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β-catenin signaling
Chemico-Biological Interactions 305 (2019) 148–155
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ATDC contributes to sustaining the growth and invasion of glioma cells through regulating Wnt/β-catenin signaling
T
Yidong Caob, Luoning Shic, Maode Wanga, Juanru Houd, Yanqiang Weid, Changwang Dua,* a
Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China Department of Neurosurgery, Tangdu Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China c Department of Kidney Transplantation, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China d Department of Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: ATDC β-catenin Dvl2 Glioma Wnt
Accumulating evidence has documented that ataxia-telangiectasia group D complementing gene (ATDC) is aberrantly expressed in various cancers and is associated with cancer development and progression. However, little is known about the role of ATDC in glioma tumorigenesis. In this study, we aimed to explore the biological function and regulatory mechanism of ATDC in glioma. We found that ATDC expression was highly upregulated in glioma cell lines. Knockdown of ATDC significantly inhibited the growth and invasion of glioma cells. In contrast, overexpression of ATDC markedly promoted the growth and invasion of glioma cells. Moreover, our results showed that inhibition of ATDC reduced the expression levels of Dishevelled 2 (Dvl2) and β-catenin and impeded the activation of Wnt/β-catenin signaling, whereas overexpression of ATDC showed the opposite effect. Knockdown of Dvl2 significantly blocked the promotion effect of ATDC overexpression on activation of Wnt/βcatenin signaling. In addition, silencing of β-catenin partially reversed the oncogenic effect of ATDC overexpression in glioma cells. Taken together, out study reveals an oncogenic role of ATDC that drives the growth and invasion of glioma by modulating the Wnt/Dvl2/β-catenin signaling pathway, suggesting a potential therapeutic target for treatment of glioma.
1. Introduction Glioma is one of the most common primary malignant tumors of the central nervous system, with increasing incidence and mortality worldwide [1]. Despite advances in cancer diagnosis and treatment over the past decades, glioma is still incurable, and the survival rate of glioma remains low [2]. Glioma is resistant to conventional chemotherapy and radiotherapy, and the efficacy of current therapies is limited [3]. Therefore, it is essential to gain a better understanding of the molecular mechanism that governs glioma growth and metastasis, which will help to improve therapeutic approaches for the treatment of glioma. Ataxia-telangiectasia group D complementing gene (ATDC), also known as tripartite motif (TRIM) 29, is a member of the TRIM family that plays an important role in tumor progression [4–6]. ATDC is located at chromosome 11q23 and was originally described in the search
for the genes responsible for the genetic disorder ataxia-telangiectasia [7]. ATDC is normally expressed in various tissues, including the lung, thymus, prostate, testis, and placenta, but its expression is undetectable in the brain, heart, pancreas, ovary, and spleen [8]. Increasing studies have shown that ATDC is aberrantly expressed in a diverse number of human malignant tumors and plays a pivotal role in tumorigenesis [9,10]. High expression of ATDC correlates with poor prognosis, tumor metastasis, and poor survival of cancer patients [11–13]. Functional research has documented that ATDC regulates the proliferation, migration/invasion, and radioresistance of cancer cells by intervening in various signaling pathways, including Wnt/β-catenin, nuclear factor (NF)-κB, Akt, and p53 [14–17]. Therefore, ATDC is a promising molecular target for cancer treatment. The canonical Wnt/β-catenin signaling pathway is a conserved molecular mechanism that broadly regulates a variety of genes that are involved in numerous physiological and pathological processes [18].
Abbreviations: ATDC, ataxia-telangiectasia group D complementing gene; Dvl2, Dishevelled 2; TCF, T cell factor; TRIM, tripartite motif; GSK-3β, glycogen synthase kinase-3β; CCK-8, cell counting kit-8; FBS, fetal bovine serum; RT-qPCR, real-time quantitative polymerase chain reaction * Corresponding author. Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, No.255 Wild Goose Pagoda Road, Xi'an, Shaanxi Province, 710061, China. E-mail address: [email protected] (C. Du). https://doi.org/10.1016/j.cbi.2019.03.033 Received 19 October 2018; Received in revised form 7 March 2019; Accepted 26 March 2019 Available online 29 March 2019 0009-2797/ © 2019 Elsevier B.V. All rights reserved.
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Under resting conditions, β-catenin protein is degraded by the destruction complex APC/Axin/glycogen synthase kinase (GSK)-3β, and this regulatory effect is dismissed when Wnt ligand binds to LRP and Frizzled co-receptors to recruit Dishevelled (Dvl) and Axin [19]. Subsequently, β-catenin transfers to the nucleus and activates T cell factor (TCF)-dependent transcription. Aberrant activation of Wnt/β-catenin signaling is oncogenic in various cancers, including glioma [20,21]. Therefore, targeting key molecules that interfere with Wnt/β-catenin signaling may present a potential therapeutic strategy for glioma treatment [22]. Although ATDC is emerging as a critical regulator in tumorigenesis of various tumors, its relevance in glioma remains largely unknown. In the present study, we aimed to explore the biological function and regulatory mechanism of ATDC in glioma. We found that ATDC expression was highly upregulated in glioma cell lines. Functional experiments showed ATDC knockdown inhibited the growth and invasion of glioma cells, while ATDC overexpression promoted the growth and invasion of glioma cells. Moreover, we found that inhibition of ATDC reduced the expression levels of Dvl2 and β-catenin and impeded the activation of Wnt/β-catenin signaling, while ATDC overexpression showed the opposite effect. Knockdown of Dvl2 significantly blocked the promotion effect of ATDC-induced activation of Wnt/β-catenin signaling. Additionally, silencing of β-catenin partially reversed the oncogenic effect of ATDC overexpression in glioma cells. Overall, out study reveals an oncogenic role of ATDC that drives the growth and invasion of glioma by modulating the Wnt/β-catenin signaling pathway, suggesting a potential therapeutic target for treatment of glioma.
was used as the internal referee for normalization of gene expression. Relative gene expression was calculated by the 2−ΔΔCt method. 2.4. Western blot analysis Cellular extracts were prepared with a lysis buffer containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in cell lysates were determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels and separated following electrophoresis. The separated proteins were transferred to a PVDF membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The membrane was then blocked with 5% dried milk in Tris-buffered saline with Tween (TBST) at 37 °C for 45 min. Afterwards, the membrane was incubated with primary antibodies against ATDC (Abcam, Cambridge, MA, USA), β-catenin (Cell Signaling Technology, Danvers, MA USA), Dvl2 (Abcam), and GAPDH (Abcam) at 4 °C. After incubation overnight, the membrane was washed thrice with TBST and probed with HRP-labeled secondary antibody (Abcam) for 1 h at room temperature. Thereafter, protein bands were visualized using Pierce ECL Western Blotting Kit (Pierce) and band intensity was analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). 2.5. Cell proliferation assay Cell proliferation was detected by using the Cell Counting Kit-8 (CCK-8) assay. In brief, cells were seeded into 96-well plates and cultured overnight. A solution of CCK-8 reagents (Beyotime Biotechnology, Shanghai, China) was added to each well at 10 μL/well. After incubation for 2 h at 37 °C, the absorbance at 450 nm (OD 450 nm) was assessed with a microplate reader (Bio-Rad, Hercules, CA, USA).
2. Materials and methods 2.1. Cell culture Human glioma cell lines A172, U-118, U-87, and SW-1088 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured per the manufacturer's recommended culture methods. Briefly, glioma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Rockville, MD, USA) in supplement with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mix. Human normal astrocyte cell line HA1800 was purchased from BeNa Culture Collection (BNCC, Beijing, China) and grown in DMEM containing 10% FBS and 1% penicillin/streptomycin mix. Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.
2.6. Colony formation assay
2.2. Cell transfection
Cell invasion ability was evaluated by transwell invasion assay. In brief, the upper surfaces of 24-well transwell inserts (8 μm pore size) were precoated with Matrigel. Cells were plated into the upper chamber with serum-free medium, while the lower chamber was filled with normal medium containing FBS. After 24 h incubation at 37 °C, the cells remaining in the upper chamber were wiped off by cotton swabs, and cells that had invaded on the lower membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Invasive cells were observed and counted under an inverted microscope.
Cells (1000 cells) were suspended in top soft agar layer (0.3% soft agar) and then seeded into 6-well plates precoated with 0.6% base agar. Cells were allowed to grow at 37 °C for 14 days. Until that time, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies were then observed and counted under an inverted microscope. 2.7. Cell invasion assay
ATDC shRNA expression vectors were purchased from GenePharma (Shanghai, China). Dvl2 and β-catenin shRNA expression vectors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The cDNA fragments of ATDC expression sequences were inserted into pcDNA3.1 vector to generate expression vectors. These vectors were transfected into cells using Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocols. Transfection efficacy was determined by Western blot analysis.
2.8. Luciferase reporter assay 2.3. RNA extraction, reverse transcription, and real-time quantitative polymerase chain reaction (RT-qPCR)
The activation of Wnt/β-catenin signaling was determined by measuring TCF-mediated transcriptional activity using the TOPflash reporter assay. In brief, cells were seeded in 12-well plates and cotransfected with TOPflash reporter vector contained β-catenin/TCFbinding sites or the FOPflash reporter plasmid contained mutant β-catenin/TCF-binding sites, Renilla-TK luciferase vector, and ATDC shRNA vector or ATDC expression vector. After incubation of 48 h at 37 °C, the luciferase activity was quantified with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
Total RNA samples were prepared using TRIzol reagents (Invitrogen, Carlsbad, CA, USA) per the manufacturer's protocols. The cDNA was obtained by reverse transcription using the PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). RT-qPCR was carried out using SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems) following these thermal cycling parameters: 95 °C, 10 min; 40 cycles of 95 °C, 15 s; and 60 °C, 1 min. GAPDH 149
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Fig. 1. Expression of ATDC in glioma cell lines. (A) Relative mRNA expression of ATDC was measured by RT-qPCR in human glioma cell lines and a normal cell line. (B) Protein expression of ATDC was detected by Western blot in human glioma cell lines and a normal cell line. Human glioma cell lines, including A172, U-118, U-87, and SW-1088, were used in this study. Normal astrocyte cell line HA1800 served as control. *p < 0.05 vs. HA1800.
invasive ability of U-87 and SW-1088 cells (Fig. 3G and H). Collectively, these data indicate an oncogenic role of ATDC by promoting glioma cell growth and invasion.
2.9. Data analysis Data are presented as the mean ± standard deviation (SD). Significance of differences between two groups were analyzed with Student's t-test. Significance of differences among multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple-comparison tests. Statistical analysis was performed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was accepted at p < 0.05.
3.4. ATDC is involved in regulating Wnt/β-catenin signaling in glioma cells To further explore the molecular basis of ATDC in regulating glioma cell growth and invasion, we detected the regulatory effect of ATDC on Wnt/β-catenin signaling, an important downstream signal of ATDC in regulating tumorigenesis [24]. The results showed that ATDC knockdown significantly downregulated the protein expression of β-catenin in A172 and U-118 cells and decreased Wnt/β-catenin-dependent TCF transcriptional activity (Fig. 4A and B). In contrast, overexpression of ATDC showed the opposite effect (Fig. 4C and D). These results suggest that ATDC is involved in regulating Wnt/β-catenin signaling in glioma cells.
3. Results 3.1. ATDC is overexpressed in glioma cell lines Accumulating evidence has documented that ATDC is overexpressed in various human cancers [14,23]. However, its expression in human glioma remains unclear. Herein, we first examined the expression levels of ATDC in a panel of human glioma cell lines, including A172, U-118, U-87, and SW-1088. The results showed that ATDC was significantly upregulated in glioma cell lines compared with the normal astrocyte cell line HA1800 at mRNA and protein levels (Fig. 1A and B). These data indicate that ATDC is overexpressed in glioma cell lines.
3.5. ATDC promotes Wnt/β-catenin signaling via upregulation of Dvl2 expression To elucidate the molecular mechanism of ATDC in regulating Wnt/ β-catenin signaling, we detected the regulatory effect of ATDC on Dvl2, an upstream regulator of β-catenin. We found that silencing of ATDC significantly downregulated the expression of Dvl2 in A172 and U118 cells (Fig. 5A). In contrast, overexpression of ATDC markedly upregulated the expression of Dvl2 in U-87 and SW-1088 cells (Fig. 5B). Notably, knockdown of Dvl2 significantly reversed the promotion effect of ATDC overexpression on activation of Wnt/β-catenin signaling (Fig. 5C–E). These results suggest that ATDC regulates Wnt/β-catenin signaling via mediating Dvl2 expression.
3.2. ATDC knockdown suppresses the growth and invasion of glioma cells in vitro To investigate the biological effect of ATDC in glioma cells, we performed loss-of-function experiments of ATDC in A172 and U118 cells, which showed higher expression of ATDC. Transfection of ATDC shRNA expressing vector significantly downregulated the expression of ATDC in A172 and U-118 cells (Fig. 2A and B). We then detected the effect of ATDC knockdown on the growth of glioma cells by CCK-8 and colony formation assays. The CCK-8 results showed that ATDC knockdown resulted in a significant decrease in glioma cell proliferation (Fig. 2C and D). In addition, ATDC knockdown suppressed the colony-formation ability of A172 and U-118 cells (Fig. 2E and F). Moreover, the invasive ability of A172 and U-118 cells was also markedly decreased by ATDC knockdown (Fig. 2G and H). These results suggest a tumor suppressive effect of ATDC knockdown by inhibiting the growth and invasion of glioma cells in vitro.
3.6. Inhibition of β-catenin suppresses ATDC-mediated oncogenic effects To confirm activation of Wnt/β-catenin signaling contributes to ATDC-mediated oncogenic effects, we examined the effect of the inhibition of Wnt/β-catenin signaling on ATDC-induced cell proliferation and invasion. We showed that transfection of β-catenin shRNA vector significantly decreased the expression of β-catenin in ATDC expression vector-transfected cells (Fig. 6A). Moreover, ATDC-induced upregulation of TCF transcriptional activity was significantly abolished by βcatenin knockdown (Fig. 6B). As expected, blocking Wnt/β-catenin signaling partially reversed the oncogenic effect of ATDC on promoting glioma cell proliferation and invasion (Fig. 6C–E). These results suggest that ATDC promotes the proliferation and invasion through regulating Wnt/β-catenin signaling.
3.3. ATDC overexpression promotes the growth and invasion of glioma cells in vitro To confirm the biological function of ATDC in regulating glioma cell growth and invasion, we performed gain-of-function experiments of ATDC in U-87 and SW-1088 cells, which showed lower expression of ATDC. The overexpression of ATDC in ATDC expression vector-transfected cells was confirmed by RT-qPCR and Western blot (Fig. 3A and B). We found that ATDC overexpression significantly promoted the proliferation and colony formation of U-87 and SW-1088 cells (Fig. 3C–F). Additionally, overexpression of ATDC upregulated the
4. Discussion The overexpression of ATDC has been found to be associated with many types of human cancers. However, little is known about the role of ATDC in glioma. The present study for the first time reports an important role of ATDC in glioma. Our findings demonstrate that ATDC is 150
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Fig. 2. ATDC knockdown inhibited glioma cell growth and invasion. A172 and U-118 cells were transfected with ATDC shRNA vector and incubated for 48 h. (A) Relative ATDC mRNA expression was detected by RT-qPCR. (B) Protein expression of ATDC was determined by Western blot. (C) Effect of ATDC knockdown on A172 cell proliferation was detected by CCK-8 assay. (D) Effect of ATDC knockdown on U-118 cells was assessed by CCK-8 assay. (E, F) Effect of ATDC knockdown on colony formation of A172 and U-118 cells was evaluated by colony formation assay. (G, H) Effect of ATDC knockdown on invasive ability of A172 and U-118 cells was examined by transwell invasion assay. *p < 0.05 vs. control shRNA.
and prognosis. ATDC promotes the growth and metastasis of pancreatic cancer in vitro and in vivo [14]. Moreover, overexpression of ATDC in Kras-mice accelerates the formation of pancreatic intraepithelial neoplasia and the development of invasive and metastatic cancers in vivo [24]. In addition, multiple evidence shows that ATDC drives the proliferation, migration, and invasion of various cancers, including lung cancer, osteosarcoma, colorectal cancer, and bladder cancer [11,17,29–31]. Notably, ATDC has radioprotective function, which promotes radioresistance through an interaction with RNF8 ubiquitin ligase [5,32,33]. ATDC reportedly inhibits ionizing radiation-induced DNA damage by assembling DNA repair proteins into damaged chromatin [34]. Interestingly, knockdown of ATDC increases the chemosensitivity of lung cancer cells to cisplatin [35]. Collectively, all these findings suggest an oncogenic role of ATDC in tumorigenesis. Consistently, our results showed a high expression pattern of ATDC in glioma cell lines and demonstrated that ATDC promoted the growth and
upregulated in glioma cells, and functional experiments showed that knockdown of ATDC suppresses the growth and invasion of glioma cells; while overexpression of ATDC promotes glioma cell growth and invasion of glioma cells, indicating an oncogenic role of ATDC in glioma. Moreover, we elucidated that the underlying mechanism is associated with its regulatory effect on Dvl2, which promotes the accumulation of β-catenin and activation of TCF-dependent transcription (Fig. 7). ATDC plays an important role in various biological processes, such virus infection and immune response [25–27]. Particularly, the role of ATDC in tumorigenesis has been highlighted in recent years [9,10]. High expression levels of ATDC have been detected in tumor tissues of various cancers, including gastric cancer, esophageal squamous cell carcinoma, and pancreatic cancer, and it correlates with tumor progression, lymph node metastasis, and a lower survival rate [12,13,28]. Thus, ATDC is suggested as a potential biomarker for cancer diagnosis 151
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Fig. 3. ATDC overexpression promoted glioma cell growth and invasion. U-87 and SW-1088 cells were transfected with ATDC expression vectors and incubated for 48 h. (A) Relative ATDC mRNA expression was detected by RT-qPCR. (B) Protein expression of ATDC was determined by Western blot. (C) Effect of ATDC overexpression on U-87 cell proliferation was examined by CCK-8 assay. (D) Effect of ATDC overexpression on SW-1088 cell proliferation was detected by CCK-8 assay. (E, F) Effect of ATDC overexpression on colony formation of U-87 and SW1088 cells was assessed by colony formation assay. (G, H) Effect of ATDC overexpression on invasive ability of U-87 and SW-1088 cells was measured by transwell invasion assay. *p < 0.05 vs. vector.
Fig. 4. ATDC regulates Wnt/β-catenin signaling in glioma cells. (A) Effect of ATDC knockdown on βcatenin expression was detected by Western blot in A172 and U-118 cells. *p < 0.05 vs. control shRNA. (B) Effect of ATDC knockdown on TCF transcriptional activity was determined by β-catenin-responsive TOPflash reporter assay. *p < 0.05 vs. control shRNA. (C) Effect of ATDC overexpression on β-catenin expression was examined by Western blot in U87 and SW-1088 cells. *p < 0.05 vs. vector. (D) Effect of ATDC overexpression on TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. *p < 0.05 vs. vector.
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Fig. 5. ATDC promotes Wnt/β-catenin signaling via upregulation of Dvl2 expression. (A) Effect of ATDC knockdown on Dvl2 expression was detected by Western blot in A172 and U-118 cells. *p < 0.05 vs. control shRNA. (B) Effect of ATDC overexpression on Dvl2 expression was detected by Western blot in U-87 and SW1088 cells. *p < 0.05 vs. vector. (C) Western blot detection of Dvl2 expression in U-87 and SW-1088 cells cotransfected with ATDC expression vector and Dvl2 shRNA expression vector. (D) Effect of Dvl2 knockdown of ATDC-induced β-catenin expression was detected by Western blot. (E) Effect of Dvl2 knockdown on ATDCinduced TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. *p < 0.05.
inhibitory effect on the tumor suppressors p53 and p63 [16,39,40]. In addition, ATDC is also involved in activation of NF-κB, extracellular signal-regulated protein kinase/c-Jun N-terminal kinase, and Janus kinase 2/signal transducer and activator of transcription 3 signaling pathways that are oncogenic in tumor progression [29,41,42]. Notably, high expression of ATDC drives the activation of the oncogenic Wnt/βcatenin signaling pathway [14,24,43]. ATDC expression is positively correlated with abnormal expression of β-catenin in squamous cell carcinoma tissues of non-small cell lung cancer [44]. Interestingly, ATDC is shown to promote β-catenin accumulation via induction of Dvl2, which inhibits GSK-3β-mediated β-catenin degradation [14]. However, whether ATDC exerts oncogenic function in glioma through mediating Wnt/β-catenin signaling remains unclear. Herein, we found that ATDC promotes the accumulation of β-catenin and increases the activation of Wnt/β-catenin signaling. Importantly, we identified that the underlying mechanism was associated with its regulatory effect on Dvl2. Dvl2 is a key component of the Wnt signaling pathway, which binds to the Axin/GSK-3β complex and restricts GSK-3β-dependent phosphorylation and degradation of β-catenin. Study shows that Dvl2 is overexpressed in human glioma and promotes the proliferation of glioma cells [45]. Our results indicate that ATDC contributes to the regulation of Dvl2 expression in glioma. Importantly, we demonstrated that inhibition of Dvl2 or β-catenin significantly reversed the oncogenic effect of ATDC in glioma cells, implying that the ATDC/Dvl2/β-catenin axis is involved in regulating glioma cell growth and invasion. In conclusion, our results demonstrate that ATDC promotes the
invasion of glioma cells in vitro, supporting an oncogenic role of ATDC. Our study highlights an important role of ATDC in tumorigenesis and suggests ATDC as a potential therapeutic target for cancer treatment. Although the oncogenic effect of ATDC receives wide attention, a tumor suppressive role of ATDC is also reported by several studies. It is reported that ATDC is undetectable in multiple cancer cell lines, including osteosarcoma, breast cancer, and retinoblastoma [36]. Overexpression of ATDC inhibits the colony-forming efficiency of these cancer cell lines in soft agar in vitro [36]. ATDC expression is decreased in primary breast tumor tissues and reduced ATDC expression is associated with increased tumor size, grade, and metastatic characteristics and reduced relapse-free survival [37]. ATDC suppresses the invasive behavior of breast cancer by inhibiting TWIST1, a critical driver of the epithelial-mesenchymal transition [37]. In addition, loss of ATDC expression promotes the malignant transformation of normal breast luminal cells and contributes to breast cancer progression in premenopausal women [38]. Therefore, these findings indicate that ATDC modulates breast cancer progression in a tumor-suppressive mechanism distinct from its regulatory mechanism in regulating oncogenic progression. ATDC has been shown to regulate tumor progression through intervening in a diverse number of signaling pathways. ATDC exerts its oncogenic effects in bladder cancer by silencing of the tumor suppressor PTEN via a series of signal transductions [23]. Thereby, ATDC promotes the activation of PI3K/Akt signaling, the downstream target of PTEN [15]. Moreover, the oncogenic role of ATDC is also associated with its 153
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Fig. 6. Knockdown of β-catenin abrogates ATDC-mediated oncogenic effects in glioma cells. U-87 and SW-1088 cells were cotransfected with ATDC expression vector and β-catenin shRNA vector and incubated for 48 h. (A) Protein expression of β-catenin was examined by Western blot. (B) TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. (C) Effect of β-catenin knockdown on ATDC-induced glioma cell proliferation was detected by CCK-8 assay. (D, E) Effect of β-catenin knockdown on ATDC-induced glioma cell invasion was measured by transwell invasion assay. *p < 0.05.
as a promising target for the development of novel therapeutic approaches for gliomas. Conflicts of interest The authors declare that they have no conflict of interest. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.cbi.2019.03.033. References [1] D. Ricard, A. Idbaih, F. Ducray, M. Lahutte, K. Hoang-Xuan, J.Y. Delattre, Primary brain tumours in adults, Lancet 379 (2012) 1984–1996. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA A Cancer J. Clin. 68 (2018) 7–30. [3] D.L. Schonberg, D. Lubelski, T.E. Miller, J.N. Rich, Brain tumor stem cells: molecular characteristics and their impact on therapy, Mol. Aspect. Med. 39 (2014) 82–101. [4] S. Hatakeyama, TRIM family proteins: roles in autophagy, immunity, and carcinogenesis, Trends Biochem. Sci. 42 (2017) 297–311. [5] E.A. Leonhardt, L.N. Kapp, B.R. Young, J.P. Murnane, Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC), Genomics 19 (1994) 130–136. [6] S. Hatakeyama, TRIM proteins and cancer, Nat. Rev. Canc. 11 (2011) 792–804. [7] L.N. Kapp, R.B. Painter, L.C. Yu, N. van Loon, C.W. Richard 3rd, M.R. James, D.R. Cox, J.P. Murnane, Cloning of a candidate gene for ataxia-telangiectasia group D, Am. J. Hum. Genet. 51 (1992) 45–54. [8] Y. Hosoi, L.N. Kapp, Expression of a candidate ataxia-telangieetasia group D gene in cultured fibroblast cell lines and human tissues, Int. J. Radiat. Biol. 66 (1994) S71–S76.
Fig. 7. Diagram representation of ATDC-mediated Dvl2/β-catenin/TCF signaling in glioma cells. High expression of ATDC upregulates Dvl2 expression, resulting in accumulation of β-catenin in the nucleus and subsequent activation of TCF-dependent transcription, which contributes to the development and progression of glioma.
growth and invasion of glioma cells through upregulating Dvl2 and βcatenin expression, indicating an important role of ATDC in regulating Wnt/β-catenin signaling in glioma. These findings of the present study provide novel mechanistic insights into understanding the function of ATDC in glioma cell growth and invasion. Therefore, ATDC may serve 154
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ATDC contributes to sustaining the growth and invasion of glioma cells through regulating Wnt/β-catenin signaling
T
Yidong Caob, Luoning Shic, Maode Wanga, Juanru Houd, Yanqiang Weid, Changwang Dua,* a
Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China Department of Neurosurgery, Tangdu Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China c Department of Kidney Transplantation, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China d Department of Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: ATDC β-catenin Dvl2 Glioma Wnt
Accumulating evidence has documented that ataxia-telangiectasia group D complementing gene (ATDC) is aberrantly expressed in various cancers and is associated with cancer development and progression. However, little is known about the role of ATDC in glioma tumorigenesis. In this study, we aimed to explore the biological function and regulatory mechanism of ATDC in glioma. We found that ATDC expression was highly upregulated in glioma cell lines. Knockdown of ATDC significantly inhibited the growth and invasion of glioma cells. In contrast, overexpression of ATDC markedly promoted the growth and invasion of glioma cells. Moreover, our results showed that inhibition of ATDC reduced the expression levels of Dishevelled 2 (Dvl2) and β-catenin and impeded the activation of Wnt/β-catenin signaling, whereas overexpression of ATDC showed the opposite effect. Knockdown of Dvl2 significantly blocked the promotion effect of ATDC overexpression on activation of Wnt/βcatenin signaling. In addition, silencing of β-catenin partially reversed the oncogenic effect of ATDC overexpression in glioma cells. Taken together, out study reveals an oncogenic role of ATDC that drives the growth and invasion of glioma by modulating the Wnt/Dvl2/β-catenin signaling pathway, suggesting a potential therapeutic target for treatment of glioma.
1. Introduction Glioma is one of the most common primary malignant tumors of the central nervous system, with increasing incidence and mortality worldwide [1]. Despite advances in cancer diagnosis and treatment over the past decades, glioma is still incurable, and the survival rate of glioma remains low [2]. Glioma is resistant to conventional chemotherapy and radiotherapy, and the efficacy of current therapies is limited [3]. Therefore, it is essential to gain a better understanding of the molecular mechanism that governs glioma growth and metastasis, which will help to improve therapeutic approaches for the treatment of glioma. Ataxia-telangiectasia group D complementing gene (ATDC), also known as tripartite motif (TRIM) 29, is a member of the TRIM family that plays an important role in tumor progression [4–6]. ATDC is located at chromosome 11q23 and was originally described in the search
for the genes responsible for the genetic disorder ataxia-telangiectasia [7]. ATDC is normally expressed in various tissues, including the lung, thymus, prostate, testis, and placenta, but its expression is undetectable in the brain, heart, pancreas, ovary, and spleen [8]. Increasing studies have shown that ATDC is aberrantly expressed in a diverse number of human malignant tumors and plays a pivotal role in tumorigenesis [9,10]. High expression of ATDC correlates with poor prognosis, tumor metastasis, and poor survival of cancer patients [11–13]. Functional research has documented that ATDC regulates the proliferation, migration/invasion, and radioresistance of cancer cells by intervening in various signaling pathways, including Wnt/β-catenin, nuclear factor (NF)-κB, Akt, and p53 [14–17]. Therefore, ATDC is a promising molecular target for cancer treatment. The canonical Wnt/β-catenin signaling pathway is a conserved molecular mechanism that broadly regulates a variety of genes that are involved in numerous physiological and pathological processes [18].
Abbreviations: ATDC, ataxia-telangiectasia group D complementing gene; Dvl2, Dishevelled 2; TCF, T cell factor; TRIM, tripartite motif; GSK-3β, glycogen synthase kinase-3β; CCK-8, cell counting kit-8; FBS, fetal bovine serum; RT-qPCR, real-time quantitative polymerase chain reaction * Corresponding author. Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, No.255 Wild Goose Pagoda Road, Xi'an, Shaanxi Province, 710061, China. E-mail address: [email protected] (C. Du). https://doi.org/10.1016/j.cbi.2019.03.033 Received 19 October 2018; Received in revised form 7 March 2019; Accepted 26 March 2019 Available online 29 March 2019 0009-2797/ © 2019 Elsevier B.V. All rights reserved.
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Under resting conditions, β-catenin protein is degraded by the destruction complex APC/Axin/glycogen synthase kinase (GSK)-3β, and this regulatory effect is dismissed when Wnt ligand binds to LRP and Frizzled co-receptors to recruit Dishevelled (Dvl) and Axin [19]. Subsequently, β-catenin transfers to the nucleus and activates T cell factor (TCF)-dependent transcription. Aberrant activation of Wnt/β-catenin signaling is oncogenic in various cancers, including glioma [20,21]. Therefore, targeting key molecules that interfere with Wnt/β-catenin signaling may present a potential therapeutic strategy for glioma treatment [22]. Although ATDC is emerging as a critical regulator in tumorigenesis of various tumors, its relevance in glioma remains largely unknown. In the present study, we aimed to explore the biological function and regulatory mechanism of ATDC in glioma. We found that ATDC expression was highly upregulated in glioma cell lines. Functional experiments showed ATDC knockdown inhibited the growth and invasion of glioma cells, while ATDC overexpression promoted the growth and invasion of glioma cells. Moreover, we found that inhibition of ATDC reduced the expression levels of Dvl2 and β-catenin and impeded the activation of Wnt/β-catenin signaling, while ATDC overexpression showed the opposite effect. Knockdown of Dvl2 significantly blocked the promotion effect of ATDC-induced activation of Wnt/β-catenin signaling. Additionally, silencing of β-catenin partially reversed the oncogenic effect of ATDC overexpression in glioma cells. Overall, out study reveals an oncogenic role of ATDC that drives the growth and invasion of glioma by modulating the Wnt/β-catenin signaling pathway, suggesting a potential therapeutic target for treatment of glioma.
was used as the internal referee for normalization of gene expression. Relative gene expression was calculated by the 2−ΔΔCt method. 2.4. Western blot analysis Cellular extracts were prepared with a lysis buffer containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in cell lysates were determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels and separated following electrophoresis. The separated proteins were transferred to a PVDF membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The membrane was then blocked with 5% dried milk in Tris-buffered saline with Tween (TBST) at 37 °C for 45 min. Afterwards, the membrane was incubated with primary antibodies against ATDC (Abcam, Cambridge, MA, USA), β-catenin (Cell Signaling Technology, Danvers, MA USA), Dvl2 (Abcam), and GAPDH (Abcam) at 4 °C. After incubation overnight, the membrane was washed thrice with TBST and probed with HRP-labeled secondary antibody (Abcam) for 1 h at room temperature. Thereafter, protein bands were visualized using Pierce ECL Western Blotting Kit (Pierce) and band intensity was analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). 2.5. Cell proliferation assay Cell proliferation was detected by using the Cell Counting Kit-8 (CCK-8) assay. In brief, cells were seeded into 96-well plates and cultured overnight. A solution of CCK-8 reagents (Beyotime Biotechnology, Shanghai, China) was added to each well at 10 μL/well. After incubation for 2 h at 37 °C, the absorbance at 450 nm (OD 450 nm) was assessed with a microplate reader (Bio-Rad, Hercules, CA, USA).
2. Materials and methods 2.1. Cell culture Human glioma cell lines A172, U-118, U-87, and SW-1088 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured per the manufacturer's recommended culture methods. Briefly, glioma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Rockville, MD, USA) in supplement with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mix. Human normal astrocyte cell line HA1800 was purchased from BeNa Culture Collection (BNCC, Beijing, China) and grown in DMEM containing 10% FBS and 1% penicillin/streptomycin mix. Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.
2.6. Colony formation assay
2.2. Cell transfection
Cell invasion ability was evaluated by transwell invasion assay. In brief, the upper surfaces of 24-well transwell inserts (8 μm pore size) were precoated with Matrigel. Cells were plated into the upper chamber with serum-free medium, while the lower chamber was filled with normal medium containing FBS. After 24 h incubation at 37 °C, the cells remaining in the upper chamber were wiped off by cotton swabs, and cells that had invaded on the lower membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Invasive cells were observed and counted under an inverted microscope.
Cells (1000 cells) were suspended in top soft agar layer (0.3% soft agar) and then seeded into 6-well plates precoated with 0.6% base agar. Cells were allowed to grow at 37 °C for 14 days. Until that time, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies were then observed and counted under an inverted microscope. 2.7. Cell invasion assay
ATDC shRNA expression vectors were purchased from GenePharma (Shanghai, China). Dvl2 and β-catenin shRNA expression vectors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The cDNA fragments of ATDC expression sequences were inserted into pcDNA3.1 vector to generate expression vectors. These vectors were transfected into cells using Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocols. Transfection efficacy was determined by Western blot analysis.
2.8. Luciferase reporter assay 2.3. RNA extraction, reverse transcription, and real-time quantitative polymerase chain reaction (RT-qPCR)
The activation of Wnt/β-catenin signaling was determined by measuring TCF-mediated transcriptional activity using the TOPflash reporter assay. In brief, cells were seeded in 12-well plates and cotransfected with TOPflash reporter vector contained β-catenin/TCFbinding sites or the FOPflash reporter plasmid contained mutant β-catenin/TCF-binding sites, Renilla-TK luciferase vector, and ATDC shRNA vector or ATDC expression vector. After incubation of 48 h at 37 °C, the luciferase activity was quantified with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
Total RNA samples were prepared using TRIzol reagents (Invitrogen, Carlsbad, CA, USA) per the manufacturer's protocols. The cDNA was obtained by reverse transcription using the PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). RT-qPCR was carried out using SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems) following these thermal cycling parameters: 95 °C, 10 min; 40 cycles of 95 °C, 15 s; and 60 °C, 1 min. GAPDH 149
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Fig. 1. Expression of ATDC in glioma cell lines. (A) Relative mRNA expression of ATDC was measured by RT-qPCR in human glioma cell lines and a normal cell line. (B) Protein expression of ATDC was detected by Western blot in human glioma cell lines and a normal cell line. Human glioma cell lines, including A172, U-118, U-87, and SW-1088, were used in this study. Normal astrocyte cell line HA1800 served as control. *p < 0.05 vs. HA1800.
invasive ability of U-87 and SW-1088 cells (Fig. 3G and H). Collectively, these data indicate an oncogenic role of ATDC by promoting glioma cell growth and invasion.
2.9. Data analysis Data are presented as the mean ± standard deviation (SD). Significance of differences between two groups were analyzed with Student's t-test. Significance of differences among multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple-comparison tests. Statistical analysis was performed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was accepted at p < 0.05.
3.4. ATDC is involved in regulating Wnt/β-catenin signaling in glioma cells To further explore the molecular basis of ATDC in regulating glioma cell growth and invasion, we detected the regulatory effect of ATDC on Wnt/β-catenin signaling, an important downstream signal of ATDC in regulating tumorigenesis [24]. The results showed that ATDC knockdown significantly downregulated the protein expression of β-catenin in A172 and U-118 cells and decreased Wnt/β-catenin-dependent TCF transcriptional activity (Fig. 4A and B). In contrast, overexpression of ATDC showed the opposite effect (Fig. 4C and D). These results suggest that ATDC is involved in regulating Wnt/β-catenin signaling in glioma cells.
3. Results 3.1. ATDC is overexpressed in glioma cell lines Accumulating evidence has documented that ATDC is overexpressed in various human cancers [14,23]. However, its expression in human glioma remains unclear. Herein, we first examined the expression levels of ATDC in a panel of human glioma cell lines, including A172, U-118, U-87, and SW-1088. The results showed that ATDC was significantly upregulated in glioma cell lines compared with the normal astrocyte cell line HA1800 at mRNA and protein levels (Fig. 1A and B). These data indicate that ATDC is overexpressed in glioma cell lines.
3.5. ATDC promotes Wnt/β-catenin signaling via upregulation of Dvl2 expression To elucidate the molecular mechanism of ATDC in regulating Wnt/ β-catenin signaling, we detected the regulatory effect of ATDC on Dvl2, an upstream regulator of β-catenin. We found that silencing of ATDC significantly downregulated the expression of Dvl2 in A172 and U118 cells (Fig. 5A). In contrast, overexpression of ATDC markedly upregulated the expression of Dvl2 in U-87 and SW-1088 cells (Fig. 5B). Notably, knockdown of Dvl2 significantly reversed the promotion effect of ATDC overexpression on activation of Wnt/β-catenin signaling (Fig. 5C–E). These results suggest that ATDC regulates Wnt/β-catenin signaling via mediating Dvl2 expression.
3.2. ATDC knockdown suppresses the growth and invasion of glioma cells in vitro To investigate the biological effect of ATDC in glioma cells, we performed loss-of-function experiments of ATDC in A172 and U118 cells, which showed higher expression of ATDC. Transfection of ATDC shRNA expressing vector significantly downregulated the expression of ATDC in A172 and U-118 cells (Fig. 2A and B). We then detected the effect of ATDC knockdown on the growth of glioma cells by CCK-8 and colony formation assays. The CCK-8 results showed that ATDC knockdown resulted in a significant decrease in glioma cell proliferation (Fig. 2C and D). In addition, ATDC knockdown suppressed the colony-formation ability of A172 and U-118 cells (Fig. 2E and F). Moreover, the invasive ability of A172 and U-118 cells was also markedly decreased by ATDC knockdown (Fig. 2G and H). These results suggest a tumor suppressive effect of ATDC knockdown by inhibiting the growth and invasion of glioma cells in vitro.
3.6. Inhibition of β-catenin suppresses ATDC-mediated oncogenic effects To confirm activation of Wnt/β-catenin signaling contributes to ATDC-mediated oncogenic effects, we examined the effect of the inhibition of Wnt/β-catenin signaling on ATDC-induced cell proliferation and invasion. We showed that transfection of β-catenin shRNA vector significantly decreased the expression of β-catenin in ATDC expression vector-transfected cells (Fig. 6A). Moreover, ATDC-induced upregulation of TCF transcriptional activity was significantly abolished by βcatenin knockdown (Fig. 6B). As expected, blocking Wnt/β-catenin signaling partially reversed the oncogenic effect of ATDC on promoting glioma cell proliferation and invasion (Fig. 6C–E). These results suggest that ATDC promotes the proliferation and invasion through regulating Wnt/β-catenin signaling.
3.3. ATDC overexpression promotes the growth and invasion of glioma cells in vitro To confirm the biological function of ATDC in regulating glioma cell growth and invasion, we performed gain-of-function experiments of ATDC in U-87 and SW-1088 cells, which showed lower expression of ATDC. The overexpression of ATDC in ATDC expression vector-transfected cells was confirmed by RT-qPCR and Western blot (Fig. 3A and B). We found that ATDC overexpression significantly promoted the proliferation and colony formation of U-87 and SW-1088 cells (Fig. 3C–F). Additionally, overexpression of ATDC upregulated the
4. Discussion The overexpression of ATDC has been found to be associated with many types of human cancers. However, little is known about the role of ATDC in glioma. The present study for the first time reports an important role of ATDC in glioma. Our findings demonstrate that ATDC is 150
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Fig. 2. ATDC knockdown inhibited glioma cell growth and invasion. A172 and U-118 cells were transfected with ATDC shRNA vector and incubated for 48 h. (A) Relative ATDC mRNA expression was detected by RT-qPCR. (B) Protein expression of ATDC was determined by Western blot. (C) Effect of ATDC knockdown on A172 cell proliferation was detected by CCK-8 assay. (D) Effect of ATDC knockdown on U-118 cells was assessed by CCK-8 assay. (E, F) Effect of ATDC knockdown on colony formation of A172 and U-118 cells was evaluated by colony formation assay. (G, H) Effect of ATDC knockdown on invasive ability of A172 and U-118 cells was examined by transwell invasion assay. *p < 0.05 vs. control shRNA.
and prognosis. ATDC promotes the growth and metastasis of pancreatic cancer in vitro and in vivo [14]. Moreover, overexpression of ATDC in Kras-mice accelerates the formation of pancreatic intraepithelial neoplasia and the development of invasive and metastatic cancers in vivo [24]. In addition, multiple evidence shows that ATDC drives the proliferation, migration, and invasion of various cancers, including lung cancer, osteosarcoma, colorectal cancer, and bladder cancer [11,17,29–31]. Notably, ATDC has radioprotective function, which promotes radioresistance through an interaction with RNF8 ubiquitin ligase [5,32,33]. ATDC reportedly inhibits ionizing radiation-induced DNA damage by assembling DNA repair proteins into damaged chromatin [34]. Interestingly, knockdown of ATDC increases the chemosensitivity of lung cancer cells to cisplatin [35]. Collectively, all these findings suggest an oncogenic role of ATDC in tumorigenesis. Consistently, our results showed a high expression pattern of ATDC in glioma cell lines and demonstrated that ATDC promoted the growth and
upregulated in glioma cells, and functional experiments showed that knockdown of ATDC suppresses the growth and invasion of glioma cells; while overexpression of ATDC promotes glioma cell growth and invasion of glioma cells, indicating an oncogenic role of ATDC in glioma. Moreover, we elucidated that the underlying mechanism is associated with its regulatory effect on Dvl2, which promotes the accumulation of β-catenin and activation of TCF-dependent transcription (Fig. 7). ATDC plays an important role in various biological processes, such virus infection and immune response [25–27]. Particularly, the role of ATDC in tumorigenesis has been highlighted in recent years [9,10]. High expression levels of ATDC have been detected in tumor tissues of various cancers, including gastric cancer, esophageal squamous cell carcinoma, and pancreatic cancer, and it correlates with tumor progression, lymph node metastasis, and a lower survival rate [12,13,28]. Thus, ATDC is suggested as a potential biomarker for cancer diagnosis 151
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Fig. 3. ATDC overexpression promoted glioma cell growth and invasion. U-87 and SW-1088 cells were transfected with ATDC expression vectors and incubated for 48 h. (A) Relative ATDC mRNA expression was detected by RT-qPCR. (B) Protein expression of ATDC was determined by Western blot. (C) Effect of ATDC overexpression on U-87 cell proliferation was examined by CCK-8 assay. (D) Effect of ATDC overexpression on SW-1088 cell proliferation was detected by CCK-8 assay. (E, F) Effect of ATDC overexpression on colony formation of U-87 and SW1088 cells was assessed by colony formation assay. (G, H) Effect of ATDC overexpression on invasive ability of U-87 and SW-1088 cells was measured by transwell invasion assay. *p < 0.05 vs. vector.
Fig. 4. ATDC regulates Wnt/β-catenin signaling in glioma cells. (A) Effect of ATDC knockdown on βcatenin expression was detected by Western blot in A172 and U-118 cells. *p < 0.05 vs. control shRNA. (B) Effect of ATDC knockdown on TCF transcriptional activity was determined by β-catenin-responsive TOPflash reporter assay. *p < 0.05 vs. control shRNA. (C) Effect of ATDC overexpression on β-catenin expression was examined by Western blot in U87 and SW-1088 cells. *p < 0.05 vs. vector. (D) Effect of ATDC overexpression on TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. *p < 0.05 vs. vector.
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Fig. 5. ATDC promotes Wnt/β-catenin signaling via upregulation of Dvl2 expression. (A) Effect of ATDC knockdown on Dvl2 expression was detected by Western blot in A172 and U-118 cells. *p < 0.05 vs. control shRNA. (B) Effect of ATDC overexpression on Dvl2 expression was detected by Western blot in U-87 and SW1088 cells. *p < 0.05 vs. vector. (C) Western blot detection of Dvl2 expression in U-87 and SW-1088 cells cotransfected with ATDC expression vector and Dvl2 shRNA expression vector. (D) Effect of Dvl2 knockdown of ATDC-induced β-catenin expression was detected by Western blot. (E) Effect of Dvl2 knockdown on ATDCinduced TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. *p < 0.05.
inhibitory effect on the tumor suppressors p53 and p63 [16,39,40]. In addition, ATDC is also involved in activation of NF-κB, extracellular signal-regulated protein kinase/c-Jun N-terminal kinase, and Janus kinase 2/signal transducer and activator of transcription 3 signaling pathways that are oncogenic in tumor progression [29,41,42]. Notably, high expression of ATDC drives the activation of the oncogenic Wnt/βcatenin signaling pathway [14,24,43]. ATDC expression is positively correlated with abnormal expression of β-catenin in squamous cell carcinoma tissues of non-small cell lung cancer [44]. Interestingly, ATDC is shown to promote β-catenin accumulation via induction of Dvl2, which inhibits GSK-3β-mediated β-catenin degradation [14]. However, whether ATDC exerts oncogenic function in glioma through mediating Wnt/β-catenin signaling remains unclear. Herein, we found that ATDC promotes the accumulation of β-catenin and increases the activation of Wnt/β-catenin signaling. Importantly, we identified that the underlying mechanism was associated with its regulatory effect on Dvl2. Dvl2 is a key component of the Wnt signaling pathway, which binds to the Axin/GSK-3β complex and restricts GSK-3β-dependent phosphorylation and degradation of β-catenin. Study shows that Dvl2 is overexpressed in human glioma and promotes the proliferation of glioma cells [45]. Our results indicate that ATDC contributes to the regulation of Dvl2 expression in glioma. Importantly, we demonstrated that inhibition of Dvl2 or β-catenin significantly reversed the oncogenic effect of ATDC in glioma cells, implying that the ATDC/Dvl2/β-catenin axis is involved in regulating glioma cell growth and invasion. In conclusion, our results demonstrate that ATDC promotes the
invasion of glioma cells in vitro, supporting an oncogenic role of ATDC. Our study highlights an important role of ATDC in tumorigenesis and suggests ATDC as a potential therapeutic target for cancer treatment. Although the oncogenic effect of ATDC receives wide attention, a tumor suppressive role of ATDC is also reported by several studies. It is reported that ATDC is undetectable in multiple cancer cell lines, including osteosarcoma, breast cancer, and retinoblastoma [36]. Overexpression of ATDC inhibits the colony-forming efficiency of these cancer cell lines in soft agar in vitro [36]. ATDC expression is decreased in primary breast tumor tissues and reduced ATDC expression is associated with increased tumor size, grade, and metastatic characteristics and reduced relapse-free survival [37]. ATDC suppresses the invasive behavior of breast cancer by inhibiting TWIST1, a critical driver of the epithelial-mesenchymal transition [37]. In addition, loss of ATDC expression promotes the malignant transformation of normal breast luminal cells and contributes to breast cancer progression in premenopausal women [38]. Therefore, these findings indicate that ATDC modulates breast cancer progression in a tumor-suppressive mechanism distinct from its regulatory mechanism in regulating oncogenic progression. ATDC has been shown to regulate tumor progression through intervening in a diverse number of signaling pathways. ATDC exerts its oncogenic effects in bladder cancer by silencing of the tumor suppressor PTEN via a series of signal transductions [23]. Thereby, ATDC promotes the activation of PI3K/Akt signaling, the downstream target of PTEN [15]. Moreover, the oncogenic role of ATDC is also associated with its 153
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Fig. 6. Knockdown of β-catenin abrogates ATDC-mediated oncogenic effects in glioma cells. U-87 and SW-1088 cells were cotransfected with ATDC expression vector and β-catenin shRNA vector and incubated for 48 h. (A) Protein expression of β-catenin was examined by Western blot. (B) TCF transcriptional activity was measured by β-catenin-responsive TOPflash reporter assay. (C) Effect of β-catenin knockdown on ATDC-induced glioma cell proliferation was detected by CCK-8 assay. (D, E) Effect of β-catenin knockdown on ATDC-induced glioma cell invasion was measured by transwell invasion assay. *p < 0.05.
as a promising target for the development of novel therapeutic approaches for gliomas. Conflicts of interest The authors declare that they have no conflict of interest. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.cbi.2019.03.033. References [1] D. Ricard, A. Idbaih, F. Ducray, M. Lahutte, K. Hoang-Xuan, J.Y. Delattre, Primary brain tumours in adults, Lancet 379 (2012) 1984–1996. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA A Cancer J. Clin. 68 (2018) 7–30. [3] D.L. Schonberg, D. Lubelski, T.E. Miller, J.N. Rich, Brain tumor stem cells: molecular characteristics and their impact on therapy, Mol. Aspect. Med. 39 (2014) 82–101. [4] S. Hatakeyama, TRIM family proteins: roles in autophagy, immunity, and carcinogenesis, Trends Biochem. Sci. 42 (2017) 297–311. [5] E.A. Leonhardt, L.N. Kapp, B.R. Young, J.P. Murnane, Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC), Genomics 19 (1994) 130–136. [6] S. Hatakeyama, TRIM proteins and cancer, Nat. Rev. Canc. 11 (2011) 792–804. [7] L.N. Kapp, R.B. Painter, L.C. Yu, N. van Loon, C.W. Richard 3rd, M.R. James, D.R. Cox, J.P. Murnane, Cloning of a candidate gene for ataxia-telangiectasia group D, Am. J. Hum. Genet. 51 (1992) 45–54. [8] Y. Hosoi, L.N. Kapp, Expression of a candidate ataxia-telangieetasia group D gene in cultured fibroblast cell lines and human tissues, Int. J. Radiat. Biol. 66 (1994) S71–S76.
Fig. 7. Diagram representation of ATDC-mediated Dvl2/β-catenin/TCF signaling in glioma cells. High expression of ATDC upregulates Dvl2 expression, resulting in accumulation of β-catenin in the nucleus and subsequent activation of TCF-dependent transcription, which contributes to the development and progression of glioma.
growth and invasion of glioma cells through upregulating Dvl2 and βcatenin expression, indicating an important role of ATDC in regulating Wnt/β-catenin signaling in glioma. These findings of the present study provide novel mechanistic insights into understanding the function of ATDC in glioma cell growth and invasion. Therefore, ATDC may serve 154
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