TOPflash reporter plasmid, pRL-TK reporter plasmid, and DIXDC1 siRNA or DIXDC1 expression plasmid using RNAiMAX Reagent. After transfection for 48 h, relative luciferase activity was analyzed by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
3.3. Overexpression of DIXDC1 promotes the growth of AML cells To validate the role of DIXDC1 in regulating AML cell growth, we performed gain-of-function experiments of DIXDC1 by using DIXDC1 expression plasmid. Transfection with the DIXDC1 expression plasmid significantly increased the expression of DIXDC1 in HL-60 and Kasumi1 cells (Fig. 3A). We then found that DIXDC1 overexpression significantly promoted proliferation (Fig. 3B and C) and colony formation (Fig. 3D) in AML cells. Moreover, DIXDC1 overexpression decreased the number of cells in the G0/G1 phases (Fig. 3E) and reduced apoptosis in HL-60 and Kasumi-1 cells (Fig. 3F). Overall, these results suggest that DIXDC1 promotes the growth of AML cells.
2.10. Statistical analysis The mean values and corresponding standard deviations (SD) were calculated from the results. Data are presented as mean ± SD. Oneway analysis of variance (ANOVA) and Student’s t-test were used to determine statistically significant differences. Statistical analyses were carried out by using SPSS version 19.0 software (SPSS Inc., Chicago, IL, USA). Differences with p < 0.05 were regarded as statistically significant.
3.4. DIXDC1 has a regulatory effect on Wnt/β-catenin signaling in AML cells
3. Results
DIXDC1 functions as a positive regulator of oncogenic Wnt/β-catenin signaling during tumor development [19,21]. To further investigate the molecular basis of DIXDC1 in regulating AML cell growth, we detected the regulatory effect of DIXDC1 on Wnt/β-catenin signaling in AML cells. Our results showed that DIXDC1 knockdown significantly decreased β-catenin expression (Fig. 4A) and inhibited TCFdependent transcriptional activity (Fig. 4B). Moreover, the mRNA and protein expression of cyclin D1 and c-myc, two oncogenes of Wnt/βcatenin signaling downstream targets, were also decreased by DIXDC1 silencing (Fig. 4C-F). By contrast, DIXDC1 overexpression showed a promotional effect on the activation of Wnt/β-catenin signaling and the expression of downstream target genes (Fig. 5A-F). Collectively, these results indicate that DIXDC1 promotes the activation of Wnt/β-catenin signaling in AML cells.
3.1. DIXDC1 is overexpressed in AML blasts and cell lines To investigate whether DIXDC1 is involved in the development of AML, we first detected the expression of DIXDC1 in blast derived from AML patients and AML cell lines. We found that the mRNA expression of DIXDC1 was significantly upregulated in primary AML blasts and AML cell lines compared with normal blasts (Fig. 1A). Consistent with this result, western blot analysis showed that DIXDC1 protein expression was also markedly increased in primary AML blasts and AML cell lines (Fig. 1B). These results indicate that DIXDC1 may play an important role in the progression of AML. 3.2. Silencing of DIXDC1 impedes the growth of AML cells To investigate the precise role of DIXDC1 in AML, we examined the regulatory effect of DIXDC1 on AML cell growth by gene silencing of DIXDC1 using siRNAs specifically targeting DIXDC1. The results showed that transfection with DIXDC1 siRNAs significantly decreased the expression of DIXDC1 in HL-60 and Kasumi-1 cells (Fig. 2A). We then detected the effect of DIXDC1 silencing on AML cell growth. The CCK-8 assay showed that DIXDC1 silencing significantly reduced the proliferation of HL-60 and Kasumi-1 cells (Fig. 2B and C). The colony formation assay showed that DIXDC1 knockdown significantly decreased colony formation in HL-60 and Kasumi-1 cells (Fig. 2D). Moreover, DIXDC1 knockdown induced a significant increase in the number of cells in the G0/G1 phases (Fig. 2E). In addition, DIXDC1 silencing promoted apoptosis in HL-60 and Kasumi-1 cells (Fig. 2F). These results suggest that DIXDC1 knockdown inhibits the growth of
3.5. Knockdown of β-catenin reverses the oncogenic effect of DIXDC1 in AML cells To investigate whether DIXDC1 regulates AML cell growth through acting on Wnt/β-catenin signaling, we examined the effect of β-catenin silencing on DIXDC1-induced effect. We found that transfection of βcatenin siRNA into DIXDC1-overexpressing cells significantly decreased the expression of β-catenin and blocked the promotional effect of DIXDC1 overexpression on Wnt/β-catenin signaling (Fig. 6A and B). As expected, the promotional effect of DIXDC1 on the growth of AML cells was also partially reversed by β-catenin knockdown (Fig. 6C and D). Overall, these results suggest that DIXDC1 promotes the growth of AML cells by upregulating Wnt/β-catenin signaling. Fig. 1. DIXDC1 was highly expressed in AML cells. (A) Relative mRNA expression of DIXDC1 in primary AML blasts and AML cell lines (HL60, Kasumi-1, and AML-193) was detected by RT-qPCR analysis. Relative DIXDC1 mRNA expression was calculated by normalizing against GAPDH expression. (B) Protein expression of DIXDC1 in primary AML blasts and AML cell lines was determined by western blot analysis. Normal blasts were used as control. *p < 0.05 vs. normal blasts.
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Fig. 2. DIXDC1 Knockdown represses the growth of AML cells. (A) Relative protein expression of DIXDC1 in DIXDC1 siRNA-transfected cells was detected by western blot. HL-60 and Kasumi-1 cells were transfected with DIXDC1 siRNAs (si-DIXDC1-1 or si-DIXDC1-2) or control siRNA for 48 h. The effect of DIXDC1 silencing on cell proliferation of HL-60 (B) and Kasumi-1 (C) cells was examined by using the CCK-8 assay after siRNA transfection for 24, 48, and 72 h. (D) The effect of DIXDC1 silencing on colony formation was evaluated by the colony formation assay. (E) Flow cytometric analysis of cell cycle distribution in the G0/G1 phases 48 h after DIXDC1 siRNA transfection. (F) Effect of DIXDC1 silencing on cell apoptosis was determined by measuring caspase-3 activity. *p < 0.05 vs. control siRNA.
4. Discussion
microRNA-582-5p/3p shows promising therapeutic effects on the inhibition of bladder cancer progression associated with inhibition of DIXDC1 expression [29]. Moreover, miR-186, miR-539 and miR-1271 are also suggested as tumor-suppressive miRNAs that are capable of restricting the progression of multiple tumors by targeting and inhibiting DIXDC1 [30–32]. These reports suggest that DIXDC1 may be used as potential target for development of anticancer drugs. In line with these reports, our study suggests that DIXDC1 may serve as a target gene for treating AML. In addition to its oncogenic role, the tumor-suppressor role of DIXDC1 is also reported by several studies. DIXDC1 has been reported to inhibit the migration and invasion of lung cancer cells by downregulation of Snail expression [9]. Interestingly, DIXDC1 was found to be decreased in hepatocellular carcinoma tissues, and reduced DIXDC1expression was correlated with tumor size, tumor differentiation, TNM stage, and poor survival of hepatocellular carcinoma patients [33]. The precise regulatory role of DIXDC1 in tumorigenesis may differ profoundly among cancer types or experimental conditions. Therefore, the precise role of DIXDC1 in tumorigenesis needs further investigation. Nevertheless, our study suggests a promotional role of DIXDC1 in AML progression. DIXDC1 has been reported as a positive regulator of DIXDC1 Wnt/βcatenin signaling. It has been shown that DIXDC1 promotes the activation of Wnt/β-catenin signaling by interacting with Dishevelled and Axin [11,12]. DIXDC1 contributes to regulation of neural patterning and psychiatric pathogenesis through regulation of Wnt/β-catenin signaling [11,34]. Notably, DIXDC1-medated Wnt/β-catenin signaling plays an important role in tumor progression. It is reported that DIXDC1 is co-localized with β-catenin in intestinal-type gastric carcinoma [21]. Tan et al. reported that DIXDC1 promotes the invasion and metastasis of gastric cancer cells by enhancing β-catenin accumulation and nuclear
In the present study, we provide the first compelling evidence that DIXDC1 expression is elevated in AML cells and may play a key role in AML progression. We showed that DIXDC1 overexpression promoted AML cell growth, while DIXDC1 silencing impeded AML cell growth. Moreover, our study reveals that the underlying mechanism may be associated with its regulatory effect on Wnt/β-catenin signaling (Fig. 7), which highlights an importance of DIXDC1/Wnt/β-catenin signaling axis in the progression of AML. Currently, the role of DIXDC1 in cancers has been widely studied. High expression of DIXDC1 has been detected in clinical tumor specimens and is correlated with tumor stage, lymph node metastasis, and poor prognosis [19,21]. DIXDC1 overexpression promotes the proliferation of colon cancer cells in vitro and in vivo [18]. DIXDC1 has been reported to regulate the growth, migration, and invasion of gastric cancer cells [19,27]. Moreover, a recent study reports that DIXDC1 promotes tumor proliferation and drug resistance in non-Hodgkin's lymphomas [28]. These findings indicate that DIXDC1 plays an oncogenic role in tumorigenesis. However, whether DIXDC1 is involved in the progression of AML remains unknown. In this study, we demonstrated that DIXDC1 expression was increased in AML cells. Functional experiments revealed that DIXDC1 knockdown inhibited AML cell growth, whereas DIXDC1 overexpression accelerated AML cell growth in vitro, indicating a critical role of DIXDC1 in AML. Targeting DIXDC1 has been suggested as a potential therapeutic approach for cancer therapy. Inhibiting DIXDC1 by siRNA in human gliomas has been shown to inhibit cell proliferation and migration [7]. Interestingly, microRNAs have been suggested as promising targets to modulate DIXDC1 expression, which can be used as promising agents in treatment of DIXDC1-related malignancies. Uchino et al. reported that targeting
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Fig. 3. DIXDC1 overexpression promotes the growth of AML cells. (A) Relative protein expression of DIXDC1 was detected by western blot. HL-60 and Kasumi-1 cells were transfected with pcDNA3.1/DIXDC1 vector or pcDNA3.1 empty vector (vector) for 48 h. The effect of DIXDC1 overexpression on proliferation of HL-60 (B) and Kasumi-1 (C) cells was monitored by CCK-8 assay at 24, 48, and 72 h after transfection with the DIXDC1 expression vector. (D) The effect of DIXDC1 overexpression on colony formation was detected by colony formation assay. (E) Flow cytometric analysis of cell cycle distribution in the G0/G1 phases 48 h after DIXDC1 expression vector transfection. (F) The effect of DIXDC1 overexpression on cell apoptosis was determined by measuring caspase-3 activity. *p < 0.05 vs. vector.
of DIXDC1 remains undetermined. High expression of DIXDC1 has been frequently detected in clinical specimens of gastric cancer, colon cancer, pancreatic cancer and lung cancers [8,18–21]. These studies reveal that high expression of DIXDC1 is correlated with high cell proliferation index, tumor size, tumor node metastasis and poor outcome, indicating a diagnostic and prognostic role of DIXDC1. In this study, we show that DIXDC1 is overexpressed in blasts from AML patients. Therefore, further studies should be performed to assess the relationship between DIXDC1 expression and clinicopathologic progress of AML patients. In conclusion, our results demonstrated that DIXDC1 expression was upregulated in AML cells and that high expression of DIXDC1 contributes to AML cell growth in vitro by promoting Wnt/β-catenin signaling. Our study indicates that DIXDC1 may play a critical role in the development and progression of AML. These findings provide novel insights into understanding the molecular pathogenesis of AML and suggest that DIXDC1 may be used as a potential target for AML therapy.
translocation to activate Wnt signaling [19]. Additionally, DIXDC1mediated Wnt/β-catenin signaling is also involved in the development and progression of glioma, retinoblastoma, and prostate cancer [30–32]. Consistent with these findings, our results showed that DIXDC1 overexpression increased β-catenin accumulation and expression of Wnt target genes, including cyclin D1 and c-myc. Notably, blockade of Wnt signaling by silencing of β-catenin partially reversed the DIXDC1-induced effect in AML cells, indicating that DIXDC1 regulates the growth of AML cells through acting on Wnt/β-catenin signaling. Aberrant activation of Wnt/β-catenin signaling and downstream effectors is frequently occurred in AML [26]. Therefore, high expression of DIXDC1 may contribute to aberrant activation of Wnt/β-catenin signaling during AML development and progression. Blockade of Wnt/ β-catenin signaling through targeting DIXDC1 may have potential application for treatment of AML. Interestingly, DIXDC1 regulates tumorigenesis also through other oncogenic signaling pathways, such as the Akt signaling pathway. DIXDC1 enhances the expression level of phosphorylated Akt (pAkt) to promote colon cancer cell proliferation [18]. Xu et al. reported that DIXDC1 promoted the migration and invasion of non-small-cell lung cancer cells through upregulation of pAkt expression [8]. Moreover, DIXDC1 upregulation contributes to tumor proliferation and drug resistance of non-Hodgkin's lymphomas by enhancing pAkt expression [28]. It's worth noting that Akt signaling regulates the activation of Wnt/β-catenin signaling. Therefore, Akt signaling may be involved in DIXDC1-mediated Wnt/β-catenin signaling. In this study, we focused on investigating the biological function of DIXDC1 using AML cell lines in vitro. However, the clinical significance
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgments This study was supported by Foundation Project of Shaanxi Provincial Department of Education (12JK0760). 1552
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Fig. 4. DIXDC1 knockdown inhibits Wnt/β-catenin signaling in AML cells. (A) Protein expression of β-catenin in HL-60 and Kasumi-1 cells transfected with siDIXDC1 or control siRNA for 48 h was detected by western blot. (B) TCF-dependent transcription activity was measured by using the TOPflash reporter assay. HL-60 and Kasumi-1 cells were cotransfected with the TOPflash reporter vector and si-DIXDC1 for 48 h. The effect of DIXDC1 silencing on the mRNA expression of cyclin D1 (C) and c-myc (D) was detected by RT-qPCR. The effect of DIXDC1 silencing on the protein expression of cyclin D1 (E) and c-myc (F) was detected by Western blot. *p < 0.05 vs. control siRNA.
Fig. 5. DIXDC1 overexpression promotes activation of Wnt/β-catenin signaling in AML cells. (A) Protein expression of β-catenin in HL-60 and Kasumi-1 cells transfected with pcDNA3.1/DIXDC1 vector or control vector for 48 h was detected by western blot. (B) TCF-dependent transcription activity was measured by using the TOPflash reporter assay. HL-60 and Kasumi-1 cells were cotransfected with the TOPflash reporter vector and pcDNA3.1/DIXDC1 vector for 48 h. The effect of DIXDC1 overexpression on the mRNA expression of cyclin D1 (C) and c-myc (D) was evaluated by RT-qPCR. The effect of DIXDC1 overexpression on the protein expression of cyclin D1 (E) and c-myc (F) was detected by Western blot. *p < 0.05 vs. control siRNA. 1553
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Fig. 6. Blockade of Wnt/β-catenin signaling reverses DIXDC1-meidated effect in AML cells. HL-60 and Kasumi-1 cells were cotransfected with the DIXDC1 expression vector and β-catenin siRNA for 48 h, and (A) the protein expression of β-catenin was detected by western blot. (B) Wnt/β-catenin signaling was detected by measuring TCF-dependent transcription activity by using the TOPflash reporter assay. The effect of β-catenin silencing on AML cell growth was assessed by CCK-8 (C) and colony formation (D) assays. *p < 0.05.
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Fig. 7. Diagrammatic representation of DIXDC1-mediated Wnt/β-catenin signaling in AML. DIXDC1 promotes the accumulation of β-catenin in the nucleus and activates TCF/β-catenin-dependent transcription of cyclin D1 and c-myc, thus participating in the development and progression of AML.
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retinoblastoma cells, J. Mol. Neurosci. 64 (2018) 252–261. [31] J. Quan, J. Qu, L. Zhou, MicroRNA-539 inhibits glioma cell proliferation and invasion by targeting DIXDC1, Biomed. Pharmacother. 93 (2017) 746–753. [32] J. Zhong, Y. Liu, Q. Xu, et al., Inhibition of DIXDC1 by microRNA-1271 suppresses the proliferation and invasion of prostate cancer cells, Biochem. Biophys. Res. Commun. 484 (2017) 794–800. [33] S. Zhou, J. Shen, S. Lin, et al., Downregulated expression of DIXDC1 in hepatocellular carcinoma and its correlation with prognosis, Tumour Biol. 37 (2016) 13607–13616. [34] P.M. Martin, R.E. Stanley, A.P. Ross, et al., DIXDC1 contributes to psychiatric susceptibility by regulating dendritic spine and glutamatergic synapse density via GSK3 and Wnt/beta-catenin signaling, Mol. Psychiatry 23 (2018) 467–475.
[26] J.H. Mikesch, B. Steffen, W.E. Berdel, et al., The emerging role of Wnt signaling in the pathogenesis of acute myeloid leukemia, Leukemia 21 (2007) 1638–1647. [27] J. Song, Z. Guan, M. Li, et al., MicroRNA-154 inhibits the growth and invasion of gastric cancer cells by targeting DIXDC1/WNT signaling, Oncol. Res. (2017). [28] Y. Ouyang, F. Zhong, Q. Wang, et al., DIXDC1 promotes tumor proliferation and cell adhesion mediated drug resistance (CAM-DR) via enhancing p-Akt in NonHodgkin’s lymphomas, Leuk. Res. 50 (2016) 104–111. [29] K. Uchino, F. Takeshita, R.U. Takahashi, et al., Therapeutic effects of microRNA582-5p and -3p on the inhibition of bladder cancer progression, Mol. Ther. 21 (2013) 610–619. [30] X. Che, Y. Qian, D. Li, Suppression of Disheveled-Axin domain containing 1 (DIXDC1) by microRNA-186 inhibits the proliferation and invasion of
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DIXDC1 promotes the growth of acute myeloid leukemia cells by upregulating the Wnt/β-catenin signaling pathway
T
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Hong Xina, Chengliang Lib, , Minjuan Wangc a
Department of Cardiovasology, The First Affiliated Hospital of Xi'an Medical University, No. 48 Fenghao West Road, Xi'an, 710077, China Department of Hematology, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China c Department of General Practice and Geriatrics, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute myeloid leukemia DIXDC1 Wnt/β-catenin
Accumulating evidence suggests that dysregulation of Dishevelled-Axin domain-containing 1 (DIXDC1) is involved in the progression and development of various cancers. However, little is known about the relevance of DIXDC1 in acute myeloid leukemia (AML). In this study, we aimed to investigate the expression status and potential biological function of DIXDC1 in AML. Our results showed that DIXDC1 expression was highly upregulated in AML cell lines and primary AML blasts compared with normal blasts. Knockdown of DIXDC1 by siRNA-mediated gene silencing significantly inhibited proliferation, induced cell cycle arrest, and promoted apoptosis of AML cells in vitro. By contrast, DIXDC1 overexpression promoted proliferation, accelerated cell cycle progression, and reduced apoptosis of AML cells. Moreover, we found that DIXDC1 knockdown decreased the expression of β-catenin and restricted the activation of Wnt signaling. In addition, DIXDC1 knockdown decreased the expression of Wnt/β-catenin target genes, including cyclin D1 and c-myc, while DIXDC1 overexpression had the opposite effect. Notably, β-catenin knockdown partially reversed the oncogenic effect of DIXDC1 in AML cells. Taken together, these results demonstrate that DIXDC1 promotes the growth of AML cells, possibly through upregulating the Wnt/β-catenin signaling pathway. Our study suggests that DIXDC1 may serve as a potential therapeutic target for the treatment of AML.
1. Introduction Acute myeloid leukemia (AML) is a common type of hematological malignancy, which is characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow and peripheral blood [1,2]. In China, the incidence and mortality rates of AML have increased in recent years [3]. Despite advances in cancer therapy strategies, an effective treatment for AML is still lacking [4]. Genetic alterations and environmental exposures are involved in the development and progression of AML [5,6]. However, the precise molecular mechanisms underlying AML pathogenesis remains largely unknown. Therefore, identification of novel and key molecules that drive the development and progression of AML will aid the elucidation of the molecular pathogenesis of AML and development of effective target therapies. Dishevelled-Axin domain-containing 1 (DIXDC1), the human homolog of Coiled-coil-Dishevelled-Axin1 (Ccd1), is emerging as a key
player in tumorigenesis [7,8]. DIXDC1 is identified as a scaffolding protein consisting of three protein-protein interaction domains: a DIX domain, a coiled-coil (CC) domain, and an actin-binding calponin homology domain [9,10]. An increasing number of studies have suggested that DIXDC1 is a positive regulator of Wnt/β-catenin signaling, which activates TCF-dependent transcription through interaction with Dishevelled and Axin [11,12]. DIXDC1 plays an important role in regulating neural development, neural differentiation, and nerve injury [13–17]. However, the dysregulation of DIXDC1 is associated with the development and progression of numerous cancers. A growing amount of evidence has demonstrated that DIXDC1 is highly expressed in a variety of cancer types, including colon cancer [18], gastric cancer [19], lung cancer [8], and pancreatic ductal adenocarcinoma [20]. High levels of DIXDC1 are correlated with tumor size, tumor invasion depth, and tumor node metastasis and predict poor overall survival [20,21]. Notably, DIXDC1 has been reported to promote the growth, migration, and invasion of cancer cells associated with activation of
Abbreviations: AML, acute myeloid leukemia; DIXDC1, Dishevelled-Axin domain containing 1; FBS, fetal bovine serum; CCK-8, cell counting kit-8; RT-qPCR, realtime quantitative polymerase chain reaction ⁎ Corresponding author. E-mail address: [email protected] (C. Li). https://doi.org/10.1016/j.biopha.2018.08.144 Received 19 June 2018; Received in revised form 17 August 2018; Accepted 28 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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various oncogenic signaling pathways, such as Wnt/β-catenin and PI3K/Akt [8,19]. However, whether DIXDC1 is involved in AML development and progression remains unknown. The canonical Wnt/β-catenin signaling pathway plays an important role in regulating development, morphogenesis, differentiation, and proliferation [22]. Under resting conditions, β-catenin is phosphorylated by the destruction complex APC/Axin/GSK-3β and undergoes ubiquitination and proteasome-mediated degradation [23]. When the Wnt ligand binds to the Frizzled and LRP co-receptors, Dishevelled is recruited to the plasma membrane and forms a heteromeric complex with Axin [23]. Consequently, the promotional effect of the destruction complex APC/Axin/GSK-3β on β-catenin phosphorylation is dismissed, leading to β-catenin accumulation and nuclear translocation to activate TCF-dependent transcription of Wnt target genes [23]. Wnt/β-catenin signaling is involved in the pathological processes of various diseases, including cancer [24,25]. Aberrant activation of Wnt/β-catenin signaling and its downstream effectors is frequently occurred in AML [26]. A better understanding of the regulatory mechanism of Wnt/β-catenin signaling in AML may provide novel insights into the development of effective therapies for AML. In this study, we aimed to investigate the expression, biological function, and regulatory mechanism of DIXDC1 in AML. We found that DIXDC1 expression was increased in primary AML blasts and AML cell lines. DIXDC1 Knockdown inhibited AML cell growth, while DIXDC1 overexpression promoted AML cell growth. We further demonstrated that the regulatory effect of DIXDC1 on AML cell growth was associated with its promotional effect on activation of Wnt/β-catenin signaling. Overall, these findings suggest that DIXDC1 may serve as a novel therapeutic target for AML.
concentrations were measured using a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China). Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After incubation with 5% non-fat milk in Tris-buffered Saline with Tween 20 (TBST) for 45 min at 37 °C, the membrane was incubated with primary antibodies against DIXDC1 (Abcam, Cambridge, MA, USA), β-catenin (Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-myc (Santa Cruz Biotechnology) and GAPDH (Abcam) overnight at 4 °C. Afterwards, the membrane was washed three times with TBST and incubated with HRP-conjugated secondary antibody (Abcam) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The protein band intensities were measured using Image-Pro Plus 6.0. GPADH was used as the loading control.
2. Materials and methods
Cell proliferation was measured by using a cell counting kit-8 (CCK8) assay. In brief, cells were seeded into 96-well plates at 5 × 103 cells/ well and cultured overnight. After transfection for 24, 48, and 72 h, 10 μl of CCK8 reagents (Beyotime Biotechnology) were added to each well. After incubation at 37 °C for 2 h, the absorbance at 450 nm was detected using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).
2.4. Cell transfection DIXDC1 siRNA and control siRNA were synthesized by GenePharma (Shanghai, China). β-catenin siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ORF of DIXDC1 was inserted into the pcDNA3.1 plasmid to generate a pcDNA3.1/DIXDC1 expression plasmid. Transfection of siRNA or plasmid into cells was performed using RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The transfection efficacy was determined by RT-qPCR and western blot analysis. 2.5. Cell proliferation assay
2.1. Cell culture Acute myeloid leukemia (AML) cell lines (HL-60, Kasumi-1, and AML-193) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to the manufacturer’s instructions. Briefly, HL-60, Kasumi-1, and AML-193 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. CD34+ blasts were isolated from bone marrow of AML patients and normal volunteers by mini-magnetic activated cell sorting method using CD34 MicroBead Kit (Miltenyi Biotec, Shanghai, China) according to the manufacturer’s instructions. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
2.6. Colony formation assay The 6-well plates were precoated with growth medium containing 0.6% agar. Cells (48 h post transfection) were resuspended in medium containing 0.3% agarose and seeded in 6-well plates at 1000 cells/well. After a 14-day incubation, the number of colonies was counted under an inverted microscope.
2.2. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
2.7. Cell cycle analysis
Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to cDNA by using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA, USA). RT-qPCR was carried out with Power SYBR Green PCR Master Mix (Applied Biosystems) and the following thermal cycling protocols: 95 °C, 10 min; 40 cycles of 95 °C, 15 s, and 60 °C, 1 min. The RT-qPCR primer sequences were as follows: DIXDC1 (F: 5′-TGCATGTTATGGAGACGCAG AAG-3′ and R: 5′-AGGTGCTGCTGACAGTTGGAGA-3′); Cyclin D1 (F: 5′-CGTGGCCTCTAAGATGAAGGA-3′ and R: 5′-CGGTGTAGATGCACAG CTTCTC-3′); c-myc (F: 5′-GCTGCTTAGACGCTGGATTT-3′ and R: 5′-CACCGAGTCGTAGTCGAGGT-3′); GAPDH (F: 5′-ACAGTCAGCCGCA TCTTCTT-3′ and R: 5′-GACAAGCTTCCCGTTCTCAG-3′). GAPDH was used as the internal control. Relative gene expression was calculated using the 2−ΔΔCt method.
Cells (1 × 106 cells) were harvested after 48 h of transfection and fixed with 75% cold ethanol at 4 °C for 24 h. After washing with phosphate buffer saline (PBS), 0.5 ml of PI/RNase Staining Solution (Thermo Fisher Scientific) was added to each sample and incubated for 30 min at room temperature in the dark. Afterwards, each sample was analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA), and data were analyzed by using BD CellFIT software. 2.8. Cell apoptosis assay Cell apoptosis was determined by measurement of caspase-3 activity with the Caspase 3 Activity Assay Kit (Beyotime Biotechnology). Briefly, 2 × 106 cells were collected from each sample after the indicated treatments and washed with PBS. Cells were then lysed with 100 μl of lysis buffer followed by centrifugation at 16,000 g for 15 min. The supernatants were harvested and treated with Ac-DEVD-pNA (2 mM) for 1–2 h at 37 °C. The absorbance at 405 nm was measured using an ELISA reader (Molecular Devices).
2.3. Western blot analysis Total proteins were extracted with RIPA lysis buffer, and protein 1549
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AML cells.
2.9. Luciferase reporter assay Cells were seeded into 96-well plates and cotransfected with the TOPflash reporter plasmid, pRL-TK reporter plasmid, and DIXDC1 siRNA or DIXDC1 expression plasmid using RNAiMAX Reagent. After transfection for 48 h, relative luciferase activity was analyzed by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
3.3. Overexpression of DIXDC1 promotes the growth of AML cells To validate the role of DIXDC1 in regulating AML cell growth, we performed gain-of-function experiments of DIXDC1 by using DIXDC1 expression plasmid. Transfection with the DIXDC1 expression plasmid significantly increased the expression of DIXDC1 in HL-60 and Kasumi1 cells (Fig. 3A). We then found that DIXDC1 overexpression significantly promoted proliferation (Fig. 3B and C) and colony formation (Fig. 3D) in AML cells. Moreover, DIXDC1 overexpression decreased the number of cells in the G0/G1 phases (Fig. 3E) and reduced apoptosis in HL-60 and Kasumi-1 cells (Fig. 3F). Overall, these results suggest that DIXDC1 promotes the growth of AML cells.
2.10. Statistical analysis The mean values and corresponding standard deviations (SD) were calculated from the results. Data are presented as mean ± SD. Oneway analysis of variance (ANOVA) and Student’s t-test were used to determine statistically significant differences. Statistical analyses were carried out by using SPSS version 19.0 software (SPSS Inc., Chicago, IL, USA). Differences with p < 0.05 were regarded as statistically significant.
3.4. DIXDC1 has a regulatory effect on Wnt/β-catenin signaling in AML cells
3. Results
DIXDC1 functions as a positive regulator of oncogenic Wnt/β-catenin signaling during tumor development [19,21]. To further investigate the molecular basis of DIXDC1 in regulating AML cell growth, we detected the regulatory effect of DIXDC1 on Wnt/β-catenin signaling in AML cells. Our results showed that DIXDC1 knockdown significantly decreased β-catenin expression (Fig. 4A) and inhibited TCFdependent transcriptional activity (Fig. 4B). Moreover, the mRNA and protein expression of cyclin D1 and c-myc, two oncogenes of Wnt/βcatenin signaling downstream targets, were also decreased by DIXDC1 silencing (Fig. 4C-F). By contrast, DIXDC1 overexpression showed a promotional effect on the activation of Wnt/β-catenin signaling and the expression of downstream target genes (Fig. 5A-F). Collectively, these results indicate that DIXDC1 promotes the activation of Wnt/β-catenin signaling in AML cells.
3.1. DIXDC1 is overexpressed in AML blasts and cell lines To investigate whether DIXDC1 is involved in the development of AML, we first detected the expression of DIXDC1 in blast derived from AML patients and AML cell lines. We found that the mRNA expression of DIXDC1 was significantly upregulated in primary AML blasts and AML cell lines compared with normal blasts (Fig. 1A). Consistent with this result, western blot analysis showed that DIXDC1 protein expression was also markedly increased in primary AML blasts and AML cell lines (Fig. 1B). These results indicate that DIXDC1 may play an important role in the progression of AML. 3.2. Silencing of DIXDC1 impedes the growth of AML cells To investigate the precise role of DIXDC1 in AML, we examined the regulatory effect of DIXDC1 on AML cell growth by gene silencing of DIXDC1 using siRNAs specifically targeting DIXDC1. The results showed that transfection with DIXDC1 siRNAs significantly decreased the expression of DIXDC1 in HL-60 and Kasumi-1 cells (Fig. 2A). We then detected the effect of DIXDC1 silencing on AML cell growth. The CCK-8 assay showed that DIXDC1 silencing significantly reduced the proliferation of HL-60 and Kasumi-1 cells (Fig. 2B and C). The colony formation assay showed that DIXDC1 knockdown significantly decreased colony formation in HL-60 and Kasumi-1 cells (Fig. 2D). Moreover, DIXDC1 knockdown induced a significant increase in the number of cells in the G0/G1 phases (Fig. 2E). In addition, DIXDC1 silencing promoted apoptosis in HL-60 and Kasumi-1 cells (Fig. 2F). These results suggest that DIXDC1 knockdown inhibits the growth of
3.5. Knockdown of β-catenin reverses the oncogenic effect of DIXDC1 in AML cells To investigate whether DIXDC1 regulates AML cell growth through acting on Wnt/β-catenin signaling, we examined the effect of β-catenin silencing on DIXDC1-induced effect. We found that transfection of βcatenin siRNA into DIXDC1-overexpressing cells significantly decreased the expression of β-catenin and blocked the promotional effect of DIXDC1 overexpression on Wnt/β-catenin signaling (Fig. 6A and B). As expected, the promotional effect of DIXDC1 on the growth of AML cells was also partially reversed by β-catenin knockdown (Fig. 6C and D). Overall, these results suggest that DIXDC1 promotes the growth of AML cells by upregulating Wnt/β-catenin signaling. Fig. 1. DIXDC1 was highly expressed in AML cells. (A) Relative mRNA expression of DIXDC1 in primary AML blasts and AML cell lines (HL60, Kasumi-1, and AML-193) was detected by RT-qPCR analysis. Relative DIXDC1 mRNA expression was calculated by normalizing against GAPDH expression. (B) Protein expression of DIXDC1 in primary AML blasts and AML cell lines was determined by western blot analysis. Normal blasts were used as control. *p < 0.05 vs. normal blasts.
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Fig. 2. DIXDC1 Knockdown represses the growth of AML cells. (A) Relative protein expression of DIXDC1 in DIXDC1 siRNA-transfected cells was detected by western blot. HL-60 and Kasumi-1 cells were transfected with DIXDC1 siRNAs (si-DIXDC1-1 or si-DIXDC1-2) or control siRNA for 48 h. The effect of DIXDC1 silencing on cell proliferation of HL-60 (B) and Kasumi-1 (C) cells was examined by using the CCK-8 assay after siRNA transfection for 24, 48, and 72 h. (D) The effect of DIXDC1 silencing on colony formation was evaluated by the colony formation assay. (E) Flow cytometric analysis of cell cycle distribution in the G0/G1 phases 48 h after DIXDC1 siRNA transfection. (F) Effect of DIXDC1 silencing on cell apoptosis was determined by measuring caspase-3 activity. *p < 0.05 vs. control siRNA.
4. Discussion
microRNA-582-5p/3p shows promising therapeutic effects on the inhibition of bladder cancer progression associated with inhibition of DIXDC1 expression [29]. Moreover, miR-186, miR-539 and miR-1271 are also suggested as tumor-suppressive miRNAs that are capable of restricting the progression of multiple tumors by targeting and inhibiting DIXDC1 [30–32]. These reports suggest that DIXDC1 may be used as potential target for development of anticancer drugs. In line with these reports, our study suggests that DIXDC1 may serve as a target gene for treating AML. In addition to its oncogenic role, the tumor-suppressor role of DIXDC1 is also reported by several studies. DIXDC1 has been reported to inhibit the migration and invasion of lung cancer cells by downregulation of Snail expression [9]. Interestingly, DIXDC1 was found to be decreased in hepatocellular carcinoma tissues, and reduced DIXDC1expression was correlated with tumor size, tumor differentiation, TNM stage, and poor survival of hepatocellular carcinoma patients [33]. The precise regulatory role of DIXDC1 in tumorigenesis may differ profoundly among cancer types or experimental conditions. Therefore, the precise role of DIXDC1 in tumorigenesis needs further investigation. Nevertheless, our study suggests a promotional role of DIXDC1 in AML progression. DIXDC1 has been reported as a positive regulator of DIXDC1 Wnt/βcatenin signaling. It has been shown that DIXDC1 promotes the activation of Wnt/β-catenin signaling by interacting with Dishevelled and Axin [11,12]. DIXDC1 contributes to regulation of neural patterning and psychiatric pathogenesis through regulation of Wnt/β-catenin signaling [11,34]. Notably, DIXDC1-medated Wnt/β-catenin signaling plays an important role in tumor progression. It is reported that DIXDC1 is co-localized with β-catenin in intestinal-type gastric carcinoma [21]. Tan et al. reported that DIXDC1 promotes the invasion and metastasis of gastric cancer cells by enhancing β-catenin accumulation and nuclear
In the present study, we provide the first compelling evidence that DIXDC1 expression is elevated in AML cells and may play a key role in AML progression. We showed that DIXDC1 overexpression promoted AML cell growth, while DIXDC1 silencing impeded AML cell growth. Moreover, our study reveals that the underlying mechanism may be associated with its regulatory effect on Wnt/β-catenin signaling (Fig. 7), which highlights an importance of DIXDC1/Wnt/β-catenin signaling axis in the progression of AML. Currently, the role of DIXDC1 in cancers has been widely studied. High expression of DIXDC1 has been detected in clinical tumor specimens and is correlated with tumor stage, lymph node metastasis, and poor prognosis [19,21]. DIXDC1 overexpression promotes the proliferation of colon cancer cells in vitro and in vivo [18]. DIXDC1 has been reported to regulate the growth, migration, and invasion of gastric cancer cells [19,27]. Moreover, a recent study reports that DIXDC1 promotes tumor proliferation and drug resistance in non-Hodgkin's lymphomas [28]. These findings indicate that DIXDC1 plays an oncogenic role in tumorigenesis. However, whether DIXDC1 is involved in the progression of AML remains unknown. In this study, we demonstrated that DIXDC1 expression was increased in AML cells. Functional experiments revealed that DIXDC1 knockdown inhibited AML cell growth, whereas DIXDC1 overexpression accelerated AML cell growth in vitro, indicating a critical role of DIXDC1 in AML. Targeting DIXDC1 has been suggested as a potential therapeutic approach for cancer therapy. Inhibiting DIXDC1 by siRNA in human gliomas has been shown to inhibit cell proliferation and migration [7]. Interestingly, microRNAs have been suggested as promising targets to modulate DIXDC1 expression, which can be used as promising agents in treatment of DIXDC1-related malignancies. Uchino et al. reported that targeting
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Fig. 3. DIXDC1 overexpression promotes the growth of AML cells. (A) Relative protein expression of DIXDC1 was detected by western blot. HL-60 and Kasumi-1 cells were transfected with pcDNA3.1/DIXDC1 vector or pcDNA3.1 empty vector (vector) for 48 h. The effect of DIXDC1 overexpression on proliferation of HL-60 (B) and Kasumi-1 (C) cells was monitored by CCK-8 assay at 24, 48, and 72 h after transfection with the DIXDC1 expression vector. (D) The effect of DIXDC1 overexpression on colony formation was detected by colony formation assay. (E) Flow cytometric analysis of cell cycle distribution in the G0/G1 phases 48 h after DIXDC1 expression vector transfection. (F) The effect of DIXDC1 overexpression on cell apoptosis was determined by measuring caspase-3 activity. *p < 0.05 vs. vector.
of DIXDC1 remains undetermined. High expression of DIXDC1 has been frequently detected in clinical specimens of gastric cancer, colon cancer, pancreatic cancer and lung cancers [8,18–21]. These studies reveal that high expression of DIXDC1 is correlated with high cell proliferation index, tumor size, tumor node metastasis and poor outcome, indicating a diagnostic and prognostic role of DIXDC1. In this study, we show that DIXDC1 is overexpressed in blasts from AML patients. Therefore, further studies should be performed to assess the relationship between DIXDC1 expression and clinicopathologic progress of AML patients. In conclusion, our results demonstrated that DIXDC1 expression was upregulated in AML cells and that high expression of DIXDC1 contributes to AML cell growth in vitro by promoting Wnt/β-catenin signaling. Our study indicates that DIXDC1 may play a critical role in the development and progression of AML. These findings provide novel insights into understanding the molecular pathogenesis of AML and suggest that DIXDC1 may be used as a potential target for AML therapy.
translocation to activate Wnt signaling [19]. Additionally, DIXDC1mediated Wnt/β-catenin signaling is also involved in the development and progression of glioma, retinoblastoma, and prostate cancer [30–32]. Consistent with these findings, our results showed that DIXDC1 overexpression increased β-catenin accumulation and expression of Wnt target genes, including cyclin D1 and c-myc. Notably, blockade of Wnt signaling by silencing of β-catenin partially reversed the DIXDC1-induced effect in AML cells, indicating that DIXDC1 regulates the growth of AML cells through acting on Wnt/β-catenin signaling. Aberrant activation of Wnt/β-catenin signaling and downstream effectors is frequently occurred in AML [26]. Therefore, high expression of DIXDC1 may contribute to aberrant activation of Wnt/β-catenin signaling during AML development and progression. Blockade of Wnt/ β-catenin signaling through targeting DIXDC1 may have potential application for treatment of AML. Interestingly, DIXDC1 regulates tumorigenesis also through other oncogenic signaling pathways, such as the Akt signaling pathway. DIXDC1 enhances the expression level of phosphorylated Akt (pAkt) to promote colon cancer cell proliferation [18]. Xu et al. reported that DIXDC1 promoted the migration and invasion of non-small-cell lung cancer cells through upregulation of pAkt expression [8]. Moreover, DIXDC1 upregulation contributes to tumor proliferation and drug resistance of non-Hodgkin's lymphomas by enhancing pAkt expression [28]. It's worth noting that Akt signaling regulates the activation of Wnt/β-catenin signaling. Therefore, Akt signaling may be involved in DIXDC1-mediated Wnt/β-catenin signaling. In this study, we focused on investigating the biological function of DIXDC1 using AML cell lines in vitro. However, the clinical significance
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgments This study was supported by Foundation Project of Shaanxi Provincial Department of Education (12JK0760). 1552
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Fig. 4. DIXDC1 knockdown inhibits Wnt/β-catenin signaling in AML cells. (A) Protein expression of β-catenin in HL-60 and Kasumi-1 cells transfected with siDIXDC1 or control siRNA for 48 h was detected by western blot. (B) TCF-dependent transcription activity was measured by using the TOPflash reporter assay. HL-60 and Kasumi-1 cells were cotransfected with the TOPflash reporter vector and si-DIXDC1 for 48 h. The effect of DIXDC1 silencing on the mRNA expression of cyclin D1 (C) and c-myc (D) was detected by RT-qPCR. The effect of DIXDC1 silencing on the protein expression of cyclin D1 (E) and c-myc (F) was detected by Western blot. *p < 0.05 vs. control siRNA.
Fig. 5. DIXDC1 overexpression promotes activation of Wnt/β-catenin signaling in AML cells. (A) Protein expression of β-catenin in HL-60 and Kasumi-1 cells transfected with pcDNA3.1/DIXDC1 vector or control vector for 48 h was detected by western blot. (B) TCF-dependent transcription activity was measured by using the TOPflash reporter assay. HL-60 and Kasumi-1 cells were cotransfected with the TOPflash reporter vector and pcDNA3.1/DIXDC1 vector for 48 h. The effect of DIXDC1 overexpression on the mRNA expression of cyclin D1 (C) and c-myc (D) was evaluated by RT-qPCR. The effect of DIXDC1 overexpression on the protein expression of cyclin D1 (E) and c-myc (F) was detected by Western blot. *p < 0.05 vs. control siRNA. 1553
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Fig. 6. Blockade of Wnt/β-catenin signaling reverses DIXDC1-meidated effect in AML cells. HL-60 and Kasumi-1 cells were cotransfected with the DIXDC1 expression vector and β-catenin siRNA for 48 h, and (A) the protein expression of β-catenin was detected by western blot. (B) Wnt/β-catenin signaling was detected by measuring TCF-dependent transcription activity by using the TOPflash reporter assay. The effect of β-catenin silencing on AML cell growth was assessed by CCK-8 (C) and colony formation (D) assays. *p < 0.05.
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Fig. 7. Diagrammatic representation of DIXDC1-mediated Wnt/β-catenin signaling in AML. DIXDC1 promotes the accumulation of β-catenin in the nucleus and activates TCF/β-catenin-dependent transcription of cyclin D1 and c-myc, thus participating in the development and progression of AML.
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